Dynein heavy chain 64C
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).
Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Mutations in the dynein heavy chain gene have been used to impair the motorís
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 embryoís 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, Lvaís 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).
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).
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).
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 s