] Interactive Fly, Drosophila

Dynein heavy chain 64C


ZW10 helps recruit dynactin and dynein to the kinetochore

Mutations in the Drosophila melanogaster zw10 gene, which encodes a conserved, essential kinetochore component, abolish the ability of dynein to localize to kinetochores. Several similarities between the behavior of ZW10 protein and dynein further support a role for ZW10 in the recruitment of dynein to the kinetochore: (a) in response to bipolar tension across the chromosomes, both proteins mostly leave the kinetochore at metaphase, when their association with the spindle becomes apparent; (b) ZW10 and dynein both bind to functional neocentromeres of structurally acentric minichromosomes; and (c) the localization of both ZW10 and dynein to the kinetochore is abolished in cells mutant for the gene rough deal. ZW10's role in the recruitment of dynein to the kinetochore is likely to be reasonably direct, because dynamitin, the p50 subunit of the dynactin complex, interacts with ZW10 in a yeast two-hybrid screen. Since in zw10 mutants no defects in chromosome behavior are observed before anaphase onset, these results suggest that dynein at the kinetochore is essential for neither microtubule capture nor congression to the metaphase plate. Instead, dynein's role at the kinetochore is more likely to be involved in the coordination of chromosome separation and/or poleward movement at anaphase onset (Starr, 1998).

Dynein-mediated cargo transport in vivo: a switch controls travel distance

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Mutations in the dynein heavy chain gene have been used to impair the motor’s function, and biophysical measurements have been employed to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. The progeny of mothers carrying hypomorphic mutations in Dhc64C, the single gene for the heavy chain of cytoplasmic dynein, were examined. Although all known mutations in Dhc64C are homozygous lethal, a combination of two weak alleles supports development to fertile adults. Embryos laid by these mothers develop seemingly normally through cellularization and early gastrulation. However, global transport of lipid droplets is disrupted in the Dhc64C mutant embryos. In the wild type, there are three phases of net droplet transport. At the syncytial blastoderm stage (phase I), droplets move bidirectionally, but show no net displacement. During early cycle 14 (phase II), lipid droplets display net plus end transport, whereas at the onset of gastrulation (phase III) they undergo net minus end transport. In Dhc64C mutant embryos during phase II, droplets accumulate in the center, towards the plus end of microtubules, similar to wild type. However, they fail to redistribute toward the minus ends (the periphery) during gastrulation. This depletion of lipid droplets in the periphery results in embryos that are abnormally transparent from gastrulation onward, reminiscent of the failure of net minus end droplet transport due to mutations in the klarsicht (Welte, 1998). This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows the quantitation of dynein-mediated cargo motion in vivo, providing a framework for investigating how the activity of dynein is controlled. Three distinct travel states whose general features also characterize plus end motion are identified. These states are preserved in different developmental stages. For each travel direction, single droplets are moved by multiple motors of the same type. Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, the existence of a process is proposed that ends runs before the motors fall off the microtubules. This process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport (Gross, 2000).

What type of process might end runs before the inherent motor processivity becomes limiting? A study of the in vitro behavior of motors suggests one model: namely, that reversals in direction, and thus end of runs, result from competition between opposite polarity motors. In these experiments, simultaneous activity of dynein and kinesin in a microtubule gliding assay result in back and forth motion of microtubules, with sharp reversals of direction. The underlying mechanism is unclear, but apparently involves simultaneous activity of both types of motors; relative to gliding driven by any one class of motors, velocity decreases by a factor of two if both motors are present. This situation likely does not apply to reversals following long-fast travel of lipid droplets, where plus and minus end motors do not appear to be active simultaneously. Thus, a novel mechanism must be responsible for determining travel distance on lipid droplets. The analysis presented in this study allows some of its properties to be characterized. The fact that the long-fast travel distances are described by a single exponential distribution suggests that the probability of ending a run remains constant during travel. This observation is not consistent with a mechanism that measures travel distances, e.g., one for which the likelihood of a run ending increases when the droplet has traveled a certain distance. Rather, it suggests that the mechanism that terminates a run is governed by a single rate-limiting step. When runs end, are motors simply detached from their track? In that case, runs should typically be followed by the droplet diffusing away or pausing. However, such diffusion is almost never seen and pauses are rare. Instead, when droplets reverse direction of travel, they move in the opposite direction without delay, as if the motor for the opposite direction of travel becomes active as soon as the activity of the other motor ends. This observation suggests that the activity of the opposing motors is closely linked. Therefore, it is proposed that the process responsible for ending runs is a switch, which both coordinates plus and minus end motors, and determines when runs end. The motor for plus end travel has not yet been identified. Thus, it cannot be determined if long-fast plus end runs are also shorter than predicted from motor processivity in vitro. However, because this travel state is described by a single exponential distribution and is followed by immediate reversals, it seems likely that it is also governed by a switch. Since the probability for long-fast plus end travel terminates changes during development, and since such changes control the direction of net droplet transport, the cell can apparently regulate the properties of these switches (Gross, 2000).

Dynein, klarsicht and nuclear migration within differentiating cells of the Drosophila eye

The temporally regulated, cell-type-specific transport of organelles has great biological significance, yet little is known about the regulation of organelle transport during development. The Drosophila gene klarsicht is required for temporally regulated lipid droplet transport in developing embryos and for the stereotypical nuclear migrations in differentiating cells of the developing eye. Klarsicht is thought to coordinate the function of several molecular motors bound to a single lipid droplet or to facilitate the attachment of dynein to the cargo, but it is not known whether Klarsicht affects motors directly or indirectly. The klarsicht gene has been cloned and shown to encode a unique large protein. Drosophila klarsicht null mutants are viable, with obvious defects only in adult eye morphology. Epitope-tagged Klarsicht expressed in the eye from a transgene is perinuclear. In flies carrying transgenes that express markers for microtubule plus and minus ends, microtubules in differentiating cells of the eye are oriented with their plus ends apical and their minus ends at the nucleus. Drosophila klarsicht null mutants are viable and fertile, demonstrating that klarsicht is essential only for specific motor protein functions. Perinuclear localization of Klarsicht protein indicates that Klarsicht has a direct mechanical role in nuclear migration. Taken together with the finding that the minus ends of the microtubules are associated with the photoreceptor nuclei, the observation that Klarsicht is largely perinuclear supports the idea that Klarsicht associates with dynein, consistent with a model in which Klarsicht assists dynein in 'reeling in' the nucleus (Mosley-Bishop, 1999).

Flies expressing the assembled cDNA klar1 in particular patterns (some transformant lines with two copies of glrs-klar1 (expressed in all cells posterior to the furrow) or one of the three UAS-klar1 lines driven by elav-Gal4, i.e. expressed in all R cells posterior to the furrow, or GMR-Gal4, i.e. expressed in all cells posterior to the furrow) show a rough external eye phenotype. In the retinas of these adult eyes, many facets appear to lack some photoreceptors). As elav-Gal4;UAS-klar1 expresses klar1 only in the R cells, the rough phenotype, at least in this case, is due to overexpression of klar1 in R cells rather than to expression outside R cells. The rough eye phenotypes of the transformants in which klar1 was overexpressed in R cells (glrs-klar1 and elav-Gal4; UAS-klar) were not due to the failure of the initial apical nuclear migration in developing photoreceptors or cone cells posterior to the furrow. Thus, overexpression of klar1 in photoreceptors results in a mutant phenotype that is unlike the klar loss-of-function phenotype and that may not have involved nuclear migration. It is proposed that the mutant phenotype is most likely due to the sequestration of proteins with which Klar normally interacts, thus preventing them from performing the functions that they have independent of Klar and nuclear migration. The glrs-klar1 and UAS-klar1 flies provide useful tools for performing genetic screens to identify genes encoding proteins that interact with Klar (Mosley-Bishop, 1999).

In order to understand the mechanism of Klar function, its subcellular localization in larval eye discs was investigated. Flies were transformed with a glrs-Myc6-klar1 construct, in which the glrs promoter drives the expression of klar1 with a Myc6 epitope tag located just downstream of the start codon. One copy of the transgene rescues the mutant phenotype of klarmCD4 homozygotes completely. Thus, the Myc6-Klar1 protein is functional and at least some of the protein must be localized similarly to endogenous Klar. The Myc6-Klar1 protein is detected perinuclearly (Mosley-Bishop, 1999).

Mechanisms for moving nuclei are thought be different from those that move other organelles; unlike other organelles, nuclei are usually associated with the microtubule-organizing center and thus are essentially stuck to the minus ends of microtubules. In yeast and filamentous fungi, genetic and biochemical experiments have shown that, like lipid droplet transport, nuclear transport requires dynein and microtubules. There are two simple models, generated from the results of experiments on nuclear migration in fungi, that could explain how photoreceptor nuclei might carry out their apical migration upon specification. In the 'reeling-in' model, dynein becomes attached to the apical surface of photoreceptor cells and moves along the microtubules toward their minus ends, located at the nuclei, thereby reeling in the nucleus to the apical surface of the cell. The alternative 'walking-up' model proposes that the nucleus becomes disassociated from the microtubule-organizing center and the microtubules are organized in developing photoreceptors such that their minus ends are apical. In this case, dynein, as it moves up toward the minus ends of microtubules, could transport the nucleus apically as it would any other organelle, like a lipid droplet (Mosley-Bishop, 1999).

Assuming that, as in lipid transport, Klar functions directly with dynein in nuclear transport, the fungal models for nuclear migration make distinct predictions about microtubule organization and Klar localization in the eye. The reeling-in hypothesis predicts that microtubules should be oriented with their plus ends apical and their minus ends attached to the nucleus. Also, at least some Klar should be localized apically in the cell, perhaps helping to tether dynein to the apical cell surface or to otherwise aid its function there. Conversely, the walking-up model predicts that microtubules would be oriented with their minus ends apical and that Klar should be associated with the nucleus, where it would play the same role proposed for its function in lipid droplet transport (Mosley-Bishop, 1999).

The results do not fall neatly into either set of predictions. Although it is found that the microtubules are oriented with their plus ends apical and their minus ends associated with the nucleus, as predicted by the reeling-in model, Klar mainly is associated with the nucleus, as predicted by the walking-up model. The results do suggest, however, that Klar associates with dynein and do not rule out the existence of a small amount of Klar protein (and dynein) tethered to the apical surface that could reel in the nucleus. Further experiments in which Myc6-Klar is expressed in the eye using promoters that are active at high levels in R cells before the apical migration of their nuclei may enable clear detection of any apical Klar protein. Similarly, the method for detection of Nod-beta-gal and kinesin-beta-gal proteins would not reveal small subpopulations of microtubules that run counter to the array. Further studies to determine the subcellular localization of other components of the nuclear migration pathway, including dynein and kinesin, as well as to identify other molecules that interact with Klar should help to distinguish among the possible models (Mosley-Bishop, 1999).

Guidance of bidirectional motor complexes by mRNA cargoes through control of dynein number and activity

During asymmetric cytoplasmic mRNA transport, cis-acting localization signals are widely assumed to tether a specific subset of transcripts to motor complexes that have intrinsic directionality. This study provides evidence that mRNA transcripts control their sorting by regulating the relative activities of opposing motors on microtubules. In Drosophila embryos it is shown that all mRNAs undergo bidirectional transport on microtubules and that cis-acting elements produce a range of polarized transcript distributions by regulating the frequency, velocity, and duration of minus-end-directed runs. Increased minus-end motility is dependent on the dosage of RNA elements and the proteins Egalitarian (Egl) and Bicaudal-D (BicD). These proteins, together with the dynein motor, are recruited differentially to different RNA signals. Cytoplasmic transfer experiments reveal that, once assembled, cargo/motor complexes are insensitive to reduced cytoplasmic levels of transport proteins. Thus, the concentration of these proteins is only critical at the onset of transport. This work suggests that the architecture of RNA elements, through Egl and BicD, regulates directional transport by controlling the relative numbers of opposite polarity motors assembled. The data raise the possibility that recruitment of different numbers of motors and regulatory proteins is a general strategy by which microtubule-based cargoes control their sorting (Bullock, 2006).

mRNA localization signals modulate the kinetics of microtubule-based transcript movements. In mammalian cells, uniformly distributed mRNAs undergo short, unidirectional transport events that are augmented in duration and frequency by the RNA signal from the localizing β-actin transcript. In Drosophila syncytial blastoderm embryos, individual nucleotide changes in the hairy (h) transcript slow the rate of delivery of injected fluorescently labeled mRNAs to the apical cytoplasm. However, it is unclear whether localization elements simply limit dissociation of mRNAs from their RNA binding protein(s), and hence the microtubule, or actively regulate motor movement (Bullock, 2006).

To investigate whether mRNA cargoes regulate the movement of motors on microtubules, improved microscopy and automatic tracking software has been used to analyze the detailed movements of mRNAs in the Drosophila blastoderm. Here, the microtubules have a stereotypical arrangement, with minus ends nucleated apically and plus ends extending basally (Bullock, 2006).

Particles of injected, fluorescently labeled h mRNA are transported bidirectionally, undergoing relatively long runs in the minus-end direction interspersed with shorter reversals (plus-end runs), as well as periods of little or no persistent movement (pauses). The net rate of apical transport of the h mRNA particles is ~150 nm/s, with active transport in both directions reaching velocities of up to ~1–1.5 μm/s (Bullock, 2006).

The characteristics of mRNA motion are reminiscent of those of other bidirectional cargoes. Firstly, the distances of runs in both directions approximate a decaying exponential distribution, as if each opposing motor activity ceases productive cargo transport due to a constant-probability event. Secondly, particles frequently undergo rapid switching between minus- and plus-end motility. This suggests that the mRNA binds opposite polarity motors at all times, with the net distribution determined by differences in their relative activities (Bullock, 2006).

It is not clear how control of net transport of other bidirectional cargoes is achieved, although opposing motor activities appear to be mutually dependent. Indeed, inhibition of the minus-end-directed motor dynein, which is required for apical mRNA localization, by preinjection of anti-dynein intermediate chain (Dic) antibodies or the dynein inhibitor vanadate, inhibits motility of h mRNA in both directions. The identity of the motor engaged during plus-end movement is not known, and it could even be dynein; recent work suggests that the motor with its accessory complex dynactin can undergo bidirectional motion on a single microtubule in vitro (Bullock, 2006).

To address the mechanistic role of apical localization signals, the behavior of h transcripts was contrasted to those of Krüppel (Kr), which are distributed evenly in the cytoplasm, or several heterologous mRNAs such as those derived from transcription of a plasmid backbone. Neither the Kr nor heterologous mRNA populations become enriched apically following injection, but each of these mRNAs rapidly assembles into particles with a spectrum of sizes similar to those of h. Surprisingly, the nonlocalizing transcripts undergo short bidirectional movements that initiate with similar kinetics to localizing mRNAs (Bullock, 2006).

The movements of the nonlocalizing transcript populations are indistinguishable from one another and are clearly motor driven; like those of h, persistent movements in both directions frequently reach velocities of ~1–1.5 μm/s and are sensitive to anti-Dic (anti-dynein intermediate chain) injection, as well as to hypomorphic mutations in the gene encoding dynein heavy chain (dhc64C). Consistent with a physiological function of bidirectional transport in achieving uniform spreading of mRNAs, endogenous Kr transcripts are retained in the perinuclear region upon injection of anti-Dic antibodies. Transport of uniform mRNAs presumably facilitates encounters with other posttranscriptional machinery (Bullock, 2006).

The ability of localizing and nonlocalizing mRNAs to undergo bidirectional transport led to an examination of which aspects of motion are significant for determining their different net distributions. Compared to nonlocalizing transcripts, apically localizing mRNAs spend more time undergoing minus-end transport and less time moving in a plus-end direction or pausing. There is a ~2.5-fold increase in the mean distance of minus-end runs of h compared to nonlocalizing mRNAs. This does not reflect increased dissociation of nonlocalizing mRNAs from recognition factors; for all RNAs tested, the distribution of plus-end run lengths is indistinguishable. Furthermore, minus-end runs are often immediately followed by reversals, which implies that mRNAs remain associated with a microtubule (Bullock, 2006).

Localizing mRNAs also have a subtle, but significant, increase in mean velocity of minus-end transport (10%–15%) compared to nonlocalizing RNAs, whereas plus-end velocity is not significantly different for all mRNAs tested. Localizing mRNAs are also ~1.4 times more likely than nonlocalizing mRNAs to undergo a minus-end run instead of a plus-end run following a pause. In contrast, the likelihood of a minus-end run or pause following a plus-end run is similar for localizing and nonlocalizing mRNAs (Bullock, 2006).

Overall, these data indicate that the h RNA localization signal is not obligatory for linking mRNAs to molecular motors. Instead, it gives rise to net apical localization by increasing the probability of initiation and maintenance of rapid minus-end-directed excursions of a bidirectional motor complex (Bullock, 2006).

To address whether localization signals are binary switches, the effects were tested of altering the sequence of the h element upon mRNA movement. The weak localizing h transcript 1328A→U—which has a mutated base in the first of two stem loops (SL1 and SL2a) that comprise the localization signal undergoes slightly longer runs in the minus-end direction than nonlocalizing transcripts, whereas plus-end run lengths are the same as those of nonlocalizing and wild-type h transcripts. This mode of localization is probably employed by certain endogenous mRNAs. For instance, endogenous ken transcripts are only partially enriched apically, and injected ken localizes with kinetics indistinguishable from those of h1328A→U. Conversely, replacing the h localization signal with three copies of SL1 (hSL1x3) leads to apical accumulation ~2-fold faster than h due to significant increases in the initiation, velocity, and maintenance of minus-end runs. Plus-end motility of hSL1x3 is indistinguishable from that of the other transcripts tested. These findings indicate that mRNA sequences can generate a range of motile behaviors of bidirectional transport complexes. (Bullock, 2006).

To investigate how mRNA signals regulate minus-end transport, the potential roles of Egalitarian (Egl) and Bicaudal-D (BicD) were investigated. These proteins are components of a complex required for accumulation of several mRNAs at the minus end of microtubules in Drosophila, and their recruitment to an injected localizing transcript population can be observed above normal cytoplasmic levels. Egl binds directly to Dynein light chain (Dlc), and mammalian BicD associates with components of the dynein and dynactin complexes and recruits membranous vesicles for transport (Bullock, 2006).

It is not possible to characterize transport in egl or BicD null embryos because both factors have earlier essential functions. However, hypomorphic mutations in egl or BicD strongly reduce duration and velocity of minus-end, but not plus-end, runs of both wild-type and h1328A→U mutant particles. Weak apical accumulation of endogenous ken transcripts is also abolished by these mutations (Bullock, 2006).

In order to test the full requirements for Egl and BicD in transport of mRNAs, embryos were preinjected with blocking antibodies specific to each protein. Antibodies to either protein block net asymmetric movement of mRNAs. However, whereas anti-Dic or vanadate injection results in very little movement of mRNAs, anti-Egl- or anti-BicD-injected embryos frequently display short runs of localizing and nonlocalizing mRNAs in both directions. This limited motility does not result from residual protein activity because it cannot be significantly reduced by injecting the antibodies into partial loss-of-function embryos or by coinjecting the two antibodies. Thus, Egl or BicD is not obligatory for linking mRNAs to a motor, which is compatible with the impairment of apical mRNA anchorage upon inhibition of dynein, but not Egl or BicD (Bullock, 2006).

Inhibition of Egl and BicD modulates the transitions between pauses, minus-end runs, and plus-end runs similarly to perturbation of localization signals. Thus, like RNA localization signals, Egl and BicD promote the initiation and maintenance of rapid minus-end-directed movement of mRNAs along microtubules (Bullock, 2006).

Nonlocalizing transcripts can, however, make use of the Egl/BicD machinery very occasionally; a small subset of Kr and plasmid mRNA particles undergo relatively long minus-end-directed runs that are sensitive to inhibition of either protein (Bullock, 2006).

The data demonstrate that Egl and BicD augment minus-end-directed movements of mRNAs on microtubules. Interestingly, the frequency, speed, and duration of minus-end runs are significantly reduced when the level of wild-type BicD protein is reduced to ~6% of normal (BicDHA40/r5), indicating that BicD has concentration-dependent roles in regulating motility. Indeed, overexpression of BicD results in more efficient apical transport of injected h. Egl and its binding protein Dlc also function dose-dependently; minus-end motility of h on microtubules is significantly increased upon overexpression of either protein and reduced by halving egl gene dosage (Bullock, 2006).

Interestingly, elevating Egl, BicD, or Dlc levels augments minus-end motility in different ways. Very similar to increasing the number of localization elements, overexpression of Egl increases minus-end run length and minus-end velocity and modulates the transitions between travel states. In contrast, overexpressing BicD only modulates transitions between travel states, and increasing Dlc levels only increases minus-end run length and minus-end velocity. Egl might therefore function to independently recruit the activities of BicD and Dlc to mRNA signals. Indeed, different domains of Egl mediate association with these two proteins. Consistent with a concentration-dependent role of transport proteins during localization of endogenous mRNAs, there is also a subtle increase in the apical enrichment of endogenous uniform mRNAs upon Egl or Dlc overexpression. Strikingly, there is only a 2- to 2.5-fold increase in levels of Egl upon its overexpression in all of the experiments. Thus, the distinction between net symmetric and asymmetric transcript distribution could reflect subtle differences in the affinities of mRNAs for rather nonselective recognition factors (Bullock, 2006).

Egl, BicD, and Dlc levels could be important for minus-end motility because mRNAs dissociate from them during transit and efficient transport requires reassociation, or because of a function for different numbers of molecules in the transport complex from the outset. To discriminate between these two possibilities, an investigation was carried out to see whether mRNAs assembled in an environment where there is sufficient BicD are sensitive to a subsequent drop in the levels of BicD in the cytoplasm. This is not the case; h transcripts injected into an embryo overexpressing BicD and then withdrawn ~1 min later continue to be transported efficiently through the cytoplasm of BicDHA40/r5 acceptor embryos in which BicD levels are otherwise limiting (Bullock, 2006).

This finding is associated with neither diffusion of the BicD from the donor embryo following transplantation, confirmed using an epitope tag specific to the overexpressed BicD, nor the transplantation procedure, because mRNA transferred either between BicDHA40/r5 or between wild-type embryos behaves similarly to when transcripts are simply injected into these genotypes. Likewise, the movement of h particles exposed to cytoplasm overexpressing Egl or Dlc is not sensitive to the drop in the concentration of these proteins upon transfer to a wild-type embryo (Bullock, 2006).

These experiments indicate that the only point at which levels of these three proteins is critical is at the initial assembly of transport complexes and suggest that different RNA signals modulate motor activity in a perduring fashion by recruiting different numbers of Egl, BicD, and dynein molecules to each mRNP. Indeed, both Egl and BicD are found complexed with other molecules of themselves in vivo. Higher-order assemblies of dynein also exist in the embryo; measurements of stall forces of minus-end runs of lipid droplets reveal quantized steps of 1.1 pN -- equivalent to that of a single motor -- up to ~6 pN. Furthermore, the increases in both minus-end run length and velocity observed for localizing mRNAs and upon augmenting levels of transport proteins are consistent with observations of increasing numbers of active dyneins working together in vitro (Bullock, 2006).

To directly test whether the extent of minus-end motility of cargoes is associated with the amount of transport proteins nucleated, transcripts of wild-type h or the more efficiently localizing construct hSL1x3 was injected into embryos and the amount of Egl and BicD assembled on individual mRNA particles in transit was assayed. Consistent with such a model, concentration of Egl and BicD above cytoplasmic levels can be observed on many particles of hSL1x3 mRNA, but never on particles of h. Because it is not possible to observe motor components above background levels in these injection experiments, their recruitment to different RNA signals was investigated following incubation with cytoplasmic extracts in vitro. These experiments reveal that localization efficiency of mRNAs does indeed correlate with the amount of dynein components, as well as Egl and BicD, that they assemble (Bullock, 2006).

Together, these data provide evidence of a novel mechanism in which apical localization signals bias bidirectional motor movement by controlling the number of Egl, BicD, and dynein molecules incorporated into each mRNP. Because short-range bidirectional transport can occur in the absence of RNA localization signals, it is envisaged that these signals regulate minus-end motility, at least in part, by recruiting dynein motors in addition to those involved in distributing uniform mRNAs. Egl and BicD could function as adaptors that mediate the association of these additional dyneins with localization signals. Consistent with such a role, tethering mammalian BicD sequences to cargoes is sufficient to stimulate dynein recruitment and transport, and recruitment of Dhc to localizing mRNAs in vitro is reduced, but not abolished, upon immunodepletion of BicD from extracts (Bullock, 2006).

Nonetheless, the finding that increasing levels of Egl, BicD, or Dlc can modulate transport argues against a strict linear pathway of assembly. One intriguing explanation, which could also account for the substantial differences in the relative amounts of Egl and BicD assembled on individual mRNA particles, is that not all of the binding sites within the RNA:motor assembly must be saturated before transport is initiated. Modulating the number of mRNA elements or the concentration of each of the transport proteins could therefore alter the average number of fully functional Egl/BicD/dynein complexes assembled on each mRNP. Such a probabilistic mechanism could also account for the large variation in motile behaviors exhibited by particles of the same mRNA (Bullock, 2006).

Although differential motor recruitment appears to be one important mechanism to generate different classes of mRNA motion, the data hint at the existence of additional regulatory processes. The anti-BicD antibody largely uncouples minus-end run lengths from velocity, and overexpression of BicD alters transitions between travel states, but not run lengths or velocity. Thus, BicD is likely to have additional roles in regulating dynein activity. This could be through its binding to the dynactin complex, which is likely to play a key role in coordinating minus- and plus-end-directed motor activity. Indeed, Egl and BicD levels could regulate the hypothesized switch mechanism that is proposed to coordinate opposite polarity motor activities and determine when runs end (Bullock, 2006).

In many cell types, mRNAs exhibit net transport toward the plus ends of microtubules. These distributions could result from modulation of a related bidirectional transport complex using mRNA elements that preferentially nucleate or stimulate plus-end-directed motor activity. The findings also suggest that different organelles, vesicles, and macromolecules could assume a wide range of polarized distributions within the same cell by balancing opposite polarity motor activities through numerical differences in the same repertoire of transport proteins (Bullock, 2006).

Squid/hnRNP helps Dynein switch from a gurken mRNA transport motor to an ultrastructural static anchor in sponge bodies

In Drosophila oocytes, gurken mRNA localization orientates the TGF-α signal to establish the anteroposterior and dorsoventral axes. This study has elucidated the path and mechanism of gurken mRNA localization by time-lapse cinematography of injected fluorescent transcripts in living oocytes. gurken RNA assembles into particles that move in two distinct steps, both requiring microtubules and cytoplasmic Dynein. gurken particles first move toward the anterior and then turn and move dorsally toward the oocyte nucleus. Evidence is presented suggesting that the two steps of gurken RNA transport occur on distinct arrays of microtubules. Such distinct microtubule networks could provide a general mechanism for one motor to transport different cargos to distinct subcellular destinations (Delanoue, 2007).

This study analyzed the molecular mechanism of grk mRNA transport and anchoring in the Drosophila oocyte using a number of novel methods, combining live cell imaging of oocytes with immunoelectron microscopy to covisualize grk RNA and transacting factors. grk mRNA is transported in particles containing many individual RNA molecules assembled with numerous molecules of Dynein motor components and Squid. Approximately two thirds of transport particles are in close association with MTs and are not consistently associated with membranes, such as ER or vesicles. This supports the idea that grk RNA particles are transported directly by motors on MTs. This notion is strengthened by the fact that the directed movement of the transport particles is disrupted very rapidly when MTs are depolymerized and Dhc, BicD, or Egl function is inhibited. Furthermore, the particles observed moving along MTs in live cell imaging experiments correspond well to the similar-sized grk RNA-rich particles that were visualized by EM. The direct movement of grk RNA particles along MTs is in stark contrast to the transport of yeast ASH1 RNA, which is thought to be cotransported with ER membrane (Delanoue, 2007).

Once delivered to its final destination at the oocyte dorso-anterior corner, many copies of both injected grk RNA and endogenous grk mRNA are anchored in large electron-dense structures previously described as sponge bodies, together with the same components present in the transport particles, including Dynein and Squid. Sponge bodies are distinct in appearance from transport particles and have been previously described in nurse cells and hypothesized to be RNA transport intermediates from the nurse cells to the oocyte. Although Exu-GFP was found in grk anchoring structures, these structures have been identified in the oocyte as functioning in anchoring, rather than transport, and containing components of the Dynein complex Dhc, Egl, and BicD. These data show that the endoplasmic reticulum is not involved in the transport and anchoring of grk mRNA (Delanoue, 2007).

Transport particles and sponge bodies are related to RNA particles (also termed germinal granules, P bodies, and neuronal granules) that display a large spectrum of sizes, composition, and morphology, reflecting several functions in RNA transport, storage, translational control, and processing. Nevertheless, it seems likely that the transport particles that were identified are related to bcd and osk mRNA granules as well as to neuronal RNA granules. The data demonstrate that sponge bodies play key roles in RNA anchoring, but it is not known whether they are also involved in translational control, degradation, and storage of mRNA (Delanoue, 2007).

Dynein is not only present in the sponge bodies but is also required for the static anchoring of grk RNA in the sponge bodies. However, anchoring does not require Egl or BicD, the motor cofactors that are required for RNA transport in the embryo and oocyte. While it is not certain whether Egl and BicD are required for cargo loading, transport initiation, or motor activity itself, the evidence shows that none of these functions are required for anchoring. grk RNA is therefore anchored by a similar mechanism to pair-rule and wingless transcripts in the syncytial blastoderm embryo. In both the embryo and oocyte, it is proposed that when the Dynein motor complex reaches its final destination, the motor becomes a static anchor that no longer depends on the transport activity of the motor (Delanoue, 2007).

Dynein (but not Egl and BicD) is not only a static anchor but is also required for the structural integrity of the grk RNA anchoring structures in the oocyte, the sponge bodies. Their rapid speed of disassembly upon Dynein inhibition (3-5 min) argues that Dynein has a direct role in anchoring and is required to form and maintain the large RNP complexes that constitute the sponge bodies. This evidence rules out that Dynein could indirectly be required for the delivery of anchoring components that are then used in anchoring grk when it is delivered to the sponge bodies. Dynein could also tether the cargo complex directly on the MTs when the transport particles reach their final destination, but the results show that MTs only flank the sponge bodies and are not, as predicted by this model, consistently interdigitated with most of the cargo and motor molecules that are detected in the sponge bodies. This is also consistent with the fact that disassembling MTs does not lead to a change in sponge body structure and only leads to a partial loss of endogenous grk mRNA anchoring (Delanoue, 2007).

In addition to its previously documented role in the second step of grk mRNA transport, a novel function was identified for hnRNP Squid, which plays an essential role in the anchoring of grk RNA at the dorso-anterior corner. Like Dynein, Sqd is also enriched at the site of anchoring upon injection of excess grk RNA. Inactivation of Sqd before transport begins leads to grk transport particles being present at the anterior of the oocyte in permanent anterior flux without anchoring, even for the particles that reach the dorso-anterior corner. Conversely, inactivation of Sqd after grk RNA arrives at the dorso-anterior corner leads to a breakdown of anchoring and the conversion of sponge bodies into anterior transport particles containing grk RNA. This suggests an active role for Sqd in keeping anchoring structures intact, and most likely a role for Sqd in promoting the conversion of transport particles into anchoring structures by facilitating their reorganization into anchoring complexes (Delanoue, 2007).

It is proposed that sponge bodies are assembled at the dorso-anterior corner by delivery of grk mRNA, Dynein motor component and Squid present together in transport particles. First, the same components are present in the transport particles and in the sponge bodies. Second, some transport particles containing endogenous grk mRNA are detected on dorso-anterior MTs. Third, injection of a large excess of grk RNA leads to an increase in the size and number of sponge bodies (Delanoue, 2007).

At the dorso-anterior corner, sponge bodies are maintained by both Dynein and Squid. When the Dynein motor complex reaches its final destination, the motor becomes a static anchor that no longer depends on the transport activity of the motor. Given the size of Dhc and the presence of many putative domains whose function remains elusive but that could play a central role in the switch from motor to anchor, it is proposed that Dhc can associate with many other cellular factors to form a large and immobile anchoring complex. Sqd is known to be involved in translational regulation, and it is proposed that association with this class of factors could help create a large and immobile anchoring complex. A strong link between molecular motors and hnRNPs has already been shown to control the localization of their associated mRNAs. For instance, She2 hnRNP acts as a linker between the Myosin motor complex and its mRNA cargo. Kinesin and hnRNP are present together in RNA-transporting granules in neurons. It is proposed that transport and anchoring of mRNA by molecular motors involve assembly into transport particles followed by reconfiguration of the same components into large electron-dense anchoring complexes at the final destination. Future work will establish how widely this model can be applied. It will also be interesting to determine whether the specificity of transport and anchoring of other RNA cargos in the Drosophila oocyte and embryo is also established by distinct combinations of RNA-binding proteins that influence the function of molecular motors. Time will tell what proportion of mRNA is anchored like pair-rule and grk transcripts by static functions of molecular motors, as opposed to other possible mechanisms of anchoring (Delanoue, 2007).

Localization of Dynein in the Oocyte

The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote, subsequently have been shown to regulate cellular polarity in diverse cell types by means of an evolutionarily conserved protein complex including PAR-3, PAR-6, and atypical protein kinase C (aPKC). The Drosophila homologs of par-1, par-3 (bazooka [baz]), par-6 (DmPar-6), and pkc-3 (Drosophila aPKC; DaPKC) each are known to play conserved roles in the generation of cell polarity in the germ line as well as in epithelial and neural precursor cells within the embryo. In light of this functional conservation, the potential role of baz and DaPKC in the regulation of oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of initial anterior-posterior polarity within germ-line cysts and maintenance of oocyte cell fate. Germ-line clonal analyses indicate both proteins are essential for two key aspects of oocyte determination: the posterior translocation of oocyte specification factors and the posterior establishment of the microtubule organizing center within the presumptive oocyte. Baz and DaPKC colocalize to belt-like structures between germarial cyst cells. However, in contrast to their regulatory relationship in the Drosophila and C. elegans embryos, these proteins are not mutually dependent for their germ-line localization, nor is either protein specifically required for PAR-1 localization to the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).

Oocyte differentiation requires the polarized accumulation of oocyte specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in the localization of these factors, mutant germ-line clones for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst cells in region 2a, and then both molecules are targeted first to the two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In contrast, ORB fails to translocate from the anterior to a posterior crescent in both baz and DaPKC null germ-line cysts in germarial region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in baz and DaPKC null germ-line clones in germarial region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).

Furthermore, in contrast to wild-type germ-line cysts in which DHC64C localizes to a single posterior cell, in DaPKC null germ-line clones DHC64C fails to localize to a single cell posteriorly, but rather accumulates in the two posterior-most presumptive pro-oocytes of the mutant germ-line cyst. Therefore, baz and DaPKC display essentially identical phenotypes in germ-line mutant clones with regards to oocyte differentiation and the establishment of initial A-P polarity within the oocyte. The failure to maintain oocyte identity in either baz or DaPKC mutant cysts can therefore be directly correlated with defects in the A-P translocation of oocyte specification factors within a single posterior cell of a germ-line cyst, suggesting oocyte differentiation depends on this early polarization event (Cox, 2001).

The posterior assembly of a functional MTOC has been directly implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the failure to translocate these factors to a posterior crescent in region 3 baz or DaPKC mutant cysts may result from a defect in microtubule reorganization within these mutant cysts. In contrast to wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of oocyte specification factors in these mutants are likely caused, at least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).

Drosophila Gurken mRNA localizes as particles that move within the oocyte in two Dynein-dependent steps

In Drosophila oocytes, gurken mRNA localization orients the TGF-alpha signal to establish the anteroposterior and dorsoventral axes. The path and mechanism of gurken mRNA localization has been evaluated by time-lapse cinematography of injected fluorescent transcripts in living oocytes. gurken RNA assembles into particles that move in two distinct steps, both requiring microtubules and cytoplasmic Dynein. gurken particles first move toward the anterior and then turn and move dorsally toward the oocyte nucleus. Evidence is presented suggesting that the two steps of gurken RNA transport occur on distinct arrays of microtubules. Such distinct microtubule networks could provide a general mechanism for one motor to transport different cargos to distinct subcellular destinations (MacDougall, 2003).

The organization of MTs in the oocyte was analyzed with high-resolution imaging of Tau-GFP and Nod-LacZ distributions in the oocyte. A particularly high concentration of MT minus ends is detected at the dorsoanterior corner as well as in the anterior cortex and entire anterior. The presence of an MT network associated with the oocyte nucleus explains why a higher concentration of MTs are found in the anterior than in the posterior, despite the diffuse nature of the MTOC in the oocyte. A distinct network of MTs associated with the oocyte nucleus also explains why, in merlin mutant oocytes, injected grk RNA is observed accumulating at the posterior, where the oocyte nucleus is located. A high concentration of MT minus ends is also detected in an anterior ring in addition to a lower concentration all over the anterior. Considering all these results in the context of the published data on MT distribution in the oocyte leads the authors to propose the following model for MT organization in the oocyte. In addition to MTs with their minus ends at the diffuse anterior MTOC and their plus ends at the posterior, there are some other MTs with their minus ends throughout all parts of the cortex. It is proposed that, in addition to these networks, there is also a distinct network of MTs that are specifically associated with the oocyte nucleus. These MTs form a loose basket surrounding the nucleus and radiate throughout the anterior and partly into the middle of the oocyte. Observations of Tau-GFP suggest that there are many other MTs that are more loosely organized throughout much of the oocyte (MacDougall, 2003).

The organization of MTs proposed provides a good explanation for why the grk particle movements occur in two distinct steps. It is proposed that, during the first step of movement of grk particles to the anterior of the oocyte, the RNA is likely to be moving on MTs whose plus ends are at the posterior of the oocyte and whose minus ends are along the entire anterior. The second step of movement of the particles is likely to occur on the MT network that forms a basket around the nucleus, with the MT minus ends at the dorsoanterior corner and the plus ends extending toward the anterior and, also, partly into the middle of the oocyte. This model for MT organization fits well with the fact that many grk RNA particles were observed to make sharp turns at the anterior, and some in the interior, of the oocyte (MacDougall, 2003).

The model showing that distinct classes of MTs exist within the oocyte begs a question: how does Dynein-dependent transport deliver grk RNA to a very different destination from other RNAs in the oocyte, which may also be transported to the minus ends of MTs by Dynein? It is proposed that different RNAs that are transported to the minus ends of MTs by the same Dynein motors could move on distinct networks of MTs. This would explain why the destination of injected bcd RNA (which is thought to require Dynein for its localization), depends on whether it is preexposed to nurse cell cytoplasm. bcd RNA injected into the oocyte moves to the nearest cortex along MTs whose minus ends are at the cortex. However, bcd RNA that is preexposed to nurse cell cytoplasm is able to move from the posterior to the anterior of the oocyte, apparently in a similar route to that in step 1 of grk localization, which has been defined. Step 2 of grk RNA particle movement is not shared with bcd RNA and could occur along the MT network that is specifically associated with the oocyte nucleus. Interestingly, bcd, but not grk, mRNA localization requires gamma-Tub37C and Dgrip75 (MacDougall, 2003 and references therein).

It is most likely that specific transacting factors that recognize RNA signals are responsible for determining which RNAs use which motors and also which distinct MT network is utilized during the Dynein-dependent transport to different destinations. For example, in nerve cells, the choice of cytoplasmic destination of cargo transported by Kinesin is determined by the presence or absence of a protein called GRIP. Such key transacting factors are likely to also include Squid and K10, since, in mutants of these genes, grk mRNA is localized in the anterior, rather than the dorsoanterior corner. However, in addition to the transacting factors, the different MTs are likely to differ in some way, allowing the different kinds of RNA-motor complexes to distinguish among them. Such differences could include chemical modifications of tubulin or different tubulin isoforms as well as distinct populations of MT-associated proteins (MAPs). It is also possible that alphaTub37C and Dgrip75 could be involved in selectively nucleating a subset of MTs used for bcd, but not grk, mRNA localization (MacDougall, 2003 and references therein).

Dynein-dependent motility of RNA and other cargo to the minus ends of MTs is likely to be a widely deployed mechanism within cells. Selective utilization of different MT networks would provide a nice way to sort different cellular components that are transported by the same Dynein motor to a variety of distinct minus ends in the same cell. Rapid and efficient real-time assays for mRNA localization will allow the definition of cis-acting signals and trans-acting factors that determine which subset of MTs are selected by different RNA cargos that utilize the same motors (MacDougall, 2003).

Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes

Mass movements of cytoplasm, known as cytoplasmic streaming, occur in some large eukaryotic cells. In Drosophila oocytes there are two forms of microtubule-based streaming. Slow, poorly ordered streaming occurs during stages 8-10A, while pattern formation determinants such as oskar mRNA are being localized and anchored at specific sites on the cortex. Then fast well-ordered streaming begins during stage 10B, just before nurse cell cytoplasm is dumped into the oocyte. The plus-end-directed microtubule motor kinesin-1 is required for all streaming and is constitutively capable of driving fast streaming. Khc mutations reduce the velocity of kinesin-1 transport in vitro, block streaming, yet still support posterior localization of oskar mRNA -- this suggests that streaming is not essential for the oskar localization mechanism. Inhibitory antibodies indicated that the minus-end-directed motor dynein is required to prevent premature fast streaming, suggesting that slow streaming is the product of a novel dynein-kinesin competition. Since F-actin and some associated proteins are also required to prevent premature fast streaming, these observations support a model in which the actin cytoskeleton triggers the shift from slow to fast streaming by inhibiting dynein. This allows a cooperative self-amplifying loop of plus-end-directed organelle motion and parallel microtubule orientation that drives vigorous streaming currents and thorough mixing of oocyte and nurse-cell cytoplasm (Serbus, 2005).

To address questions about microtubule-based cytoplasmic streaming in Drosophila oocytes, functional disruption approaches were combined with fixed and time-lapse fluorescence microscopy. The results confirm that plus-end-directed kinesin-1 is the primary motor for both slow and fast streaming, and, furthermore, that it is constitutively capable of driving fast streaming. The minus-end-directed motor cytoplasmic dynein does not contribute force for fast streaming; rather, dynein and a normally regulated actin cytoskeleton impede the fast streaming activity of kinesin-1, allowing only slow streaming currents prior to stage 10B (Serbus, 2005).

It is reasonable to assume that the purpose of active but random transport processes like streaming is to facilitate the dispersal of cytoplasmic components that do not diffuse fast enough to support cellular and developmental demands. However, it could also be important for asymmetric localization processes by facilitating encounters of cytoplasmic components with localized anchors. More specific insights into how microtubule-based streaming contributes to particular processes have been elusive, in part because the only means to prevent streaming was to eliminate microtubules, which are needed for many fundamental cellular processes. Identification of kinesin-1 as the motor for streaming in Drosophila provides the opportunity for more focused studies, because kinesin-1 has a narrower range of functions and is not essential for early oocyte development (Serbus, 2005).

The Khc allelic series allowed investigation of the significance of nurse cell/ooplasm mixing. Khc-null oocytes, with no streaming, usually show yolk stratification as evidence of mixing failure. Embryos developing from those oocytes arrest in early stages, suggesting that mixing may be important for subsequent development. However, hypomorphic Khc17 oocytes, which support weak fast streaming in only one-third of oocytes, allow three-fourths of the derived embryos to develop to adulthood. Yolk stratification is not seen in Khc17 oocytes, suggesting that some mixing can occur without ordered streaming. Although these observations are consistent with the hypothesis that vigorous ooplasmic mixing helps optimize development, it is likely that fast streaming is not absolutely essential (Serbus, 2005).

The Khc allelic series also allowed exploration of a role for slow ooplasmic streaming in determinant mRNA localization. The null allele Khc27 prevents streaming: it blocks oskar mRNA accumulation at the posterior pole and it blocks gurken mRNA localization to the anterodorsal corner. However, the hypomorphic alleles Khc17 and Khc23, which prevented most slow streaming, support both oskar and gurken localization. Thus, although localization of both determinants requires Khc, it does not require slow streaming (Serbus, 2005).

It has been suggested that posterior oskar localization during stages 7-10a proceeds via two phases. (1) oskar RNPs are driven by kinesin-1 away from microtubule minus ends at the anterior and lateral cortex, which leads to a transient concentration of oskar in the central region of the oocyte. (2) Then diffusion or other random forces, coupled with a dearth of minus ends at the posterior cortex, facilitates encounters of oskar RNPs with posterior anchors. Tests of Khc17 and Khc23, which slow the ATPase activity and velocity of Khc in vitro, show a delay in the central accumulation of oskar, consistent with slowed kinesin-1-driven transport away from the anterolateral cortex. Strikingly, Khc17 and Khc23 allow that central accumulation to persist through later stages, as if the shift to posterior anchors is also slowed. This correlation between slowed motor mechanochemistry and slowed oskar localization supports the hypothesis that kinesin-1 links to and transports oskar RNPs in both phases of localization (Serbus, 2005).

If microtubules are poorly ordered during oskar localization, as suggested by GFP-tubulin imaging and by studies of fixed oocytes, how could kinesin-1 accomplish such directed posterior transport? There may be a special subset of microtubules, with plus-ends oriented directly toward the posterior pole, that are difficult to distinguish among a mass of randomly oriented microtubules. However, given that the period of oskar localization spans at least 10 hours, and that the distance from the oocyte center to the posterior pole is only 25-40 µm, such perfectly oriented transport tracks should not be necessary. With microtubule minus ends most abundant at the anterior cortex and least abundant at the posterior cortex, plus ends should be somewhat biased toward the posterior. If kinesin-1 binds an oskar RNP and transports it to a plus end, then binds a neighboring microtubule and runs to its plus end, and so forth, it would accomplish a biased random walk away from the anterolateral cortex that would concentrate oskar RNPs near posterior anchors. This highlights a central question about the mechanism of localization. What is the degree of directional bias for oskar RNP transport? Advances in osk RNP imaging that allow single particle tracking will be needed to obtain clear answers to that question (Serbus, 2005).

Regarding the mechanism of streaming, a model is suggested in which kinesin-1 drives plus-end-directed motion of cargoes that act as impellers, exerting force on ooplasm that surrounds them. Concerted movement of multiple impellers along neighboring microtubules that are oriented in the same general direction creates streams of ooplasm. Prior to stage 10B, small streams occur, but are slow and not well-ordered because dynein resists both plus-end-directed transport and parallel ordering of microtubules. This resistance may be accomplished via: (1) a tug-of-war between opposing motors co-attached to individual impellers; (2) by movement of different impellers in opposite directions, imparting conflicting forces on cytoplasm; or (3) competition by dynein and kinesin for the same binding site on microtubules. Regardless of how dynein interferes with kinesin-1, just before nurse cell cytoplasm is dumped into the oocyte, dynein is suppressed. This allows kinesin-1 to generate fast plus-end-directed impeller transport that sweeps microtubules into parallel arrays that then enhance more robust currents that enhance larger arrays, and so forth, in a self-amplifying loop (Serbus, 2005).

The finding that dynein inhibition enhances a kinesin-1-driven transport process provides the first direct indication of a competitive relationship between opposing microtubule motors. Other studies have produced convincing evidence of alternating coordination between dynein and plus-end-directed motors in a number of processes, including transport of Drosophila embryo lipid droplets, Drosophila cultured cell RNPs and peroxisomes, Drosophila axonal mitochondria (A. Pilling, PhD thesis, Indiana University, 2005, cited in Serbus, 2005), and Xenopus pigment granules. In those processes, inhibition of one motor does not enhance transport in the opposite direction. In fact kinesin-1 inhibition inhibits not only plus-end transport but also dynein-driven minus-end transport. Furthermore, dynein depletion can inhibit both directions of peroxisome transport, confirming that kinesin-1 and dynein each can have positive influences on the other. The observation of competition between dynein and kinesin-1 suggests that alternating coordination and positive interactions between microtubule motors are not a uniform rule, and that some processes have evolved to take advantage of motor competition (Serbus, 2005 and references therein).

If slow streaming is a product of kinesin-dynein competition, why does Khc inhibition arrest all streaming, rather than freeing dynein to drive reverse streaming? One possibility is that although forces from impeller-bound dynein can resist kinesin-1 and confound parallel microtubule ordering, it is not sufficiently processive to generate minus-end-directed streaming currents. A second possibility is that Khc inhibition blocks minus-end as well as plus-end-directed streaming forces, similar to the processes noted above in which dynein transport activity is dependent on Khc (Serbus, 2005).

The observation that actin cytoskeleton depolymerization or mutation of certain actin-interacting proteins can induce premature kinesin-1-driven fast streaming is particularly interesting. Actin filaments are most abundant in the cortex and ring canals of the oocyte and nurse cells, but filaments probably also traverse the internal cytoplasm. An intact actin cytoskeleton could physically assist dynein in resisting kinesin-based plus-end-directed transport during slow streaming, either passively by increasing viscosity or actively by generating antagonistic forces. The active force idea is supported by reports that myosin V can alter the balance between alternating dynein and kinesin-2-driven runs of melanosomes in Xenopus. Drosophila myosin V inhibition tests have not yet been reported, but a disordered cortical actin cytoskeleton in Moesin mutant oocytes does not trigger premature fast streaming, suggesting that well-ordered actin-based forces may not be important for the streaming control mechanism. An alternative to such physical resistance is that dynein inhibitory factors are sequestered by F-actin prior to stage 10B. Then, just before dumping, those factors are released, dynein is inhibited, and kinesin-1 is freed to drive fast streaming (Serbus, 2005).

Recently, several other factors have been identified that are required for prevention of premature fast streaming. Mutations in Maelstrom (Mael), Orb and Spindle-E (Spn-E) allow premature fast streaming and parallel microtubule arrays during stages 8-10A. Orb, a CPEB homolog, is required for osk translation, spn-E is an RNA helicase, and Mael is a modifier of Vasa, which is another RNA helicase. Perhaps these proteins control expression of actin regulators or other factors needed to prevent premature activation of a dynein inhibitory signal. Future work aimed at identifying the regulatory mechanisms that control kinesin in oocytes should be an important focus in understanding the slow-fast streaming transition and also for the broader issue of how the functions of the actin and microtubule cytoskeletons are integrated (Serbus, 2005 and references therein).

Dynein anchors its mRNA cargo after apical transport in the Drosophila blastoderm embryo

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. A new RNA anchoring assay in living Drosophila blastoderm embryos has been used to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. This study proposes a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule (Delanoue, 2005).

This study has used a specific RNA anchoring assay to distinguish between the four main models that could explain how apical wg and pair-rule mRNA (runt, and fushi tarazu) are retained in the apical cytoplasm after their transport by dynein. The models that have been proposed could also apply to other molecular motors and their various cargos. (1) The dynein motor could release the RNA cargo at its final destination, allowing the RNA to bind to an actin-dependent static anchor and the motor to participate in further transport. (2) The anchor could be MT associated rather than actin based. (3) RNA could be retained in the apical cytoplasm by continuous active transport without anchoring. (4) The motor itself could retain the cargo and turn into a static anchor when it reaches the final destination (Delanoue, 2005).

At the outset of this study, it was anticipated that cargo anchoring via actin was the most likely possibility given that actin is thought to be involved in anchoring of many other RNAs. It was also thought that after a motor completes a transport cycle, it releases the cargo and is available for transport of new cargo. However, in general, there has not been very good direct evidence showing that such a model is correct because of the lack of an assay that could discriminate between the transport and anchoring steps. In this study, two specific assays were used: one for transport and another for anchoring. Both anchoring and transport were assayed at the same time in the same embryo using two distinct RNAs. These specific assays have allowed a test and refutation of the prevailing actin anchoring model at least in the case of runt, fushi tarazu and wg apical mRNA localization in the Drosophila blastoderm embryo. Against expectations, the results show that the fourth model is correct, namely that wg and pair-rule RNA are anchored by a dynein-dependent mechanism so that the motor molecules are maintained to the site of anchoring with the cargo. The data shows that the requirement for dynein to anchor the apical RNA is independent of the ATPase activity of the motor and its transport cofactors Egl and BicD, all of which are required for the active transport of the RNA. These observations are best explained by a model in which the dynein motor involved in apical transport of RNA does not release the cargo and acts as a static anchor at the final destination (Delanoue, 2005).

It is interesting to consider how a dynamic motor such as dynein could turn into a static anchor after completion of cargo transport. Dynein is a large multicomplex motor that is difficult to work with in vitro. Nevertheless, many of the subunits of dynein are defined and the force-generating protein, Dhc, is thought to contain physically distinct ATPase and MT binding domains. It is therefore easy to imagine how the motor could change to a static anchor by remaining attached to MTs via the MT binding domain and losing its ATPase force-generating capacity. Indeed, ATPase-independent MT binding has been observed with dynein under in vitro conditions. While it is difficult to compare in vitro studies with the current studies in vivo, the latter are likely to show much more complex and varied interactions with proteins in the cell. Indeed, anchoring may also involve interactions with additional components not present in vitro, such as MT-associated proteins (MAPs), which could stabilize the binding of dynein to the apical MTs or could physically obstruct the motor movement. Another possibility could be anchoring through association with ribosomes, but this can be ruled out in the case of wg and pair-rule RNA, since RNAs lacking a coding region can be transported and anchored correctly. Alternative hypotheses, which cannot be ruled out, include a change of conformation or modifications of the structure of the dynein-dynactin complex. While the data demonstrate conclusively a new RNA-anchoring function for dynein, they do not allow distinguishing between the various hypotheses of how this anchoring occurs at the molecular level, nor test definitively whether Dynactin is required for anchoring. p50/dynamitin is present with the anchored RNA, and overexpression of p50/dynamitin and a Glued/p150 allele cause a partial inhibition of RNA localization with no obvious effects on anchoring. These results suggest, but do not demonstrate conclusively, that Dynactin is not required for anchoring. Furthermore, while it is shown that the ATPase activity of the motor is not required for anchoring, this observation does not test whether dynactin is required in addition to dynein for anchoring (Delanoue, 2005).

Whatever the molecular basis for the dynein anchoring function that was uncovered, it seems likely that the described anchoring does not involve a single dynein molecule anchoring a single RNA molecule. Instead, the RNA cargo is likely to consist of particles containing many RNA molecules and probably many motor complexes. The cargo is thus likely to remain strongly attached to at least some motor molecules throughout transport and anchoring. However, it is not yet known what the linkers between the RNA and motors are (Delanoue, 2005).

Little is also known about the mechanism of anchoring of other dynein cargos, although the mechanism of transport of RNA by dynein could be very similar to other cargos such as lipid droplets. Dynein is also required for nuclear positioning and tethering in many systems, so its role as a static anchor may be widespread. Furthermore, some kinesin-like proteins are also thought to interact with static cell components, and recent in vitro studies show that myosin VI can switch from a motor to an anchor under tension. This process has been proposed to stabilize actin cytoskeletal structures and link protein complexes to actin structures. It is therefore proposed that myosins, kinesins, and dynein may all be able to switch under certain circumstances from dynamic motors to static anchors and that the observations of this study may represent a general principle for anchoring of some cargos following transport to their final cytoplasmic destination (Delanoue, 2005).

Assembly of endogenous oskar mRNA particles for motor-dependent transport in the Drosophila oocyte

oskar mRNA localization at the oocyte posterior pole is essential for correct patterning of the Drosophila embryo. This study shows, at the ultrastructural level, that endogenous oskar ribonucleoprotein complexes (RNPs) assemble sequentially with initial recruitment of Hrp48 (Heterogeneous nuclear ribonucleoprotein at 27C) and the exon junction complex (EJC) to oskar transcripts in the nurse cell nuclei, and subsequent recruitment of Staufen and microtubule motors, following transport to the cytoplasm. oskar particles are non-membrane-bound structures that coalesce as they move from the oocyte anterior to the posterior pole. This analysis uncovers a role for the EJC component Barentsz in recruiting Tropomyosin II (TmII) to oskar particles in the ooplasm and reveals that TmII is required for kinesin binding to the RNPs. Finally, it was shown that both kinesin and dynein associate with oskar particles and are the primary microtubule motors responsible for transport of the RNPs within the oocyte (Trucco, 2009).

This study shows that osk mRNA is synthesized in the nurse cell nuclei, where it assembles into small particles comprising Hrp48 and the Berentz-containing EJC, which are recruited independently to the RNA. The particles are then exported into the nurse cell cytoplasm where, upon loading of Stau, whose association is partially EJC dependent, they recruit both dynein and kinesin and associate with MTs. Upon transport into the oocyte, the small particles transiently reside at the anterior where they are remodeled, in a process requiring Hrp48, Btz, and Stau. During this process, the osk RNPs presumably lose their association with the MTs that mediated their transport from the nurse cells into the oocyte and bind to oocyte MTs for their subsequent transport (Trucco, 2009).

From the anterior, small osk particles are transported by kinesin toward the center of the oocyte, where a mixed population of small and more abundant large particles are observed. It is likely that these large particles arise through coalescence of small particles, as a result of their concentration in the center. Once formed, large particles do not appear to shuttle back to the anterior, as they were not observed in this area. Although a transient accumulation of osk mRNA in the oocyte center has been described as an important step prior to posterior transport, a recent live-cell imaging study of transgenic tagged osk mRNA argued against its existence. A probable explanation for this discrepancy lies in the different stages of oogenesis at which the analyses were performed. A central enrichment of osk mRNA is indeed detected at stage 8, whereas the in vivo analysis was restricted to stage 9 oocytes. It is possible that the transient central enrichment of osk particles reflects the dynamic organization of the MTs in the oocyte at stage 8 (Trucco, 2009).

From the center, large osk particles are transported along MTs toward the posterior pole where they form aggregates. Although a detailed understanding of MT organization will require more sophisticated ultrastructural approaches, such as EM tomography, this study has revealed that MTs are present throughout the oocyte, including the posterior cytoplasm. Indeed, osk RNPs are associated with MTs even in this region, suggesting that osk mRNA is actively transported on MT from the center of the oocyte to the posterior pole (Trucco, 2009).

At the posterior pole, osk aggregates form a continuum interspersed among abundant endocytic tubules where Long Osk has been shown to bind, suggesting that these membranous structures are involved in anchoring the mRNA at the posterior pole. However, endogenous osk RNPs detected throughout the egg chamber are non-membrane-bound structures that are not associated with any specific intracellular organelle, excluding a direct involvement of membrane traffic in osk localization (Trucco, 2009).

This analysis has shown that the loading of the shuttling proteins Hrp48 and Btz on osk mRNA is independent yet that both are required to generate mature particles that can associate with motor proteins and MTs. In particular, EJC components enhance the association between osk RNPs and TmII, which in its turn promotes the loading or stable association of Khc on osk transport particles. Moreover, in hrp48 and EJC mutants, osk is present in small particles uniformly distributed in the oocyte. It is likely that in these mutants the bulk of the mRNA in the oocyte is transported by cytoplasmic flows, explaining its homogeneous distribution and its failure to coalesce into larger particles. This also explains the 5-fold decrease in the number of actively moving particles observed in EJC mutant oocytes. The current findings demonstrate that the nuclear history of the mRNA is critical for the loading of motor proteins to RNPs (Trucco, 2009).

Surprisingly, the association of osk particles with motors and MTs is better preserved in the nurse cells than in the oocytes of hrp48 and EJC mutants, suggesting that the RNPs are remodeled upon entry into the oocyte. Indeed, RNP remodeling in the oocyte is probable, as incoming RNPs must switch from dynein-dependent to kinesin-dependent transport, and presumably detach from the MTs mediating their nurse cell-to-oocyte transport, to oocyte MTs mediating their transport to the posterior pole (Trucco, 2009).

The ultrastructural approach of this study has also provided insight into the function of the cytoplasmic actin-binding protein TmII in osk mRNA transport. TmII associates with osk particles mainly in the ooplasm, consistent with the idea that the RNPs are remodeled upon entry into the oocyte. Surprisingly, in TmII mutant oocytes the association of Khc with osk particles is reduced, suggesting that TmII promotes the recruitment or stability of Khc on osk transport particles (Trucco, 2009).

Ultrastructural analysis shows that Staufen is required for efficient recruitment or stabilization of the MT motor proteins on osk transport particles, explaining the reduced frequency of movements observed during in vivo imaging. The residual motors associated with osk RNPs may mediate their binding to the dense MT network at the oocyte anterior but not support their efficient transport, leading to the observed retention of the mRNA in this region. Alternatively, Staufen may have a role in the switching of osk RNPs from MTs mediating their transport from the nurse cells into the oocyte to the oocyte MTs responsible for the final transport of the mRNA to the posterior pole. A failure to dissociate from nurse cell-to-oocyte MTs would result in retention of the mRNA at the oocyte anterior. A similar, but less pronounced accumulation of osk mRNA is observed in btz mutant oocytes, where the loading of Staufen is affected (Trucco, 2009).

In WT egg chambers nearly 90% of osk RNPs are associated with both Khc and Dhc in the nurse cell cytoplasm and throughout the oocyte. This can explain why Exu-GFP particles, which are thought to contain osk mRNA, show accelerated movement in the nurse cell cytoplasm of khc null mutants. Indeed, it is likely that kinesin, which is present on the same osk particles as dynein, counteracts the dynein-mediated transport of osk RNPs from the nurse cells to the oocyte (Trucco, 2009).

This analysis of khc and TmII mutants has shown that when osk particles fail to associate with Khc, they also fail to accumulate in the oocyte center at stage 8, undergo only partial coalescence, and mostly accumulate around the oocyte cortex at stage 9. This indicates that Khc is a key motor transporting osk to the posterior pole. Interestingly, however, in both khc and TmII mutants, osk RNPs retain their association with Dhc and MTs, suggesting that dynein links osk RNPs to MTs also in the oocyte. Experiments involving live-cell imaging, antibody injection, and MT depolymerization confirm this and show that Khc and Dhc are the primary MT motors actively transporting osk particles. This analysis further suggests that during a single minute, Khc is responsible for active movement of nearly 75% of the particles, whereas dynein mediates the movement of the remaining 25% of particles in the oocyte. It is therefore concluded that in khc mutant oocytes, Dhc most likely transports osk particles to the minus ends of MTs at the lateral cortex, and that in WT oocytes, the activity of dynein in osk transport is masked by that of kinesin. Consistent with this, the speed of osk particle transport is greater in dhc hypomorphic than in WT oocytes, and previous studies have proposed a role of dynein in restricting kinesin activity (Trucco, 2009).

In situ hybridization coupled with immuno-EM is now an established technique that has been successfully used in the present study to visualize the assembly of osk transport particles in the Drosophila oocytes and to reveal the function of the different osk RNP components in this process. As a mechanism for localized protein expression, RNA localization is most powerful when tightly coupled to translational control. Future ultrastructural analysis combined with live-cell imaging is bound to provide new insight into the relationship between the RNA transport and translational control machineries (Trucco, 2009).

Single-molecule assays reveal that RNA localization signals regulate dynein-dynactin copy number on individual transcript cargoes

Subcellular localization of mRNAs by cytoskeletal motors plays critical roles in the spatial control of protein function. However, optical limitations of studying mRNA transport in vivo mean that there is little mechanistic insight into how transcripts are packaged and linked to motors, and how the movement of mRNA-motor complexes on the cytoskeleton is orchestrated. This study has reconstituted transport of mRNPs containing specific RNAs in vitro. It was shown directly that mRNAs that are either apically localized or non-localized in Drosophila embryos associate with the dynein motor and move bidirectionally on individual microtubules, with localizing mRNPs exhibiting a strong minus-end-directed bias. Single-molecule fluorescence measurements reveal that RNA localization signals increase the average number of dynein and dynactin components recruited to individual mRNPs. It was found that, surprisingly, individual RNA molecules are present in motile mRNPs in vitro, and evidence is provided that this is also the case in vivo. Thus, RNA oligomerization is not obligatory for transport. These findings lead to a model in which RNA localization signals produce highly polarized distributions of transcript populations through modest changes in motor copy number on single mRNA molecules (Amrute-Nayak, 2012).

This study has reconstituted transport of specific mRNA species along individual microtubules in vitro and employed single-molecule-resolution measurements to shed light on the composition of transport complexes. An in vivo study of oskar mRNA transport in Drosophila oocytes has demonstrated that asymmetric RNA localization can be achieved by a random walk of a single motor species along a weakly polarized microtubule cytoskeleton. The current findings provide direct evidence for an additional mechanism for RNA targeting in which localization signals control sorting by regulating the net directionality of bidirectional motor complexes on individual microtubules. It is proposed that this is associated with modest differences in the number of motors assembled on individual mRNA molecules. These findings raise fascinating questions about how dynein-dynactin and the unidentified plus-end motor(s) are bound to localizing and non-localizing mRNA molecules and how their activities are orchestrated in time and space (Amrute-Nayak, 2012).

Germ plasm anchoring is a dynamic state that requires persistent trafficking

Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP) particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, RNP particle motility was shown to be required for long-term germ plasm retention. It is proposed that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations (Sinsimer, 2013).

To determine whether motors mediate microtubule-dependent germ plasm RNP particle motility in late-stage oocytes, advantage was taken of mutations that disrupt motor protein activity. The initial localization of osk mRNA during midoogenesis is mediated by the plus-end motor kinesin, and a null mutation in Kinesin heavy chain (Khc), the force-generating component of kinesin, causes mis-localization of germ plasm around the entire oocyte cortex during midoogenesis. Visualization of GFP-Vas in Khc mutant germline clones showed that this aberrant pattern persisted in late-stage oocytes. Despite the paucity of GFP-Vas particles at any one cortical location, examples were observed of dynamic behavior, and these occurred regardless of where on the cortex the particles were located. Quantification of the small population of GFP-Vas particles at the posterior cortex of Khc mutant oocytes showed a 1.7-fold reduction in the motile fraction as compared to wild-type oocytes and a 36% reduction in the median velocity of the motile particles (Sinsimer, 2013).

The finding that germ plasm RNP particle motility is reduced but not abolished in the complete absence of kinesin function suggested the involvement of a second motor. Therefore whether the minus-end motor dynein might be required was tested. Unlike kinesin, dynein is essential during early oogenesis, and complete abrogation of dynein activity precludes egg development. Consequently, the effect of hypomorphic mutations in Dynein heavy chain (Dhc) was examined; these eggs have reduced dynein activity but still complete oogenesis. Quantification of GFP-Vas in late-stage Dhc mutant oocytes showed a 1.6-fold decrease in the motile fraction (19%) as compared to wild-type. Moreover, the median velocity of motile particles in Dhc mutants was half that of wild-type oocytes. In a second approach, dynein activity was disrupted acutely in late-stage oocytes by heat shock-inducible expression of p50/Dynamitin (Dmn), a component of the dynactin complex that interferes with dynein activity when overexpressed. Although heat shock alone had a minor effect on GFP-Vas particle motility, there was a 2.4-fold decrease in the motile fraction when dynein was inactivated as compared to heat-shocked wild-type controls. Moreover, the median velocity of the motile population in late oocytes overexpressing Dmn was reduced by 30% compared to control oocytes. Thus, it is concluded that both kinesin and dynein contribute to germ plasm RNP motility. The comparable loss of motility in null kinesin and hypomorphic dynein mutants, however, suggests that dynein-mediated transport predominates (Sinsimer, 2013).

Both of these motors are also involved in transport events during midoogenesis: dynein for movement of mRNAs from nurse cells to oocyte and anteriorly directed transport within the oocyte, kinesin for posterior transport of osk. Previous work indicated that the bulk of germ plasm mRNA localization occurs not by motor-dependent transport, however, but by diffusion and entrapment of transcripts that enter the oocyte during nurse cell dumping. It has not been possible to resolve particles containing kinesin or dynein in late oocytes using current GFP fusions. Thus, the important question of when and where the association of motors with germ plasm RNP particles occurs awaits the development of new methods for visualization of motors in Drosophila oocytes (Sinsimer, 2013).

The association of localized germ plasm RNP complexes with dynein in late-stage oocytes may serve a second purpose, providing preassembled transport particles for germ cell inheritance in the early embryo. The process of germ cell formation initiates when centrosomes and/or astral microtubules associated with nuclei that migrate to the posterior of the syncytial embryo induce release of germ plasm from the posterior cortex. Recruitment of germ plasm to the centrosomes by dynein-dependent transport on astral microtubules is required for these nuclei to induce germ cell formation and for the inheritance of the germ plasm by the newly formed germ cells. The prior coupling of germ plasm RNP particles to dynein in the oocyte may allow their rapid accumulation on astral microtubules upon release from the cortex (Sinsimer, 2013).

Under conditions of stress such as nutrient deprivation or in the absence of potential mates, female flies will hold mature eggs until conditions improve to increase the likelihood of survival for their progeny. Notably, females can hold mature eggs for at least 15 days without consequence to the viability or fertility of their progeny). Thus, sustaining germ plasm localization through such a delay of fertilization and the onset of embryogenesis is biologically crucial. It is hypothesized that the persistent trafficking of germ plasm might provide a mechanism for retaining germ plasm at the posterior over long periods of time (Sinsimer, 2013).

Because dynein is a major mediator of germ plasm RNP motility and can be manipulated acutely, whether compromising dynein function in held eggs by inducible Dmn overexpression would lead to a progressive loss of germ plasm from the posterior was tested. Advantage was taken of a single-molecule fluorescent in situ hybridization (smFISH) method to detect endogenous nos and osk mRNAs in mature oocytes. smFISH provides a major advance for mRNA analyses during the vitellogenic stages of Drosophila oocytes, which are largely impervious to standard molecular probes. The amount of localized germ plasm was quantified by measuring fluorescence intensity for each probe (Sinsimer, 2013).

More than 70% of mature oocytes from wild-type control or Dmn-overexpressing females, dissected immediately following heat shock, exhibited robust germ plasm accumulation. A spreading of nos and osk along the cortex was observed in some oocytes overexpressing Dmn, likely due to an immediate effect of dynein inhibition on germ plasm retention following the 2 hr heat shock regimen. Examination of wild-type control oocytes held for 18 hr showed little effect of the holding period alone on localization of the germ plasm RNAs. In contrast, inhibition of dynein function in held oocytes led to a dramatic loss of both nos and osk from the posterior cortex, with robust localization persisting in fewer than 30%. Thus, it is concluded that dynein-mediated motility is required for long-term retention of germ plasm at the posterior cortex of the oocyte (Sinsimer, 2013).

Similarly, the effect of MyoV loss on germ plasm components was tested in held oocytes. In mature didum mutant oocytes, nos mRNA was properly localized to the posterior, and unlike the case for Dmn overexpression, nos persisted during the holding period. Together, these data support a model in which enhanced dynein-mediated motility facilitates nos RNP particle accumulation at the posterior. In contrast, osk mRNA was no longer confined to the posterior cortex in didum mutant oocytes but often had a graded or diffuse distribution. Thus, a requirement for MyoV function in entrapment and/or retention of osk extends into late oogenesis. Strikingly, a tight cortical distribution of osk was largely restored in didum mutant oocytes held for 18 hr. This suggests that given sufficient time, microtubule-based motility of osk RNP particles allows localization to recover in the absence of MyoV (Sinsimer, 2013).

The golgin Lava lamp mediates dynein-based Golgi movements during Drosophila cellularization

Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells. Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. These results provide new evidence that golgins promote dynein-based motility of Golgi membranes (Papoulas, 2005).

Following thirteen mitotic nuclear divisions, Drosophila syncytial embryos undergo a form of cytokinesis called cellularization. The process occurs during the interphase of nuclear cycle 14 over a 1-h period and relies on both microtubules and Golgi-based secretion. During cellularization, microtubules emanate from apically positioned centrosomes to form 'inverted baskets' around each nucleus at the embryo’s surface. The furrows form between adjacent nuclei, generating a honey comb pattern, and ultimately expand laterally at their base and fuse to seal each nucleus off from the inner yolk, creating a multicellular embryo. Cellularization requires an increase in cell surface area of approximately 20-fold, and because the disruption of Golgi function inhibits furrowing, a significant amount of this membrane is believed to come from de novo secretion. A portion of the Golgi undergoes two waves of apically directed movements during furrow formation that are coordinated with the dynamic changes in microtubule organization (Papoulas, 2005).

Golgi body movements can be visualized by injecting live embryos with dilute Cy5-tagged anti-Lava-lamp antibody, which fluorescently labels a small number of Golgi near the site of injection and does not affect Golgi movement or cellularization. Injection of colchicine to depolymerize microtubules blocks the Golgi movements, whereas microfilament depolymerization with cytochalasin D does not, consistent with the idea that microtubule-based Golgi movements support active membrane secretion (Papoulas, 2005).

The mechanism that drives the microtubule-dependent Golgi movements is unknown; however, the trajectory of the Golgi movements suggests that cytoplasmic dynein is involved. Cytoplasmic dynein is a multisubunit minus-end-directed microtubule motor that is known to transport a variety of cargos in animal cells. Several studies have suggested that dynein can associate with mammalian Golgi through Golgi-associated spectrin and dynactin, a protein complex that is required for processive movement of dynein cargo. α-spectrin is present on Drosophila Golgi, raising the possibility that it could recruit dynein/dynactin (Papoulas, 2005).

To determine whether cytoplasmic dynein is responsible for the apically directed Golgi movements, Dynein heavy chain (Dhc) activity was inhibited and the effects on Golgi movement were monitored. The use of transgenic flies that express green fluorescent protein (GFP)-tagged Myosin II (MyoII-GFP) permitted the simultaneous visualization of the furrow tip. Injection of function-blocking anti-Dhc antibodies into live MyoII-GFP embryos disrupted Golgi movements, reducing the number of Golgi that move processively. In addition, disruption of Dhc function blocks the progression and organization of the furrow front. Injection of a control [anti-glutathione-Stransferase (GST)] antibody had no effect. Next, embryos were collected from wild-type or Dhc6 mutant females, fixed, and prepared for indirect immunofluorescence using antibodies against tubulin and Lva. Embryos derived from mutant females that develop to nuclear cycle 14 fail to undergo normal furrow formation. These mutant embryos also accumulate fewer Golgi bodies in the apical cytoplasm compared with wild-type embryos at the same stage, despite the fact that microtubules seem abundant and oriented appropriately to support apically directed Golgi movement (Papoulas, 2005).

To explore the possibility that dynein directly mediates the Golgi movements, it was determined whether Dhc localizes to Golgi bodies. Fixed cellularizing embryos were prepared for immunofluorescence using antibodies against Lva and Dhc. Dhc is abundant throughout the cortical cytoplasm, but areas of Dhc enrichment colocalize with Golgi-associated Lva staining. Corroborating the immunofluorescence results, a portion of membrane-associated dynein and a significant fraction of α-spectrin in membrane extracts are present in Golgi-enriched membrane fractions with Lva on density gradients. Some membrane-associated dynein also sediments with an unknown membrane population that is not associated with Lva (Papoulas, 2005).

The role of spectrin and the dynactin complex in the recruitment of dynein to mammalian Golgi and the previous observation that the golgin Lva and Golgi spectrin associate in Drosophila, prompted an investigation of whether Lva might have a direct role in recruiting and/or regulating dynein function. To determine whether Lva associates with dynein, immunoprecipitations were performed using protein extracts prepared from embryos. Anti-Lva antibodies coimmunoprecipitate α-spectrin, as expected, and also coimmunoprecipitate Dhc, whereas control immunoprecipitations do not. These data demonstrate that a portion of the endogenous Lva and dynein associate in soluble embryo extracts. In conjunction with the colocalization and membrane cofractionation results, these data suggest that Lva and dynein may interact on the surface of Golgi in vivo (Papoulas, 2005).

So far, nearly all dynein-based motility of membrane vesicles requires the dynactin complex, and may also be facilitated by microtubule plus-end tracking proteins (+TIPs). +TIPs bind growing microtubule plus-ends and are believed to regulate microtubule dynamics and the docking of membranes to microtubules. Consistent with this model, the +TIPs p150Glued and CLIP-190 were detected in the Golgi-enriched membrane fractions. p150Glued, a subunit of the dynactin complex, extensively cosediments with Lva; and although anti-p150Glued antibodies coimmunoprecipitate Lva and Dhc, anti-Lva antibodies do not detectably coimmunoprecipitate p150Glued under the conditions used. These observations are consistent with the possibility that antibody binding to Lva disrupts adjacent binding of dynactin and previous data that show that anti-Lva antibody injections disrupt Golgi movements in live embryos. CLIP-190 is the Drosophila orthologue of mammalian CLIP-170, which has been implicated in linking vesicles to microtubules for dynein-dependent transport. CLIP-190 is known to cofractionate and colocalize with Lva, and as expected, CLIP-190 and Lva coimmunoprecipitate. Taken together, the association of Lva with dynein, p150Glued, CLIP-190 and spectrin strongly suggests a role for Lva in the microtubule-dependent movement of Golgi during cellularization (Papoulas, 2005).

The ability of different portions of Lva to interact with these microtubule motility factors and with Golgi membrane was tested. Seven contiguous fragments spanning the full length of Lva were expressed as GST-Lva fusion proteins and used to make affinity columns. Chromatography was performed with native extracts that were derived from cellularizing embryos. Dynein exclusively binds the globular carboxyl terminus of Lva (Lva5), whereas both p150Glued and CLIP-190 bind the coiled-coil central portion of Lva (Lva3) and the globular C terminus. Furthermore, α-spectrin binds to Lva3, but not to the dynein-binding C terminus. Therefore, spectrin and dynactin by themselves are insufficient to recruit dynein under the in vitro conditions. Moreover, dynein binds the C terminus of Lva in the absence of spectrin. Interestingly, Drosophila BicD fails to bind any of the seven Lva segments or colocalize with Golgi-associated Lva by immunofluorescence, despite the established role of its orthologues in dynein recruitment to mammalian Golgi membrane. Membrane-binding assays were used to map the region(s) of Lva that are required for Golgi association. Golgi-enriched membrane preparations were incubated with each of the GST-Lva fusion proteins and then collected by centrifugation and assayed for the presence of Lva fusion proteins by anti-GST immunoblotting. Only GST-Lva2A and 2B were found to associate with Golgienriched membranes with high affinity. No fusion proteins pelleted in the absence of membrane (Papoulas, 2005).

To test the functional significance of the Lva interactions with microtubule motility factors Lva fusion proteins were injected into live D. melanogaster embryos and the effects on Golgi movement and furrow formation were monitored. Injection of GST or Lva1, which do not bind the microtubule motility factors, does not inhibit Golgi movement or furrowing. In contrast, injection of Lva3 or Lva5 severely impairs the Golgi movements and furrowing. The number of Golgi bodies that move is significantly reduced by Lva5 and virtually eliminated by Lva3. The Golgi movements that remain in each case are significantly delayed, occurring exclusively during the time period when the second wave of apical movement is normally observed. The impaired furrowing caused by injection of Lva3 and Lva5 occurs primarily during the late (fast) phase, suggesting that the early Golgi movements are a prerequisite for rapid membrane growth during this later phase. Injection of Brefeldin A (BFA), a potent inhibitor of membrane transport, has a similar effect on the fast phase of membrane growth3. Staining with the lectin concanavalin A reveals normal plasma membrane ruffles at the apical margin of furrows and some subcortical membrane during the slow phase in GST-injected embryos. This plasma membrane topology has been previously described in greater detail and coincides with proposed sites of exocytosis. However, this plasma membrane structure is severely disrupted in Lva3-injected embryos and discontinuity in plasma membrane furrows is evident. These results are consistent with the idea that Golgi targeting to the cell surface is required for new plasma membrane secretion to form cleavage furrows (Papoulas, 2005).

These biochemical experiments suggest that the dominant-negative effect of Lva3 on Golgi body movement might result from displacing endogenous dynactin and CLIP-190 from the Golgi surface. To test this the subcellular localizations of Dhc, CLIP-190 and Lva were studied in fixed embryos after injection with GST or Lva3. The uniform expression of p150Glued in embryos prevented identification of any potential effect on dynactin localization, and Dhc localization was indistinguishable between GST- and Lva3-injected embryos, consistent with the absence of dynein binding to Lva3 in vitro. By contrast, the normal CLIP-190 Golgi-association is disrupted in Lva3-injected embryos, particularly at the furrow front during the slow phase of cellularization. GST-injected embryos are unaffected, and Lva remains Golgi-associated in Lva3-injected embryos; although, Golgi are more dispersed (Papoulas, 2005).

Thus, biochemical and functional analysis of Lva suggests that it has a unique function among golgin proteins. The data are consistent with an adaptor model in which Lva stimulates dynein-dependent Golgi movement by binding cytoplasmic dynein in association with promoters of dynein-dependent motility. This model is based in part on the observation that dynein-dependent Golgi movement and furrow formation are significantly inhibited by injections of Lva3, which has no detectable affinity for dynein. This dominant-negative effect can be explained as a consequence of displacing CLIP-190 from Golgi bodies, but is likely to also displace dynactin, resulting in diminished dynein function. The microtubule motility factors bound to the central domain of Lva could facilitate dynein function indirectly by capturing microtubule plus ends, as has been proposed for dynactin7. Alternatively, Lva’s predicted hinge region could facilitate interactions between microtubule motility factors that are bound to the central region of Lva and the dynein motor that is bound to the C terminus, and/or modulate dynein catalytic activity or microtubule binding. These possibilities are not mutually exclusive, and whether Lva contributes to dynein recruitment in vivo remains an open question, as does the possibility that two distinct Lva/dynein complexes exist, one containing dynactin and the other CLIP-190. Future studies will be required to distinguish between these possibilities and to assess the role of Lva in other developmental contexts (Papoulas, 2005).

APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila

In a genetic screen for Kinesin heavy chain (Khc)-interacting proteins, APLIP1, a neuronally expressed Drosophila homolog of JIP-1, a JNK scaffolding protein (Taru, 2002), was discovered. JIP-1 and its homologs have been proposed to act as physical linkers between kinesin-1, which is a plus-end-directed microtubule motor, and certain anterograde vesicles in the axons of cultured neurons (Verhey, 2001). Mutation of Aplip1 causes larval paralysis, axonal swellings, and reduced levels of both anterograde and retrograde vesicle transport, similar to the effects of kinesin-1 inhibition. In contrast, Aplip1 mutation causes a decrease only in retrograde transport of mitochondria, suggesting inhibition of the minus-end microtubule motor cytoplasmic dynein (Pilling, 2005). Consistent with dynein defects, combining heterozygous mutations in Aplip1 and Dynein heavy chain (Dhc64C) generate synthetic axonal transport phenotypes. Thus, APLIP1 may be an important part of motor-cargo linkage complexes for both kinesin-1 and dynein. However, it is also worth considering that APLIP1 and its associated JNK signaling proteins could serve as an important signaling module for regulating transport by the two opposing motors (Horiuchi, 2005).

To identify proteins that influence kinesin-1-based axonal transport, genetic interaction tests were done to search for mutations that act as dominant enhancers of Kinesin heavy chain. A number of such E(Khc) mutations were found that caused synthetic axonal transport phenotypes (i.e., larval paralytic 'tail flipping' and organelle-filled 'axon swellings') when combined with a Khc null (Khc27/+; E(Khc)/+). Tail flipping was not seen and swellings were rare in Khc27/+ or E(Khc)/+ single heterozygotes. A subset of E(Khc) loci cause tail flipping and swellings when homozygous mutant in a wild-type Khc background, suggesting that the products of those loci have direct roles in axonal transport. That subset includes Kinesin light chain (Klc), Dynein heavy chain 64C (Dhc64C), Glued and an unknown locus on chromosome 3 initially designated E(Khc)ek4 (abbreviated as ek4) (Horiuchi, 2005).

To gain more insight into the functions of ek4 products, a number of phenotypic tests were done. Homozygous ek4 mutant larvae showed classic posterior paralysis and axonal swelling phenotypes with severities similar to those caused by strong hypomorphic Khc genotypes. However, in contrast to such Khc mutants, which die during larval and pupal stages of development, ek4 mutants survive to become active, fertile adults. Severity comparisons with a null [Df(3L)Fpa2] indicate that the ek4 mutation is a strong hypomorphic allele, causing nearly a complete loss of function. These observations suggest that wild-type products of the ek4 locus have important axonal transport functions in larvae and that they have a positive functional relationship with kinesin-1. However, ek4 is not itself essential, suggesting that its products contribute to only a subset of kinesin-1 functions (Horiuchi, 2005).

To test the effects of ek4 mutations on kinesin-1-dependent fast axonal transport, time-lapse confocal microscopy was used. GFP-neuronal synaptobrevin (GFP-nSyb) was used to image transport vesicles, while cytochrome c oxidase-GFP (mito-GFP) (Pilling, 2005) was used to image mitochondria. These constructs were expressed in motoneurons of larvae by virtue of Gal4-UAS promoters that were activated by P[GawB]D42-Gal4 (abbreviated D42), a motor neuron Gal4 driver. With this system, it has been shown that hypomorphic Khc mutations cause anterograde and retrograde flux reductions for GFP-nSyb (60%–70%) and for mito-GFP (75% and 90%), supporting the hypothesis that normal dynein function in some processes depends on kinesin-1 (Pilling, 2005). Both anterograde and retrograde GFP-nSyb flux were reduced ~35% in ek4 mutant axons, supporting the idea that wild-type ek4 products facilitate some kinesin-1 functions. Surprisingly, ek4 mutant axons showed no change in anterograde mito-GFP flux and a 60% reduction in retrograde flux. Currently, the only mutations known to cause a similar unidirectional inhibition of retrograde mitochondrial flux are in Dhc64C (~80%) (Pilling, 2005), which encodes the motor subunit of cytoplasmic dynein (Horiuchi, 2005).

To further test the possibility that ek4 influences dynein, additional genetic interaction tests were done. Consistent with the original genetic screen for dominant enhancers of Khc, ek4 acts as a dominant enhancer of Kinesin light chain (Klc), causing synthetic tail flipping and axonal swelling phenotypes. No such interaction was seen when ek4 was combined with a mutant allele of Klp64D, which encodes an anterograde axonal motor of the kinesin-2 family. However, when ek4 was combined with a mutant allele of Dhc64C, synthetic tail flipping and axonal swelling phenotypes were seen. In summary, these results support the hypothesis that wild-type ek4 gene products facilitate vesicle transport by kinesin-1 and mitochondrial transport by cytoplasmic dynein (Horiuchi, 2005).

To identify the ek4 locus, meiotic recombination and deletion mapping approaches were initially used. The results indicated a position near the tip of the left arm of chromosome 3 within the 61F3-4 cytological region. That interval included APP-like interacting protein 1 (Aplip1), a gene that encodes a neuronally expressed Drosophila homolog of c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1), a scaffolding protein that has been shown to bind Kinesin light chain (KLC), a reelin receptor (ApoER2), and Alzheimer's amyloid precursor protein (APP), as well as JNK pathway kinases (Taru, 2000; Verhey; 2001; Yasuda, 1999). It has been proposed that JIP-1 and its close relative JIP-2 link kinesin-1 with axon vesicles to facilitate anterograde vesicle transport. Similar kinesin-1 linker functions have been proposed for an unrelated JNK scaffolding protein, sunday driver (syd, JSAP, JIP-3), and for APP, although the APP-kinesin relationship may be mediated by APLIP1/JIP-1. A P element transgene that included Aplip1 and flanking sequences fully rescued the tail flipping and partially rescued the axonal swelling phenotypes of larvae that were doubly heterozygous for Khc27 and ek4. Finally, sequencing of the Aplip1 locus from ek4 mutant animals revealed a single base change that converts a conserved proline at position 483 to leucine. This proline is within a conserved 11 amino acid C-terminal region (KBD) that has been shown to be important for binding of mammalian JIP-1 to KLC (Verhey, 2001). The transgenic rescue and sequencing results confirm that ek4 is a mutant allele of the Aplip1 gene, and hence it will be referred to as Aplip1ek4 (Horiuchi, 2005).

To determine whether the P483L mutation affects KLC-APLIP1 binding, epitope-tagged versions of KLC and APLIP1 were used for immunoprecipitation studies. After coexpression of Myc-KLC and wild-type Flag-APLIP1 in S2 cultured cells, anti-Myc antibody precipitated both proteins. Removal of the 11 amino acid KBD from Flag-APLIP1 eliminated detectable binding to Myc-KLC. Furthermore, changing proline 483 to either leucine or alanine substantially reduced KLC binding. This shows that P483 is indeed important for KLC binding, which suggests that at least some of the Aplip1ek4 mutant phenotypes are due to poor association of APLIP1 and kinesin-1 (Horiuchi, 2005).

If APLIP1 links kinesin-1 to anterograde transport vesicles in Drosophila axons, as has been proposed for JIP-1 in vertebrates (Verhey, 2001), APLIP1 should localize in axons and such localization should depend on its ability to bind KLC. To test those predictions, flies were transformed with P elements that carried either full-length UAS-Flag-Aplip1 or UAS-Flag-Aplip1ΔKBD. When driven by D42-Gal4, the two constructs produced equivalent levels of mRNA, which were many times in excess relative to the endogenous gene in larvae. Western blots of larvae with anti-Flag were not successful, but both the full-length and the ΔKBD Flag-tagged proteins were seen at equivalent levels in Westerns of transfected S2 cells, suggesting that both were stable. Interestingly, D42-Gal4-driven expression of one copy of full-length UAS-Flag-Aplip1 in motoneurons causes dramatic tail flipping and nearly 100% lethality during late larval and pupal stages. In larval nerves, it causes axon swellings that stain intensely for vesicles (anti-Syt) and APLIP1 (anti-Flag) . D42-Gal4-driven expression of the deletion construct caused no tail flipping or lethality. It did cause some axon swellings in larval nerves, and Flag-APLIP1ΔKBD staining was visible in those swellings. However, the overall amount of staining in nerves was substantially reduced relative to the amount seen after expression of the full-length protein (Horiuchi, 2005).

The presence of residual Flag-APLIP1ΔKBD in larval nerves indicates that some is transported into axons despite the fact that its binding to kinesin-1 is compromised. JIP-1 as well as APLIP1 is known to form multimers (Taru, 2002; Yasuda, 1999). Indeed, immunoprecipitation tests indicate that tagged APLIP1 and APLIP1ΔKBD can form stable multimers with one another. Thus, it is possible that in larval neurons, endogenous wild-type APLIP1 mediates linkage of some transgenic Flag-APLIP1ΔKBD to kinesin-1. Overall, these results suggest that binding between APLIP1 and KLC is an important factor in the presence of APLIP1 in axons, providing in vivo support for the hypothesis that APLIP1 is transported anterograde by kinesin-1 (Horiuchi, 2005).

To test the possibility that APLIP1 is associated with dynein-driven retrograde transport as well as with kinesin-1-driven anterograde transport, transgenic flies were developed carrying a UAS-GFP-Aplip1 transgene that expressed a stable fusion protein. When combined with the D42-Gal4 driver, some transformant lines showed paralysis and GFP-filled swellings, similar to the Flag-APLIP1 lines. Time-lapse imaging did not reveal obvious transport, suggesting that the GFP-APLIP1 was transported in a form too dispersed for imaging of discrete punctate signals. Turning to a classic axonal transport approach, a method was developed for nerve ligation in Drosophila larvae. A homozygous UAS-GFP-Aplip1 D42-Gal4 transformant line was used in which there were few axonal swellings and little visible axonal GFP fluorescence, presumably because of low expression. Intact live larvae were constricted with a fine synthetic fiber midway between head and tail to compress their segmental nerves. After 4 hr, they were partially dissected, the ligation threads were cut, dissection was completed, and the nerves were imaged. Distinct compressed regions were flanked by bright accumulations of GFP-APLIP1 on both the proximal and distal sides. This provides a strong indication that APLIP1 is carried not only by anterograde, but also by retrograde axonal transport (Horiuchi, 2005).

By using an in vivo genetic approach to identify proteins that contribute to the mechanism of kinesin-1-driven anterograde axonal transport, this study has identified APLIP1, a Drosophila homolog of the JNK-interacting protein JIP-1. In vivo axonal transport analysis with intact nervous systems suggests roles for APLIP1 in anterograde and retrograde transport of nSyb-tagged vesicles and in retrograde transport of mitochondria. Similar neuronal phenotypes were seen with either Aplip1 inhibition or overexpression, suggesting that correct stoichiometry of APLIP1 and its interacting proteins is critical for normal organelle transport. The influence of APLIP1 on nSyb vesicle transport in both directions could be explained simply by its importance for kinesin-1 function. Khc is required for normal retrograde dynein activity as well as for anterograde kinesin-1 activity, probably because of a physical or regulatory relationship between the two motors. Alternatively, APLIP1 might make separate contributions to kinesin-1-driven anterograde and dynein-driven retrograde vesicle transport (Horiuchi, 2005).

The selective influence of APLIP1 on retrograde, but not anterograde, transport of mitochondria, as well as Aplip1-Dhc64C genetic interactions, suggests that APLIP1 does have distinct, kinesin-independent functions in dynein-driven transport, at least for mitochondria. Considering how APLIP1 and other JIP-1-related proteins contribute to axonal transport mechanisms, binding studies suggest they may be structural components of kinesin-1-cargo linkage complexes. However, the APLIP1 influence on retrograde mitochondria, the well-known scaffolding role of APLIP1/JIP-1 in the JNK signaling pathway, and indications that JNK may influence motor linkage must also be kept in mind. Mitochondrial transport and distribution in axons responds dramatically to extracellular signaling and may also respond to intracellular signaling stimulated by changes in mitochondrial membrane potential. APLIP1 might be important in these or in other pathways that regulate dynein-cargo linkage and/or mechanochemistry. Future tests for a physical APLIP1-dynein association and for influences of JNK signaling on axonal transport may provide important insights into the microtubule-based transport mechanisms required to sustain neurons and other large asymmetric cells (Horiuchi, 2005).

Control of a Kinesin-Cargo linkage mechanism by JNK pathway kinases

Long-distance organelle transport toward axon terminals, critical for neuron development and function, is driven along microtubules by kinesins . The biophysics of force production by various kinesins is known in detail. However, the mechanisms of in vivo transport processes are poorly understood because little is known about how motor-cargo linkages are controlled. A c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) has been identified previously as a linker between kinesin-1 and certain vesicle membrane proteins, such as Alzheimer's APP protein and a reelin receptor ApoER2. JIPs are also known to be scaffolding proteins for JNK pathway kinases. Evidence is presented that a Drosophila ubiquitin-specific hydrolase (Fat facets) and a JNK signaling pathway that it modulates can regulate a JIP1-kinesin linkage. The JNK pathway includes a MAPKKK (Wallenda/DLK), a MAPKK (Hemipterous/MKK7), and the Drosophila JNK homolog Basket. Genetic tests indicate that those kinases are required for normal axonal transport. Biochemical tests show that activation of Wallenda (DLK) and Hemipterous (MKK7) disrupts binding between kinesin-1 and APLIP1, which is the Drosophila JIP1 homolog. This suggests a control mechanism in which an activated JNK pathway influences axonal transport by functioning as a kinesin-cargo dissociation factor (Horiuchi, 2007).

Maintaining proper distributions of protein complexes, RNAs, vesicles, and other organelles in axons is critical for the development, function, and survival of neurons. The primary distribution mechanism relies on long-distance transport driven by microtubule motor proteins. Components newly synthesized in the cell body, but needed in the axon, bind kinesin motors that carry them toward microtubule plus ends and the axon terminal (anterograde transport). Neurotrophic signals and endosomes, examples of axonal components that require transport to the cell body, bind dynein motors that carry them toward minus ends (retrograde transport). The importance of these processes is highlighted by the observation that mutation of motors and other transport machinery components can cause neurodegenerative diseases in humans and analogous phenotypes in model organisms (Horiuchi, 2007).

Two key questions are (1) how do cargoes link to particular motors, and (2) how are such linkages regulated to ensure appropriate pickup and dropoff dynamics? For kinesin-vesicle linkages, scaffolding proteins have emerged as key connectors. For example, the cargo-binding kinesin light chain (Klc) subunit of kinesin-1 binds not only the kinesin-1 heavy chain (Khc) but also JNK-interacting proteins (JIPs). Vertebrate JIPs can bind multiple components of the JNK signaling pathway, e.g., JNK itself, upstream activating kinases (MAPKKs), and regulatory kinases (MAPKKKs). JIPs can also bind vesicle-associated membrane proteins, such as ApoER2, which is a reelin receptor, and APP, a key factor in Alzheimer's disease. Therefore, JIP scaffolding proteins are likely to link JNK pathway kinases and kinesin-1 to vesicles carrying these membrane proteins. This raises an interesting question: Are the JNK pathway kinases simply passive hitchhikers on the kinesin-1/JIP/vesicle complex, or can they actively regulate its transport (Horiuchi, 2007)?

A genetic screen was conducted for factors that control kinesin-JIP linkage during axonal transport. The screen was based on the previous observation that neuron-specific overexpression of Aplip1, which encodes the Drosophila JIP1, causes synaptic protein accumulation in axons, larval paralysis, and larval-pupal lethality, the classic axonal-transport-disruption phenotypes caused by Khc and Klc mutations. Why might overexpression of the JIP1 cargo linker for kinesin-1 disrupt axonal transport? The disruptive effect requires APLIP1 (JIP1)-Klc binding. It may be that excess APLIP1 (JIP1) competes with other Klc-binding proteins, for example, different linkers that may attach kinesin-1 to other cargoes. In search of factors that can disrupt or antagonize APLIP1 (JIP1)-Klc binding, a screen was performed for genes that can suppress the axonal-transport phenotypes when co-overexpressed with Aplip1. An 'EP' collection of fly strains capable of the targeted overexpression of endogenous Drosophila genes was screened and P{EP}fafEP381, a line that overexpresses fat facets (faf), was identified as a strong suppressor of the APLIP1 (JIP1)-induced lethality and other neuronal overexpression phenotypes (Horiuchi, 2007).

Faf protein antagonizes ubiquitination and proteasome-mediated degradation of its target proteins. Interestingly, Faf was recently reported to stimulate a Drosophila neuronal JNK signaling pathway that is regulated by the MAPKKK Wallenda (Wnd), a homolog of dual leucine zipper-bearing kinase (DLK) that is known to bind JIP1. Overexpression of faf leads to increased levels of Wnd (MAPKKK) protein and thereby causes excessive synaptic sprouting through a pathway that requires the Drosophila JNK homolog Basket. It was found that mutating just one copy of wnd blocked the suppression of Aplip1 overexpression by P{EP}fafEP381. This suggests that faf overexpression suppresses APLIP1 (JIP1)-Klc interaction by elevating the level of Wnd (MAPKKK). Consistent with this, direct overexpression of wnd in neurons with a wild-type transgene (UAS-wnd) was as effective as P{EP}fafEP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins. Equivalent expression of a 'kinase-dead' mutant transgene (UAS-wndKD) did not suppress the defects. Thus, Wnd (MAPKKK) and its downstream phosphorylation targets may actively regulate APLIP1 (JIP1)-Klc binding in neurons (Horiuchi, 2007).

If Wnd (MAPKKK) signaling plays a role in normal axonal transport, disrupting its function should cause axonal-transport phenotypes. Consistent with this, wnd loss-of-function mutations (wnd1/wnd2) in an otherwise wild-type background caused accumulation of synaptic proteins in axons. The accumulation phenotype was rescued by motoneuron expression of the wild-type wnd transgene but not by equivalent expression of the kinase-dead mutant transgene. The likely target of Wnd (MAPKKK) kinase activity is the Drosophila homolog of MKK7, Hemipterous (Hep), a MAPKK that activates Bsk (JNK). Mutation of hep also causes axonal accumulations, as does neuronal expression of a dominant-negative mutant bsk transgene. The results of these genetic-inhibition tests combined with those of the Aplip1-overexpression-suppression tests suggest that a Wnd (MAPKKK)-activated JNK pathway influences fast axonal transport by regulating APLIP1 (JIP1)-Klc binding (Horiuchi, 2007).

Is a Wnd (MAPKKK)-Hep (MAPKK)-Bsk (JNK) signaling module bound by APLIP1 (JIP1)? Although all three components of the homologous vertebrate module (DLK-MKK7-JNK) bind JIP1, APLIP1 (JIP1) lacks a conserved JNK-binding domain, and it does not bind directly to Bsk (JNK). However, APLIP1 (JIP1) does bind Hep (MAPKK), Klc, and the Drosophila APP homolog APPL. To determine whether Wnd (MAPKKK) associates with Hep (MAPKK) and APLIP1 (JIP1), coexpression and immunoprecipitation tests were performed in Drosophila S2 cultured cells. Wnd (MAPKKK) did not coprecipitate with APLIP1 (JIP1). However, Hep (MAPKK) did coprecipitate with APLIP1 (JIP1), and Wnd (MAPKKK) coprecipitated with Hep (MAPKK). Thus, Wnd (MAPKKK) may bind and influence the APLIP1 (JIP1)-kinesin complex via Hep (MAPKK) (Horiuchi, 2007).

Can Wnd (MAPKKK) and Hep (MAPKK) control the binding of APLIP1 (JIP1) to Klc? When expressed in S2 cells, APLIP1 (JIP1) and Klc exhibit strong binding, as assessed by coimmunoprecipitation. Coexpression of wild-type Wnd (MAPKKK) partially inhibited APLIP1 (JIP1) binding to Klc, but coexpression of a kinase-dead mutant Wnd (MAPKKK) did not. Wild-type Hep (MAPKK) also caused a partial inhibition of APLIP1 (JIP1)-Klc binding, and a constitutively active mutant Hep (MAPKK) caused nearly complete inhibition. Finally, coexpression of wild-type Wnd (MAPKKK) and Hep (MAPKK) together caused an almost complete inhibition of APLIP1 (JIP1)-Klc binding. In addition to inhibiting APLIP1 (JIP1)-Klc binding, Wnd-Hep activation in S2 cells increased the level of Bsk (JNK) activation. Hence, there is a correlation between decreased levels of APLIP1 (JIP1)-Klc binding and elevated levels of Bsk (JNK) activation. This suggests that, despite the lack of a known JNK-binding site on APLIP1 (JIP1), Bsk (JNK) may be the kinase that disrupts the APLIP1 (JIP1)-Klc complex. These results suggest that Wnd (MAPKKK) activation of Hep (MAPKK), and perhaps also Hep (MAPKK) activation of Bsk (JNK), can regulate the linkage between kinesin-1 and a cargo complex via the JIP1-like scaffolding protein, APLIP1 (Horiuchi, 2007).

Hep (MAPKK) may regulate the APLIP1 (JIP1) complex either by activating JNK or by a mechanism independent of JNK. Observations that motoneuron-specific inhibition of Bsk (JNK) caused transport defects similar to those caused by mutations in wnd and hep and that decreased APLIP1 (JIP1)-Klc binding in S2-cell lysates coincided with increased phosphorylated Bsk (JNK) support pathway 1, i.e., Hep (MAPKK) activation of Bsk (JNK), which then directly or indirectly inhibits APLIP1 (JIP1)-Klc binding. A second pathway, Pathway 2, employs an alternative mechanism in which activated Hep (MAPKK) does not need Bsk (JNK) to inhibit APLIP1 (JIP1)-Klc binding. There is little current evidence that Hep or its vertebrate MAPKK homolog MKK7 have phosphorylation targets other than Bsk (JNK). However, that does not exclude the possibility that activated Hep induces in APLIP1 (JIP1) a direct conformational change that causes Klc dissociation. Regardless of how Hep (MAPKK) disrupts binding, when kinesin-1 is not attached to cargo via JIP1, it can fold into a compact form that does not interact with microtubules. Hence, the activated Wnd (MAPKKK) pathway could both inhibit APLIP1 (JIP1)-Klc binding and cause dissociation of kinesin-1 from microtubules. Consistent with this, recent studies report that stimulation of JNK pathways in cultured cells or axoplasm can disrupt the association of kinesin-1 with microtubules (Horiuchi, 2007 and references therein).

From a broader perspective on axonal-transport regulation, it is interesting to consider that there are multiple types of kinesin-1 cargoes, that there are various JIPs that could be specific for different cargoes, and that different MAPKKKs can associate with different JIPs. By sitting at the top of a classic signaling cascade, MAPKKKs such as Wnd are in a good position to differentially control the transport of specific subsets of anterograde kinesin-1 cargoes in response to specific cellular signals. It is known in mammals that other MAPKKKs such as MLK, ASK1, and MEKK1 can bind JIP scaffolding proteins. It will be interesting to determine whether they too influence kinesin-cargo interactions (Horiuchi, 2007).

This work provides the first demonstration that a kinesin and its transport functions can be influenced by a MAPKKK. More specifically, the MAPKKK Wnd and its downstream MAPKK Hep can regulate attachment of a JIP1 cargo linker to kinesin-1. These results also provide the first indication that ubiquitination pathways, by way of MAPKKKs, could be important for proper regulation of axonal transport. Finally, these results suggest that JNK pathway kinases are not just hitchhikers on the axonal kinesin-1/JIP/cargo complex; rather, they can actively regulate its transport dynamics (Horiuchi, 2007).

Distinct mechanisms govern the localisation of Drosophila CLIP-190 to unattached kinetochores and microtubule plus-ends

CLIP-170 was the first microtubule plus-end-tracking protein to be described, and is implicated in the regulation of microtubule plus-ends and their interaction with other cellular structures. The cell-cycle-dependent mechanisms which localise the sole Drosophila melanogaster homologue CLIP-190 have been studied. During mitosis, CLIP-190 localises to unattached kinetochores independently of spindle-checkpoint activation. This localisation depends on the dynein-dynactin complex and Lis1 which also localise to unattached kinetochores. Further analysis revealed a hierarchical dependency between the proteins with respect to their kinetochore localisation. An inhibitor study also suggested that the motor activity of dynein is required for the removal of CLIP-190 from attached kinetochores. In addition, CLIP-190 association to microtubule plus-ends is regulated during the cell cycle. Microtubule plus-end association is strong in interphase and greatly attenuated during mitosis. Another microtubule plus-end tracking protein, EB1, directly interacts with the CAP-Gly domain of CLIP-190 and is required to localise CLIP-190 at microtubule plus-ends. These results indicate distinct molecular requirements for CLIP-190 localisation to unattached kinetochores in mitosis and microtubule ends in interphase (Dzhindzhev, 2005; full text of article).

The CLIP family of proteins is implicated in regulating microtubule dynamics and linking microtubule plus-ends with other cellular structures. To understand these functions, it is crucial to elucidate where and how these proteins localise within cells. This study investigated the molecular mechanisms of CLIP-190 localisation using RNAi in Drosophila cells, rather than using the expression of dominant proteins, to gain a clearer and more comprehensive view. The study revealed that distinct, cell-cycle-dependent mechanisms localise CLIP-190 to microtubule plus-ends and unattached kinetochores (Dzhindzhev, 2005).

CLIP and EB1 proteins are two major families of microtubule plus-end-tracking proteins. This study is the first demonstration of both a physical interaction and localisation-dependency between CLIP and EB1 proteins in higher eukaryotes. CLIP-190 requires EB1 to localise to microtubule plus-ends, and CLIP-190 directly interacts with EB1 through its CAP-Gly domain, which also binds to microtubules. Considering that this has previously been reported for fission yeast homologues, these findings demonstrate that this interaction and dependency are conserved among eukaryotes. The most obvious interpretation would be that EB1 simply bridges microtubule plus-ends and CLIP-190. However, this is thought not be the case. First, CLIP-170 has been shown to bind directly to microtubule plus-ends in vitro. Secondly, localisation studies show incomplete overlapping of the two proteins on microtubule plus-ends. Also, other EB1 interacting proteins, such as RhoGEF2 and the spectraplakin Short stop, which both require EB1 for their localisation to microtubule plus-ends (Rogers, 2004; Slep, 2005), show distinct localisation from that of EB1. Therefore, it is more likely that EB1 acts as a loading factor for these proteins, rather than a simple bridge. In addition, it seems that multiple microtubule plus-end-binding proteins, such as CLIPs, CLASPs, p150Glued, EB1, Lis1, Dynein, Short stop, APC and RhoGEF2, directly interact with each other and with microtubules. It is an exciting challenge to understand the regulatory network acting on microtubule plus-ends (Dzhindzhev, 2005).

In addition, it was found that the association of CLIP-190 to microtubule plus-ends is greatly reduced during mitosis. This is in contrast to EB1, which is associated with plus-ends throughout the cell cycle. This cell-cycle-regulation has not been described in other systems, possibly because of a lack of co-examination with EB1, and leads to the conclusion that it is not the consequence of a change in microtubule dynamics. It might be possible that CLIP-190 is modified during the cell cycle. Since EB1 is essential for CLIP-190 localisation to microtubule ends, EB1 activity or interaction between EB1 and CLIP-190 might also be regulated. Alternatively, other inhibitory proteins might be activated during mitosis to attenuate CLIP-190 association with microtubule ends. Phosphorylation of the EB1 homologue mal3p and its inhibitory effects on the interaction with the CLIP-190 homologue tip1p have been shown in fission yeast (Busch, 2004). It remains to be examined whether this phosphorylation is cell-cycle-regulated. Cell-cycle regulation might be important for releasing CLIP-190 for kinetochore function or preventing the plus-end-binding activity from interfering with CLIP-190 function at kinetochores. This report is the first to describe the cell-cycle regulation of the plus-end-binding of CLIP proteins. Elucidation of the precise mechanism and significance of this regulation may lead to further understanding of the temporal and spatial regulation of microtubules in cells (Dzhindzhev, 2005).

CLIP-190 localises to unattached kinetochores in mitosis. The localisation of CLIP proteins to kinetochores has been shown in mammalian, Drosophila and budding-yeast cells. Studies of CLIP-170 localisation to kinetochores in mammalian cells suggest an intricate physical and functional relationship with the dynein-dynactin complex. CLIP-170 binds directly to and requires Lis1 for kinetochore localisation. In turn, Lis1 interacts with multiple subunits of dynein-dynactin and is displaced from kinetochores when the motor complex is disrupted. Most of these studies relied on the overexpression of dominant-negative proteins (Dzhindzhev, 2005).

A previously unreported dependency was found, namely the requirement of Lis1 for dynein localisation to kinetochores. In mammalian cells, dynein is required for Lis1 localisation, whereas the overexpression of full-length or truncated Lis1 does not prevent dynein localisation to kinetochores. These results lead to the idea that Lis1 might be an auxiliary protein that bridges the dynein complex to cargo proteins. The RNAi results clearly indicate that Lis1 is required for dynein localisation to kinetochores. Combined with previous results, Lis1 and dynein seem to depend on each other for their localisation. This is the first report of such dependency in any eukaryote, and it gives the Lis1 protein a more integral part in dynein function (Dzhindzhev, 2005).

The results also suggest that microtubule attachment directly removes CLIP-190 from kinetochores rather than through spindle-checkpoint signalling. Dynein seems to be responsible for the removal of CLIP-190 from kinetochores in addition to its role in localising CLIP-190 to kinetochores. It has been shown that dynein removes several kinetochore proteins along microtubules upon the attachment of microtubules. This study provides the first evidence that a member of the CLIP family also utilises dynein-motor-activity to leave attached kinetochores. Interestingly, it was found that unlike in interphase, EB1 is not required for the mitotic localisation of CLIP-190 to unattached kinetochores. This is intriguing in the light of recent evidence (Tirnauer, 2002) that EB1 associates with attached kinetochores when the kinetochore microtubules are polymerizing (Dzhindzhev, 2005).

In conclusion, these results indicate that CLIP-190 localisation is regulated during the cell cycle and requires distinct mechanisms in mitosis and interphase. Spatial and temporal regulation of CLIP-190 localisation probably play crucial roles in the regulation of microtubule dynamics and their interaction with other cellular structures (Dzhindzhev, 2005).

The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport

Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. This study shows that mutations in drop out (dop; CG6498) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. This study shows that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein (Hain, 2014).

This study is the first mutational analysis of a MAST kinase in any organism and demonstrates that the MAST kinase Dop plays an important role in plasma membrane cortex compartmentalisation during the generation of epithelial polarity in the fly. The results reported in this study demonstrate a requirement of Dop in the establishment of the furrow canal and the bAJ at the cycle 14 transition. The defect in bAJ formation is likely to be a consequence of a failure in the initial specification of the incipient furrows. It is proposed that Dop acts upstream in furrow canal formation by controlling the formation of F-actin-rich foci, which initiate the assembly of a specific furrow membrane cortex (Hain, 2014).

In mid-cellularisation stages, dop mutant phenotypes are reminiscent of embryos lacking the early zygotic gene bottleneck (bnk). In bnk mutants the initial formation of the cleavage furrows is normal, but then furrows close prematurely. Although it cannot be excluded that bnk might play a role in later defects associated with dop mutations, the primary defect in dop mutants concerned the lack of regular F-actin-rich furrows during the onset of cellularisation. Another early zygotic gene, nullo, is required for the proper recruitment of F-actin during furrow canal formation. Nullo and the actin regulator RhoGEF2 have been proposed to act in parallel pathways controlling processes that are distinct but both essential for F-actin network formation during the establishment of the furrow canal. Since early F-actin rearrangements are largely normal in nullo and RhoGEF2 single mutants, it is proposed that Dop is essential for the initial early focussing of F-actin, whereas Nullo and RhoGEF2 are required to elaborate and maintain F-actin levels to stabilise the furrows. The actin regulator enabled (ena) has been shown to act downstream of Abelson tyrosine kinase (Abl) in the redistribution of F-actin from the plasma membrane cortex into the furrows in both syncytial stages and cellularisation. Although ena would provide a good candidate for acting downstream of dop in the redistribution of F-actin, ena is already required for syncytial cleavages and the F-actin phenotypes in Abl mutants are much more severe than those that were found for dop mutants (Hain, 2014).

The similarity of syncytial cleavage furrows and the cleavage furrows at cellularisation raises the question of how they differ from each other. The molecular basis of the hexagonal pattern of the F-actin-rich cell cortex at the cleavage furrow relies upon the recycling endosome components Rab11 and Nuclear fallout (Nuf) and the actin polymerisation factors Dia and Scar/Arp2/3. In contrast to dop mutants, nuf, dia or Scar mutants indicate that these genes are required also for the dynamic redistribution of F-actin during syncytial development. Since Dop is a maternally supplied protein, its activity might be regulated by events triggered during the cycle 13-14 transition. The major difference between the furrows in syncytial stages and cellularisation is that metaphase furrows are formed during M phase, whereas cellularisation furrows are formed during G2 phase. Since Dop is a maternally supplied gene product, one would have to implicate regulation of Dop by zygotic factors to explain its phenotype at the cycle 13-14 transition. An alternative possibility is that Dop is regulated by phosphorylation or other post-translational modification through the cell cycle machinery and that, in the absence of Cdk1-dependent phosphorylation, its phosphorylation state is changed. This study provided evidence that Dop is indeed differentially post-translationally modified during syncytial versus cellular blastoderm stages. It is proposed that such cell cycle-dependent regulation of Dop may be crucial in transforming syncytial cleavages into persistent cellularisation furrows. Furthermore, the data suggest that this transition could require Dop-dependent regulation of dynein-associated microtubule transport (Hain, 2014).

The mechanisms for the initial localisation of Baz and E-cadherin are still unclear but, interestingly, dop is required for the localisation of both proteins. At the cycle 14 transition, E-cadherin and Arm puncta are associated with apical membrane projections and the homophilic association of these cadherin puncta is strengthened by membrane flow and is dependent on actin. Baz function allows these puncta to become tightened into sAJs. Thus, Dop might affect the stabilisation of the weakly interacting puncta either through cortical actin organisation or membrane flow. In addition to this early requirement for Baz localisation, Dop is also involved in clearing Baz from the basal cytoplasm during late cellularisation. The mechanism that eventually clears Baz from the basal cytoplasm depends on dynein-based transport. Therefore, Dop is required for dynein-based transport of different cargoes during cellularisation: lipid droplets, mRNA particles, Golgi and Baz. It is proposed that the main function of Dop in cellularisation is in regulating dynein-mediated transport of important cargos along microtubules (Hain, 2014).

This study presents the first evidence for regulation of dynein-mediated transport by a MAST family kinase. Dop is shown to controls phospho-Dic levels in a direct or indirect manner. The data are consistent with a model in which the initiation of furrow formation involves dynein-dependent transport that is controlled by Dop. In support of a role in membrane formation, this study found defects in the distribution of the recycling endosome and Golgi compartments in dop mutants. Interference with Rab11 function causes similar defects in Slam distribution as those shown by dop mutants. Therefore, Dop might control the transport of endomembrane compartments, which drive membrane growth. In addition, F-actin redistribution plays a major role in membrane cortical compartmentalisation in the initial stages of cellularisation. The focussing of F-actin to incipient furrows might involve a dynein-dependent shift of actin regulators or existing actin filaments to the furrow. An attractive hypothesis is that the translocation of F-actin and/or its regulators is coupled to an endomembrane compartment that is transported via microtubules towards the incipient furrow canals. Future studies should aim to determine which dynein cargos contribute to furrow formation and how Dop regulates Dic phosphorylation at the molecular level (Hain, 2014).

The influence of dynein processivity control, MAPs, and microtubule ends on directional movement of a localising mRNA

Many cellular constituents travel along microtubules in association with multiple copies of motor proteins. How the activity of these motors is regulated during cargo sorting is poorly understood. This study addressed this issue using a novel in vitro assay for the motility of localising Drosophila mRNAs bound to native dynein-dynactin complexes. High precision tracking reveals that individual RNPs within a population undergo either diffusive, or highly processive, minus end-directed movements along microtubules. RNA localisation signals stimulate the processive movements, with regulation of dynein-dynactin's activity rather than its total copy number per RNP, responsible for this effect. These data support a novel mechanism for multi-motor translocation based on the regulation of dynein processivity by discrete cargo-associated features. Studying the in vitro responses of RNPs to extrinsic microtubule-associated proteins (MAPs) and microtubule ends provides insights into how an RNA population could navigate the cytoskeletal network and become anchored at its destination in cells (Soundararajan, 2014).

Several previous studies have manipulated the copy numbers of cargo-associated motors in order to elucidate how multiple motors orchestrate sorting. One experimental approach has been to alter the numbers of isolated motors or motor domains attached to artificial cargos such as beads or DNA origami. Although very informative, these studies did not include physiological cargo, cargo adaptors, and motor co-factors that could potentially modulate motor behaviour. Other studies have used genetic manipulations to alter the numbers of motor complexes available to cargos in vivo. These approaches are physiologically relevant but cannot rule out influences from the cellular environment, including possible indirect effects of altered motor concentration on other processes that impinge on cargo motility. This study has manipulated the copy number of native motor complexes on a physiological type of cargo, by incubating cellular extracts with RNA variants, and studied the consequences on motility in a defined in vitro setting using high spatiotemporal resolution imaging (Soundararajan, 2014).

hairy (h) wild-type ribonucleoprotein complexes (RNPs) associate with dynein-dynactin and can undergo either unidirectional motion in the minus end direction that is highly processive or bidirectional motion that has characteristics of a diffusive process. The experiments indicate that unidirectional RNP movement is driven by active, ATP hydrolysis-mediated translocation of dynein along the microtubule. The most parsimonious explanation for the bidirectional motion is that it is also due to dynein undergoing back-and-forth movements along the microtubule, a behaviour that has been observed in several studies of the purified motor in vitro and also appears to occur in vivo. Indeed, bidirectional movement was observed of a significant subset of RNPs containing only the h localisation element HLE (~30% in the absence of MAPs and ~60% in their presence), on which binding of a kinesin family member was not observed under conditions in which dynein and dynactin were readily detected. Interestingly, bidirectional motion of RNPs is not overtly sensitive to inhibition of dynein's ability to hydrolyse ATP. Thus, these findings are compatible with those of of a previous study that reported passive diffusion along microtubules of dynein in complex with a dynactin component in the presence of ATP or ATP-vanadate. No evidence was fpimd that bidirectional RNPs can undergo long, ATP hydrolysis-dependent runs in both directions akin to those documented for individual, GFP-labelled dynein-dynactin complexes purified from mouse brain (Soundararajan, 2014).

Varying the number of SL1 elements within the context of the h 3'UTR revealed a correlation between the total copy number of dyneins per RNP and the probability of entering into the unidirectional, minus end-directed state. Previous studies using purified dynein bound to artificial cargos have demonstrated that increasing motor copy number is sufficient to stimulate processive movement towards minus ends. However, experiments with the isolated HLE signal indicated that total motor number is not the key determinant of the unidirectional mode of RNP movement. The HLE alone has a statistically indistinguishable copy number of dynein components to the hΔLE RNA, in which the localisation signal has been replaced by a heterologous sequence in the context of the h 3'UTR, yet only the former RNA is capable of unidirectional motion. These data suggest that features associated with the RNA signal are sufficient to increase the probability of processive movement of the associated dynein. It has recently been shown in Schizosaccharomyces pombe that microtubule-associated dynein can switch from diffusive to processive behaviour upon contacting cortical anchors, an event that regulates the generation of pulling forces on the microtubul. The current data suggest that regulation of dynein processivity by associated factors may be a widespread phenomenon (Soundararajan, 2014).

Collectively, these results support a novel model in which the same cargo species can interact with processive or non-processive dynein, with discrete cargo-associated features regulating the probability of switching between the two behaviours. Thus, the regulatory mechanisms underpinning sorting of RNPs in this system appear distinct from those of other well-studied bidirectional cargos, which involve the interplay of opposite polarity force-generating motors, such as dyneins and kinesins. How might localisation signals regulate dynein processivity? One possibility is that proteins recruited by the localisation signal directly regulate the activity of the motor. Two candidates to serve such a role are the adaptor proteins Egl and BicD, which are associated only with the dynein-dynactin bound to the localisation signals. It is also conceivable that the structure or rigidity of the RNA signal plays an architectural role in presenting dynein-dynactin to the microtubule in a manner that favours processive movement (Soundararajan, 2014).

This study also sheds light on how physiological cargo-motor complexes respond to extrinsic factors within the cytoskeletal environment. RNPs frequently reverse when encountering the regions of microtubules bound by puncta of both MAPs studied, rigor kinesin and tau23. The behaviour of RNPs at MAPs is therefore highly reminiscent of that seen for individual, purified dynein-dynactin complexes in vitro. Interestingly, comparison of the behaviour of the h RNA variants reveals that the number of dynein-dynactins associated with an RNP does not increase the probability of passing microtubule-associated obstacles (Soundararajan, 2014).

The ability of RNPs to reverse at MAPs may help them navigate to their destination in vivo. For example, reversals upon meeting an obstacle may facilitate encounters of RNPs with intersecting microtubules. Switching of RNPs between microtubules, a behaviour observed when intersections occur in in vitro assays, could allow these complexes to explore alternative routes to their destination. Reversals of RNPs at a MAP may also give dynein space to switch to a different lateral position on the same microtubule and thereby provide another opportunity to pass the obstacle following resumption of movement in the previous direction. Compatible with this notion, RNPs were seen moving on single microtubules that can pass obstacles after multiple attempts (Soundararajan, 2014).

Analysis of the behaviour of RNPs at microtubule ends indicates that plus end encounters always result in a reversal. The behaviour may also be advantageous in a cellular environment by preventing detachment of RNPs at this point, an outcome that would necessitate a rebinding event before motion on the microtubule network can resume. Interestingly, a subset of RNPs undergoes pausing at the minus ends of microtubules, with a mean dwell time of ~1 min for bidirectional complexes. These findings demonstrate that the probability of changing directions is different at the minus end and the plus end of the microtubule and that additional in vivo features, such as the γ-tubulin ring complex or other centrosome-associated factors, are not obligatory for long-term retention of RNPs at the minus end. Intrinsic behaviours of RNPs upon reaching the minus end of the α/β-tubulin polymer may therefore contribute to the dynein-dependent anchorage of RNAs in the vicinity of minus ends in vivo. Analysis of hΔLE, hWT, and hSL1x3 RNAs demonstrates that the probability of an RNP undergoing minus end pausing in vitro, as well as the duration of such events, is not influenced by the addition of more dynein-dynactins through localisation elements. This finding offers an explanation for why inhibition of Egl and BicD following translocation of localising RNAs to the apical cytoplasm of the embryo does not affect dynein-dependent anchorage (Soundararajan, 2014).

It is intriguing that the average dwell time of pausing events of unidirectional hWT and hSL1x3 RNPs at minus ends is ~ fivefold less than that of bidirectional RNPs harbouring the same RNA species. It was also found that the unidirectional HLE RNPs, which lack features within the h 3′UTR that can recruit non-processive dynein-dynactin, dwell at the minus end for significantly less time than the unidirectional hWT and hSL1x3 RNPs. One explanation for these findings is that the ability of dynein bound to localisation signals to walk processively off the minus end is antagonised by interactions with the microtubule mediated by non-processive dynein bound at other sites in the RNA. Additional, long-term experiments will be required to test this hypothesis. Nonetheless, the data suggest more generally that features that promote bidirectional motion could assist in the retention of RNPs at minus ends (Soundararajan, 2014).

Collectively, this analysis of encounters of RNPs with MAPs and microtubule ends raises the possibility that the co-existence of unidirectional or bidirectional modes of movement facilitates efficient navigation of an RNA population to its destination in vivo. Processive, unidirectional movement in the minus end direction could be beneficial for rapid, directional movement along regions of the microtubule that are not rich in MAPs. Diffusive motion along microtubules may be valuable for movement of RNPs through an obstacle rich environment and could still contribute to asymmetric sorting as it is associated with long-term retention of complexes at microtubule minus ends. This strategy appears analogous to that used by DNA enzymes and kinesins that depolymerise microtubules, which can employ one-dimensional diffusion to search for their specific target sites (Soundararajan, 2014).

This in vitro work on RNA motility has provided several new insights into how cargo-motor complexes operate and how their behaviour is modulated by encounters with the environment. The data lead to a model in which discrete cargo-associated features regulate motor processivity, a phenomenon that could not have been recapitulated using minimal motor elements coupled to artificial cargos. The results also illustrate that intrinsic motile properties of cargo populations in vivo are likely to be obscured by the influence of extrinsic factors including MAPs and microtubule ends. Further exploitation of the RAT-TRAP assay is likely to be an effective strategy for shedding light on molecular mechanisms that underpin intrinsic and extrinsic regulation of cargo motility, particularly when combined with powerful Drosophila gene perturbation techniques. In the longer term, it will be important to understand how the behaviours defined in vitro are integrated during sorting of single RNA molecules in vivo, a goal that necessitates the development of new methods to visualise movement of transcripts in the optically challenging embryo system (Soundararajan, 2014).

Protein Interactions

Dynein light chains of Drosophila

The molecular and genetic characterization of the cytoplasmic dynein light-chain gene, ddlc1, from Drosophila melanogaster is reported. ddlc1 encodes the first cytoplasmic dynein light chain identified, and its genetic analysis represents the first in vivo characterization of cytoplasmic dynein function in higher eukaryotes. The ddlc1 gene maps to 4E1-2 and encodes an 89-amino-acid polypeptide with a high similarity to the axonemal 8-kDa outer-arm dynein light chain from Chlamydomonas flagella. Developmental Northern (RNA) blot analysis and ovary and embryo RNA in situ hybridizations indicate that the ddlc1 gene is expressed ubiquitously. Anti-DDLC1 antibody analyses show that the DDLC1 protein is localized in the cytoplasm. P-element-induced partial-loss-of-function mutations cause pleiotropic morphogenetic defects in bristle and wing development, as well as in oogenesis, and hence result in female sterility. The morphological abnormalities found in the ovaries are always associated with a loss of cellular shape and structure, as visualized by a disorganization of the actin cytoskeleton. Total-loss-of-function mutations cause lethality. A large proportion of mutant animals degenerate during embryogenesis, and the dying cells show morphological changes characteristic of apoptosis, namely, cell and nuclear condensation and fragmentation, as well as DNA degradation. Cloning of the human homolog of the ddlc1 gene, hdlc1, demonstrates that the dynein light-chain 1 is highly conserved in flies and humans. Northern blot analysis and epitope tagging show that the hdlc1 gene is ubiquitously expressed and that the human dynein light chain 1 is localized in the cytoplasm. hdlc1 maps to 14q24 (Dick, 1996b).

Mutations in an 8 kDa (8x10(3) Mr) cytoplasmic dynein light chain disrupt sensory axon trajectories in the imaginal nervous system of Drosophila. Weak alleles are behaviorally mutant, female-sterile and exhibit bristle thinning and bristle loss. Null alleles are lethal in late pupal stages and alter neuronal anatomy within the imaginal CNS. P[Gal4] inserts were used to examine the axon projections of stretch receptor neurons and an engrailed-lacZ construct was used to characterize the anatomy of tactile neurons. In mutant animals both types of sensory neurons exhibit altered axon trajectories within the CNS, suggesting a defect in axon pathfinding. However, the alterations in axon trajectory did not prevent these axons from reaching their normal termination regions. In the alleles producing these neuronal phenotypes, expression of the cytoplasmic dynein 8 kDa light chain gene is completely absent. These results demonstrate a new function for the cytoplasmic dynein light chain in the regulation of axonogenesis and may provide a point of entry for studies of the role of cellular motors in growth cone guidance (Phillis, 1996).

The molecular motor dynein is involved in targeting Swallow and bicoid mRNA to the anterior pole of Drosophila oocytes

Localization of Bicoid messenger RNA to the anterior pole of the Drosophila oocyte requires the exuperantia, swallow and staufen genes. swa encodes a protein of 548 amino acids that contains a coiled-coil domain and a region with remote similarity to an RNA-recognition motif. SWA mRNA shows an even distribution during oogenesis in the nurse cells as well as the oocyte, with higher levels being found in the nurse cells. Swa protein transiently co-localizes with BCD mRNA in mid-oogenesis. Swa also localizes to the anterior pole of the oocyte in the absence of BCD mRNA. This localization does not require Exu, but depends on intact microtubules. In mutant ovaries with duplicated polarity of microtubules, Swa and BCD mRNA are ectopically localized at the posterior pole, as well as being present at the anterior pole. Dynein light chain-1 (Ddlc-1), a component of the minus-end-directed microtubule motor cytoplasmic dynein, has been identified as a Swa-binding protein. It is proposed that Swa acts as an adaptor for the dynein complex and thereby enables dynein to transport BCD mRNA along microtubules to their minus ends at the anterior pole of the oocyte (Schnorrer, 2000).

Electron-microscopic studies have revealed a concentration of Exu in discrete subcellular particles, called sponge bodies. These structures consist of endoplasmic-reticulum-like cisterna, embedded in an amorphous electron-dense mass. Swa protein alone is not sufficient to concentrate BCD mRNA anteriorly, since in exu mutants BCD mRNA reaches the oocyte but is not localized correctly, even though the localization of Swa appears normal. Thus, Exu may be involved in preparing or modifying BCD mRNA, thereby allowing the later anterior localization of this RNA while Exu itself is not concentrated anteriorly but is dispersed throughout the oocyte. Although it has not yet been shown directly that BCD mRNA is indeed concentrated in the sponge bodies, the strikingly similar pattern of localization of Exu protein and BCD mRNA in patches in the nurse cells strongly suggests that Exu targets BCD mRNA to the sponge bodies. In the current model, during this Exu-dependent phase of BCD mRNA localization, the RNA assembles, together with unknown components, into ribonucleoprotein particles (RNPs) in the sponge bodies. These RNPs are released into the oocyte. The BCD mRNA localization step in the oocyte crucially depends on Swa. Swa interacts with the BCD mRNA , which is complexed with trans-acting factors, and thus allows the transport of both Swa protein and BCD mRNA by the dynein motor along microtubules to the anterior pole of the oocyte. The Swa-dependent transport does not occur until BCD mRNA has arrived in the oocyte, since Swa cannot be detected in the nurse cells and the transport of BCD mRNA from nurse cells to the oocyte is intact in swa mutants. Binding of Swa to Ddlc-1 appears to be required for the localization of Swa, since a mutant Swa construct that lacks the dynein-binding domain is not transported. However, the binding of Swa to the dynein light chain alone seems not to be sufficient for the localization of Swa, as mutations in the C-terminal part of Swa abolish correct Swa localization but not Ddlc-1 binding. The results of pulldown experiments indicate that the Swa C terminus may contain a regulatory region or recruit a binding partner that is involved in regulating the dynein-dependent transport. One possible additional factor involved in the transport process is the dynactin complex, which has been suggested to be an essential co-factor for dynein (Schnorrer, 2000 and references therein).

Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes

To establish the major body axes, late Drosophila oocytes localize determinants to discrete cortical positions: bicoid mRNA to the anterior cortex, oskar mRNA to the posterior cortex, and gurken mRNA to the margin of the anterior cortex adjacent to the oocyte nucleus (the 'anterodorsal corner'). These localizations depend on microtubules that are thought to be organized such that plus end-directed motors can move cargoes, like oskar mRNA, away from the anterior/lateral surfaces and hence toward the posterior pole. Likewise, minus end-directed motors may move cargoes toward anterior destinations. Contradicting this, cytoplasmic Dynein, a minus-end motor, accumulates at the posterior. Disruption of the plus-end motor kinesin I causes a shift of dynein from posterior to anterior. This provides an explanation for the dynein paradox, suggesting that dynein is moved as a cargo toward the posterior pole by kinesin-generated forces. However, other results present a new transport polarity puzzle. Disruption of kinesin I causes partial defects in anterior positioning of the nucleus and severe defects in anterodorsal localization of gurken mRNA. Kinesin may generate anterodorsal forces directly, despite the apparent preponderance of minus ends at the anterior cortex. Alternatively, kinesin I may facilitate cytoplasmic dynein-based anterodorsal forces by repositioning dynein toward microtubule plus ends (Brendza, 2002).

To better understand microtubule-based localization processes in Drosophila oocytes, the localization of kinesin I was studied with an antiserum that binds its motor subunit, kinesin heavy chain (Khc). An even distribution of Khc is seen throughout the germline cells of the germarium and early egg chambers. Staining was usually more intense in the somatic follicle cells that enclose the egg chambers and was particularly strong in polar follicle cells. Beginning in stage 8 and continuing through stage 10A, Khc is most concentrated at the posterior pole of the oocyte. A small concentration also appears in the anterodorsal corner. Disruption of Khc expression in clones of cells by mitotic recombination with a null allele of the Khc gene showed that the posterior Khc is a product of the germline and not of the posterior follicle cells (Brendza, 2002).

To test the possibility that dynein is carried toward the posterior pole by kinesin I, the distribution of cytoplasmic dynein heavy chain (cDhc) and Khc was compared in late-stage Khc mutant oocytes, produced by Khc null germline clones. In the Khc mutants, cDhc staining shows little or no posterior localization; rather, it accumulates strongly at the anterior. Anti-tubulin staining indicates that the anterior-posterior gradient of microtubules is not disrupted in Khc null oocytes. Therefore, the shift of dynein to the anterior in Khc mutants suggests that kinesin I is responsible for moving cytoplasmic dynein away from minus ends at the anterior and thus moving it toward the posterior pole (Brendza, 2002).

In summary, the results provide several insights into localization processes during mid-late oogenesis: (1) kinesin I colocalizes at the posterior pole with cytoplasmic dynein; (2) kinesin I is required for the posterior localization of cytoplasmic dynein; (3) kinesin I is required for the dorsal localization of gurken mRNA, and (4) kinesin I contributes to the proper anterior positioning of the oocyte nucleus. A role for kinesin in moving dynein toward the posterior pole provides a solution to the paradox of the accumulation of a minus-end motor in an area thought to be a destination for plus end-directed transport. However, a role for kinesin in anterodorsal localization is surprising because of evidence that minus ends are most concentrated there. In particular, a Nod:ß-galactosidase fusion protein that is targeted to microtubule minus ends accumulates around the nucleus and at the anterior margin during stages 8–10. How might a plus end-directed motor participate in localization toward an area dominated by microtubule minus ends (Brendza, 2002)?

Previous reports and recent results suggest that dorsal pole specification requires the minus end-directed motor, cytoplasmic dynein. Hypomorphic mutations that impair the function of Drosophila Lis1, which is known to be required in various systems for dynein/dynactin function in nuclear migration and other motility processes, can cause ventralization of chorions, mislocalization of the nucleus, and failure of anterodorsal gurken localization. Conditional overexpression of a protein that disrupts the dynein/dynactin complex has been shown to cause equivalent, though more severe, defects in those same dorsal specification processes. The fact that the same dorsal pathway phenotypes are caused by germline Khc disruption suggests that kinesin I and cytoplasmic dynein both are required for nuclear positioning and anterodorsal gurken mRNA localization (Brendza, 2002).

The following model is proposed to explain these results. Dynein, which is synthesized in nurse cells, walks along microtubules from nurse cells through connecting ring canals toward microtubule minus ends at the oocyte posterior until stage 4. After the microtubule cytoskeleton reorganizes during stage 7, concentrating minus ends at the anterior cortex, dynein-generated movements are redirected away from the posterior. This drives the nucleus and gurken mRNA to the anterior margin. Materials like dynein and determinant mRNAs, moved by unknown forces, continue to enter the oocyte from nurse cells through the anterior ring canals. Those that need to be distributed toward the posterior and are too large to diffuse efficiently are moved by kinesin I, either directly or by means of cytoplasmic flows. As the oocyte enlarges during late stages, diffusion of the large cytoplasmic dynein/dynactin complex away from anterior minus ends becomes limiting. Thus, active transport of dynein away from the anterior by kinesin or by kinesin-generated cytoplasmic flows becomes critical. In stage-9 and -10 Khc mutant oocytes, dynein is trapped near minus ends at the anterior cortex. Anterior-directed dynein-based forces that act on gurken mRNA, the nucleus, and/or nuclear anchors are reduced, disrupting their normal positioning mechanisms (Brendza, 2002).

If this dynein recycling model is correct, why does a loss of Khc influence nuclear position and disrupt anterodorsal gurken localization but not other putative dynein functions, such as the anterior localization of bicoid mRNA? As with the initial localization of gurken mRNA, dynein-based forces toward the anterior margin may not be sensitive to poor recycling while the oocyte is small. Subsequent anterior localization of bicoid, as the oocyte enlarges, may be relatively insensitive to a decline in long-range, anterior-directed forces because its requirements for such forces are less than those of the nucleus and gurken mRNA (Brendza, 2002).

Localization of bicoid mRNA in late oocytes is maintained by continual active transport

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential to produce the Bicoid protein gradient that patterns the anterior-posterior axis of the embryo. Previous studies have characterized a microtubule-dependent pathway for bicoid mRNA localization during midoogenesis, when bicoid first accumulates at the anterior. The majority of bicoid is actually localized later in oogenesis, when the only known mechanism for mRNA localization is based on passive trapping. Through live imaging of fluorescently tagged endogenous bicoid mRNA, a temporally distinct pathway has been identified for bicoid localization in late oocytes that utilizes a specialized subpopulation of anterior microtubules and dynein. The directional movement of bicoid RNA particles within the oocyte observed in this study is consistent with dynein-mediated transport. Furthermore, the results indicate that association of bicoid with the anterior oocyte cortex is dynamic and support a model for maintenance of bicoid localization by continual active transport on microtubules (Weil, 2006).

Bcd patterning activity in the early embryo depends on the efficacy of bcd mRNA localization during oogenesis. Through live imaging of bcd mRNA fluorescently labeled in vivo, direct evidence is provided for a distinct, late bcd localization pathway that initiates with nurse cell dumping and is responsible for the majority of bcd present at the anterior of the embryo. Since specification of cell fates along the anterior-posterior axis is sensitive to single changes in bcd gene dosage, the predominant late source of localized bcd mRNA most likely makes the primary contribution to the Bcd protein gradient. Thus, it is proposed that although bcd localization also occurs during midoogenesis, the late pathway is the relevant one for anterior-posterior patterning. A new role has been identified for Stau as a component of this pathway and it is shown that localization of bcd/Stau complexes during late stages of oogenesis depends on the integrity of a subpopulation of microtubules that are anchored at the anterior cortex by the actin cytoskeleton. Movement of bcd RNA particles within the oocyte is consistent with anteriorly directed transport along these microtubules. Moreover, the results reveal dynamic behavior of bcd that does not fit the prevailing two-step transport and anchoring paradigm for mRNA localization. Rather, they support a model for steady-state localization of bcd at the anterior cortex by continual active transport (Weil, 2006).

Evidence for microtubule-directed transport in late-stage oocytes, when the cytoskeletal polarity thought to underlie transport along the anterior-posterior axis is no longer apparent, has been lacking. Indeed, previous studies have shown that posterior localization of mRNAs like nos and osk after stage 10 does not depend directly on microtubules, but occurs by a diffusion/trapping mechanism that is facilitated by ooplasmic streaming. In contrast, results presented in this study suggest that microtubules emanating from the anterior cortex support transport of bcd mRNA to the anterior. Since it has not been possible to follow the movement of bcd particles as they enter the oocyte during nurse cell dumping, the possibility cannot be excluded that some bcd is simply trapped by anterior microtubules as it enters, in a manner analogous to trapping of nos mRNA by germ plasm at the posterior. However, the requirement for microtubules, dynein, and ATP to maintain bcd mRNA localization after nurse cell dumping and ooplasmic streaming, together with the anteriorly directed movement of bcd particles near the anterior cortex, implicates these microtubules in active transport of bcd. Although ooplasmic streaming is capable of distributing mRNA localization complexes to the posterior pole, no bcd particles are detected in the posterior half of the oocyte. Thus, bcd must associate rapidly with these anterior microtubules upon entry into the oocyte. In the early embryo, nearly all bcd mRNA resides at the anterior cortex, whereas only a small fraction of nos mRNA is localized at the posterior pole. The existence of a microtubule-dependent pathway specific for anterior transport in late-stage oocytes could account for the dramatic difference in the efficiencies with which bcd and nos mRNAs are localized (Weil, 2006).

Anterior microtubules that mediate bcd mRNA localization in late-stage oocytes are most likely nucleated by a MTOC formed at the anterior cortex during stage 10. Analysis of mutants γ-Tub37C and Dgrip-75 implicate this MTOC in the transition of bcd mRNA from its ring-like distribution in stages 8–9 to a disc-like distribution in stage 10. Previously localized bcd mRNA is subsequently released from the anterior cortex in γ-Tub37C and Dgrip-75 mutants during nurse cell dumping, whereas other microtubule-dependent processes, such as ooplasmic streaming, are unaffected. These defects suggest that a specific reorganization of microtubules at the anterior cortex is responsible for maintaining bcd localization, while the majority of microtubules are reorganized for ooplasmic streaming (Weil, 2006).

Microtubules present at the anterior cortex in late-stage oocytes are distinct from microtubules that mediate bcd mRNA localization during midoogenesis and from cortical microtubules that mediate ooplasmic streaming by their dependence on the actin cytoskeleton. The results suggest that association with the actin cytoskeleton enables microtubules nucleated from the anterior MTOC during stage 10 to survive the dramatic changes in the oocyte that occur with nurse cell dumping and ooplasmic streaming and persist to later stages. These microtubules serve multiple functions in late-stage oocytes, as their selective perturbation disrupts both bcd mRNA localization and oocyte nucleus positioning (Weil, 2006).

Localization of bcd mRNA during mid- and late oogenesis can also be distinguished by a requirement for Stau. Stau participates in both the transport and anchoring of osk mRNA during midoogenesis, and Stau homologs have been implicated in microtubule-dependent transport of mRNAs in mammalian hippocampal neurons and Xenopus oocytes. This evidence, together with the presence of Stau in neuronal RNA granules, suggests a common function for Stau in coupling mRNAs to motor proteins for transport. Stau's function in bcd mRNA localization is not limited to anchoring bcd during the transition from oogenesis to embryogenesis as previously thought; rather, Stau plays an important role from the onset of nurse cell dumping. Although the stau mutant used in these experiments is a null allele, bcd mRNA localization is not completely eliminated in all stau mutant oocytes. Similarly, posterior localization of osk mRNA is greatly reduced, but it is not abolished in stau null mutant oocytes. It is possible that bcd mRNA localized during midoogenesis can persist at the anterior cortex in the absence of Stau. Alternatively, an as yet unidentified factor could act redundantly with Stau in late bcd localization. Redundant recognition elements are indeed present within the bcd mRNA localization signal (Weil, 2006).

Colocalization of Stau in particles with bcd mRNA suggests that it is an integral component of a bcd localization RNP. Individual bcd RNA particles that exhibit directional movement range in size from 0.3 to 1 μm, indicating that they consist of multiple mRNA and protein molecules. These particles are similar in size to the particles that form after injection of synthetic bcd 3′UTR RNA into embryos and become associated with astral microtubules. Formation of bcd 3′UTR particles requires Stau as well as intermolecular interactions between two or more bcd 3′UTRs. Similar assembly of large particles through interactions among bcd mRNA molecules during oogenesis would enable Stau, or another factor, to couple many bcd molecules to a single dynein motor. Concurrent transport of multiple mRNAs may contribute to the efficiency of bcd localization (Weil, 2006).

Current models for mRNA localization invoke independent transport and anchoring steps. Evidence for distinct steps comes from the differential effects of cytoskeletal inhibitors applied during and after translocation of RNAs to their destinations. In this manner, a kinesin- and microtubule-dependent transport step is paired with an actin-dependent anchoring step for localization of Vg1 and osk mRNAs, whereas a dynein- and microtubule-dependent transport step is paired with a dynein-dependent anchoring step for Drosophila pair-rule transcripts. The results indicate that bcd localization does not fit neatly into this two-step model. FRAP and FLIP experiments show turnover in the population of bcd mRNA at the anterior cortex even after dumping and streaming have ended and accumulation is maximal. This turnover suggests that bcd/Stau RNPs transported to the anterior cortex are unable to make stable associations with cortical components. Upon release from dynein, bcd/Stau RNPs may interact transiently with the cortex or may be released directly into the ooplasm, where they are reloaded onto dynein for another round of transport. Continual active transport may be critical for anterior localization of mRNAs like bcd, which occurs at a time when the rapid growth and movement of the oocyte cortex may inhibit or delay the establishment of a static anchoring mechanism. It will now be of interest to determine whether other localized mRNAs, particularly those in cell types that undergo rapid morphological changes such as migrating growth cones, behave similarly to bcd (Weil, 2006).

A Dynein-dependent shortcut rapidly delivers axis determination transcripts into the Drosophila oocyte

The primary axes of Drosophila are set up by the localization of transcripts within the oocyte. These mRNAs originate in the nurse cells, but how they move into the oocyte remains poorly understood. This study investigates the path and mechanism of movement of gurken RNA within the nurse cells and towards and through ring canals connecting them to the oocyte. gurken transcripts, but not control transcripts, recruit the cytoplasmic Dynein-associated co-factors Bicaudal D (BicD) and Egalitarian in the nurse cells. gurken RNA requires BicD and Dynein for its transport towards the ring canals, where it accumulates before moving into the oocyte. The results suggest that bicoid and oskar transcripts are also delivered to the oocyte by the same mechanism, which is distinct from cytoplasmic flow. It is proposed that Dynein-mediated transport of specific RNAs along specialized networks of microtubules increases the efficiency of their delivery, over the flow of general cytoplasmic components, into the oocyte (Clark, 2007).

Within nurse cells, a new path of Dynein-dependent transport to the ring canals has been identified that links the nurse cells to the oocyte. grk RNA moves along this route, and the data suggest that bcd and osk transcripts also follow the same path. This intracellular shortcut requires BicD and is distinct from the route taken by general cytoplasmic components and control RNAs, which move into the oocyte less effectively. The data suggest that the distinction between RNA components that follow this direct path and those that do not is the ability to recruit the Dynein-associated co-factors BicD and Egl. It is proposed that Dynein-dependent transport of grk, bcd and osk transcripts towards the ring canals follows a MT network, which is distinct from other networks in the nurse cells (Clark, 2007).

It was not possible to determine the proportion of grk RNA particles that move compared with ones that were stationary because it is hard to distinguish stationary particles from autofluorescence. By contrast, rapidly moving RNA particles of the same intensity are easy to distinguish from background. By showing directly that Dynein is required for the transport of axis specification transcripts from the nurse cells to the oocyte, this work explains previous work on this topic that did not directly address the mechanism of transport from the nurse cells into the oocyte. The results also explain why the movement of bcd and osk mRNA into the oocyte is MT dependent, and why pair-rule transcripts, which are transported in the blastoderm embryo in a MT-dependent manner, by Dynein, are also transported into the oocyte when exogenously expressed in the nurse cells. It is suggested that nurse cell-to-oocyte transport is likely to be a fairly promiscuous transport system that can deliver any transcript that has the capacity for transport by the Dynein motor complex along MTs to their minus ends. It is therefore likely that the Dynein-dependent shortcut is deployed by many other transcripts that are localized in the oocyte during mid-oogenesis, such as orb, K10 and nanos (nos). In fact, given that the oocyte nucleus is largely transcriptionally inactive, it is possible that up to 10% of all transcripts thought to be localized in the oocyte could first be transported by the same Dynein-dependent mechanism into the oocyte (Clark, 2007).

The Dynein-dependent transport route uncovered within the nurse cells is likely to allow transcripts encoding axis specification determinants to be delivered rapidly at key times in oogenesis. In particular, cytoplasmic transport during stages 5-8 is likely to be relatively slow and non-specific, so delivery of transcripts from the nurse cell nuclei to the oocyte cytoplasm is likely to be very slow, if it involves an undirected diffusion-based process. Certainly, osk and bcd mRNA and other transcripts are thought to form large multimeric complexes in the nurse cells, so are unlikely to be easily dispersed within the cytoplasm by free diffusion. osk and grk are transported into the oocyte at the same stages of oogenesis, and both require Bruno and Hrp48 (also known as Hrb27C - FlyBase); however, it is unclear whether they are transported within the same complexes into the oocyte. At stage 10B, the mechanism this paper has described is not required, because the rapid dumping of all of the cytoplasmic contents of the nurse cells into the oocyte occurs. However, by stage 10B, most of the major patterning transcripts have probably been localized in the oocyte (Clark, 2007).

This work does not address directly the speed of passive diffusion of RNA into the oocyte or the mechanism of cytoplasmic flow and dumping in stage 10B. Although Kinesin 1 is required for cytoplasmic movements within the oocyte, it is not required for the general growth of the oocyte or for the presence of mRNAs in the oocyte. These observations suggest that Kinesin 1 is not important for cytoplasmic transport or for specific mRNA transport into the oocyte (Clark, 2007).

The existence of a specific intracellular route for the transport of transcripts in nurse cells adds to existing evidence that there are various minus-end destinations to which different cargos are delivered by Dynein within the same cell. For example, within the oocyte, bcd RNA is transported to the cortex if injected into the oocyte, but to the anterior, after transport into the oocyte, following injection into the nurse cells. grk RNA is transported in two steps, both of which depend on Dynein. The second step is towards the oocyte nucleus, and is unique to grk and I factor RNA, but is not shared with bcd and K10 transcripts, despite the fact that all of these transcripts are probably being transported by Dynein. There are, therefore, likely to be several distinct MT routes along which Dynein can transport cargos within egg chambers. In neurons, choices between distinct MT routes are made by Kinesin-dependent vesicle transport depending on the presence of a specific neurotransmitter-receptor-interacting protein, GRIP1. How Dynein chooses between distinct MT networks is less clear, but could be based on distinct isoforms of the motor complex, on distinct kinds of MTs with different tubulin isoforms, or on their decoration with different MT-associated proteins. In addition, there is evidence that cargos can influence the behaviour of their motor, raising the interesting possibility that cargos could also influence the choice of MT route adopted by their motors. This work suggests that the presence of BicD and Egl could also influence the choice of MT route adopted by motors. Future work, including new approaches for co-visualizing MTs and RNAs in living cells, will be required to distinguish between all of these possible ways of selecting intracellular routes. Whatever the basis of such distinct routes, they are likely to exist for various kinds of molecular motors and to be functionally important for a wide range of tissues and cargos (Clark, 2007).

Distinguishing direct from indirect roles for bicoid mRNA localization factors

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. This study used live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. Swallow and bicoid mRNA are shown to be transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, evidence is presented that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. These results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead a model is proposed for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport (Weil, 2010).

A direct role for Swa in either transport or anchoring of bcd mRNA predicts and requires that the protein be colocalized with bcd mRNA during transport or anchoring. This study tested this prediction conclusively in three ways. First, an OMX microscope with highly sensitive and rapid multi-channel imaging was used to study to movement of Swa and bcd RNA particles simultaneously. Second, advantage was taken of the increased resolution of the OMX microscope with fixed material to analyze the precise locations of Swa and bcd at the anterior oocyte cortex. Importantly, the results show conclusively that Swa and bcd mRNA move independently to the anterior and occupy distinct domains at the anterior cortex. Third, the subcellular distributions of Swa and bcd was determined at EM resolution, demonstrating that bcd is mostly in particles near the anterior cortex whereas Swa is mostly confined to the plasma membrane of the entire oocyte. How the membrane association of Swa, which does not contain a transmembrane domain and is not predicted to harbor lipid modifications, is mediated remains to be investigated (Weil, 2010).

At the plasma membrane, Swa is found in close proximity to the cortical actin cytoskeleton. Together with the defects in the cortical actin cytoskeleton observed in swa mutants, this indicates a role for Swa in organization of the actin cytoskeleton. Whereas the actin cytoskeleton is not required for anchoring bcd at the anterior cortex until the very end of oogenesis (Weil, 2008), it plays an indirect role in bcd localization by anchoring the anterior microtubules required for the continual transport of bcd during stages 11-13. The results indicate that, in swa mutants, these microtubules remain intact but are not properly attached to or organized at the cortex, such that bcd transport is non-productive (Weil, 2010).

Swa is not limited to the anterior, however, and can be detected along the entire cortex of the oocyte. Moreover, in swa mutants, the cortical actin cytoskeleton is disrupted throughout the entire oocyte. Accordingly, some swa alleles show defects in posterior localization of osk mRNA at late stages of oogenesis (Pokrywka, 2004). Since actin is required for osk mRNA anchoring, this phenotype could be a result of disruption of the actin cytoskeleton. Clues as to how Swa regulates the actin cytoskeleton are not readily apparent from the Swa protein sequence, and an understanding of this mechanism awaits further biochemical analysis of Swa (Weil, 2010).

IEM results provide direct evidence that bcd mRNA is packaged with Stau and dynein into RNPs that are enriched at, and presumably transported to, the anterior after stage 10b. Indeed, live imaging using the OMX system showed co-transport of bcd and Stau in the same dynamic particles. In addition to its role in bcd localization, Stau is a key component of osk RNPs and is required for kinesin-dependent transport of osk to the oocyte posterior. Thus, Stau is a component of two independent transport RNPs, each associated with a different motor protein for transport to distinct locations within the oocyte. Whether Stau, which contains five double-stranded RNA-binding domains, interacts directly with bcd and osk mRNAs or indirectly by association with sequence-specific mRNA-binding proteins, remains to be determined. However, structure/function analysis of Stau suggests that different Stau double-stranded RNA-binding domains may determine which transport factors are linked to each mRNA. Furthermore, as Stau functions in osk localization during stages 8-9 but is required for bcd localization only after stage 10b, the assembly of Stau/bcd transport complexes must be temporally regulated. Stau is also required during the oocyte-to-embryo transition, for the redistribution of bcd from its tight cortical distribution in the oocyte to its more diffuse anterior distribution in the early embryo. Stau has been shown to colocalize with bcd mRNA when it is anchored to the actin cytoskeleton at the latest stages of oogenesis and is retained by bcd particles in the early embryo (Weil, 2008). Thus, Stau remains an integral component of bcd RNPs as they are remodeled from transport to anchoring complexes and finally to their translationally active state in the embryo. In the future, detailed biochemical analysis combined with advanced imaging methods that permit detection of in vivo RNA-protein and protein-protein interactions will be necessary to ascertain how specificity is conferred on localization and anchoring (Weil, 2010).

Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate

In many cell types, polarized transport directs the movement of mRNAs and proteins from their site of synthesis to their site of action, thus conferring cell polarity. The cytoplasmic dynein microtubule motor complex is involved in this process. In Drosophila, the Egalitarian (Egl) and Bicaudal-D (BicD) proteins are also essential for the transport of macromolecules to the oocyte and to the apical surface of the blastoderm embryo. Hence, Egl and BicD, which have been shown to associate, may be part of a conserved core localization machinery in Drosophila, although a direct association between these molecules and the dynein motor complex has not been shown. This study reports that Egl interacts directly with Drosophila dynein light chain (Dlc), a microtubule motor component, through an Egl domain distinct from that which binds BicD. It is proposed that the Egl-BicD complex is loaded through Dlc onto the dynein motor complex thereby facilitating transport of cargo. Consistent with this model, point mutations that specifically disrupt Egl-Dlc association also disrupt microtubule-dependant trafficking both to and within the oocyte, resulting in a loss of oocyte fate maintenance and polarity. These data provide a direct link between a molecule necessary for oocyte specification and the microtubule motor complex, and supports the hypothesis that microtubule-mediated transport is important for preserving oocyte fate (Navarro, 2004).

The novel zinc finger protein dASCIZ regulates mitosis in Drosophila via an essential role in dynein light chain expression

The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. This study reports the identification of the Drosophila orthologue of ASCIZ (dASCIZ) and shows that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, it was shown that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp-LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. It is speculated that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, these data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light chain levels and a novel regulator of mitosis in flies (Zaytseva, 2013).

spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfα-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. The ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. spn-F was clond and found to encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. Spn-F interacts directly with the Dynein light chain Ddlc-1 (Cut up). These results show that this novel protein affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis (Abdu, 2006; full text of article).

In a global two-hybrid screen, Spn-F (CG12114) was found to interact with the Ik2 (CG2615) protein. Mutations in Ik2 have been isolated and characterized, ik2 mutants share many phenotypes with spn-F, including a very similar bristle phenotype and specific effects on MT organization in oogenesis. However, ik2 mutants are lethal, whereas spn-F homozygotes survive. In addition, whereas spnF mutations have only mild effects on Oskar protein localization and a low frequency of bicaudal phenotypes, such effects are more pronounced in the ik2 mutants. Nevertheless, the striking similarities strongly suggest that the two genes function in a common pathway that affects certain types of MT more strongly than others. In normal mitotic cells, the minus ends of MTs are usually focused by the centrosomes in the interior of the cell, and plus ends contact the cortex. However, in specialized cells, such as the Drosophila oocyte, there are minus ends that make contact with the cortex. It is therefore possible that Spn-F and Ik2 are required for providing a stable connection between such cortical MT minus ends and cortical actin for subsets of MTs involved in specialized transport processes. Future experiments will address the interactions of Spn-F and Ik2 directly, and will determine whether, for instance, Spn-F might be a target of Ik2 (Abdu, 2006).

The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor

Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila Anastral spindle-2 (Ana2) is a conserved centriole duplication factor. While recent work demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. This study examined the Ana2-LC8 interaction and mapped two LC8-binding sites within Ana2's central region, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 mμM) while site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 mμM). Both LC8-binding sites flank a predicted ~34-residue alpha-helix. Two independent atomic structures are presented of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form beta-strands that extend a central composite LC8 beta-sandwich. LC8 recognizes the signature TQT motif in Ana2's first LC8 binding site, forming extensive van der Waals contacts and hydrogen bonding with the peptide, while the Ana2 site 2 TQC motif forms a uniquely extended beta-strand, not observed in other dynein light chain-target complexes. Size-exclusion chromatography coupled with multi-angle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization likely enhances Ana2's avidity for centriole binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis (Slevin, 2014).

Dynein intermediate chains of Drosophila

The intermediate chains (ICs) are the subunits of the cytoplasmic dynein that provide binding of the complex to cargo organelles through interaction of their N termini with dynactin. In Drosophila, the IC subunits are represented by at least 10 structural isoforms, created by the alternative splicing of transcripts from a unique Cdic gene. The splicing pattern is tissue specific. A constitutive set of four IC isoforms is expressed in all tissues tested; in addition, tissue-specific isoforms are found in the ovaries and nervous tissue. The structural variations between isoforms are limited to the N terminus of the IC molecule, where the interaction with dynactin takes place. This suggests differences in the dynactin-mediated organelle binding by IC isoforms. Accordingly, when transiently expressed in Drosophila Schneider-3 cells, the IC isoforms differ in their intracellular targeting properties from each other. A mechanism is proposed for the regulation of dynein binding to organelles through the changes in the content of the IC isoform pool (Nurminsky, 1998).

The microtubule motor cytoplasmic dynein performs multiple cellular functions; however, the regulation and targeting of the motor to different cargoes is not well understood. A biochemical interaction between the dynein intermediate chain subunit and the p150-Glued component of the dynein regulatory complex, dynactin, has supported the hypothesis that the intermediate chain is a key modulator of dynein attachment to cellular cargoes. Multiple intermediate chain polypeptides have been identified that cosediment with the 19S dynein complex. Two differentially expressed transcripts derive from the single cytoplasmic dynein intermediate chain (Cdic) gene that differ in the 3' untranslated region sequence. These results support previous observations of multiple Cdic gene products that may contribute to the specialization of dynein function. Most significantly, genetic evidence is provided that the interaction between the dynein intermediate chain and p150-Glued is functionally relevant. A genomic Cdic transgene has been used to show that extra copies of the dynein intermediate chain gene act to suppress the rough eye phenotype of the mutant Glued1, a mutation in the p150-Glued subunit of dynactin. Furthermore, the interaction between the dynein intermediate chain and p150-Glued has been shown to be dependent on the dosage of the Cdic gene. This result suggests that the dynein intermediate chain may be a limiting component in the assembly of the dynein complex and that the regulation of the interaction between the dynein intermediate chain and dynactin is critical for dynein function (Boylan, 2000).

The gene for the intermediate chain subunit of cytoplasmic dynein is essential in Drosophila

The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, mutations in the gene for Cytoplasmic dynein intermediate chain, Dic19C, has been identified. A functional Dic transgene has been used in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued1, a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, complementation analysis was used to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene; they reveal a novel role for dynein function during wing development (Boylan, 2002).

Cytoplasmic dynein is a minus-end-directed microtubule motor involved in numerous intracellular motility events including retrograde axonal transport, the transport and positioning of vesicles and organelles, spindle assembly and morphogenesis, and nuclear migration. The dynein motor is a large complex composed of two heavy chain polypeptides and numerous intermediate and light chain subunits. The heavy chains compose the ATPase portion of the molecule, providing energy for movement along microtubules through the binding and hydrolysis of ATP. Electron microscopy analysis has shown that the heavy chains form two globular heads connected by thin stalks. The intermediate and light chain subunits are present as a complex at the base of the heavy chain stalk where they are in a position to interact with other cellular components and may participate in targeting the motor to specific cargoes (Boylan, 2002).

A role for the intermediate chain (IC) subunit in the attachment of dynein to cargo was first suggested by structural analysis of axonemal outer arm dynein. In the flagellar axoneme, dynein motor activity drives the sliding of adjacent outer doublet microtubules. As the heavy chain motor subunit moves along one outer doublet, the base of the motor complex remains attached to the adjacent outer doublet. Thus the transported cargo for axonemal dynein is another doublet microtubule attached through the base of the motor complex. Chemical crosslinking studies show that attachment through the base is mediated by direct binding of the intermediate chain subunit and alpha-tubulin within the A-tubule lattice of the outer doublet microtubule (Boylan, 2002).

The homology between axonemal and cytoplasmic dynein intermediate chains has suggested a similar cargo-binding function for the IC subunit of cytoplasmic dynein. Subsequently, in vitro binding of the cytoplasmic dynein intermediate chain to the p150-Glued subunit of dynactin was demonstrated in rat brain extracts. Dynactin, initially identified because of its ability to stimulate dynein-mediated vesicle motility, may act to couple dynein to cellular cargoes. The interaction between the dynein intermediate chain and p150-Glued and the association of the Arp1 subunit of dynactin with the membrane skeleton component spectrin have suggested a model in which dynactin serves as a cargo adapter molecule for dynein attachment to vesicular cargo. Additional studies showing that dynactin function is required during mitosis present the possibility that dynactin may also serve as a cargo adapter for dynein during cell division. Alternatively, the interaction of dynein IC and p150-Glued may affect motor processivity (Boylan, 2002).

The diversity of cytoplasmic dynein heavy chains is limited, but the multiplicity of accessory subunits is proposed to modulate specific dynein functions. Evidence for the assembly of functionally different dynein complexes has been demonstrated for the dynein light intermediate chain (LIC) and light chain subunits. Two LIC genes have been identified in rat: one that binds to pericentrin and one that does not. In triple overexpression studies it has been shown that the dynein heavy chain can bind to either LIC1 or LIC2, but not to both. Multiple alternatively spliced isoforms of the dynein intermediate chain have been identified and it has been suggested that this isoform diversity contributes to functional specificity, perhaps by the formation of distinct dynein complexes with specific intermediate chain isoforms. Recent reports have also implicated dynein light chain subunits in binding directly to specific cargoes, suggesting that the dynein intermediate chain may act indirectly to modulate cargo attachment by association with specific light chain subunits. For example, the 14-kD light chain was found to bind rhodopsin in the rod cells of the vertebrate retina and may function in turnover of photoreceptor membrane. Despite these leads, the functional analysis of how accessory subunits might contribute to specifying dynein functions is limited (Boylan, 2002).

As part of a systematic approach to understanding the functions of the intermediate chain subunit in the attachment of dynein to specific cargoes, the gene Dic19C from Drosophila has been cloned and characterized (Boylan, 2000). Similar to the dynein heavy chain, the dynein intermediate chain is present as a single gene that is expressed throughout Drosophila development. In addition, evidence has been found for a dosage-sensitive interaction between the intermediate chain gene and a mutation in the p150/Glued subunit of dynactin. The Dic transgene has been used in a screen to identify mutations in the dynein intermediate chain gene and investigate functions of the dynein intermediate chain during Drosophila development through analysis of the mutant phenotypes, both alone and in combination with the mutant Glued1 (Boylan, 2002).

These results provide the first direct evidence of an essential function for the intermediate chain subunit of cytoplasmic dynein. Previous analysis of dynein heavy chain mutations in mouse and Drosophila has demonstrated that dynein function is essential in these organisms. Similar to the yeast mutations in the heavy chain, mutations in the Saccharomyces cerevisiae dynein intermediate chain gene (pac11) are not lethal, but are synthetic lethal in combination with mutations in the kinesin gene, cin8. Attempts to generate dynein heavy chain and intermediate chain knockouts by homologous recombination in Dictyostelium have failed, consistent with an essential function for both subunits. In Dictyostelium, mutants overexpressing truncations of the Dic gene by a conditional promoter exhibit defects in Golgi dispersion, interphase microtubule organization, cell division, and centrosome replication and separation. Whether the intermediate chain subunit is required for all dynein functions is not known (Boylan, 2002).

Lethal phase analysis shows that the Dic mutations result in larval lethality. A similar lethal phase has been observed for mutations in the dynein heavy chain. Strong alleles of the dynein heavy chain (Dhc64C) die as first instar larvae, and somatic clone analysis of Dhc64C mutations demonstrates that dynein function is required for cell viability. These results suggest that the maternal contribution of dynein is sufficient to allow the completion of embryogenesis without a zygotic contribution of gene product. Although all three Dic alleles die as larvae, the weakest Dic allele (Dic2) lives to a late larval stage, while two Dic alleles (Dic1 and Dic3) appear to die as first instar larvae. Although none of the Dic alleles identified appears to be a null allele, the relative efficiency of screening for Dic mutants should allow for identification of additional alleles (Boylan, 2002).

In addition to lethality, one of the Dic mutations displays a larval crawling defect. This may result from progressive larval paralysis, since the mutant larvae become stiff with their heads poking out of the food like spikes. Similar crawling and paralysis phenotypes have been identified in mutations in the kinesin heavy chain and kinesin light chain genes. The kinesin mutant larvae show axonal organelle jams, suggesting that the larval paralysis is due to a defect in axonal transport. Mutations in the dynein heavy chain (Dhc64C) have also been shown to disrupt axonal transport, causing larval paralysis. Additionally, screens for mutants with sluggish larval crawling behavior identified a gene, roadblock, in which the larval crawling phenotype was shown to be due to a mutation in the dynein light chain LC7. The roadblock mutants also accumulate axonal cargo and, additionally, have severe axonal loss and nerve degeneration (Bowman, 1999). Not surprisingly, also observed were accumulations of cargo in the axons of Dic mutant larvae, suggesting that the larval paralysis is due to a defect in axonal transport (Boylan, 2002).

The genetic analysis of the mutant sw strongly suggests that it represents a viable allele of the cytoplasmic dynein intermediate chain gene, Dic19C. sw fails to complement the lethal Dic alleles in a temperature-sensitive manner. A copy of the wild-type Dic transgene rescues the lethal and visible phenotypes resulting from noncomplementation of sw and the Dic alleles. In addition, the complementation behavior of sw with the lethal Dic alleles provides a way to gauge the relative strength of the lethal alleles. For example, there is a range of wing phenotypes for the combinations of sw with the lethal Dic alleles from mild (Dic2/sw) to severe (Dic1/sw). The weakest lethal allele, Dic2, fully complements sw at 25°, but fails to complement the sw wing and eye phenotype at 28°. The stronger alleles, Dic3 and Dic1, fail to complement sw for viability at 25°. At lower temperatures, Dic1/sw and Dic3/sw adults are viable, but exhibit the sw eye phenotype and display severe defects in wing development. By this test, the allele Dic1 is the strongest of the lethal alleles, although comparison of Dic1/sw to Df/sw suggests that Dic1 is not a null allele (Boylan, 2002).

Using a deficiency that removes the intermediate chain locus and a Dic genomic transgene (Boylan, 2000), it has been shown that the rough eye phenotype of the Glued1 mutation is sensitive to the dosage of the dynein intermediate chain gene. The Glued1 mutation was initially identified on the basis of the dominant rough eye phenotype and was subsequently shown to be due to a truncation of the p150 subunit of dynactin caused by a transposon insertion in the Glued gene. The truncated Glued polypeptide is unable to assemble into a functional dynactin complex; however, it retains the region identified as important for interaction with the dynein intermediate chain. Consequently the Glued1 truncation could act as a 'poison' to dynein function, by uncoupling dynein from its cargo. The interaction between Glued1 and Dic depends on the dosage of Dic, suggesting a model where Glued1 acts by reducing the level of dynein intermediate chain available for cargo binding below a threshold required for normal eye development. Similar to a deficiency for the intermediate chain locus, the lethal dynein intermediate chain alleles identified in the screen and the viable allele sw all dominantly enhance the rough eye phenotype of Glued1. This result indicates that the intermediate chain alleles are all loss-of-function mutations that reduce the level of wild-type intermediate chain available to interact with the dynactin complex, causing an enhanced rough eye phenotype. Additional evidence for a dosage-sensitive interaction between the dynein intermediate chain and Glued1 comes from the observation that in males, the combination of the viable Dic allele, sw, with Glued1 is lethal. Moreover, this result shows that the interaction between the dynein intermediate chain and Glued is essential for viability and is not restricted to eye development (Boylan, 2002).

The wing defects present in the sw;Dic mutant identify a novel dynein phenotype. Analysis of mutations in the dynein heavy chain (Dhc64C) has revealed heteroallelic combinations of alleles that complement for viability but have phenotypes in the eye and bristles and during oogenesis, but no wing phenotypes have been observed. A major question raised by this observation is whether the wing phenotype reflects a tissue-specific function for the dynein intermediate chain and dynein transport. This seems unlikely since sw/sw females fail to exhibit a wing phenotype. An alternative explanation is that different tissues require different levels of dynein function during development. However, if this were the case one might expect the mutant phenotypes to 'accumulate' on the basis of the level of dynein function provided by a particular mutant allele. For example, if the level of dynein function required for oogenesis is higher than that required for proper eye development, then all dynein mutants with a rough eye phenotype might also be expected to exhibit female sterility. This is not the case, suggesting that the different dynein mutant alleles affect different aspects of dynein function. Consistent with the explanation that levels of dynein function account for different phenotypes in different tissues would be the prediction that noncomplementation may arise between Dhc and Dic mutations. However, so far no such genetic interactions between Dic mutations and mutations in other dynein subunits have been found. Further experiments will be necessary to establish whether, in addition to its essential functions, the dynein intermediate chain subunit serves tissue-restricted functions. The identified Dic alleles will provide new tools to identify interacting components that, similar to Glued (dynactin), play a role in regulating dynein function and its interaction with specific cargoes (Boylan, 2002).

Glued functions in Drosophila

The complete cDNA sequence for the Glued gene of wild-type Drosophila melanogaster contains an open reading frame encoding 1319 amino acids, which constitute the Glued polypeptide. The secondary structure predicted from the deduced sequence of the Glued polypeptide has extensive alpha-helical internal domains, which contain heptad-repeat sequences characteristic of an elongated coiled-coil conformation. There are striking sequence and conformation similarities between the Glued alpha-helical domains and those found in certain filamentous proteins from various organisms, particularly in muscle fibers and intermediate filaments. The possible role of the Glued polypeptide as an architectural filamentous component of Drosophila cells and tissues is discussed. Two of the five Glued exons are located in the 5' untranslated region of the cDNA. One of the introns interrupting the Glued open reading frame encodes at least two polyadenylylated transcripts, suggesting that other genes might map within the span of the Glued gene (Swaroop, 1987).

p150Glued is the largest polypeptide in the dynactin complex, a protein heteromultimer that binds to and may mediate the microtubule-based motor cytoplasmic dynein. The Drosophila Glued gene has been extensively characterized by traditional genetic approaches. The original dominant Glued mutation, Gl, reported by Plough and Ives in 1934, results in abnormal eye formation in heterozygotes, with a reduced number of facets and a smooth shiny surface. These macroscopic aberrations result from gross disruptions of the optic lobe, as well as disorganization of axonal projections between the retinal cells and the medulla neuropil. Homozygotes for Gl as well as null mutants at the Glued locus die by the first instar stage of development. The ability of embryos to survive to this stage is probably due to maternal contribution of Glued transcripts to the zygote. In situ hybridization studies showed that Glued is transcribed in virtually all tissues and at multiple stages of Drosophila development. In addition, mosaic analyses of mutations induced at the Glued locus by somatic recombination showed a failure to recover homozygous cells in heterozygous flies, implying that Glued is essential for the viability of individual cells (Waterman-Storer, 1996 and references). Cloning of a cDNA encoding p150Glued from rat revealed 31% amino acid sequence identity with the product of the Drosophila gene, Glued. A dominant Glued mutation results in neuronal disruption; null mutations are lethal. However, the Glued gene product has not been characterized. To determine whether the Glued polypeptide is functionally similar to vertebrate p150Glued, the Glued protein was characterized in the Drosophila S-2 cell line. Antibodies raised against Glued were used to demonstrate that this protein sediments exclusively at 20 S, and associates with microtubules in a salt- and ATP-dependent manner. Immunoprecipitations from S-2 cytosol with the anti-Glued antibody results in the co-precipitation of subunits of both cytoplasmic dynein and the dynactin complex. An affinity column with covalently bound Glued protein retains cytoplasmic dynein from S-2 cytosol. Based on these observations, it is concluded that Glued is a component of a dynactin complex in Drosophila and binds to cytoplasmic dynein, and therefore the mutant Glued phenotypes can be interpreted as resulting from a disruption in the function of the dynactin complex (Waterman-Storer, 1996).

A dominant negative mutation, Glued1, that codes for a component of the dynactin complex, disrupted the axonal anatomy of leg sensory neurons in Drosophila. To examine neuron structure in mutant animals, a P[Gal4] enhancer trap targeted expression of lacZ to the sensory neurons and thereby labeled neurons in the femoral chordotonal organ and their axons within the central nervous system. When these sensory axons were examined in the Glued1 mutant specimens, they were observed to arborize abnormally. This anatomical disruption of the sensory axons is associated with a corresponding disruption in a reflex. Normally, the tibial extensor motor neurons are excited when the femoral-tibial joint is flexed, but this resistance reflex is nearly absent in mutant animals. P[Gal4] insertion strains were used to target expression of tetanus toxin light chain to these sensory neurons in wild-type animals. This blocks the resistance reflex and produces a phenocopy of the Glued result. It is concluded that disruption of the dynein-dynactin complex disrupts sensory axon path finding during metamorphosis, and this in turn disrupts synaptic connectivity (Reddy, 1997).

A C-terminal truncation of Glued, the Drosophila homolog of the cytoplasmic dynein activating protein, dynactin, results in a severe and complex retinal phenotype, including a roughening of the facet array, malformation of the photosensitive rhabdomeres, and a general deficit and disorder of retinal cells. The developmental phenotype in Glued1 has been characterized and defects are found in multiple stages of eye development, including mitosis, nuclear migration, cell fate determination, rhabdomere morphogenesis and cell death. Mitosis is delayed in the second mitotic wave in Glued eyes Transgenic flies that express dominant negative Glued under heat-shock control reproduce distinct features of the original Glued1 phenotype depending on the stage of development. The multiple phenotypes effected by truncated Glued point to the multiple roles served by dynactin/dynein during eye development (Fan, 1997).

The connection between second wave mitoses and nuclear mispositioning is notable; blocking the second mitotic wave using the cyclin-dependent kinase inhibitor, p21, substantially rescues nuclear mispositioning in Glued1. If nuclear migration in photoreceptors is keyed to the cell cycle, mitotic delays in the second wave in Glued1 may disrupt this coordination. The basal displacement of nuclei in cells expressing truncated Glued may be related to the polarity of the longitudinal microtubules spanning the cells. While microtubule polarity remains to be determined in eye disc cells, microtubules in Drosophila wing disc cells are oriented with their minus ends toward the apical cell surface. If nuclear position depends upon minus-end directed microtubule motor activity, compromise of this system could lead to a failure of nuclei to achieve their normal apical position (Fan, 1997).

Mutations in the genes for components of the dynein-dynactin complex disrupt axon path finding and synaptogenesis during metamorphosis in the Drosophila central nervous system. In order to better understand the functions of this retrograde motor in nervous system assembly, the path finding and arborization of sensory axons during metamorphosis was analyzed in wild-type and mutant backgrounds. In wild-type specimens the sensory axons first reach the CNS 6-12 h after puparium formation and elaborate their terminal arborizations over the next 48 h. In Glued1 and Cytoplasmic dynein light chain mutants, proprioceptive and tactile axons arrive at the CNS on time but exhibit defects in terminal arborizations that increase in severity up to 48 h after puparium formation. The results show that axon growth occurs on schedule in these mutants but the final process of terminal branching, synaptogenesis, and stabilization of these sensory axons requires the dynein-dynactin complex. Since this complex functions as a retrograde motor, it is suggested that a retrograde signal needs to be transported to the nucleus for the proper termination of some sensory neurons (Murphey, 1999).

Glued1 (Gl1) mutants produce a truncated protein that acts as a poison subunit and disables the cytoplasmic retrograde motor dynein. Heterozygous mutants have axonal defects in the adult eye and the nervous system. Selective expression of the poison subunit in neurons of the giant fiber (GF) system disrupts synaptogenesis between the GF and one of its targets, the tergotrochanteral motorneuron (TTMn). Growth and pathfinding by the GF axon and the TTMn dendrite are normal, but the GF axon terminal fails to develop normally and becomes swollen with large vesicles. This is a presynaptic defect because expression of truncated Glued restricted to the GF results in the same defect. When tested electrophysiologically, the flies with abnormal axons show a weakened or absent GF-TTMn connection. In Glued1 heterozygotes, GF-TTMn synapse formation appears morphologically normal, but adult flies show abnormal responses to repetitive stimuli. This physiological effect is also observed when tetanus toxin is expressed in the GFs. Because the GF-TTMn is thought to be a mixed electrochemical synapse, the results show that Glued has a role in assembling both the chemical and electrical components. It is speculated that disrupting transport of a retrograde signal disrupts synapse formation and maturation (Allen, 1999).

The BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those observed in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth (McCabe, 2003).

The retrograde requirement for Gbb in synapse structural growth is further supported by experiments inhibiting dynein motor function by overexpression of dominant-negative Glued protein (ΔGl). RNAi-mediated depletion of Arp-1/centractin as well as overexpression of dominant P150/Glued reveal a requirement for dynactin to stabilize NMJ synapses (Eaton, 2002). The degree of net synapse growth appears to be determined by a balance of synapse expansion and retraction (Eaton, 2002). Expression of a dominant-negative Glued protein in the presynaptic cell reduces synaptic bouton number and produces synaptic ultrastructure defects, which are remarkably similar to those described for mutations in the BMP signaling pathway. These ultrastructure defects include membrane detachments along active zones and increased numbers of large vesicles in the presynaptic nerve (Eaton, 2002). Two possible models have been put forth to explain the requirement for dynactin at nerve termini: either it affects local properties, perhaps by altering microtubule stability and dynamics, or it interferes with a retrograde signal. These models are not mutually exclusive and, as described in this study, presynaptic expression of ΔGlu interferes with accumulation of P-Mad in motoneurons. This led to the conclusion that at least some portion of the ΔGl overexpression phenotype is attributable to interference with the retrograde BMP signal. It is suggested that perhaps ΔGl-induced retraction defects result from local disruptions in microtubule stability as suggested by (Eaton, 2002), while other phenotypes, such as reduced bouton number and active zone defects, are the result of disruption in BMP signaling. When synaptic function was examined in animals overexpressing dominant P150/Glued, quantal content was found to be reduced by 40%; however, mEJP amplitude and frequency was unaffected (Eaton, 2002). This contrasts with the findings for mutants of gbb where an 85% reduction in neurotransmitter release is found but also a 3-fold decrease in mEJP frequency. This is consistent with a disconnect between the retrograde requirement for Gbb in synapse structural growth and the requirement for Gbb in neurotransmitter release (McCabe, 2003).

In the case of the BMP signal described in this study, the finding that a high accumulation of P-Mad is detectable in motoneuron nuclei when Gbb is resupplied to nerve terminals from the postsynaptic muscle cell implies that a retrograde signal likely contributes to P-Mad nuclear localization. Consistent with this view is the observation that blocks in the dynein/dynactin motor complex also disrupt P-Mad accumulation similar to what has been reported for transport of activated Trks. Since Mad and Medea mutants also display NMJ defects that are very similar to those exhibited by receptor and ligand mutants, it seems likely that the majority of these defects result from the lack of the retrograde signal itself as opposed to some being caused by the lack of a hypothetical local signal. As is the case for Trks, a signaling endosome consisting of activated heteromeric receptor complexes containing Gbb, Wit, Tkv, and Sax might be transported back to the cell body where these complexes would phosphorylate cytoplasmic Mad, resulting in its translocation to the nucleus. Alternatively, nonphosphorylated Mad may first be transported anterogradely to the nerve. Subsequent to phosphorylation at the NMJ, it may then be selectively transported in a retrograde fashion back to the cell body (McCabe, 2003).

A modifier screen in the Drosophila eye reveals that aPKC interacts with Glued during central synapse formation

The Glued gene of Drosophila encodes the homologue of the vertebrate p150Glued subunit of dynactin. The Glued1 mutation compromises the dynein-dynactin retrograde motor complex and causes disruptions to the adult eye and the CNS, including sensory neurons and the formation of the giant fiber system neural circuit. A 2-stage genetic screen was performed to identify mutations that modified phenotypes caused by over-expression of a dominant-negative Glued protein. Over 34,000 flies were screened and 41 mutations were isolated that enhanced or suppress an eye phenotype. Of these, 12 were assayed for interactions in the giant fiber system by which they altered a giant fiber morphological phenotype and/or altered synaptic function between the giant fiber and the tergotrochanteral muscle motorneuron. Six showed interactions including a new allele of atypical protein kinase C (aPKC). This cell polarity regulator interacts with Glued during central synapse formation. The five other interacting mutations were mapped to discrete chromosomal regions. This study has used a novel approach to screen for genes involved in central synapse formation by performing a primary screen, using a sensitized background, on the adult eye and then a secondary screen, on the isolated mutations, for synaptic phenotypes. This study shows that forward genetic screens are powerful tools for identifying genes with roles in CNS development. This has highlighted a role for aPKC in the formation of an identified central synapse (Ma, 2009).

The success of the two-stage screening approach may have been facilitated by the fact that Glued has a plethora of distinct roles during eye development, including organizing optic neural architecture and an involvement in the formation of sensory neuronal circuits. Therefore an eye phenotype was available on which to base the screen. However, this does not preclude such a method being used for identifying genes involved in other aspects of neural differentiation. It was found that 50% (6/12) of the isolated mutation-containing chromosomes that altered the eye phenotype also altered GFS phenotypes when tested (Ma, 2009).

The over-expression of the truncated Glued protein caused strong phenotypes in both the eye and GF neurons, greater than those caused by heterozygosity for the dominant Gl1 allele. This is likely to be due to the GAL4-UAS system producing many more molecules of the truncated product than Gl1/+ cells in which, theoretically, a maximum of half of the Glued molecules will be truncated. Consistent with this observation, both the suppressors and enhancers isolated during this screen showed stronger effects on GlDN eye phenotypes than on those produced by Gl1. Determining interactions with the Gl1allele also allowed confirmation of GAL4-independent interactions with the Glued locus. For all of the mutations (with the exception of EG162), the alterations of the weaker Gl1/+ eye phenotype were not obvious, however, SEM and sectioning was performed to show interactions with two of the mutations (EG37 & SG13) (Ma, 2009).

Two different disruptions of Glued function, one strong and the other weaker, were used to assay successfully the effects of both enhancer and suppressor mutations in the giant fiber system (GFS) using both morphological and electrophysiological criteria. The severe disruptions of GF morphology and synaptic function enabled the effects of suppressor mutations to be clearly observed. This was less reliable when assaying the effects of mutations isolated as enhancers as either no increase of the already severe phenotype was seen or the interaction was lethal. For the enhancers, therefore, double heterozygotes were generated with Gl1/+. As was the case in the eye, interactions were less pronounced and only two enhancers, EG37/+ and EG162/+ showed enhancement of the Gl1/+ electrophysiological phenotype. Indeed, the subtlety of some interactions with Gl1/+ may have resulted in the analyses being unable to detect some positive interacting loci in the GFS that altered the eye phenotype caused by GlDN (Ma, 2009).

Some EMS alleles were generated, two of which were mapped to known genetic loci and four of which were mapped to discrete chromosomal locations. However, these four complement all the available lethal alleles in these regions indicating that the mutations lie in loci for which there are few or no lethal alleles available. Identification of the location of these new alleles will require either new rounds of mutagenesis, such as via P-element excision in the mapped regions, finer mapping using SNPs or custom made deficiencies using stocks from the DrosDel project. Completion of the BDGP Gene Disruption Project may also enable mapping of the lesions along with more recent approaches using other transposable elements that may disrupt genes refractory to P-element disruption. Interestingly, no mutations were isolated in genes that encode known components of the retrograde motor complex including any further alleles of Glued. During some of the early genetic analysis of the Glued locus, dominant second-site suppressors of the Gl1 eye phenotype were isolated and reported. Of these, two were mapped to the X chromosome (Su [Gl]27 &Su [Gl]57, and the others, Su(Gl)77 &Su(Gl)102 are alleles of Dynein heavy chain 64C (Ma, 2009).

Two new alleles of known genes, Su(H) and aPKC were isolated. Of the two, this study showned that alleles of aPKC genetically interact with Glued in the GFS and suppress the abnormalities in GF-TTMn synapse formation seen when the retrograde motor complex is compromised by GlDN. These abnormalities are: a lack of the presynaptic 'bends'; a branching event that takes place after the two neurons have met; swollen axon tips and a weak or absent functional synaps. aPKC is part of a protein complex, with PAR-3 (Bazooka in Drosophila) and PAR6 that regulates cell polarity in a number of different tissues/cells of Drosophila and vertebrates including neurons. So what is the role of aPKC in the GF neuron? In vertebrate neurons aPKC is needed for neurite outgrowth. In contrast, aPKC in flies is an essential part of the machinery that polarizes dividing neuroblasts but is not needed postmitotically for outgrowth. The data also indicate that aPKC is not needed for neurite extension since the introduction of aPKC mutations into the sensitized background has no effect on GF outgrowth. aPKC is involved in memory formation in Drosophila and at the developing larval NMJ it regulates microtubules (MTs) both pre- and postsynaptically during synapse formation. Indeed MTs are one of the major targets of the PAR-3/PAR-6/aPKC complex in several contexts. aPKC regulates MT orientation in fibroblasts and MT organization in the early embryo. At the NMJ it controls MT stability with a reduction in aPKC activity causing a decreased association of MTs with the microtubule associated protein Futsch and MT fragmentation. Dynein-dynactin is known to be involved in MT organization during growth cone remodeling as well as polarizing MTs in axons. The data indicate that dynein-dynactin and aPKC are acting antagonistically during formation of the GF presynaptic structure and suggest that both are needed to control microtubule organization and dynamics in synapse formation but have opposing roles. One simple explanation is that one of the roles of dynein-dynactin in the GF is to alter MT dynamics at the tip of the axon, when it has reached its post-synaptic target, so that they are more mobile enabling the presynaptic bend to be formed. aPKC regulates the stability of MTs thereby confining axon branching to a single bend. Blocking dynein-dynactin function prevents the MT re-organization needed for formation of the bends and this is ameliorated when aPKC function is reduced (Ma, 2009).

Lis1, a component of the Dynein complex

Lissencephaly is a severe congenital brain malformation resulting from incomplete neuronal migration. Lissencephaly-1 (Lis1), the Drosophila homolog of a human lissencephaly disease gene, is required for germline cell division and oocyte differentiation. One causal gene, LIS1, is homologous to nudF, a gene required for nuclear migration in A. nidulans. nudF was isolated in a screen for nuclear migration mutants in Aspergillus nidulans. NudF interacts genetically with nudC, another nuclear migration gene in A. nidulans. NUDC is required to maintain a normal concentration of NUDF protein. The murine NUDC homolog and Lis1 are co-expressed in the ventricular zone of the forebrain and in the cortical plate, and they also interact in a two-hybrid system, suggesting that nuclear migration may play an important role for neuronal or cell migration. Components of the dynein complex have also been identified as nuclear migration mutants in the filamentous fungi A. nidulans and Neurospora crassa. These components include: cytoplasmic dynein heavy chain (NUDA and RO-1); cytoplasmic dynein light chain (NUDG); p150 Glued (RO-3), the largest polypeptide in the dynactin complex that stimulates vesicle movement by dynein, and centractin (RO-4), the most abundant component in the dynactin complex. PAC1, sharing significant identity with LIS1, is one of the components of the cytoplasmic dynein pathway in Saccharyomyces cerevisiae. These results support the notion that LIS1 and the dynein complex function together to regulate nuclear movement in fungi (Liu, 1999).

The Drosophila homolog of LIS1 (Lis1) is essential for fly development. Analysis of ovarian Lis1 mutant clones demonstrates that Lis1 is required in the germline for synchronized germline cell division, fusome integrity and oocyte differentiation. Abnormal packaging of the cysts is observed in Lis1 mutant clones. These results indicate that LIS1 is important for cell division and differentiation and the function of the membrane cytoskeleton. Lis1 mutant larvae die at the second instar larval stage, indicating that Lis1 is an essential gene in Drosophila as it is in vertebrates. Analysis of Lis1 ovarian mutant clones demonstrates that Lis1 is required in the germline for synchronized cystocyte division and oocyte differentiation. Furthermore, fusomes are aberrantly formed in Lis1 mutant cysts similar to the phenotypes observed in Dhc64C mutants. These results suggest that Lis1 is involved in the formation and maintenance of fusomes, important for regulating cystocyte division and oocyte differentiation. This study also supports the notion that LIS1 interacts with the dynein complex to regulate the function of membrane skeletons, necessary for nuclear and neuronal migration. They support the notion that LIS1 functions with the dynein complex to regulate nuclear migration or cell migration (Liu, 1999).

Roadblock interacts with Dynein

To identify new genes involved in the regulation of axonal transport in Drosophila, a screen was undertaken based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported in this study. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. It is proposed that roadblock/LC7 family members may modulate specific dynein functions (Bowman, 1999).

Disruption of axonal transport by expression of Huntingtin and other pathogenic polyQ proteins in Drosophila

Pathogenic polyQ proteins cause axonal transport defects and neuronal apoptosis:
To address whether axonal transport defects are selective to the pathogenic Huntingtin protein, or whether they are a feature of polyQ proteins in general, the effects were examined on axonal transport of various proteins containing polyQ tracts of different lengths and in different contexts. 179Y-GAL4 and APPL-GAL4 were crossed to lines encoding proteins with either a 'normal' length, nondisease-causing polyQ repeat region or proteins with an expanded, disease-causing polyQ repeat region. These proteins consisted either entirely of polyQ repeats (20Q, 22Q, 108Q, 127Q) or polyQ repeats embedded in the C-terminal region of the polyQ disease protein Machado-Joseph disease (MJD) protein (MJD-27Q, MJD-78Q). Proteins with normal length polyQ regions (22Q, MJD-27Q) were found to be present within axons, based upon the smooth staining seen with either an antibody against polyQ or an antibody against the HA tag, and these proteins were found to accumulate at neuromuscular junctions, suggesting that they are normally transported within larval axons. In contrast, proteins with expanded polyQ repeats (MJD-78Q, 127Q) exhibited prominent polyQ staining within organelle blockages, while reduced staining was observed at the neuromuscular junctions, suggesting impaired transport of pathogenic polyQ proteins (Gunawardena, 2003).

The extent of axonal accumulations induced by polyQ repeats was length dependent, since a correlation was observed between the number of polyQ repeats and the amount of axonal accumulations. Larvae expressing 20Q, 22Q, or MJD-27Q were similar to wild-type in that they exhibited no axonal accumulations. Larvae expressing the pathogenic proteins MJD-78Q, 108Q, or 127Q exhibited a severe sluggish larval movement phenotype, with prominent axonal accumulations observed in all instances. The expression of MJD-78Q and 127Q at 29°C was also observed to be very toxic such that larvae expressing these proteins never survived to adulthood and died at second or third instar larval stage. Western blot analysis ruled out a general expression difference between proteins with normal length polyQ repeats and proteins with expanded polyQ repeats since, if anything, more MJD-27Q expression was observed compared to MJD-78Q. To reduce the level of toxicity, the amount of MJD-78Q and 127Q made was reduced by growing animals at 25°C (GAL4 activity is temperature dependent). Organelle accumulations were still reduced although these larvae now survived much longer, dying at early pupal stages (Gunawardena, 2003).

To confirm the results seen by immunofluorescent staining, EM analysis was conducted on larvae expressing 127Q and on severe genotypes in which expression of MJD-78Q or 127Q was combined with a 50% reduction in KHC gene dose. Prominent axonal blockages were observed characteristic of those observed in homozygous mutations of motor protein genes. Mutant larval nerves also contained enlarged axons, some almost four or five times the diameter of those observed in wild-type. Sometimes 'holes' were observed lacking organelles within the nerve, perhaps indicative of degeneration (Gunawardena, 2003).

To test directly whether pathogenic polyQ proteins block transport by inducing nonmoving blockages in axonal processes, live analysis was performed of vesicular movement within whole-mount larval axons. YFP-tagged human amyloid precursor protein (APP-YFP) was expressed either in the presence or absence of MJD-78Q, using the GAL4 driver pGAL4-62B SG26-1, which is expressed in only a small population of motor neurons. Neurons expressing only APP-YFP contained many actively motile vesicles moving at velocities of approximately 1 microm/s; large bright nonmotile accumulations such as those seen in the presence of MJD-78Q were never observed. Neurons coexpressing MJD-78Q and APP-YFP revealed nonmoving large, bright aggregates of APP-YFP. Thus, polyQ expression can interfere with transport of APP-YFP vesicles (Gunawardena, 2003).

TUNEL analysis was used to test whether proteins with expanded polyQ repeats can induce neuronal apoptosis. A large increase in neuronal apoptosis was observed in lines expressing MJD-78Q and 127Q, but not in lines expressing the nonpathogenic control proteins 22Q or MJD-27Q. Anti-polyQ staining revealed obvious nuclear inclusions within larval brain cells. Many stained nuclei were obviously enlarged and may be undergoing apoptosis. Expression during embryonic cycle 14 revealed smooth cytoplasmic staining for MJD-78Q protein, while staining for 127Q revealed obvious punctate cytoplasmic aggregates with some nuclear aggregates. At later cycles, both MJD-78Q and 127Q were observed as punctate cytoplasmic and nuclear aggregates. In addition, embryos expressing both MJD-78Q and 127Q died soon after they hatched into larvae, indicating substantial polyQ toxicity on normal development. Taken together, these results confirm that proteins with expanded polyQ repeats cause axonal transport defects, perhaps by blocking axonal processes by polyQ accumulations, neuronal cell death, and neurodegeneration (Gunawardena, 2003).

It is possible that polyQ proteins bind and deplete critical components of the molecular motor machinery, perhaps via a Drosophila version of HAP1. This hypothesis makes two predictions: (1) genetic reduction of motor protein dosage should worsen the phenotypes caused by proteins with polyQ expansions by further depleting the motor protein supply, and (2) motor protein depletion should be observable with biochemical methods. To test this hypothesis, proteins with expanded polyQ repeats were expressed and levels of dynein and kinesin were reduced. While a 50% reduction in the dose of KHC has no significant phenotype on its own, when combined with pathogenic polyQ repeats, it dramatically enhances the axonal organelle accumulation phenotype. Similarly, while a 50% reduction in the dose of DLC or components of the dynactin complex (p150Glued, Arp1, and dynamitin) also normally have no significant phenotypes on their own, when combined with pathogenic polyQ proteins, these reductions substantially enhance the organismal phenotype leading to early larval lethality. This finding is consistent with the observation that the neuronal APPL-GAL4 driver turns on during embryonic stage 15 as observed by the expression pattern of UAS-GFP. The enhanced lethality precluded analysis of axonal transport in these genotypes. Interestingly, organelle accumulations now appeared in transgenic lines expressing normally nonpathogenic poly Q repeats (22Q and MJD-27Q) with a 50% reduced dose of dynein, consistent with the hypothesis that all of these proteins may titrate motor proteins, but to varying extents. To test directly for motor protein depletion mediated by expression of proteins with expanded polyQ regions, early embryos expressing httex1-20Q (Huntingtin with non-expanded polyQ repeats), MJD-27Q, MJD-78Q, httex1-93Q, and 127Q (Huntingtin proteins with expanded polyQ repeats), were examined using the early embryonic GAL4 driver da-GAL4, which turns on at the blastoderm stage based on its UAS-GFP expression pattern (Gunawardena, 2003).

Two considerations led to an evaluation of the effects of polyQ proteins on available motor protein pools by assessing soluble levels of motor proteins: (1) if motor proteins are titrated from normal cargoes by binding to large aggregates, it may be difficult to distinguish motor proteins bound to sedimentable cargoes from sedimentable aggregates; (2) it is not possible to measure the amount of each motor protein associated with normal cargoes owing to the lack of information about such cargoes and how such cargoes fractionate relative to polyQ aggregates. Thus, soluble levels of motor proteins were assessed under the hypothesis that aggregated polyQ proteins may bind motor proteins and deplete both soluble and cargo bound pools in parallel (Gunawardena, 2003).

At 6 hr of development, no significant change in the amount of total motor protein present in these embryos was observed. In contrast to the normal amounts of total motor proteins, an obvious reduction was observed in the amount of soluble motor proteins in embryos expressing MJD-27Q, MJD-78Q, and 127Q compared to wild-type embryos (i.e., da-GAL4 alone and yw). The amount of soluble DHC, DIC, p150Glued, KHC, and KLC were reduced, with no change observed in tubulin, actin, HDAC3, and Rab8. However, for reasons that are not clear, syntaxin was upregulated. Similar observations were evident from 12 and 16 hr embryo collections. The effect of httex1-93Q expression on levels of soluble motor proteins was not obvious at 6 hr of development. However, at 18 hr of development, there is an obvious reduction in soluble p150Glued and KLC in embryos expressing httex1-93Q but not httex1-20Q or wild-type. Expression of polyQ proteins in embryos was obvious as detected by anti-HA antibody and confirmed by anti-polyQ antibody. The level of 127Q was difficult to evaluate, perhaps due to the formation of aggregates, and was convincingly observed only after immunoprecipitation with anti-HA antibody. These observations indicate that expanded polyQ proteins can deplete or sequester available soluble motor proteins, perhaps into polyQ aggregates. The high expression level of MJD-27Q in embryos and the observed depletion of soluble motor proteins in these embryos are consistent with the finding that axonal blockages can be observed in larvae expressing MJD-27Q when motor protein gene dose is reduced by 50%. This finding is also consistent with the proposal that motor titration and aggregation may act in concert to poison axonal transport (Gunawardena, 2003).

Enhanced expression of chaperones restores neuronal transport and suppresses cell death caused by pathogenic polyQ proteins
The neurodegenerative adult eye phenotype caused by polyQ expansion proteins in Drosophila is suppressed by excess chaperone proteins (Warrick, 1999). This suppression has been proposed to occur by modulating soluble properties of pathogenic polyQ proteins, by preventing abnormal interactions with other proteins, or by rescuing chaperone depletion (Bonini, 2002). Whether expression of excess HSC70 protein would suppress axonal blockages and neuronal cell death was tested. Expression of UAS-HSC70 using APPL-GAL4 in the presence of MJD-78Q and 127Q restores axonal transport within larval nerves and suppresses neuronal death. PolyQ accumulations were absent within larval nerves, while cytoplasmic and punctate polyQ staining was present within larval brains. However, while these larvae were now able to pupate (expression of MJD-78Q or 127Q alone causes death at second or third instar larval stages), they still failed to eclose, suggesting that polyQ toxicity was still sufficient to cause lethality. Expression of HSC70 by itself did not cause axonal blockages or neuronal cell death. These results suggest that chaperones could 'clear' larval axons of blockages caused by polyQ proteins and suppress cell death within the larval brain, although organismal toxicity was not completely suppressed (Gunawardena, 2003).

Do axonal defects instigate neuronal dysfunction?
The pathogenic polyQ proteins accumulate in both axonal and nuclear inclusions. To dissect the relative contributions of nuclear and axoplasmic inclusions to the phenotype, transgenes were used that expressed proteins that had different subcellular localizations. One transgene encoded a protein with an expanded polyQ repeat with a nuclear localization sequence (MJD-65QNLS). While expression of MJD-65QNLS within the larval brain caused neuronal apoptosis as observed by TUNEL staining, organelle accumulations within larval axons were absent. These larvae pupated but failed to eclose. While reduction in dynein dose by 50% with excess MJD-65QNLS had no effect, reduction in kinesin dose by 50% with excess MJD-65QNLS caused a small number of accumulations, perhaps due to continued motor titration by these proteins even when targeted to nuclei (Gunawardena, 2003).

To test further if dying neuronal cells induce axonal transport defects, the axonal transport phenotype was analyzed of the cell death gene reaper, which also induces neuronal apoptosis. Transport following reaper expression in these genotypes appeared to be normal based on immunostaining with synaptic vesicle markers even though high levels of neuronal apoptosis were induced. In addition, a 50% reduction in KHC combined with excess reaper expression had no effect on axonal transport. These findings emphasize that not all neuronal death is associated with axonal accumulations, and that the axonal transport defects induced by pathogenic polyQ proteins may be specific to cytoplasmic aggregations of expanded polyQ proteins (Gunawardena, 2003).

To test for cytoplasmic or axoplasmic toxicity, a protein with an expanded polyQ repeat with a nuclear export sequence (MJD-77QNES) was studied. Expression of MJD-77QNES within larval neurons caused large numbers of synaptotagmin-containing organelle accumulations within larval nerves. Consistent with primarily cytoplasmic localization of this protein, polyQ/HA staining was absent from cell nuclei within the larval brain, with bright anterior staining (just distal to the brain) present within larval nerves. Neuronal apoptosis as determined by TUNEL staining was completely absent and these larvae died at second or third instar, similar to MJD-78Q. Quantitative analysis indicates that the extent of organelle accumulations within MJD-77QNES is comparable to accumulations observed in mutations of motor proteins, suggesting that perhaps the extent of accumulations causes lethality. Similar to MJD78Q, a 50% reduction in the dose of KHC with MJD-77QNES enhanced organelle blockages, while 50% reduction in DLC combined with MJD-77QNES caused early larval lethality. Additionally, expression of MJD-77QNES in the adult eye using GMR-GAL4 caused a severe degenerative eye phenotype, indicating that cytoplasmic polyQ protein can cause degeneration of adult neurons (Gunawardena, 2003).

To directly test if excess MJD-77QNES sequestered motor proteins, embryos expressing MJD-27Q, MJD-78Q, and MJD-77QNES were compared with wild-type (da-GAL4). While embryos expressing both MJD-78Q and MJD-77QNES showed normal levels of total motor proteins, they exhibited a striking reduction in the amount of soluble motor proteins. MJD-77QNES also exhibited high molecular weight aggregates, which could be immunoprecipitated with the HA antibody. These high molecular weight aggregates also contained sequestered DHC. Although phenotypically normal when expressed in larvae, MJD-27Q appears to titrate more DHC into high molecular weight aggregates than do MJD-78Q, MJD-77QNES, or 127Q. It is conceivable that pathogenic MJD-78Q, MJD-77QNES, and 127Q form high molecular weight aggregates that were not possible trap or to solubilize using current protocols. Indeed, dramatic phenotypes are only observed in MJD-78Q, MJD-77QNES, and 127Q. Additionally, similar to embryos expressing MJD-78Q or 127Q, embryos expressing MJD-77QNES died soon after they hatch into larvae, indicating significant polyQ toxicity on normal development. It is possible that the 'soluble' polyQ aggregates observed in embryos expressing MJD-77QNES represent a subclass of misfolded proteins, while the class of insoluble aggregates, which were not possible to observe directly on SDS-PAGE, may be responsible for polyQ toxicity (Gunawardena, 2003).

To distinguish if MJD-78Q blockages and MJD-77QNES blockages are comparable, whether blockages caused by MJD-77QNES expression can be suppressed by excess HSC70 was tested. Expression of MJD-77QNES with excess HSC70 completely suppressed axonal accumulations, polyQ aggregates, and rescued larval lethality to pupae, suggesting that axonal blockages caused by either MJD-78Q or MJD-77QNES were comparable. PolyQ-containing accumulations were also absent in larval nerves. However, excess HSC70 was not sufficient to suppress organismal lethality (Gunawardena, 2003).

Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150Glued suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ('organelle jams' or 'clogs') caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. It is speculated that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration (Pilling, 2006).

The presence of different types of motor proteins on one organelle raises interesting questions about how they are coordinated to accomplish proper cargo transport and distribution. Coordination of cytoplasmic dynein and an unidentified plus-end motor on lipid droplets in Drosophila embryos is sensitive to the dominant-negative Gl1allele, implicating the P150Glued component of dynactin in the coordination mechanism. The current results are consistent with cohabitation of dynein and kinesin-1 on axonal mitochondria and with their activities being coordinated to avoid antagonism, but the coordination mechanism is insensitive to either the Gl1 allele or neuron-targeted Gl RNAi. Both those inhibition approaches enhance, rather than reduced, plus- and minus-end mitochondrial runs in motor axons. When a more complete disruption of P150Glued in larval neurons becomes possible, perhaps a role in the coordination mechanism will become evident. However, the current data suggest that axonal P150Glued slows mitochondrial runs in both directions. It is an elongated protein with a microtubule-binding site at one end that is thought to facilitate dynein processivity by acting as a tether to hold the dynein and its attached cargo near the microtubule. Because mitochondria are sufficiently large to accommodate many motor complexes and they are confined near microtubules by the small diameter of axons, processivity in axons may not need assistance from P150Glued. In the confines of the axon, perhaps the microtubule-binding action of P150Glued actually generates drag that retards rather than facilitates mitochondrial runs (Pilling, 2006).

The results are consistent with a model in which directional programming is controlled by signals that partition mitochondria into distinct anterograde, stationary, and retrograde classes. It is suggested that mitochondria with fresh, cell body-derived components generate a local signal that represses cytoplasmic dynein, activates kinesin-1, and thus dictates anterograde transport toward the terminal. In the axon, areas requiring ATP generate local signals that convert anterograde mitochondria to stationary by activating static cross-links with the cytoskeleton. Over time, damage to mitochondrial components from reactive oxygen species causes a decline in energy-producing capacity that triggers release from cytoskeletal anchoring, activation of dynein, and repression of kinesin-1. After retrograde transport to the cell body, senescent mitochondria are enclosed in membranes by autophagocytosis and then are degraded by fusion with lysosomes. Regulatory mechanisms that control transitions between the three states may be triggered by extracellular signals, local ATP and ion concentrations, mitochondrial inner membrane potential, or other internal cues from mitochondria. Definition of those control mechanisms should be an exciting endeavor in the coming years (Pilling, 2006).

The genesis and nature of axonal swellings is of great interest because they are a prominent pathological feature of Alzheimer's and some motor neuron diseases. The discovery that kinesin-1 and dynein mutations in Drosophila cause axonal swellings led to the proposal that they are a consequence of traffic jams, i.e., halted organelles impede passage of others causing stochastic pile-ups that locally block transport and force axon swelling. However, it is clear from the direct observations presented in this study that organelles in swellings can be quite mobile and that swellings do not cause a general blockade of mitochondrial transport (Pilling, 2006).

If axonal swellings are not caused by localized transport blockades, what causes them? One answer is suggested by the prominence of large autophagosomes and lysosomal organelles within swellings. Mitochondria with damaged proteins and weak membrane potential undergo a membrane permeability transition that allows release of signals for apoptotic and necrotic cell death. That permeability transition also triggers rapid autophagocytosis and lysosomal degradation of mitochondria that presumably help suppress the cell death signaling. It is suggested that mitochondria stranded by failed retrograde transport pass the permeability transition in the axon, release cell death signals and stimulate local axonal autophagocytosis. Structural and physiological changes that accompany the autophagocytosis cause local enlargement of axon diameter. If this hypothesis is true, then the swellings associated with some neurodegenerative diseases may be triggered by failed retrograde transport and the lingering presence of spent mitochondria in axons (Pilling, 2006).

Support for this stranded mitochondria hypothesis comes from classic studies of physical blockades of axonal transport. Anterograde organelles accumulate on the proximal (cell body) side of a blockade, and retrograde organelles accumulate on the distal side. Proximal organelles can return to the cell body by switching to retrograde transport, but distal organelles are trapped in the axon. Among the normal-looking organelles on the distal side, many autophagosomes and lysosomal organelles accumulate. The source of those organelles has been puzzling, because they are not common in normal axons. As proposed for autophagocytic/lysosomal organelles in the axonal swellings of Drosophila kinesin-1 and dynein mutants, those distal blockade-induced organelles could reflect local autophagy induced by trapped mitochondria that have passed the membrane permeability transition. Further study of the relationship between mitochondrial transport behavior, physiology, and autophagy may provide new insight into the genesis of axonal swellings and contribute to understanding of the pathology of neurodegenerative diseases (Pilling, 2006).

GSK-3β-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome

Microtubule arrays direct intracellular organization and define cellular polarity. This study shows a novel function of glycogen synthase kinase-3β (GSK-3β) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. The data revealed that GSK-3β is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3β or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3β knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3β and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization (Fumoto, 2006).

Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore

The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod-Zw10-Zwilch complex (RZZ) from aligned kinetochores. Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, a new protein (Spindly) has been identified that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. A human homologue of Spindly serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. These findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores (Griffis, 2007).

The spindle assembly checkpoint (SAC) is critical for preventing the onset of anaphase until all chromosomes are aligned on the metaphase plate. A single misaligned kinetochore is sufficient to generate a wait anaphase signal, thereby ensuring that all sister chromatids segregate to opposite ends of the spindle and are equally distributed to the daughter cells. Failure of the SAC can lead to premature anaphase onset and aneuploidy. Such defects can have consequences for a whole organism; mice that lack a full complement of SAC genes have more frequent DNA segregation errors and are more susceptible to tumor development (Griffis, 2007).

The presence of the SAC was initially inferred from observations that cells delay in metaphase when meiotic sex chromosomes fail to pair and align or after the spindle is perturbed by either microtubule poisons or microsurgery. Molecules responsible for the SAC were later identified in yeast genetic screens and named Mad1, -2, and -3 (Mad for mitotic arrest deficient) and Bub1, -2, and -3 (Bub for budding unperturbed by benzimidazole). Subsequent work showed that these proteins together with the MPS1 kinase form distinct complexes that target to the kinetochore. Two additional metazoan checkpoint proteins, Zw10 and Rough Deal (Rod), were later isolated as cell cycle mutants in Drosophila melanogaster. These two proteins, together with a third protein called Zwilch (for review see Karess, 2005), form a complex (Rod-Zw10-Zwilch complex [RZZ]) that regulates the levels of Mad1 and Mad2 on the kinetochore (Griffis, 2007).

Ultimately, the SAC pathway must lead to inhibition of the anaphase-promoting complex (APC), a multisubunit ubiquitin E3 ligase that targets multiple mitotic regulators (e.g., mitotic cyclins as well as the securin protein that inhibits the cleavage of cohesin molecules) for proteosome degradation to allow mitotic exit. Several studies have shown that localization of the checkpoint proteins to misaligned kinetochores is essential for establishing the SAC and keeping the APC inhibited, most likely by generating a diffusible signal that inhibits the APC. The nature of the diffusible signal is still subject to debate. However, a current model (for review see Musacchio, 2007) suggests that the kinetochore-bound Mad1-Mad2 complex acts as a template that coverts the free, inactive Mad2 to an active form that can diffuse away from the kinetochore and bind to and sequester Cdc20, a regulatory component of the APC (Griffis, 2007).

The capture of microtubules by the kinetochore and the downstream activity of two different microtubule motors are required for silencing the SAC in metazoans. One of these motors is the kinesin centromere protein (CENP) E, which may act as a tension sensor that, when stretched, inactivates the BubR1-dependent inhibition of Cdc20 (Chan, 1999; Mao, 2005). The second motor is dynein, which transports Mad1, Mad2, and RZZ from the kinetochore to the spindle pole. Dynein-based removal of Mad1 and Mad2 from the kinetochore may disrupt the template mechanism that generates the active Mad2 that inhibits the APC (for review see Musacchio, 2007). After inhibition or depletion of dynein or its cofactors, metazoan cells arrest in metaphase with correctly aligned chromosomes and high levels of kinetochore-bound Mad1, Mad2, and RZZ (Griffis, 2007).

Resolving the mechanism of dynein recruitment to kinetochores is important for understanding how kinetochore-microtubule binding ultimately leads to inactivation of the SAC. Currently, it is thought that dynein is brought to the kinetochore by binding directly to dynactin (a multisubunit complex required for multiple dynein functions), which, in turn, binds to the Zw10 subunit of the RZZ complex. Lis1, another dynein cofactor, also has been proposed to play a role in targeting dynein to kinetochores. Dynactin, Lis1, and Zw10 are not kinetochore-specific factors, as they are involved in targeting dynein to multiple other locations in the cell. It has not been clearly established whether dynactin and Lis1 are sufficient for targeting dynein to kinetochores or whether other proteins might be involved (Griffis, 2007).

To find new proteins that might participate in the SAC, an automated 7,200 gene mitotic index RNAi screen was undertaken in S2 cells. This screen uncovered a novel gene, which was also identified in an independent screen of genes involved in S2 cell spreading and morphology. This protein (termed Spindly) localizes to microtubule plus ends in interphase and to kinetochores during mitosis. Cells depleted of Spindly arrest in metaphase with high levels of Mad2 and Rod on aligned kinetochores, a defect caused by a failure to recruit dynein to the kinetochore. However, Spindly is not required for other dynein functions during interphase and mitosis. A human homologue of Spindly, which is similarly involved in recruiting dynein to kinetochores, was identifed. Thus, these results have uncovered a novel conserved dynein regulator that is involved specifically in dynein's function in silencing the SAC (Griffis, 2007).

An RNAi screen has identified Spindly as an essential factor for docking dynein to the kinetochore. Spindly is recruited to the kinetochore in an RZZ-dependent manner, and there, together with dynactin, Spindly recruits dynein to the outermost region of the kinetochore. The dynein motor complex then transports Spindly along with Mad2 and the RZZ complex to the spindle poles to inactivate the SAC. A Spindly homologue plays a similar role in human cells, revealing a conserved dynein kinetochore targeting mechanism in invertebrates and vertebrates. These data provide new insight into the mechanism and importance of recruiting dynein to the kinetochore to inactivate the SAC. Spindly also plays a role in maintaining S2 cell morphology during interphase and localizes to the growing ends of microtubules (Griffis, 2007).

The depletion of Spindly creates several mitotic defects that appear to reflect a loss of dynein activity exclusively at the kinetochore. Metaphase arrest is the most evident defect observed after the RNAi-mediated depletion of Spindly in Drosophila or human cells. This metaphase arrest phenotype is most likely explained by the absence of kinetochore-bound dynein in Spindly-depleted cells, and, indeed, the data support a model proposes that kinetochore-bound dynein is required for transporting Mad2 from the kinetochore to inactivate the SAC. Nevertheless, the possibility that the mitotic delay seen after dynein or Spindly depletion is caused by another kinetochore aberration that keeps the checkpoint activated. However, Spindly-depleted cells ultimately overcome metaphase arrest, as seen in live cell imaging experiments and by the modest increases in the mitotic indices of Spindly-depleted S2 and HeLa cells (three- to seven-fold and two-fold, respectively). The mechanism of slippage from this metaphase arrest is not clear, but it might involve proteins (e.g., p31 comet) that silence the SAC by disrupting the interaction between Mad2 and Cdc20 (Griffis, 2007).

In addition to mitotic arrest, chromosomes in Spindly- and dynein-depleted S2 cells require a longer time to align on the metaphase plate. This result may be attributable either to the displacement of CLIP-190 (a microtubule tip-binding protein) from kinetochores after Spindly or dynein depletion or the loss of dynein-mediated lateral attachments to microtubules in early prometaphase. In HeLa cells, a defect in chromosome alignment was noticed after Hs Spindly depletion, which also has been observed after the depletion of dynein (perhaps mediated through a loss of kinetochore-bound CLIP-170) (Griffis, 2007).

Thus, the spectrum of mitotic defects observed in Spindly-depleted cells is consistent with a loss of dynein function specifically at the kinetochore. Spindly depletion does not produce any other defects seen after dynein depletion, such as centrosome detachment and spindle defocusing. Dynactin is another protein that is required for recruiting dynein to kinetochores, but it is important for other mitotic and interphase dynein functions. Depletion of the RZZ complex inhibits the kinetochore recruitment of dynein, but this also prevents Mad1 and Mad2 recruitment and reduces kinetochore tension to a greater degree than Spindly or dynein depletion alone. Thus, Spindly depletion appears to be the most specific means identified to date for interfering with dynein function only at the kinetochore (Griffis, 2007).

These findings provide new insight into how dynein localizes to kinetochores. Previous studies have led to a model in which dynactin binds to the RZZ complex and then, either alone or in collaboration with Lis1, recruits dynein to the kinetochore. Because it was found that both dynactin and Spindly are required for dynein localization to kinetochores, an updated model is proposed in which Spindly and dynactin target to the kinetochore independently and work together to recruit dynein (Griffis, 2007).

Thus, dynein recruitment to the kinetochore may involve multiple weak interactions. Consistent with the possibility of weak interactions, endogenous dynein, dynactin, and Rod did not coprecipitate with GFP in pull-down experiments, and Spindly did not coenrich with these proteins in sucrose gradient fractions. Lis1 is not included in the dynein localization model, since it was found that Lis1 RNAi does not block dynein recruitment to the kinetochore (using a colchicine treatment localization assay), although Lis1 depletion does cause a mitotic delay and substantial increase in GFP-Spindly on aligned kinetochores. Thus, a role is favored for Lis1 in dynein activity but not in recruiting dynein to the kinetochore (Griffis, 2007).

Spindly's role in the spreading morphology of S2 cells makes it unusual among proteins involved in silencing the SAC (including dynein and dynactin), which did not produce phenotypes in the interphase morphology screen. The Spindly RNAi interphase phenotype of defective actin morphology and the formation of extensive microtubule projections is still not understood. However, a clue may be Spindly's dynamic localization to the growing microtubule plus end. Other plus end-binding proteins (+TIPs) interact with signaling molecules that regulate cell shape, one example being the binding and recruitment of RhoGEF2 to the microtubule plus end by EB1. Spindly may similarly interact with and carry an actin regulatory molecule to the cortex, but this hypothesis will require identifying proteins that interact with Spindly during interphase (Griffis, 2007).

The mechanism of Spindly recruitment to the microtubule plus end also warrants further investigation. This interaction must be regulated by the cell cycle because GFP-Spindly no longer tracks along microtubule tips in prometaphase. Seven consensus CDK1 phosphorylation sites are present in the positively charged C-terminal repeats of Spindly, and phosphorylation of these sites could reverse the charge of these repeats and regulate the transition from microtubule tip binding to kinetochore binding at the onset of mitosis (Griffis, 2007).

Motor proteins must be guided to the correct subcellular site to execute their biological function. To carry out the multitude of transport activities required in eukaryotic cells, metazoans have evolved numerous kinesin motors (25 genes in Drosophila) with distinct domains that dictate their localization and regulation. In contrast, a single cytoplasmic DHC performs numerous roles in interphase and mitosis, suggesting that additional regulatory factors guide dynein to specific cargoes (e.g., organelles, mRNAs, and vesicles). The main dynein-associated proteins (the dynactin complex, Lis1, and NudEL) are involved in dynein function at many sites and, thus, do not appear to be cargo specific. Zw10 was initially thought to specifically regulate the recruitment of dynein-dynactin to the kinetochore, but it now also appears to play an essential role in targeting dynein to membrane-bound organelles. Bicaudal D is another multifunctional adaptor molecule that has a role in the dynein-based transport of multiple cargoes such as RNA, vesicles, and nuclei. Perhaps the most site-specific dynein recruitment factor is the Saccharomyces cerevisiae Num1 protein that binds to the DIC Pac11p to target the motor to the cortex of daughter cells, where it pulls the nucleus into the bud neck. However, dynein only serves this one function in yeast compared with its plethora of activities in metazoans, and Num1p homologues have yet to be identified in higher eukaryotes (Griffis, 2007 and references therein).

Spindly appears to be a highly selective dynein-recruiting factor, and, unlike other dynein cofactors, it does not appear to be involved in the motor's nonkinetochore functions in mitosis (e.g., pole focusing) or in interphase (e.g., endosome transport). However, the mechanism by which Spindly recruits dynein to the kinetochore remains to be elucidated. Observations that Spindly moves from kinetochores to the spindle poles as discrete punctae strongly suggests that it may incorporate into a large and somewhat stable particle that contains the RZZ complex, Mad1-Mad2, dynein, and likely additional proteins. Therefore, Spindly not only serves to recruit dynein to the kinetochore but also is part of a cargo that dynein transports. Future studies will be needed to better understand the protein composition of these transport particles and the contacts that Spindly makes within them (Griffis, 2007).

The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation

Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, this study demonstrates that microtubules are required for proper metaphase furrow formation. It is proposed that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows (Riggs, 2007; full text of article).

Microtubule-based motility has been implicated in many steps in endocytosis, and there is increasing evidence that it influences the distribution and activity of endocytic organelles. The work presented in this study suggests that motor-based movement of Rab effectors may be another means of regulating endosomal activity. Previous studies have shown that the Drosophila Rab11 effector, Nuf, is required for stable Rab11 localization at the RE and thus RE activity. Nuf concentrates at the MTOC during interphase through prophase and disperses into the cytoplasm at metaphase. This study demonstrates that Nuf relies on microtubules and minus-end microtubule motor Dynein both for its accumulation and maintenance at the MTOC. This raises the possibility that the Dynein-dependent delivery of Nuf to the RE may play a role in regulating Rab11 activity at the RE. Significantly maximal localization of Nuf at the MTOC-associated RE occurs during late interphase and prophase. This is the time of the establishment and formation of the metaphase furrows, which rely on RE-based vesicle delivery (Riggs, 2007).

Immunoprecipitation data demonstrates a physical interaction between Nuf and Dynein. This raises the possibility that the cell cycle-regulated localization of Nuf at the MTOC is mediated by a corresponding cell cycle-regulated interaction between Nuf and Dynein. Support for this idea comes from a study in vertebrate cells, demonstrating that Polo-like kinase (Plk) mediated phosphorylation of Ninein-like protein (Nlp), a microtubule-nucleating protein, directly determines its cell cycle-regulated localization at the centrosome. Like Nuf, Nlp localizes to the centrosome by associating with the minus-end-directed motor protein Dynein. As cells progress into metaphase, Plk is activated and phosphorylates Nlp on sites that are required for its association with Dynein. This disrupts Nlp ability to associate with Dynein and results in loss of Nlp from the centrosome (Riggs, 2007).

There is a strong correlation between maximal Nuf localization at the MTOC and furrow invagination. During the cortical divisions, furrow invagination and maximal Nuf concentration at the MTOC occurs during prophase. During cellularization, furrow invagination and maximal Nuf concentration at the MTOC occurs during interphase. Stable localization of Nuf and Rab11 at the MTOC during cellularization enabled a demonstration that microtubules are continuously required for maintaining Nuf and Rab11 at the MTOC. Colchicine-induced disruption of the interphase microtubules results in the rapid loss of Nuf from the MTOC. One interpretation of this result is that colchicine disrupts MTOC organization, which is required for maintaining Nuf at the MTOC. In contrast to the colchicine injections, injecting anti-Dynein antibody does not alter microtubule organization and results in a slow steady decrease of Nuf at the MTOC. This result suggests that the steady-state level of Nuf at the MTOC is maintained by continuous Dynein-dependent recruitment of Nuf to the MTOC. This also implies that Nuf is continuously released from the MTOC as well. The mechanism driving the release is unclear. Previous live analysis revealed vectorial movement of Nuf away from the centrosome, suggesting that it may rely on a kinesin, a plus-end-directed microtubule motor. If kinesin is involved, this implies that the balance between plus- and minus-end motor activities dictates whether Nuf is concentrated at the MTOC or dispersed in the cytoplasm. Recent work indicates that the positioning and activity of the early endosome is mediated through a balance of plus- and minus-end motor activities. In addition, investigations into cellular furrow elongation demonstrated that Lava lamp, a Golgi-associated protein, is complexed with Dynein and is responsible for Golgi-based movements necessary for latter half of furrow elongation (Riggs, 2007 and references therein).

The above studies demonstrate that microtubules are continuously required for proper Nuf localization at the MTOC. This raises the possibility that microtubule-based localization of Nuf at the MTOC is necessary for its association with the Rab11 and proper RE function. Because RE function is necessary for metaphase furrow formation, this predicts that microtubules are required for proper metaphase furrow formation. However previous studies did not observe defects in furrow formation when embryos were treated with microtubule inhibitors. It has been concluded that microtubules are dispensable for proper metaphase furrow formation in the early embryo. This issue was reexamined by injecting microtubule inhibitors at precise times throughout the cell cycle during the syncytial divisions. Because disrupting the microtubules at metaphase activates the spindle assembly checkpoint, the embryos were injected immediately after entry into anaphase. In these experiments, the nuclear cycle progressed normally but formation of the metaphase furrows were profoundly disrupted. Incorporation of GFP-tagged Moesin into the furrows that form at the next prophase completely fails. Thus these experiments define anaphase as a key time in which microtubules are required for recruiting actin to the furrows that form in the following prophase. The previous study failed to appreciate the role of microtubules in metaphase furrow formation because it was not possible to produce disruptions in the microtubule network at defined stages of the cell cycle (Riggs, 2007).

These studies also revealed that injecting colchicine at telophase produced no defects in actin recruitment. Similar injections at interphase through prophase also produced no defects in actin recruitment to the metaphase furrows. One interpretation of these results is that microtubules are specifically required during anaphase but not telophase or later for furrow formation in the next prophase. However it must be pointed the different classes of microtubules are differentially sensitive to microtubule inhibitors. Thus this differential sensitivity may contribute to the observed cell phase sensitivity of metaphase furrow formation to colchicine (Riggs, 2007).

That microtubules are required during anaphase for metaphase furrow formation in the following prophase is significant for a number of reasons. First, these studies support, although certainly do not prove, a model in which microtubule-based transport of Nuf to the MTOC is necessary for normal metaphase furrow formation. Second, anaphase/telophase is the point at which the metaphase furrows begin to regress. Thus the timing of furrow regression corresponds to the time at which microtubules are involved in establishing the next round of furrow formation. This indicates that the speed of the cortical divisions is not only achieved by an accelerated nuclear cycle but also by overlapping furrow regression with furrow formation. During anaphase, the replicated centrosomes possess robust astral arrays and the midbody has not yet fully formed. It is hypothesized that the plus ends of these overlapping arrays from neighboring centrosomes define the position of the metaphase furrow in the next cell cycle. This readily explains why furrows encompass the spindle and do not form at the midzone microtubules. Finally, although the furrows form at prophase, these studies identify anaphase as a critical time in which furrow is established. This also corresponds to the time at which microtubules are required during conventional furrow formation (Riggs, 2007).

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell. It is unknown whether spindle alignment requires both apical and basal cues, nor have molecular motors been identified that regulate spindle movement. Using live imaging of neuroblasts within intact larval brains, independent movement of both apical and basal spindle poles is detected, suggesting that forces act on both poles. Reducing astral microtubules decreases the frequency of spindle movement, but not its maximum velocity, suggesting that one or few microtubules can move the spindle. Mutants in the Lis1/dynactin complex strongly decrease maximum and average spindle velocity, consistent with this motor complex mediating spindle/cortex forces. Loss of either astral microtubules or Lis1/dynactin leads to spindle/cortical polarity alignment defects at metaphase, but these are rescued by telophase. It is proposed that an early Lis1/dynactin-dependent pathway and a late Lis1/dynactin-independent pathway regulate neuroblast spindle orientation (Siller, 2008).

This study shows that spindle/cortical polarity alignment is established at prophase in Drosophila larval neuroblasts, and that both apical and basal spindle poles move independently, as if spindle/cortex forces are applied to both poles. Reducing astral microtubule number reduces the frequency of spindle pole movements, but that maximum spindle pole velocity is unaffected, suggesting that maximum velocity may occur when only one or a few microtubules are simultaneously contacting the cortex. Yhe Lis1/dynactin complex is required for spindle pole movement; reducing Lis1/dynactin complex activity reduces the maximum and average spindle velocity, even though astral microtubules still contact the cortex. This suggests that Lis1/dynactin is required to translate microtubule-cortex contact into spindle movement. Finally, this study shows that Lis1/dynactin is required for spindle orientation at metaphase but not at telophase (Siller, 2008).

Lis1-dependent dynamic microtubule-cortex interactions were observed at both apical and basal spindle poles, as well as asynchronous movements of apical and basal spindle poles. What are the candidate apical or basal cortical proteins that might regulate spindle pole movement? Insight into the role of cortical proteins in regulating spindle movement has been made in both C. elegans and mammals, and can be used to model spindle dynamics in Drosophila. Apical proteins in neuroblasts known to regulate spindle force in C. elegans include Gαi, Pins and Mud. During the first division of the C. elegans zygote, enrichment of the Gα-binding and activating Pins-related GPR1/2 proteins at the posterior cortex leads to increased Gα activity, resulting in higher cortex-spindle force generation, spindle pole rocking, and posterior spindle displacement. This suggests that Gαi/Pins/Mud may promote movement of the apical spindle pole in Drosophila neuroblasts, which is supported by the finding that reducing Gαi can decrease spindle rocking (Siller, 2008).

In C. elegans, Lin-5 mediates the physical interaction of Lis1/dynein/dynactin with the cortical Gα and the Pins-related GPR1/2 proteins, and reduction of dynein or Lis1 function also reduces spindle pole rocking and posterior spindle displacement. Furthermore, in mammalian tissue culture cells Gαi overexpression can induce robust spindle rocking that requires LGN (a Pins/GPR-related protein) and NuMA (a Mud/Lin-5-related protein that binds dynein/dynactin). An attractive model is that Gαi/LGN activates NuMA, which interacts with dynein/dynactin/Lis1-loaded astral microtubules. In Drosophila neuroblasts, Gαi, Pins (LGN-related) and Mud (NuMA-related Pins-binding protein) are all enriched at the apical cortex and required for proper metaphase spindle orientation. Thus, it is tempting to propose that apical Gαi/Pins/Mud interacts with dynein/dynactin/Lis1-loaded astral microtubules to center the apical spindle pole with the apical cortical domain. Identifying a physical link between Mud and dynein/dynactin/Lis1, and determining its functional importance in spindle orientation, would be a good test of this model (Siller, 2008).

Surprisingly, it was found that third instar larval neuroblasts have more vigorous basal spindle pole rocking than apical spindle pole rocking, revealing a Gαi-independent spindle force generation mechanism at the basal cortex. Basal cortical proteins include Armadillo, DE-cadherin, β-catenin, APC2, and Mud. Components of the APC2/DE-cadherin/α-catenin/β-catenin complex physically interact with the dynein complex in mammalian cells, and are required for spindle positioning in the Drosophila pre-cellular embryo, epithelial cells, and germline stem cells. Previous studies indicated no spindle positioning defects in neuroblasts after reduction of APC2 function, however these findings do not rule out a role for APC2 in spindle orientation because it may function redundantly with an apical cue, such as the Gαi/Pins/Mud pathway (Siller, 2008).

This study has demonstrated that both apical and basal spindle pole movements are greatly diminished in Lis1 mutant larval neuroblasts (even in those with well-formed bipolar spindles and asters), providing first evidence that Lis1/dynactin is a critical component in the regulation of both apical and basal cortex-spindle forces. How does Lis1 regulate cortex-spindle forces? One possibility is that translocation of cortically associated motor proteins towards microtubule(-) ends results in movement of the microtubule towards the cortex. Consistent with this hypothesis, Lis1 colocalizes with and binds the microtubule minus-end motor dynein/dynactin complex. Specifically, the budding yeast Lis1 homologue (Pac1) targets dynein to astral microtubule plus-ends where it promotes movement of astral microtubules towards the cortex, resulting in translocation of the spindle apparatus through the bud neck. By analogy, Lis1 may regulate spindle pole movement in neuroblasts by promoting dynein-dependent movement of astral microtubules towards the cortex. Alternatively, Lis1 may modulate the polymerization/depolymerization cycle (dynamic instability) of cortically-attached astral microtubules or the duration of astral microtubule-cortex interactions. In support of this latter hypothesis, loss of Lis1 or dynein function in Aspergillus nidulans or budding yeast results in reduced microtubule catastrophe and/or decreased shrinkage rates, thereby promoting assembly of overly long microtubules. Currently, it was not possible to visualize astral microtubule plus-ends with sufficient spatial and temporal resolution to distinguish between these models for Lis1 function (Siller, 2008).

Both models for Lis1 function described above would require association of Lis1 protein with astral microtubules and/or the neuroblast cortex. Indeed, Lis1/dynactin complex proteins have been detected on astral microtubule plus-ends or at the cortex in mammalian, nematode, and yeast cells. The localization of HA-tagged Lis1, GFP-tagged Lis1, endogenous Lis1, and endogenous dynactin protein distribution were analyzed using various fixation and live imaging methods in embryonic and larval neuroblasts, but no enrichment of Lis1/dynactin at the cortex or at astral microtubule plus-ends was found. The most likely explanation is that Lis1/dynactin at these sites is masked by the high level of cytoplasmic protein present in neuroblasts (Siller, 2008).

The Lis1/dynactin complex is required for reliable spindle orientation with the apical/basal polarity axis in metaphase neuroblasts. These spindle orientation defects may be due in part to failure in anchoring one centrosome at the apical cortex during interphase, as reported for wild type neuroblasts; this study observed mis-positioned interphase centrosomes in Lis1 mutants, but this this phenotype was not analyzed in detail. It was surprising to find that spindle orientation was essentially normal at telophase in Lis1 and dynactin (Gl) mutant neuroblasts, despite severe defects at metaphase. This indicates that there are two pathways for regulating spindle orientation: an early Lis1/dynactin-dependent pathway (prophase/metaphase), and a late Lis1/dynactin-independent pathway (anaphase/telophase). There are several models consistent with these findings: (1) Lis1 and dynactin mutants have a delay in anaphase onset which allows sufficient time for 'telophase rescue' to occur. (2) A spindle orientation checkpoint -- analogous to the yeast spindle orientation checkpoint -- may delay cytokinesis until proper spindle orientation has occurred. These first two hypotheses are disproven by the finding that Lis1 rod double mutants have normal metaphase progression but still show metaphase defects and 'telophase rescue' of spindle orientation. (3) The cleavage furrow may be positioned by cortical polarity cues, such that cell elongation at early anaphase may mechanically re-orient the spindle along the long axis of the neuroblast. This model is unlikely because it is commonly accepted that the position of the cleavage furrow is determined by the position of the mitotic spindle and not by cortical cues. (4) Additional microtubule-cortex regulators unrelated to Lis1/dynactin promote telophase spindle orientation (Siller, 2008).

The fourth model is the most likely, except that microtubule-cortex regulators unrelated to Lis1/dynactin have not yet been identified in Drosophila neuroblasts. Help may come from analysis of budding yeast spindle orientation pathways, where Lis1/dynactin-dependent and -independent pathways have been identified. Several components of the yeast Lis1/dynactin-independent pathway are evolutionarily conserved, including the microtubule plus-end binding protein Bim1p, called EB1 in Drosophila. It is tempting to speculate that these proteins may regulate the Lis1/dynactin-independent pathway in Drosophila neuroblasts (Siller, 2008).

The auto-inhibitory domain and ATP-independent microtubule-binding region of Kinesin heavy chain are major functional domains for transport in the Drosophila germline

The major motor Kinesin-1 provides a key pathway for cell polarization through intracellular transport. Little is known about how Kinesin works in complex cellular surroundings. Several cargos associate with Kinesin via Kinesin light chain (KLC). However, KLC is not required for all Kinesin transport. A putative cargo-binding domain was identified in the C-terminal tail of fungal Kinesin heavy chain (KHC). The tail is conserved in animal KHCs and might therefore represent an alternative KLC-independent cargo-interacting region. By comprehensive functional analysis of the tail during Drosophila oogenesis, an understanding was gained of how KHC achieves specificity in its transport and how it is regulated. This is the first in vivo structural/functional analysis of the tail in animal Kinesins. The study shows that the tail is essential for all functions of KHC except Dynein transport, which is KLC dependent. These tail-dependent KHC activities can be functionally separated from one another by further characterizing domains within the tail. In particular, the data show the following. First, KHC is temporally regulated during oogenesis. Second, the IAK domain has an essential role distinct from its auto-inhibitory function. Third, lack of auto-inhibition in itself is not necessarily detrimental to KHC function. Finally, the ATP-independent microtubule-binding motif is required for cargo localization. These results stress that two unexpected highly conserved domains, namely the auto-inhibitory IAK and the auxiliary microtubule-binding motifs, are crucial for transport by Kinesin-1 and that, although not all cargos are conserved, their transport involves the most conserved domains of animal KHCs (Williams, 2013).

The oocyte allows the analysis of the C-terminal region of KHC in an in vivo context. The results show that the interaction of Kinesin with its cargos and/or the regulation of the motor is complex and relies on more than one region. The tail (aa 850-975) is essential for all functions of KHC in the st9 oocyte except Dynein transport. These functions include the positioning of the nucleus and Gurken protein (and consequently establishment of the DV axis), the localization of oskar, the induction of streaming, and the distribution of actin-recruiting vesicles. Most of these tail-dependent KHC activities can be functionally separated from one another by further characterizing the conserved domains within the tail. The various functional domains are not necessarily involved in cargo binding, but their presence is required for wild-type cargo transport. In particular, the data show the following: (1) a temporal regulation of the impact of KHC activity on cytoplasmic streaming during oogenesis; (2) a novel essential role for the IAK that is distinct from its auto-inhibitory function; (3) that lack of auto-inhibition in itself is not necessarily detrimental to KHC function; and (4) that the AMB motif is required for oskar RNA localization (Williams, 2013).

The localization of Dynein to the posterior requires Kinesin. This study shows that deletion of the tail has a weak effect on the transport of Dynein, whereas further deletion of the region covering coil3 and half of coil2 renders a motor unable to localize Dynein. This observation correlates with the finding that KLC, which together with Pat1 mediates Dynein localization, interacts with coil3 of KHC in a tail-independent manner. It is then likely that Dynein is a posterior cargo of KHC, and that the Dynein complex interacts with KHC via KLCs. In C. elegans, the KLC-binding protein Jip3 binds Dynein light intermediate chain (Dlic). However, jip3/syd mutant oocytes show no defects in the posterior localization of Dynein. Alternatively, KLC might bind the Dynein intermediate chain (DIC), as in mammals. This observation, together with the fact that amino acids 795-839 (including coil3) are conserved in animal KHCs, makes it plausible that, in the oocyte, KHC localizes Dynein via a coil3-dependent KLC-DIC complex (Williams, 2013).

It is important to keep in mind that even though KLC and the KLC-like protein Pat1 are not essential for the localization of oskar and the nucleus, or for the induction of flows, they still contribute to these KHC-dependent processes, albeit in a minor manner. Pat1 mutants have slightly slower flows, and Pat1,Klc double mutants show mild oskar and nucleus localization defects in 78% and 9%, respectively, of the mutant oocytes. These nucleus anchoring defects might correlate with those seen in KHC1-700 oocytes, since KHC1- 700 does not contain the KLC-binding domain; however, the nucleus phenotypes in KHC1-700 may not be statistically significantly different from those observed in KHC1-849 oocytes. (Williams, 2013).

oskar RNA is found at the anterior/lateral regions of the Khc mutant oocyte. Similarly, Khc27 st9 oocytes show a mispositioned nucleus and an aberrant distribution of Gurken protein. Consequently, embryos resulting from Khc27 oocytes have an aberrant anterior-posterior (AP) and DV body plan. Deletion of the tail produces a motor that is unable to localize oskar RNA and thus is unable to support the establishment of the AP axis. Further characterization of the function of conserved domains within the tail suggests that RNA transport activity relies on the AMB site. In addition, 96.5% of the embryos resulting from tailless KHC oocytes have aberrant dorsal appendage (DA) formation. This DV axis defect might be due to more than the tail function in nucleus positioning, since the nucleus is not positioned in 60% of tailless KHC1-849 oocytes. KHC1-849 oocytes are defective for Gurken protein localization, even when the nucleus seems properly positioned. Given that the oocyte nucleus is associated with one of the MT-organizing centers, it is possible that the defects in Gurken signaling, and thus DV axis, in Kinesin mutants are a result of both nucleus mispositioning and the misorganization of the anterior MTs. This is consistent with MT defects observed at the anterior of KHC1-849 and KHC1-700 oocytes. In wild-type and KHC1-975 oocytes, there is an obvious AP gradient of MTs, with a population of enriched MTs close to the anterior/lateral cortex. This gradient can also be seen in some KHC1-849 and KHC1-700 oocytes. However, most of these mutant oocytes show an extension of this anterior 'bright' MT network towards the posterior around the nucleus, as well as the misorganization of MTs in a pattern that resembles the aberrant vesicles often detected at the anterior of Khc mutant oocytes. The region encompassing the KLC-binding domain might also contribute to the establishment of the DV axis, since the number of oocytes with Gurken in an anterior-dorsal crescent drops from 14% in KHC1-849 oocytes to 0% in KHC1-700 oocytes (Williams, 2013).

At first glance, it is unclear why there are nucleus and Gurken localization defects in the Khc null, when plus ends are biased towards the posterior. As nucleus positioning requires the Dynein complex, it follows that KHC function could be indirect for the anterior cargos, for example via the recycling of Dynein. However, it is thought that KHC could be acting directly on nucleus positioning. First, it cannot be discounted that Dynein and Kinesin act independently: Dynein localization to the posterior is abolished in Pat1,Klc double mutants, whereas nucleus positioning is only weakly affected, suggesting that the coordinated action of the two motors is not necessarily required. Second, the MT network is complex, and there seem to be some plus ends towards the anterior cortex that Kinesin may harness. Third, KHC localizes at the nuclear envelope. Fourth, when KHC is missing, alpha-tubulin and Jupiter-GFP [a MT-associated protein fused to GFP are found in dots at the nuclear envelope in a similar punctate pattern to that displayed by KHC (Williams, 2013).

All these preliminary observations might suggest that KHC is acting on a set of MTs that allows positioning of the nucleus in close proximity to the anterior membrane: when KHC is missing, these MTs seem to 'collapse' to the nuclear envelope and their stable existence is not maintained. Taking work on cultured cells into consideration, Kinesin might well bind to the nuclear envelope and transport the nucleus towards the plus ends. However, it is likely that the relative importance of different molecular links between the nuclear envelope and motors depends on the cell type. For example, Drosophila SUN/KASH proteins (Msp-300, Klarsicht and Klaroid) have no essential functions during oogenesis. Mammalian KHC is also known to bind directly to the nucleoporin Ranbp2 via its tail (Williams, 2013).

There are other mutants that show nucleus positioning defects, including skittles (which encodes phosphatidylinositol 4,5- bisphosphate-synthesizing enzyme), trailer hitch (tral) and Bicaudal C (BicC). Among these, tral and BicC mutants have abnormal actin-covered vesicles that look similar to those present in Khc oocytes. This similarity, together with the data showing that Rabenosyn-5 is present in Khc mutant vesicles, suggest that KHC is required for membrane trafficking in the oocyte. This correlates with the function of KHC in other cells and with the observation that, in Khc oocytes, Rab6 vesicles aggregate abnormally around the mispositioned nucleus. The ectopic vesicles that were observe in Khc oocytes seem to nucleate actin, as seen in time-lapse movies of Utrophin-GFP. As suggested for tral and BicC, the formation of 'actin spheres' (as a readout of vesicle trafficking problems) in Khc oocytes might cause defects in Gurken signaling. In fact, Gurken is detected in close proximity to actin-recruiting vesicles in KHC1-938 oocytes (Williams, 2013).

These data stress that the anterior phenotypes observed in Khc mutant oocytes are likely to be the result of a complex relationship between vesicle trafficking, MTs and nucleus location. Ectopic sites of actin also appear in Rab5, Rab6, IKK-related kinase (IκB kinase-like 2 - FlyBase) and spn-F oocytes. They were interpreted as cytoskeleton defects, but might also be the result of ectopic actin nucleation by aberrantly distributed vesicles (Williams, 2013).

Auto-inhibition to limit the consumption of ATP/GTP by motors not bound to cargos is conserved in Myosins and Kinesins. As both protein families share a common ancestor, it is not unexpected that there is a common mechanism to this auto-inhibition, in which the tail folds back to the motor domain. It is clear from research on affecting auto-inhibition in vivo that these motors cannot function correctly, leading to detrimental transport. What was still unknown is whether the defects in transport are a consequence of a lack of inhibition or are due to alternative functions of the motifs involved. This study has compared these two hypotheses directly for the first time. Recently, the stoichiometry of the interaction between the IAK and motor domains has been determined, with one IAK motif per motor dimer required. This has led to the suggestion that the other motif could be free to bind cargo or other regulators of KHC. A mutant IAK with two individual point mutations (IAKPIRS to IAKSIRS, IAKPIRS to IAKPIRSF) shows weak defects in oskar transport and DA formation that are similar to those of Khc hypomorphic alleles, suggesting that these mutations result in inhibition rather than overactivation of transport. Similarly, the IAK seems to facilitate, rather than downregulate, axonal transport of mitochondria. However, these IAK point mutants did not constitute a full null of IAK activity, since when the entire motif are mutagenized the DA defects are much stronger than those observed in the point mutants. In addition, deletion of the IAK phenocopies the deletion of the tail regarding the formation of dorsal structures, with only a slight increase in the number of normal DAs in IAK mutant oocytes (Williams, 2013).

A hypothesis to explain the cargo transport defects observed in KHC1-975ΔIAK oocytes, which are not observed in KHC1- 975ΔHinge2 oocytes, is that KHC1-975ΔIAK has reduced function for these cargoes. That is to say, the IAK motif has an essential activity that is independent of its auto-inhibition function. Interestingly, the streaming speed of KHC1-975ΔIAK is faster than wild type, suggesting that KHC1-975ΔIAK is not defective for all KHC functions. Instead, the increased streaming speed might be due to the number of motors that are active at any one time being higher. This correlates with many more particles of KHC1-849 than of KHC1-975 moving in in vitro assays (Williams, 2013).

The results with KHCΔHinge2 show that auto-inhibition does not play a major role in transport during oogenesis. However, there seems to be a small contribution of auto-inhibition to DA formation, in accordance with work on fungal kinesin showing that maintenance of the folded conformation partially contributes to growth rates. In conclusion, KHC autoinhibition might not be such an important driving factor as previously thought, especially not in the oocyte. It might be interesting to analyze how a lack of auto-inhibition affects KHC function in other cells, such as neurons (Williams, 2013).

The region 850-910 is conserved between all animal and fungal KHCs and contains the N. crassa putative cargo-binding domain. However, KHC1-910 does not support wild-type localization of oskar or wild-type streaming. It is however possible that KHC1-910 is able to bind cargo but is somehow unable to transport it. This could be the case for oskar RNA, since there is a weak accumulation of the transcript at the posterior in KHC1-910 oocytes. If KHC1-910 binds oskar, but its action is constrained, one would expect an enrichment of KHC1-910 at the anterior/lateral cortex, where oskar accumulates. Higher levels are noy detected of KHC1-910 than of KHC1-975 in that region, and thus it is uncertain how small amounts of oskar reach the posterior in KHC1-910 oocytes. The localization of wild-type amounts of oskar RNA to the posterior is rescued when the AMB site is restored in KHC1-938, demonstrating a key role of this domain in cargo localization. This supports the observation that mutations in this region (four arginines) render a severe Khc allele with reduced motor function in neurons. This AMB region binds to MTs in vitro and in cells, perhaps via electrostatic interactions, and seems responsible for an MT polymerization activity of the tail. Furthermore, KHC slides and bundles MTs in cells and, in the case of fungal Kinesin, this MT bundling activity depends on the tail. How does this MT regulatory function of the domain relate to the capacity of KHC1-938 to localize oskar? The MTs of KHC1-938 and KHC1-910 oocytes still form an AP gradient, seemingly of wild-type topology, supported by the posterior accumulation of KHC1-910GFP. Thus, it could be that the AMB site is affecting oskar RNP binding specifically and not via any MT-related activity. This hypothesis is supported by the fact that there are several proteins that interact with the KHC tail, including Kv3.1 (Shaw - FlyBase), which binds the region containing the AMB domain and requires it for its transport (Williams, 2013).

The localization of oskar RNA in KHC1-938 and KHC1-975ΔIAK oocytes is not completely wild type, since 'dots/clouds' of the transcript are observed in close proximity to the posterior. Dots/clouds of oskar at the posterior is a phenotype observed in oocytes with minor MT defects, oskar translation defects. It is not known why there is an oskar 'dot' phenotype in KHC1-938 and KHC1-975ΔIAK oocytes. There are no obvious MT defects at the posterior of these mutants, although the mutant motors are found in the oskar dots, which might suggest the presence of plus ends. This dots phenotype is also seen in Rab6 and Rab11 mutants and it might thus be related to a vesicle trafficking function of KHC. This idea is supported by the findings, since KHC1-975ΔIAK and KHC1-938 oocytes show aberrant actin spheres/vesicles (84% and 45% of KHC1-975ΔIAK and KHC1-938 oocytes, respectively) and dots/clouds of oskar adjacent to the posterior crescent. The relationship between oskar localization, MTs and endocytosis at the posterior is complex, involving various feedback loops. It is possible that defects in vesicle trafficking result in mild defects in cytoskeleton organization, since Rab11 and Rab6 mutant oocytes show mispolarized MTs. Thus, this inefficient oskar localization to a posterior crescent in mutant oocytes might indirectly result from mild cytoskeleton defects at the posterior. Alternatively, KHC1-975ΔIAK-dependent or KHC1-938- dependent ectopic oskar protein and/or ectopic MT plus ends might result in aberrant endocytosis at the posterior (Williams, 2013).

It is interesting to note that although oskar RNA is not a conserved cargo its transport involves a highly conserved domain, i.e. the AMB domain. This, and the findings concerning the IAK domain, show that although not all cargos are conserved their transport involves the most conserved domains of animal KHCs. Thus, both the IAK and AMB domains might play a crucial role in the transport of cargos in other cell types and organisms (Williams, 2013).

A NudE/14-3-3 pathway coordinates dynein and the kinesin Khc73 to position the mitotic spindle

Mitotic spindle position is controlled by interactions of cortical molecular motors with astral microtubules. In animal cells, Partner of Inscuteable (Pins) acts at the cortex to coordinate the activity of Dynein and Kinesin-73 (Khc73; KIF13B in mammals) to orient the spindle. Though the two motors move in opposite directions, their synergistic activity is required for robust Pins-mediated spindle orientation. This study identified a physical connection between Dynein and Khc73 that mediates cooperative spindle positioning. Khc73's motor and MBS domains link Pins to microtubule plus ends, while its stalk domain is necessary for Dynein activation and precise positioning of the spindle. A motif in the stalk domain binds, in a phospho-dependent manner, 14-3-3ζ, which dimerizes with 14-3-3ε. The 14-3-3ζ/ε heterodimer binds the Dynein adaptor NudE to complete the Dynein connection. The Khc73 stalk/14-3-3/NudE pathway defines a physical connection that coordinates the activities of multiple motor proteins to precisely position the spindle (Lu, 2013).

Mitotic spindle orientation requires the coordination of several pathways that act on astral microtubules. These pathways may establish cortical-microtubule connections and generate the forces necessary for movement of this large cellular structure with metaphase spindle lengths varying from 2 mm in yeast to 60 mm of a Xenopus single-cell stage. The spindle-orientation protein Pins has a domain that has been thought to capture microtubules (Pinslinker), and another that generates force (PinsTPR). This study attempted to understand how these two pathways function together by taking advantage of an induced polarity system in cultured S2 cells in which the two pathways can be selectively activated. This system allowed for the identification of the Khc73 stalk domain as a critical element that links PinsTPR and Pinslinker pathways. This observation was used as a platform for establishing a complete physical connection between the two pathways. This study has also clarified the role of 14-3-3 proteins in spindle orientation, establishing that their interaction with Pins is likely to be indirect (through Dlg and Khc73) (Lu, 2013).

Khc73 performs two functions in Pins-mediated spindle positioning. First, it functions in the Pinslinker pathway to mediate cortical microtubule capture through its MBS and motor domains, respectively. The N-terminal portion of Khc73 is sufficient for linker activity, which is likely occurring through a DlgGK/Khc73MBS interaction at the cortex and a microtubule/ Khc73motor interaction at the spindle. This suggests that Khc73's motor domain could function at the cortex by itself, however, Ed:Khc73motor did not have spindle positioning activity, indicating that other factors could be required or the motor domain is not functional in this context (e.g., as a monomer with the coiled-coil stalk). Khc73 must therefore rely on Dlg as an adaptor to target it to the cortex, which is where it can potentially function to facilitate the initial contact of astral microtubules (Lu, 2013).

Although Khc73's MBS domain directly interacts with Dlg, Khc73 is not seen to colocalize with cortical Pins, even though Dlg robustly localizes to Pins crescents. Instead, the motor protein is seen distinctly at the ends of microtubule, suggesting that Khc73 moves to the plus ends where it may be poised for capture by the cortical Pinslinker/Dlg complex. Thus, Khc73's N-terminal domains are likely to facilitate cortical microtubule capture by linking microtubule plus ends to cortical Dlg (Lu, 2013).

In addition to facilitating cortical microtubule capture, this study found that Khc73 also forms a physical connection to the PinsTPR/Mud/Dynein pathway with its stalk region, which is essential for the synergistic function of the two pathways. Khc73 may activate Dynein by delivering NudE to the cortex, where Dynein is presumably localized by PinsTPR/Mud. Although it is not possible to observe the localization of Dynein in S2 cells for technical reasons, there is good evidence that it is cortically localized by way of PinsTPR/Mud. In HeLa cells, Dynein localizes to the cortex with the mammalian homolog of Mud, NuMA, along with mPins, during mitosis (Lu, 2013).

It is proposed that a 14-3-3 motif in Khc73's stalk region activates an 'idling' cortically localized Dynein by cargoing NudE. Interestingly, although the Khc73 14-3-3 motif mutant Khc73S1374A has a distribution of spindle-orientation angles that isn't random, the distribution is bimodal such that the spindle angles are either fully aligned or orthogonal to the polarity axis. The bimodal phenotype is distinct from the Khc73motor+MBS fragment, which has a canonical intermediate distribution of spindle angles, suggesting that there may be additional regions or domains in the stalk that are contributing to the bimodal phenotype. It is hypothesized that an element within Khc73's stalk region is required for the proper application of the forces generated from by two motor proteins to properly orient the mitotic spindle. Nevertheless, biochemical and genetic studies demonstrate that the 14-3-3 binding motif is, at the very least, required for proper Pins-mediated spindle positioning and required for Khc73's interaction with the 14-3-3 proteins and NudE (Lu, 2013).

Pins mediates spindle positioning by coordinating two motor proteins that, as a pair, facilitate the cortical capture of microtubules and also provide pulling forces to robustly orient the mitotic spindle. A model is proposed in which orientation occurs through an ordered series of events, beginning with the initial polarization of Pins, followed by recruitment of Mud through its PinsTPR domain and Dlg through Pinslinker region. Cortical Mud then recruits cytoplasmic Dynein, which is not yet active and will remain inert, but poised at the cortex. Khc73 localizes to the plus ends of microtubules, where it establishes cortical-microtubule contacts through direct binding to Dlg and also delivers NudE to cortical Dynein, thereby activating it. As astral microtubules enter the proximity of the Dynein complex, Dynein can generate specifically timed cortical pulling forces necessary for robust spindle positioning. Future work will be directed at dissecting the precise timing of these synergistic events that underlie differentiation and tissue architecture (Lu, 2013).

Spindle-F is the central mediator of Ik2 kinase-dependent dendrite pruning in Drosophila sensory neurons

During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. This study identifies a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. These findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons (Lin, 2015)

The precise assembly of neural circuits is crucial for the nervous system to function properly. The developing nervous systems often start with a primitive prototype, characterized by exuberant branches and excessive connections. Thus, further remodeling is required to refine the developing nervous systems to maturity. Neuronal pruning, one such remodeling mechanism, is a highly regulated self-destruct process that eliminates excessive neuronal branches in the absence of cell death. Pruning is widely observed in the nervous systems of both vertebrates and invertebrates, that not only ensures precise wiring during development, but also allows for adjustment of neuronal connections in response to injury and disease. Various studies have shown that defects in developmental pruning affect the function of the nervous systems in C. elegans and Drosophila. Moreover, a progressive loss of neurites far ahead of cell death is commonly observed in many neurodegenerative disorders. Thus, any dysregulation of pruning activity even at the level of individual neurons would bring catastrophic consequences to the nervous systems. Although the primary triggers for developmental pruning and pruning that ensues upon neuronal injury and disease are diverse, the downstream machinery that eliminates neuronal processes shared some common features. For example, microtubule disruption is the earliest cellular event observed in all types of pruning, and the ubiquitin-proteasome system is required in all circumstances (Lin, 2015)

During Drosophila metamorphosis, substantial neuronal remodeling takes place in both the central and peripheral nervous systems. Most of the larval peripheral neurons die during metamorphosis, whereas few, including some class IV dendritic arborization (C4da) neurons, survive and undergo large-scale dendrite pruning. Dendrite pruning of the dorsal C4da neuron ddaC starts with severing of the proximal dendrites at 4-6 h APF (after puparium formation). Subsequently these disconnected dendrites become fragmented and eventually eliminated by the surrounding epidermal cells by 16-18 h APF. In contrast to the central brain mushroom body (MB) gamma neurons where both larval dendrites and axons are pruned during development, the peripheral C4da neurons specifically prune their dendrites keeping the axons intact. The molecular basis for how the pruning activity is confined to the dendrites of C4da neurons remains unknown. It was reasoned that molecular differences between dendrites and axons should be considered for such differential pruning activity in C4da neurons. It is known that microtubule polarity is different in the dendrites and axons of neurons, including in the Drosophila sensory neurons. For example, C4da neurons have polarized microtubules in their proximal dendrites predominantly with microtubule minus end pointing away from the cell body, but have an opposite polarity in their axons. This difference in microtubule polarity is essential for maintaining the proper function and compartmental identities of dendrites and axons, and might be an important determinant for spatially restricting pruning activity in the dendritic compartments of C4da neurons. Based on this assumption, some molecules are required to connect the pruning activity with the distinctive microtubule polarity of the dendrites in C4da neurons during dendrite pruning (Lin, 2015)

Previous studies have shown that dendrite pruning in C4da neurons is initiated by the steroid hormone ecdysone and its heterodimeric receptors, ecdysone receptor B1 (EcR-B1) and Ultraspiracle (Usp). Through transcriptional regulation of sox14, ecdysone signaling activates the Sox14 target gene mical, which encodes a cytoskeletal regulator, to regulate dendrite pruning. A few other molecules mediating specific cellular activities have been shown to participate in dendrite pruning of C4da neurons, such as the ubiquitin-proteasome system, caspases, matrix metalloproteases, microtubule severing proteins and mediators of dendritic calcium transients. Previous studies identified Ik2 kinase, a homologue of vertebrate IKK-ε in Drosophila, that plays an essential role in dendrite pruning of pupal neurons, and further demonstrated that Ik2 is sufficient to induce precocious dendrite severing in larval neurons. Ik2 is the only known molecule sufficient to induce premature dendrite severing in larvae, reflecting a central role of Ik2 kinase in dendrite pruning. Therefore, this study aimed to elucidate the mechanism by which Ik2 kinase signaling is transduced and regulated in Drosophila sensory neurons during dendrite pruning (Lin, 2015)

To elucidate the mechanism of Ik2 kinase signaling, candidate molecules were sought that mediate Ik2 signals during dendrite pruning. Several lines of evidence suggested that Spn-F, a coil-coiled protein, is a good candidate. Firstly, spn-F mutant flies showed defects in developing oocytes and bristles (Abdu, 2006), similar to the phenotypes observed in ik2 mutants (Shapiro, 2006). Secondly, Spn-F physically interacts with Ik2 (Dubin-bar, 2008). It implied that ik2 and spn-F may act in the same pathway during oogenesis and bristle morphogenesis, and the possibility has been raised that a similar pathway might also be involved in dendrite pruning of C4da neurons. This study demonstrates Spn-F plays a key role in linking Ik2 kinase to microtubule motor dynein complex for dendrite pruning. Spn-F acts downstream of Ik2 kinase in the same pathway for dendrite pruning. Spn-F is shown to displays a punctate pattern in larval neurons and these Spn-F puncta become dispersed in pupal cells. The redistribution of Spn-F from puncta is essential for dendrite pruning, and depends on the activity of Ik2 kinase and the function of microtubule motor dynein complex. These data also demonstrate that Spn-F not only links Ik2 to dynein motor complex, but also mediates the formation of Ik2/Spn-F/dynein complex, that is critical for Spn-F punctum disassembly and dendrite pruning (Lin, 2015)

In addition to apoptosis, neurons have a second self-destruct program in their axons for axonal pruning during development and in response to neuronal injury and disorders. This study proposes a third self-destruct program, which is mediated by Ik2 kinase activity in Drosophila sensory neurons, specific for dendrite pruning. Ik2 is essential for dendrite severing in pupal C4da neurons (Lee, 2009), and currently is the only known molecule sufficient to cause precocious dendrite severing in larval cells (Lee, 2009), indicating that Ik2 activation must be regulated temporally. For temporal regulation, ecdysone signaling plays a key role in dendrite pruning (Williams, 2005; Lee, 2009). These studies show that no Ik2 activation is detected in pupal C4da neurons with impaired ecdysone signaling and thus places Ik2 kinase downstream of ecdysone signaling. Microarray studies have identified ik2 as one of the ecdysone/EcR up-regulated genes in brain MB γ neurons during axon pruning (Hoopfer, 2008). This suggests one possible mechanism where ecdysone/EcR regulates Ik2 activation through increasing ik2 expression in C4da neurons. Although Ik2 kinase activity is crucial for oogenesis and bristle morphogenesis (Shapiro, 2006; Otani, 2011), the activation mechanisms of Ik2 kinase in both processes remain unknown. Since pruning activity is considered as a self-destruct program, how to regulate this activity spatially in subcellular compartments within individual neurons is an intriguing issue to investigate. This study identifies Spn-F and cytoplasmic dynein complex as critical regulators of Ik2-mediated dendrite pruning activity in C4da neurons (Lin, 2015)

It is known that endogenous Spn-F exhibits a punctate pattern in nurse cells (Abdu, 2006), consistent with the observation of punctate Spn-F-GFP in larval C4da neurons. The formation of Spn-F puncta in cells is through self-association, and does not depend on the integrity of microtubule network or the function of cytoplasmic dynein. Since Ik2 could form oligomers in cells, the interaction between Ik2 and Spn-F might also play a role in Spn-F puncta formation. Indeed, it was observed that SpnF-ΔCC3-GFP has normal interaction with either SpnF-ΔCC3 or full-length Spn-F, but formed fewer puncta than the wild type Spn-F-GFP did in larval neurons. Therefore, the Spn-F puncta formation could be attributed not only to Spn-F self-association, but also to Ik2/Spn-F interaction and Ik2 oligomerization (Lin, 2015)

In larval C4da neurons, Ik2 kinase is inactive and associates with Spn-F as puncta in the cytosol. After puparium formation, Ik2 kinase becomes activated promptly and phosphorylates Spn-F in C4da neurons. This Ik2-dependent phosphorylation on Spn-F decreases Spn-F self-association, and subsequently the numbers and sizes of Spn-F puncta were reduced. One may question that protein degradation might contribute to decrease the numbers and sizes of Spn-F puncta in C4da neurons during dendrite pruning. It was known that Ik2 promotes caspase inhibitor DIAP1 degradation via proteasomes during the development of sensory organ precursors; therefore, Ik2 might promote Spn-F degradation in C4da neurons during dendrite pruning. However, Ik2 overexpression does not alter the protein level of Spn-F in either S2 or germline cells (Dubin-Bar, 2008). Thus, protein degradation by proteasomes is unlikely the mechanism leading to decreased Spn-F puncta after Ik2 activation. Since P-Ik2 signals were indistinguishable between wild-type and Dhc64C RNAi neurons, it is reasonable to presume that both Ik2 activation and Spn-F phosphorylation occur normally in dynein mutant neurons. No significant differences were found between the pruning defects of C4da neurons in spn-F mutants and that in spn-F mutants with Dhc-RNAi, and between the pruning phenotypes observed in Dhc mutants and that in Dhc mutants with ik2-RNAi. These findings further support that Ik2, Spn-F and dynein complex function together in the same pathway in dendrite pruning of C4da neurons. However, the finding of Spn-F puncta in mutant pupal neurons with impaired dynein function indicated that dynein is required for Spn-F redistribution after Ik2 activation. Furthermore, Spn-F remains punctate in S2 cells with Ik2 overexpression even after microtubule depolymerization and inhibition of dynein function, suggesting that dynein might redistribute Ik2/Spn-F complexes via transporting complexes toward the minus ends of microtubules in C4da neurons during dendrite pruning. The results in this study and studies in germline cells, (the fact that more Spn-F puncta accumulated in nurse cells with colchicine treatment and with Dhc mutation) (Abdu, 2006), favor the mechanism of protein redistribution for Spn-F punctum reduction in dendrite pruning of C4da neurons (Lin, 2015)

It has been shown that during Drosophila bristle elongation, directional transport of activated Ik2 and of Spn-F to the bristle tips, where the microtubule minus ends are concentrated, requires the function of cytoplasmic dynein, and Spn-F acts as an adaptor to link Ik2 to dynein complexes. These are similar to the current findings that both Ik2 activation and dynein complex are essential for Spn-F redistribution, and Spn-F plays a central role in the formation of Ik2/Spn-F/dynein complex, which is crucial for Spn-F redistribution and for dendrite pruning in C4da neurons. However, the studies in bristle elongation indicating that spn-F acts upstream of ik2 (Otani, 2015) disagree with the current finding that ik2 acts upstream of spn-F in dendrite pruning. The discrepancy between the epistasis analyses of ik2 and spn-F in these two different processes might be due to different cell-type specific factors in these two types of cells that affect the morphological readouts in genetic studies. Moreover, this study demonstrated that Ik2-dependent phosphorylation of Spn-F decreases Spn-F self-association, promotes Spn-F redistribution, and finally leads to dendrite pruning in C4da neurons (Lin, 2015)

The activated Ik2 signals accumulate at the microtubule minus ends in cells with polarized microtubule distribution, such as oocytes, follicle cells and bristles. This is consistent with the conclusion that dynein transports activated Ik2 toward microtubule minus ends in C4da neurons. Since Drosophila sensory neurons have polarized microtubules in their proximal dendrites predominantly with microtubule minus end pointing away from the cell body, these studies revealed a possible mechanism that Spn-F and minus-end directed motor dynein complex confine Ik2-dependent pruning activity to the somatodendritic compartments of C4da neurons. During Drosophila bristle elongation, the accumulation of endogenous Spn-F observed at the bristle tip, where the microtubule minus ends are enriched, led to an examination of Spn-F-GFP signals along the dendrites of C4da neurons during dendrite pruning. However, no enriched of Spn-F-GFP signals in the proximal dendrites, where dendrite severing is expected to occur, was observed, by live imaging during pruning. Previous studies (Lee, 2009) showed that microtubules are first disassembled in the proximal dendrites of C4da neurons during dendrite severing. This local disassembly of microtubules is suppressed in ik2 mutant neurons. Since the current genetic studies indicate that both ik2 and spn-F act in the same pathway of dendrite pruning, tests were performed to see whether local microtubule disassembly happens normally in spn-F mutants. Local breakage of microtubules found in the proximal dendrites of C4da neurons was also suppressed in spn-F RNAi mutants, suggesting that Spn-F, like Ik2, plays a role in dendrite severing that involves local microtubule disassembly. However, the molecular mechanisms by which activated Ik2 and Spn-F lead to dendrite severing in the proximal dendrites of C4da neurons will be an important question for future studies (Lin, 2015)

It is known that there is no decrease in cell death in wing discs with ik2 knockdown and in ik2 mutant embryos (Kuranaga, 2006), indicating that the primary function of Ik2 is not involved in the apoptotic pathway during development. However, ectopic Ik2 activation by overexpression leads to cell death in fly compound eyes (Kuranaga, 2006) and in C4da neurons (Lee, 2009), suggesting that excessive Ik2 kinase signaling could trigger a crosstalk with signaling molecules in apoptotic pathways and result in apoptosis. It is known that Ik2 kinase regulates the nonapoptotic function of caspase through promoting DIAP1 degradation (Kuranaga, 2006). In a similar manner, the confinement of activated Ik2 kinase in the dendritic compartments might restrict the detected caspase activity in the degenerating dendrites after separating from the soma of C4da neurons during dendrite pruning. Therefore, this raises a possibility that de-regulation of pruning activity in neurons may trigger a crosstalk with molecules in apoptotic pathways and lead to undesired cell death during neuronal injury and disorders. Recently, a caspase cascade, including caspase 3 and 6, was identified in mice to play a role in developmental axon pruning and in sensory axon pruning after trophic factor withdrawal. Moreover, activated caspase 6 was detected in human patient brains of Alzheimer and Huntington diseases long before cell death, highlighting a critical role in regulating caspase activity in both diseases. Understanding the regulatory mechanisms that confine pruning activity into proper subcellular compartments of the neuron might provide molecular insights into the pathogenesis of neural disorders (Lin, 2015)

A transport and retention mechanism for the sustained distal localization of Spn-F-IKKε during Drosophila bristle elongation

Stable localization of the signaling complex is essential for the robust morphogenesis of polarized cells. Cell elongation involves molecular signaling centers that coordinately regulate intracellular transport and cytoskeletal structures. In Drosophila bristle elongation, the protein kinase IKKε is activated at the distal tip of the growing bristle and regulates the shuttling movement of recycling endosomes and cytoskeletal organization. However, how the distal tip localization of IKKε is established and maintained during bristle elongation is unknown. This study demonstrates that IKKε distal tip localization is regulated by Spindle-F (Spn-F), which is stably retained at the distal tip and functions as an adaptor linking IKKε to cytoplasmic dynein. Javelin-like (Jvl) is a key regulator of Spn-F retention. In jvl mutant bristles, IKKε and Spn-F initially localize to the distal tip but fail to be retained there. In S2 cells, particles that stain positively for Jvl or Spn-F move in a microtubule-dependent manner, whereas Jvl and Spn-F double-positive particles are immobile, indicating that Jvl and Spn-F are transported separately and, upon forming a complex, immobilize each other. These results suggest that polarized transport and selective retention regulate the distal tip localization of the Spn-F-IKKε complex during bristle cell elongation (Otani, 2015).

Highly polarized cells, such as neurons and epithelial cells, rely heavily on intracellular transport mechanisms for their functional differentiation. Disrupted intracellular transport systems lead to a variety of diseases, including neurodegeneration and microvillus inclusion diseases. Accurate intracellular transport is ensured by the polarized cytoskeleton and by the adaptor protein-mediated recognition of specific cargoes by molecular motors. Molecular motors play central roles in intracellular transport, and have diversified through evolution. However, the diversity of molecular motors is not sufficient to explain transport specificity, as various cargoes often share the same motor while being transported to distinct locations. For example, cytoplasmic dynein is the major microtubule minus-end motor and transports a variety of cargoes including the Golgi apparatus, endosomes and RNAs. Evidence suggests that the fate of cargo is determined not only by cargo-motor recognition, which occurs upon cargo loading, but also at the cargo destination site. For instance, in axonal transport some cargoes, such as dense core vesicles and synaptic vesicles, are inefficiently captured at synaptic boutons and circulate within the axon, whereas others, such as mitochondria, are stably retained at synapses. Although the precise regulation of cargo transport is important for the functional differentiation of various polarized cells, the underlying molecular mechanisms remain poorly understood (Otani, 2015).

Cell elongation is a widely observed morphogenetic event that requires the coordinated input of intracellular transport, the cytoskeleton and cell polarity. Drosophila bristles, which are hair-like unicellular structures that function as external sensory organs, are formed by the elongation of trichogen cells, which can grow up to 350 μm in 1 day during the pupal stage. IκB kinase ε [IKKε; also known as IκB kinase-like 2 (Ik2)] acts at the distal tip of growing bristles and functions as a signaling center to regulate the bidirectional shuttling of Rab11-positive recycling endosomes during bristle elongation. Rab11-positive vesicles are transported to the distal tip by interacting with cytoplasmic dynein via an adaptor protein Nuf/Rab11FIP3. At the distal tip, IKKε phosphorylates Nuf to inactivate dynein-dependent trafficking, thereby promoting the directional switching of the recycling endosomes. In addition to its role in endosome trafficking, IKKε regulates the organization of both actin and microtubules. However, how IKKε is localized to the distal tip of growing bristles is unknown (Otani, 2015).

Spindle-F (Spn-F) is a coiled-coil protein that interacts with IKKε and has been implicated in regulating IKKε polarized activation. In oocytes, the intracellular localizations of Spn-F and IKKε depend on each other, and spn-F and ikkε mutants show similar bristle morphology and oocyte polarization phenotypes, suggesting that they function together. Several proteins other than IKKε, including Cut up (Ctp)/dynein light chain (LC8) and Javelin-like (Jvl), are reported to interact with Spn-F. It was proposed that Spn-F interacts with cytoplasmic dynein via Ctp to localize the Spn-F-IKKε complex to microtubule minus ends. However, subsequent structural studies indicated that Ctp/LC8 cannot simultaneously bind dynein and cargo molecules, challenging this model. On the other hand, IKKε can phosphorylate Spn-F, suggesting that Spn-F might act downstream of IKKε. Interestingly, another Spn-F-interacting protein, Jvl, was recently shown to regulate the polarized activation of IKKε in oocytes. Although Jvl can interact with microtubules, how it regulates the polarized activation of IKKε is unknown (Otani, 2015).

This study demonstrated that the bristle tip is a sorting station for cytoplasmic dynein-dependent cargoes. The IKKε-Spn-F complex, which acts as the signaling center in bristle cell elongation, localizes to the distal tip by dynein-dependent polarized transport and Jvl-dependent selective retention. By contrast, Rab11-positive recycling endosomes undergo both dynein-dependent distal transport and proximal transport, which is probably mediated by kinesins (Otani, 2015 and references therein).

The distinct transport characteristics at the distal tip are specified by the nature of the adaptor proteins. IKKε is transported to the distal tip by dynein via the adaptor protein Spn-F, and the IKKε-Spn-F complex is stably retained at the distal tip by Jvl, a Spn-F-interacting protein. By contrast, Rab11-positive recycling endosomes are transported to the distal tip by dynein via the adaptor protein Nuf, where it is phosphorylated by IKKε. This phosphorylation inactivates the dynein-dependent transport of Rab11-positive recycling endosomes, thereby promoting their transport back to the cell body. Thus, the IKKε-Spn-F complex stably localizes to the distal tip by polarized transport followed by selective retention, whereas Rab11-positive recycling endosomes bidirectionally shuttle by polarized transport and motor switching. The pivotal step in this sorting decision is the specific recognition of the cargo adaptor proteins (Spn-F and Nuf) by their regulatory proteins (Jvl and IKKε) at the distal tip. These results support the emerging concept that cargo adaptor proteins are not merely physical linkers between cargoes and motors, but act as regulatory hubs where various signals converge (Otani, 2015).

This study identified Jvl as a key regulator of IKKε-Spn-F retention at the distal tip. Jvl interacts with microtubules, and binding Spn-F promotes the microtubule binding activity of Jvl and induces microtubule bundling in S2 cells. Full-length Jvl localizes to punctate structures that were located along microtubules, whereas the C-terminal half of Jvl uniformly decorated microtubules. These results imply that Jvl microtubule binding activity is repressed by its N-terminal region, and that binding Spn-F could relieve this inhibition. Oligomerization of Spn-F could promote the formation of higher-order Spn-F-Jvl complexes to generate multivalent microtubule-binding sites, thereby increasing the microtubule binding activity of Jvl (Otani, 2015).

Spn-F and Jvl are independently transported to the distal tip in elongating bristles, indicating that their interaction occurs upon arrival at the tip. This interaction presumably activates Jvl microtubule binding activity, which then serves as a molecular brake to immobilize the complex on microtubules. Similar mechanisms have been proposed for the anchoring of mitochondria by Syntaphilin and Kinesin-1 in axonal mitochondrial transport, and for the immobilization of lysosomes in dendrites by the interaction of TMEM106B and MAP6. The coupling of cargo adaptor proteins with microtubule-binding proteins might be a general mechanism for regulating the transport of a particular cargo in a spatiotemporally controlled manner. As Spn-F and Jvl are also involved in the polarized activation of IKKε during oogenesis, similar mechanisms might help generate and maintain cell polarity in various cell types (Otani, 2015).

As an alternative to the molecular brake model, Jvl could act as a scaffolding protein to recruit enzymes that modify the IKKε-Spn-F complex to promote its retention, or as a regulator of microtubule organization at the distal tip to maintain the polarized organization of the cytoskeleton during bristle elongation. Further analysis of the molecular functions of Jvl will help in elucidating the mechanisms of IKKε-Spn-F retention (Otani, 2015).

The results suggest that Spn-F functions as a cargo adaptor for IKKε and cytoplasmic dynein. Structure-function analysis of Spn-F demonstrated that its dynein-binding region is required for localizing IKKε to the distal tip and for bristle morphogenesis. In contrast to the dynein-binding-deficient Spn-F mutant, which partially suppressed the spn-F mutant bristle morphology phenotype, a mutant lacking the IKKε-binding region completely failed to rescue, indicating that, in addition to its function as a cargo adaptor, Spn-F has a role in regulating IKKε activity. This role could involve regulating IKKε kinase activity or protein stability, or in scaffolding the components of the IKKε signaling pathway. IKKε overexpression could partially suppress the spn-F mutant bristle morphology phenotype despite IKKε delocalization from the distal tip, suggesting that increasing the dosage of IKKε can compensate for the loss of Spn-F to some extent. It is likely that the delocalized IKKε can phosphorylate some of its downstream target molecules (such as Nuf and Diap1) to partially support bristle morphogenesis (Otani, 2015).

In summary, this study has demonstrated that the signaling center for bristle elongation is localized to the distal tip by polarized transport and selective retention mechanisms. The distal tip of bristles acts as a sorting center for cytoplasmic dynein cargoes, where regulatory proteins recognize cargo adaptor proteins and determine whether cargo is retained or sent back to the cell body. These findings support the idea that cargo adaptor proteins act as regulatory hubs where various signals converge. It would be interesting to test whether the differential regulation of cargo-motor interactions contributes to the formation of signaling centers during the morphogenesis of mammalian cells of complex shape, such as neurons and podocytes (Otani, 2015).

Dynein heavy chain 64C: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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