Dynein heavy chain 64C

DEVELOPMENTAL BIOLOGY

Embryonic

Dhc64C, encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development; the highest levels are detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. Before bulk transport of nurse cell cytoplasm to the oocyte, an increase is observed in the amount of dynein antigen present in the nurse cell cytoplasmic domain of stage 10 egg chambers. Dynein is also detected at a low and uniform intensity in the follicle cells surrounding early egg chambers. At later stages this follicle cell dynein appears to be concentrated to the apical region of each follicle cell that surrounds the oocyte. This apical pattern of dynein distribution in follicle cells could reflect the role that these cells play in polarizing secretion of components required for the formation of several shell layers that encapsulate the mature egg. In the oocyte, a bright focus of dynein staining is closely apposed to the oocyte nuclei in early chambers; and a perinuclear concentration of the dynein antigen is frequently apparent in stage 6-8 egg chambers. During stage 9, an accumulation of dynein antigen is seen at the posterior end of the oocyte, suggesting that dynein may play a role in the assembly of the pole plasm during stages 8 and 9 of oogenesis. The posterior concentration of dynein appears to diminish at later stages, while an elevated level of dynein appears in the nurse cell cytoplasm. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte (Li, 1994).

A cytoplasmic dynein motor isoform that is present in extracts of Drosophila embryos has been characterized. A prominent high molecular weight (HMW) polypeptide (> 400 kDa) is enriched in microtubules prepared from nucleotide-depleted embryonic extracts. Based on its ATP-sensitive microtubule binding activity, 20 S sedimentation coefficient, sensitivity to UV-vanadate and nucleotide specificity, the HMW polypeptide resembles cytoplasmic dyneins prepared from other organisms. The Drosophila cytoplasmic dynein acts as a minus-end motor that promotes microtubule translocation in vitro. A polyclonal antibody raised against the dynein heavy chain polypeptide was used to localize the dynein antigen in whole-mount preparations of embryos by immunofluorescence microscopy. These studies show that the dynein motor is associated with microtubules throughout embryogenesis, including mitotic spindle microtubules and microtubules of the embryonic nervous system (Hays, 1994).

Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, the mitotic functions were examined of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. Four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, Dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Dhc64C (cytoplasmic dynein) may control the timing of anaphase onset, possibly by transporting Rod or other checkpoint proteins away from kinetochores as proposed for the fly embryo. A similar checkpoint inactivation model was proposed for mammalian dynein/dynactin based on inhibition analyses. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From this phenotypic data, a model is constructed for the distinct roles of molecular motors during mitosis in a single metazoan cell type (Goshima, 2003).

Dynactin is necessary for synapse stabilization

Evidence is presented that synapse retraction occurs during normal synaptic growth at the Drosophila neuromuscular junction (NMJ). An RNAi-based screen to identify the molecular mechanisms that regulate synapse retraction identified Arp-1/centractin, a subunit of the dynactin complex. Arp-1 dsRNA enhances synapse retraction, and this effect is phenocopied by a mutation in P150/Glued, also a dynactin component. The Glued protein is enriched within the presynaptic nerve terminal, and presynaptic expression of a dominant-negative Glued transgene enhances retraction. Retraction is associated with a local disruption of the synaptic microtubule cytoskeleton. Electrophysiological, ultrastructural, and immunohistochemical data support a model in which presynaptic retraction precedes disassembly of the postsynaptic apparatus. These data suggests that dynactin functions locally within the presynaptic arbor to promote synapse stability (Eaton, 2002).

Evidence is presented that regulated synaptic growth at the Drosophila NMJ includes synaptic retraction events in addition to the well-characterized addition of new synaptic boutons. Synaptic retraction events (footprints) are defined as the withdrawal of presynaptic antigens (synapsin, HRP, Futsch) from clearly defined regions of postsynaptic specialization defined by Discs-large immunoreactivity. Multiple lines of evidence, using a variety of analytic tools, including light level and ultrastructural analysis, are presented that a footprint represents a site where the nerve terminal once resided and has since retracted. Previous reports have likely failed to identify synaptic retraction as an important element during synapse development in this system because postsynaptic markers were employed to study synapse development. Synapse retraction events are more frequent at early developmental stages which correlate with the more rapid phase of synapse growth. These data suggest that regulated synaptic growth is achieved by a balance of synaptic growth and retraction at the Drosophila NMJ. Such a balance of growth and retraction may represent a general principle of synaptic growth control in this and other systems (Eaton, 2002).

Employing a functional genomic strategy, the dynactin protein complex has been identified as an essential component of the machinery that achieves synapse stabilization at the Drosophila NMJ. Disruption of the dynactin complex using any of three different perturbations, including Arp-1 RNAi, the Glued1 mutation, or presynaptic overexpression of the DN Glued transgene all result in an increase in the frequency and extent of synaptic retraction events at the NMJ. Presynaptic, but not postsynaptic, overexpression of the DN Glued transgene enhances synapse retraction, phenocopying genetic and RNAi perturbation of the dynactin complex. Consistent with this observation, P150/Glued protein is present at the NMJ and enriched in the presynaptic nerve terminal (Eaton, 2002).

Several lines of evidence suggest that dynactin functions locally within the presynaptic nerve terminal to control synapse stability. Retraction events do not result in the complete elimination of a synapse, but are generally restricted to a specific branch or portion of a branch within the synaptic arbor of a single motoneuron. In addition, MN 6/7 innervates multiple muscle targets, and retraction events are often specific to only one or a few of the muscle targets of this neuron, demonstrating that retraction events can be branch specific on different muscle targets (Eaton, 2002).

An obvious concern is that inhibition of dynactin function sufficiently impairs the health of the motoneuron to cause a secondary retraction of the synapse. Ultimately, an assessment of 'health' can only be achieved by assaying a number of independent variables such as electrophysiology, ultrastructure, morphology, and cell death. Synapse retraction events are not correlated with increased cell death in the motoneurons nor are they correlated with gross changes in the motoneuron microtubule cytoskeleton. Synapse retraction events can be local, affecting only a portion of a motoneuron arbor (demonstrated by analysis at both the light and ultrastructural level). In addition, motoneuron transmitter release properties are normal at a portion of the synapses that overexpress the DN Glued transgene (the most severe manipulation used). This is consistent with the observation that only a portion (40%) of the synapses reveal a retraction event despite the fact that DN Glued is expressed pan neuronally. These data argue against DN Glued simply poisoning the cell. Finally, retraction events are observed in the wild-type animal and occur with higher frequency during early larval development. Taken together, these data suggest that retraction is associated with synapse development rather than impaired health (Eaton, 2002).

The data indicate that increased synapse retraction caused by impaired dynactin activity has a functional consequence for the synapse. Increased synaptic retraction results in fewer synaptic boutons and decreased synaptic efficacy. This is predicted if synapse retraction helps to shape the outcome of synapse development. Thus, the increased frequency of retraction events observed during early synapse development (rapid growth) may be an important aspect of synaptic growth control. It is hypothesized, therefore, that presynaptic dynactin-mediated synapse stabilization may help set the balance between growth and retraction during normal synaptic development (Eaton, 2002).

Synaptic retraction caused by disruption of the dynactin complex has been characterized at the light level, ultrastructurally and electrophysiologically. At each level of analysis, the data support the conclusion that the presynaptic nerve terminal withdraws, followed by the disassembly of the postsynaptic apparatus. At the light level, retraction of presynaptic markers precedes the elimination of postsynaptic Discs-large staining and GluR clusters. In addition, no change was observed in quantal size when DN Glued is driven presynaptically, despite often severely impaired presynaptic release. By comparison, acetylcholine receptors are removed postsynaptically prior to presynaptic elimination at the vertebrate NMJ, and a decrease in quantal size is observed. Finally, it was observed ultrastructurally that the presynaptic membrane appears to separate from the postsynaptic density with high frequency at active zones when dynactin is disrupted presynaptically. This is again consistent with presynaptic retraction preceding disassembly of the postsynaptic apparatus (Eaton, 2002).

The dynactin protein complex binds microtubules and has been shown to bind a number of proteins that localize to the plus end of microtubules including Lis1, Eb1, and Clip170. In addition to their localization, these proteins have also been shown to effect microtubule stability and dynamics. In budding yeast, live imaging studies of microtubules have demonstrated that mutations in dynactin and EB1 lead to altered microtubule structure and defects in cortical association. Based on these observations, one hypothesis is that dynactin and associated proteins participate in the process of microtubule capture at the cell cortex. Alternatively, dynactin could be involved in more complex regulation of microtubule dynamics via the localization or trafficking of microtubule regulators (Eaton, 2002).

Dynactin regulation of the microtubule cytoskeleton is thought to occur at sites of intercellular adhesion and signaling. In epithelial cells, microtubules and cytoplasmic dynein have been observed to contact adherens junctions. In addition, overexpression of the p62 component of the dynactin complex localizes strongly to sites of focal adhesion. Furthermore, dynactin has been shown to bind proteins such as β-catenin and spectrin, which are known to be involved in cell adhesion (Eaton, 2002).

In Drosophila, dynactin has been studied during axon guidance. In dynactin mutations, axons reach their target regions but fail to branch normally. It was not determined whether synapses failed to form in these studies or whether synapse stability was disrupted. These data demonstrate that dynactin is not necessary for growth cone motility. The interpretation of these data has been a failure to transport important trophic molecules in a retrograde manner to the sensory neuron cell body via the dynein retrograde motor. However, these data are equally consistent with a failure of sensory neuron synapse stability within the central nervous system (Eaton, 2002).

The data at the Drosophila NMJ are consistent with an essential role for the dynactin complex in maintaining the appropriate microtubule architecture and, as a consequence, synapse stability. The possibility cannot be ruled out that an essential retrograde signal fails to reach the motoneuron soma. However, since retraction events are observed to affect only portions of a single synapse and since no change is seen in motoneuron cell death, it is suspected that failure to traffic an essential retrograde signal is not the primary cause of synapse retraction at the Drosophila NMJ (Eaton, 2002).

An important aspect of the current data is a possible link between synapse retraction and neural diseases with which dynactin function has been associated. Mutations in the human lis1 gene cause type 1 lissencephaly, a debilitating developmental disease of the nervous system characterized by severe mental retardation and reduced cerebral folds. Interestingly, in addition to defects in neuroblast proliferation and axonal transport, Drosophila Lis1 mutants have abnormal dendritic morphology. In addition, dynactin binds Huntingtin Associated Protein 1 (Hap-1) and is thought to play a role in the trafficking of this protein. The current data are most consistent with a local role for dynactin within the nerve terminal controlling synapse stabilization. With respect to vertebrate disease states, the altered control of synapse stability has not been fully investigated. As such, this aspect of dynactin function characterized is this study may be an important component of disease progression if affected cells lose essential target-derived trophic support as a result of impaired synapse stability (Eaton, 2002).

Dynactin/Dynein is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons

How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. The Dynactin complex (a regulator of the microtubule motor protein Dynein) has been shown to be required to maintain the position of the nucleus within post-mitotic Drosophila photoreceptor neurons. Multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. It has been found that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function (Whited, 2004).

The Dynactin complex is an assembly of 11 different subunits that functions as an activator of Dynein, serving as an adaptor for cargo and enhancing motor processivity. The Dynactin subunit Glued couples Dynactin to Dynein by binding to the Dynein intermediate chain (Dic, encoded by short wing). Overexpression of a truncated form of Glued that binds to Dic but cannot associate with the rest of the Dynactin complex acts as a powerful inhibitor of Dynein and Dynactin function. Overexpression of the Dynactin subunit Dynamitin disrupts Dynactin complex assembly and also inhibits Dynactin function. Biochemical studies have shown that the Dynactin complex also contains Capping Protein, a heterodimer composed of the Capping Protein alpha (Cpa) and Capping Protein beta (Cpb) subunits. Although best known for capping the barbed ends of filaments of actin, Capping Protein also associates with filaments of the actin-related Arp1 protein, which is a central element of the Dynactin complex (Whited, 2004 and references therein).

Patterning of the adult compound eye of Drosophila initiates during the third instar phase of larval life, and mutations in the Dynactin subunit Glued strongly disrupt eye development. Normally the nuclei of differentiating photoreceptors occupy apical regions of the eye disc. In animals heterozygous for the dominant-negative Glued allele Glued¹, many photoreceptor nuclei have been shown to accumulate within basal regions of the eye disc. The effect of Glued¹ on photoreceptor development was characterized using an antibody recognizing photoreceptor cell surfaces. In wild type, the region of the differentiating photoreceptor neuron containing the nucleus remained in the retina, while the photoreceptor axon extended through the optic stalk into the brain. However, in Glued¹ animals, while photoreceptors still extended axons into the brain, the region of the photoreceptor containing the nucleus often appeared to leave the retina and travel through the optic stalk into the brain. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in Glued¹ mutants (Whited, 2004).

To further establish that Glued¹ defects reflected disruptions in Dynactin function, two other approaches were used to disrupt the Dynactin complex. Drosophila Dynamitin, which also inhibits Dynactin function in flies, was overexpressed in photoreceptor neurons. Loss-of-function mutations in the Dynactin subunit Cpb were examined by generating animals whose visual systems contained homozygous mutant clones of the cpb strong loss-of-function mutation cpb^M143. In these cpb^M143 mosaic animals, the nuclear regions of many photoreceptors were observed in the optic stalk and brain (Whited, 2004).

To confirm that the cpb^M143 mutant photoreceptor defect was due to a loss of cpb function, an additional strong loss-of-function cpb allele, cpb^F44, was isolated from an EMS mutagenesis and a chromosomal deficiency uncovering the cpb locus, Df(2L)E.2, was obtained. When animals contained homozygous mutant clones of cpb^F44 cells or homozygous mutant clones of Df(2L)E.2, a similar movement of photoreceptor nuclear regions toward the brain was observed. cpb/Df(2L)E.2 animals did not survive to third instar, preventing the classic genetic demonstration that these cpb alleles behaved as strong loss-of-function mutations. Fortunately, it was found that the [pYES-ß] genomic transgene, which contains the CPB coding region, was able to rescue the lethality of cpb/Df(2L)E.2 animals, but did not rescue the previously described cpb bristle defect. This suggested that [pYES-ß] was a partially functional rescue construct that could be used to examine the visual systems of otherwise cpb/Df(2L)E.2 animals. It was found that [pYES-ß];cpb^M143/Df(2L)E.2 animals display a photoreceptor defect similar to that of other cpb mutants, consistent with nuclear mispositioning resulting from the loss of cpb function. It was further confirmed that the defect was due to the loss of cpb function by successfully rescuing the cpb^M143/Df(2L)E.2 photoreceptor defects (as well as the cpb bristle defects) by expression of a wild-type Cpb cDNA under the control of a heterologous promoter. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in cpb mutants (Whited, 2004).

The bifunctional nature of Cpb, which associates with filaments of actin as well as filaments of Arp1, means that loss of Cpb also increases filamentous actin levels (Hopmann, 2003). Nonetheless, previous studies have shown that increases in filamentous actin alone, such as those observed in hypomorphic cpb alleles or in actup mutants, do not cause photoreceptor nuclear mispositioning. Together with the Glued¹ and Dynamitin data, the cpb observations yield a consistent picture that alterations in Dynactin subunits cause mispositioning of photoreceptor cell bodies and nuclei, and indicate that Dynactin, and not just the Glued subunit, has an important role in photoreceptor development (Whited, 2004).

The mispositioning of photoreceptor nuclei in Dynactin mutants raised the question of whether these disruptions reflect altered positioning of the nucleus within the photoreceptor or simply migration of the entire photoreceptor. To address this question, single photoreceptors were labeled in wild type and in Glued¹ mutants. Wild-type photoreceptors exhibit a highly polarized morphology in which the region of the photoreceptor containing the nucleus lies in the apical region of the eye disc and an axon extends basally into the brain. Glued¹ mutant photoreceptors whose nuclei have entered the optic stalk had highly altered morphologies, with both leading and trailing processes extending from the regions of the cell where the misplaced nucleus was located. Leading and trailing processes of misplaced Glued¹ photoreceptors were quantified, considering only those with no other labeled cells or processes nearby. Of these 13 neurons, 12 had clearly detectable leading and trailing processes. The leading process (axon) extended into the target region and the trailing process extended back into the eye disc. These data demonstrate that inhibition of Dynactin function dramatically alters the position of the nucleus within the photoreceptor (Whited, 2004).

The Dynactin complex also controls the pattern of mitoses within the Drosophila retina. To determine whether nuclear mispositioning is a secondary consequence of the earlier mitotic requirement for Dynactin, the effects of specifically inhibiting the Dynactin complex in postmitotic photoreceptors was examined. Conditional inhibition of Dynactin function can be achieved through inducible expression of a truncated, dominant-negative form Glued (Glued^DN) that resembles the protein product of Glued¹. Glued^DN was expressed under the control of the postmitotic photoreceptor-specific Glass 38-1 promoter, which initiates expression in the photoreceptors only after their axons have entered the brain. Expression of Glued^DN under the control of Glass 38-1 caused photoreceptor nuclei to move into the optic stalk. Overexpression of Dynamitin under the control of Glass 38-1 caused similar photoreceptor nuclear positioning defects. These data demonstrate that Dynactin is required postmitotically in photoreceptors to maintain nuclear position and that the disruptions in nuclear positioning observed are not simply a secondary consequence of mitotic defects (Whited, 2004).

The displacement of photoreceptor nuclei from apical regions of the eye disc toward more basal regions could reflect an overall disruption in apical/basal polarity of the eye disc. The apical/basal polarity of developing photoreceptors was assessed by examining the distribution of the Drosophila ß-catenin Armadillo and the PDZ-domain-containing protein PATJ. Armadillo localizes to the zonula adherens separating the apical and basolateral membrane domains of developing photoreceptors, while PATJ localizes to the apical membrane domain. In wild-type eye discs, Armadillo is concentrated just beneath the apical tips of the developing photoreceptors. In Glued¹ animals Armadillo was still present near apical regions of the eye disc, even in areas completely devoid of apical photoreceptor nuclei. Thus, this marker of apical/basal polarity was retained even when photoreceptor nuclei moved basally. Similar results were obtained when Glued¹ mutants were visualized in cross-section using both Armadillo and PATJ. Apical localization of PATJ and Armadillo were observed in Glued¹ and the relative apical/basal ordering of these markers was maintained. These data suggest that the alterations in photoreceptor morphology are not caused by a loss of apical/basal polarity within the developing photoreceptors (Whited, 2004).

Dynactin has important functions in the organization of the microtubule cytoskeleton in many systems. The microtubule cytoskeleton of developing photoreceptors is highly polarized, with microtubule minus ends concentrated apical to the nucleus as detected using antisera recognizing gamma-tubulin. A similar apical focus is observed when using the fusion protein Nod:LacZ, which often co-localizes with microtubule minus ends. The relatively ubiquitous expression of gamma-tubulin in the retina complicated the analysis of gamma-tubulin localization when retinal patterning was disrupted. Therefore, the effect of Glued on factors associated with the microtubule cytoskeleton was examined by expressing Nod:LacZ specifically in postmitotic photoreceptors. In animals expressing Glued^DN in postmitotic photoreceptors as well as in Glued¹ mutants, Nod:LacZ was no longer exclusively concentrated in apical regions of photoreceptors, but rather spread into the photoreceptor axons. Thus, while the overall apical/basal polarity of the photoreceptors was not disrupted in Glued mutants, the spatial organization of the microtubule cytoskeleton-associated factor Nod:LacZ was affected (Whited, 2004).

Dynactin activates the microtubule motor Dynein, and strong loss-of-function mutations in dynein intermediate chain (dic) are dominant enhancers of the rough eye phenotype of Glued¹ mutants. Since Dynein and Dynactin may play multiple roles together during eye development, the effect of a reduction in dic gene dosage upon photoreceptor nuclear positioning was examined in Glued¹ animals. A twofold reduction in dic gene dosage caused a further decrease in the number of photoreceptor nuclei in apical regions of Glued¹ mutant eye discs. This did not reflect a simple reduction in the number of photoreceptors generated; large numbers of photoreceptor nuclei were crowded at the base of the eye disc and entered the optic stalk in both animals. Thus, a larger fraction of photoreceptor nuclei left apical positions when the level of dic gene activity was reduced, consistent with Dynein and Dynactin acting together in this process (Whited, 2004).

To identify additional factors that interact with Dynactin to control nuclear positioning, a genetic screen was performed to identify genes that dominantly enhanced or suppressed the Glued¹ external eye phenotype. From a collection of approximately 1800 stocks containing transposon-induced lethal mutations, several stocks were identified that had no dominant effect on eye development in a wild-type background, but were dominant enhancers or suppressors of Glued¹. Two dominant suppressors of Glued¹, khc^k13219 and khc^k13314, were alleles of kinesin heavy chain (khc), which encodes a subunit of the plus-end directed microtubule motor kinesin. The interaction with Glued¹ was further confirmed using the null allele khc⁸. Examination of developing eye discs demonstrated that a twofold reduction of khc gene dosage greatly increased the number of photoreceptor nuclei present in apical regions of Glued¹ mutant eye discs. This suggested that khc acts antagonistically to Glued in photoreceptor nuclear positioning (Whited, 2004).

To determine whether khc mutations interacted with Glued¹ in postmitotic photoreceptors, khc gene dosage was reduced in animals expressing dominant-negative Glued under the control of the postmitotic Glass_38-1 promoter. Wild-type animals (n >50 hemispheres) or animals containing the dominant-negative Glued transgene without the Glass 38-1 promoter never contained photoreceptor nuclei within their optic stalks. Glass_38-1:Glued^DN animals contained an average of 11±1 photoreceptor nuclei within the optic stalk. However, Glass_38-1:Glued^DN animals heterozygous for either khc^k13314 or khc⁸ showed a significant reduction in the number of photoreceptor nuclei in the optic stalk. Thus, a twofold reduction in khc gene dosage suppressed the effects of postmitotic expression of dominant-negative Glued, consistent with Glued and khc acting antagonistically within differentiated photoreceptors to regulate nuclear positioning (Whited, 2004).

The interaction between Glued and khc in other photoreceptors was studied by examining the Bolwig organ, a cluster of 12 photosensitive neurons that differentiate during embryonic development and extend axons into the brain. By second and third instar larval stages, Bolwig photoreceptor nuclei are located near the anterior tip of the larva and their axons extend over the eye/antennal disc into the brain, a distance of >200 µm. In wild-type second instar animals, photoreceptor neuron differentiation has not yet begun in the eye disc and no neuronal nuclei are present there. However, when Glued^DN was expressed in postmitotic Bolwig photoreceptors, their nuclei appeared on the surface of the eye/antennal disc. Thus, as in the photoreceptors of the adult eye, expression of Glued^DN in Bolwig photoreceptors caused their nuclei to be positioned closer to their axon termini; in many cases, the Bolwig nuclei were over 150 µm closer than normal to their axon terminals in the brain (Whited, 2004).

The interaction between Glued and khc in Bolwig photoreceptors was assessed by counting the number of Bolwig nuclei on the surface of the eye/antennal disc. While wild-type and UAS:Glued^DN animals had no neuronal nuclei in this region, Glass_38-1:Glued^DN animals contained 7±1. A reduction of khc gene dosage in Glass_38-1:Glued^DN; khc^k13314/+ and Glass_38-1:Glued^DN; khc⁸/+ animals significantly reduced this to 4±1 and 3±1, respectively. These data further support the functional antagonism of Glued and khc in photoreceptor nuclear positioning (Whited, 2004).

The Ste20-like kinase Misshapen functions together with Bicaudal-D and dynein in driving nuclear migration in the developing Drosophila eye

Nuclear translocation, driven by the motility apparatus consisting of the cytoplasmic dynein motor and microtubules, is essential for cell migration during embryonic development. Bicaudal-D (Bic-D), an evolutionarily conserved dynein-interacting protein, is required for developmental control of nuclear migration in Drosophila. Nothing is known about the signaling events that coordinate the function of Bic-D and dynein during development. This study shows that Misshapen (Msn), the fly homolog of the vertebrate Nck-interacting kinase is a component of a novel signaling pathway that regulates photoreceptor (R-cell) nuclear migration in the developing Drosophila compound eye. Msn, like Bic-D, is required for the apical migration of differentiating R-cell precursor nuclei. msn displays strong genetic interaction with Bic-D. Biochemical studies demonstrate that Msn increases the phosphorylation of Bic-D, which appears to be necessary for the apical accumulation of both Bic-D and dynein in developing R-cell precursor cells. It is proposed that Msn functions together with Bic-D to regulate the apical localization of dynein in generating directed nuclear migration within differentiating R-cell precursor cells (Houalla, 2005).

The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila requires Dynein function

Cell polarity is critical for epithelial structure and function. Adherens junctions (AJs) often direct this polarity, but it has been found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila. This prompted an investigation into how Baz is positioned and how downstream polarity is elaborated. Surprisingly, it was found that Baz localizes to an apical domain below (basally to) its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In fact, Baz positioning is independent of aPKC and PAR-6 relying instead on cytoskeletal cues, including an apical scaffold and dynein-mediated basal-to-apical transport. AJ assembly is closely coupled to Baz positioning, whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical domain with Baz and AJs localizing basally to aPKC and PAR-6, and specific mechanisms were identified that keep these proteins apart. These results reveal key steps in the assembly of the apical domain in Drosophila (Harris, 2005).

These results frame a model of apical domain assembly during epithelial polarity establishment in Drosophila. During cellularization, Baz acts as a primary polarity landmark that positions AJs and aPKC. Baz, itself, is positioned by two cues (an apical scaffold and dynein-mediated transport). Baz recruits and colocalizes with AJ proteins in a subapical region while helping direct aPKC to the extreme apical region. During gastrulation, a third cue becomes important for Baz and AJ positioning. At this stage, aPKC becomes required for maintaining Baz and AJs. PAR-6 is also recruited to the extreme apical region and maintains Baz and AJs. Although Baz can interact with aPKC and PAR-6 at this stage, Crb blocks these interactions. It is proposed that this interaction network establishes a robust, stratified apical domain from the earliest stages of epithelial development (Harris, 2005).

AJs are often key polarity landmarks. However, Baz positioning is AJ independent at the time that epithelial polarity is first established in Drosophila. Here, Baz appears to act as a primary polarity landmark, but what cues position Baz? The data indicate that Baz is initially positioned by cytoskeletal cues that support an apical Baz-binding scaffold and mediate basal-to-apical Baz transport. The apical scaffold is saturable. Its function requires actin; Baz becomes basally mislocalized after actin disruption. However, since Baz overlaps only the basal reaches of the apical actin network, it is unlikely that Baz simply binds actin. Interestingly, Baz remains largely membrane associated when actin is disrupted. One caveat is that there is some residual actin. However, the same treatment dissociates APC2 from the cortex. Actin is also required for PAR-3 cortical association in C. elegans one-cell embryos. During Drosophila cellularization, it is speculated that Baz may have other cortical anchors and that actin may control their distribution -- of course, actin is critical for many cellular processes and could play other roles in positioning Baz. It will be important to identify the apical scaffold for Baz (Harris, 2005).

Baz positioning also requires the minus-end-directed MT motor dynein. Live imaging of BazGFP revealed basal-to-apical translocation of BazGFP puncta during cellularization. Baz-GFP that diffuses to ectopic basal positions appears to engage a preexisting, dynein-based, basal-to-apical transport system. Such a system transports Golgi vesicles apically during cellularization. Baz-dynein associations appear to cease once dynein brings Baz to the apical region, where Baz presumably docks with its apical scaffold. Although BazGFP puncta move slower than in vitro dynein velocity measurements, dynein-mediated lipid droplet movements have similar speeds during Drosophila cellularization. In vivo, BazGFP puncta may be slowed because they form large cortical complexes. Indeed, DE-Cad, aPKC, and PAR-6 associate with these puncta and Baz oligomerization may promote complex assembly. Further supporting a role for dynein, endogenous Baz is positioned near MT minus ends in WT embryos, but mislocalizes basally in dhc64C^m/z mutants. dhc64C mutations also enhance the baz mutant embryonic phenotype. This is the first report of dynein positioning Baz or its homologues (Harris, 2005).

Analysis of dynein mutants also revealed a third mechanism that can reposition Baz apically during gastrulation. Perhaps the apical Baz-binding scaffold is strengthened during this stage. Alternatively, a distinct polarizing mechanism may be activated, or aPKC and PAR-6 may be involved. Having three Baz positioning mechanisms may ensure proper Baz localization for regulating downstream polarity (Harris, 2005).

Baz acts upstream of AJs as epithelial polarity is first established in Drosophila. The following model is proposed in which AJ assembly may be coupled to Baz positioning. During cellularization, AJ proteins accumulate in both apical and basal junctions. Basal junctions form transiently near the base of each invaginating furrow. Baz is not required for basal junctions, but is required for recruiting AJ proteins into apical junctions. Apical Baz may provide a landmark for apical AJ assembly (Harris, 2005).

The data also suggest that Baz may be involved in ferrying DE-Cad to the apical domain via dynein-mediated transport. Dynein is required for correct apical positioning of both Baz and DE-Cad, and their colocalization in ectopic basal complexes in dhc64C^m/z mutants suggests they may normally be transported to the apical domain together. Indeed, Baz can form complexes with DE-Cad and Arm. Although most endogenous Baz is apical during WT cellularization, its basal mislocalization in dhc64C^m/z mutants suggests that some Baz may normally move basally. In fact, excess BazGFP displaced from the apical domain preferentially accumulates at basal junctions. It is hypothesized that some Baz may normally interact transiently with basal junctions. From there, it may help ferry AJ proteins apically via dynein-mediated transport. MT motors have been implicated in AJ assembly. For example, dynein interacts with ß-catenin and may tether MTs to AJs assembling between PtK2 cells. Kinesin transports AJ proteins to nascent AJs in cell culture, and the mitotic kinesin-like protein 1 is required for apical targeting of AJs and other cues in C. elegans epithelia. It will be important to see if these targeting mechanisms have commonalities with AJ positioning in Drosophila, and if Baz homologues are involved (Harris, 2005).

Finally, it is hypothesized that the third Baz-AJ positioning mechanism revealed in dhc64C^m/z mutants might be related to the normal maturation/stabilization of AJs at gastrulation. At this stage, precursory spot AJs fuse into continuous belt junctions around the top of each cell. In mammalian cell culture, aPKC is required for such AJ maturation. Similarly, aPKC is required for proper AJ and Baz positioning during Drosophila gastrulation, as has been shown for PAR-6. Considering aPKC and PAR-6 are positioned apically as dhc64C^m/z mutants gastrulate, they might recruit Baz and AJs apically in this context as well (Harris, 2005).

Based on their shared roles in polarity in C. elegans, characterized physical interactions, and colocalization in mammalian cells, Baz, aPKC, and PAR-6 are thought to function, at least in some cases, as an obligate tripartite complex. The data suggest that the bulk of cortical Baz and aPKC/PAR-6 do not form obligate complexes during epithelial development in Drosophila. Instead, aPKC and PAR-6 localize to an apical region above Baz and AJs, and are positioned there by distinct mechanisms. Baz/PAR-3 also segregates from aPKC and PAR-6 in other cell types. In C. elegans one-cell embryos, PAR-3, aPKC, and PAR-6 each localize in clusters on the anterior cortex, but these different clusters have limited colocalization (60%-85% fail to colocalize. aPKC and PAR-6 colocalize without PAR-3 at the leading edge of migrating mammalian astrocytes. In Drosophila photoreceptors, Baz colocalizes with AJs below aPKC, PAR-6, and Crb. Even in polarized MDCK cells, aPKC and PAR-6 show some segregation above PAR-3, and although they mainly colocalize at tight junctions, mammalian PAR-3 can regulate tight junction assembly independently of aPKC and PAR-6. Thus, in many contexts interactions between Baz/PAR-3, aPKC, and PAR-6 are dynamic and/or regulated (Harris, 2005).

Baz (PAR-3), aPKC, and PAR-6 often recruit each other to the cortex, but the assembly pathways vary. In C. elegans, one-cell embryos, PAR-3, aPKC, and PAR-6 are mutually dependent for their cortical recruitment. However, in Drosophila neuroblasts, Baz can be positioned without aPKC and PAR-6. Similarly, apical Baz is positioned without aPKC and PAR-6 during Drosophila cellularization. In contrast, apical aPKC recruitment requires Baz, whereas PAR-6 is largely nonpolarized at this stage. Given the lack of extensive colocalization of Baz and aPKC in WT embryos, Baz may control aPKC positioning indirectly, perhaps regulating binding to a separate apical scaffold. Alternately, cortical recruitment might involve cytoplasmic Baz-aPKC complexes. Apical PAR-6 accumulates at gastrulation, and this appears partially Baz independent. Indeed, cdc42 recruits PAR-6 at this stage, and at the same time aPKC and PAR-6 become required for maintaining apical Baz. Thus, although Baz is first positioned independently of aPKC and PAR-6, these cues soon develop complex interdependencies (Harris, 2005).

Although Baz can directly bind both aPKC and PAR-6, at least two mechanisms keep them apart. During cellularization, Baz colocalizes with aPKC and PAR-6 when overexpressed, but normally it localizes with AJs below aPKC and PAR-6. This normal segregation may thus involve competition with other binding partners. After cellularization, Crb also becomes important for segregating Baz and AJs from aPKC and PAR-6. These segregation mechanisms help form a stratified apical domain from the earliest stages of epithelial development (Harris, 2005).

A stratified apical domain may strengthen the boundary between the apical and basolateral domains. This boundary forms via reciprocal antagonism between polarity cues. For example, aPKC phosphorylates and excludes Lethal giant larvae (Lgl) from the apical domain in Drosophila epithelia and Lgl appears to repel PAR-6 from the basolateral domain. The Crb and Dlg complexes also have mutual antagonism. It is proposed that the subapical Baz-AJ region may insulate the apical and basolateral domains. For example, it may inhibit active aPKC from moving basally. Indeed, PAR-3 binding can block mammalian aPKC kinase activity. The Baz-AJ subapical region could also block basolateral cues, since AJs are required to segregate Dlg. In this way, the Baz-AJ subapical region could help define a distinct apical-basolateral boundary (Harris, 2005).

To conclude, Baz appears to be a primary epithelial polarity landmark in Drosophila. It is positioned by multiple mechanisms, including an apical scaffold and dynein-mediated transport, and organizes a stratified apical domain, in which it colocalizes with AJs below its typical partners aPKC and PAR-6 (Harris, 2005).

aPKC controls microtubule organization to balance adherens junction symmetry and planar polarity during development

Tissue morphogenesis requires assembling and disassembling individual cell-cell contacts without losing epithelial integrity. This requires dynamic control of adherens junction (AJ) positioning around the apical domain, but the mechanisms involved are unclear. Atypical Protein Kinase C (aPKC) is required for symmetric AJ positioning during Drosophila embryogenesis. aPKC is dispensable for initial apical AJ recruitment, but without aPKC, AJs form atypical planar-polarized puncta at gastrulation. Preceding this, microtubules fail to dissociate from centrosomes, and at gastrulation abnormally persistent centrosomal microtubule asters cluster AJs into the puncta. Dynein enrichment at the puncta suggests it may draw AJs and microtubules together and microtubule disruption disperses the puncta. Through cytoskeletal disruption in wild-type embryos, a balance of microtubule and actin interactions was found to control AJ symmetry versus planar polarity during normal gastrulation. aPKC apparently regulates this balance. Without aPKC, abnormally strong microtubule interactions break AJ symmetry and epithelial structure is lost (Harris, 2007).

This study reveals the roles of aPKC during polarity establishment and elaboration in Drosophila embryos. In contrast to C. elegans, aPKC is not critical during initial polarity establishment; Baz and AJs are initially localized correctly and the embryonic epithelium can undergo initial morphogenesis. However, aPKC plays an early and striking role in maintaining the symmetrical organization of AJs, via effects on MT organization, and also plays an important later role in the elaboration of polarity (Harris, 2007).

aPKC's later role in polarity elaboration may reflect effects on multiple targets. aPKC is critical for the cortical localization of its normal binding partner PAR-6 and the apical determinant Crb. This latter effect is consistent with the fact that aPKC can phosphorylate Crb, and disruption of aPKC phosphorylation sites in Crb destabilizes Crb in the apical domain. Since Crb stabilizes AJs after gastrulation, this likely contributes to the eventual AJ breakdown in apkc^m/z mutants. Crb may act in concert with PAR-6 or in parallel. aPKC can also phosphorylate and exclude the basolateral cue Lgl from the apical domain, and consequenct failure to exclude Dlg from the apical domain in apkc^m/z mutants. Thus, apical invasion of basolateral cues may also contribute to the eventual loss of epithelial polarity in apkc^m/z mutants (Harris, 2007).

However, it is unlikely that these global changes in apical-basal cell polarity are responsible for the early clustering of AJs into planar-polarized puncta in apkc^m/z mutants. Indeed, most of these other polarity players affect polarity after gastrulation. crb mutants have normal spot junctions during gastrulation and early germband extension. Lgl and Dlg are not normally excluded from the apical domain until after gastrulation. Similarly, while mammalian aPKC can restrict PAR-1 to the basolateral domain of epithelial cells, Drosophila PAR-1 is not normally excluded from the apical domain at gastrulation. Thus, effects on Crb, Lgl/Dlg, and PAR-1 cannot easily account for the focusing of AJs and Baz into discrete planar-polarized puncta as apkc^m/z mutants gastrulate (Harris, 2007).

Instead, the data suggest that aPKC regulates AJ symmetry by regulating MTs. MT regulation may be a common aPKC function. For example, Drosophila aPKC promotes MT stability at synaptic boutons of neuromuscular junctions, where aPKC forms a complex with Futsch (a MAP1B-like protein) and tubulin, recruiting Futsch to boutons to stabilize MTs. aPKC also regulates MT orientation as mammalian astrocytes and fibroblasts undergo directed migration during wound healing, while in MDCK cells, aPKC helps organize the MT cytoskeleton during ciliogenesis (Harris, 2007).

MT organization and reorganization play important roles in epithelial morphogenesis, and the data demonstrate that loss of aPKC disrupts these events. During Drosophila cellularization, strong MT nucleation from apical centrosomes is likely necessary for assembling lateral MTs that support the apical transport of lipids and proteins to form cell membranes. These MTs also help direct the initial apical positioning of AJs and Baz. During later development, the analysis of apkc^m/z mutants indicates that centrosomal MTs can affect the symmetric positioning of AJs around the apical domain. Without aPKC activity, the centrosomes become abnormally dominant, bipolar cues, directing AJ clustering and thus disrupting AJ symmetry. Although this abnormal MT organization differs from changes to MT organization observed in AJ mutants, the possibility cannot be ruled out that there is feedback between MTs and AJs during epithelial morphogenesis and that aPKC may regulate these interactions. Indeed, such feedback is very likely and it will be critical to define MT-AJ cross talk mechanisms in future studies (Harris, 2007).

In apkc^m/z mutants, MT-associated AJ/Baz puncta assemble at the dorsal and ventral sides of the cells. This suggests that MTs may normally function in AJ assembly at these newly formed cell-cell contacts. However, these polarized AJ assembly events must also be counterbalanced to maintain AJ symmetry and proper epithelial structure. Cytoskeletal inhibitor studies suggest that AJ symmetry may normally be regulated by a balance of MT-AJ and actin-AJ interactions at this stage -- actin appears to counteract MT-based AJ assembly at dorsal and ventral cell contacts. Actin as been shown to be enriched at anterior and posterior cell contacts, suggesting that it may be an early planar polarity cue at this stage. Perhaps this planar-polarized actin stabilizes a pool of AJs at anterior and posterior cell contacts, thereby counterbalancing MT-based AJ assembly at dorsal and ventral contacts. Alternatively, lower levels of actin at dorsal and ventral cell contacts could directly counteract MT-based AJ assembly at these sites. Distinguishing these possibilities requires further study. Nonetheless, the apkc^m/z mutant phenotype appears to arise from a gain-of-function effect in which MTs become overactive and the proper balance between MT-AJ and actin-AJ interactions is lost. As a result, there is a break in AJ symmetry in apkc^m/z mutants, MT-associated AJ puncta eventually become randomly positioned, and the epithelium dissociates (Harris, 2007).

The data suggest a speculative mechanistic model by which aPKC could normally regulate MT-AJ interactions. This study shows that MT association is responsible for the abnormal AJ asymmetry seen in apkc^m/z mutants, and that Dynein accumulates at these abnormal AJ/Baz puncta. Since Dynein plays a role in apical transport of AJ/Baz proteins during cellularization, it is proposed that aPKC may normally regulate release of AJ/Baz complexes from Dynein, allowing a complete transport cycle. In the absence of this release, AJ/Baz complexes could maintain an abnormal association with MTs, and localized Dynein activity may pull the centrosomes and spot junctions together into the abnormal puncta seen in apkc^m/z mutants. This abnormal cortical Dynein activity might also stabilize MTs emanating from the centrosomes, resulting in the persistent centrosomal MTs in the mutants. Alternatively, aPKC may function at the centrosomes to decrease MT nucleation or increase MT severing. Future experiments will illuminate these mechanisms and the generality of aPKC's role in controlling MT organization and AJ positioning (Harris, 2007).

0ocyte

Microtubules and the Kinesin heavy chain (the force-generating component of the plus end-directed microtubule motor Kinesin I) are required for the localization of oskar mRNA to the posterior pole of the Drosophila oocyte, an essential step in the determination of the anteroposterior axis. The Kinesin heavy chain is also required for the posterior localization of Dynein, and for all cytoplasmic movements within the oocyte. Furthermore, the KHC localizes transiently to the posterior pole in an oskar mRNA-independent manner. Surprisingly, cytoplasmic streaming still occurs in kinesin light chain null mutants, and both oskar mRNA and Dynein localize to the posterior pole. Thus, the Kinesin heavy chain can function independently of the light chain in the oocyte, indicating that it associates with its cargoes by a novel mechanism (Palacios, 2002).

It is unclear why dynein localizes to the posterior, but one possibility is that it is needed to recycle kinesin to the minus ends of the microtubules, so that kinesin can mediate another round of posterior localization. The only known phenotype of the Dhc64C mutants that specifically disrupt the posterior localization of DHC is a reduction in the rate of cytoplasmic streaming, and this may due to the gradual depletion of the pool of KHC available for transport. However, this localization may be important for recycling dynein away from the minus ends of microtubules, so that dynein can mediate further rounds of minus end-directed transport (Palacios, 2002).

Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation

In the Drosophila oocyte, microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. mRNA localization and nuclear positioning at mid-oogenesis is shown to depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. It is concluded that the two microtubule motors, cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole (Januschke, 2002).

The localization of bcd mRNA in the oocyte occurs in multiple steps. Several of these involve active transport along microtubules. bcd mRNA coassembles into particles with Exuperantia (Exu) in the nurse cells and in the oocyte. This complex is essential for the correct localization of bcd to the anterior cortex in a microtubule-dependent manner. During mid-oogenesis, bcd maintenance at the anterior cortex is dependent on Swallow (Swa). This protein harbors a putative double-strand-RNA binding motif and a coiled-coil domain, which interacts with the Dynein light chain (Dlc-1). Swa has been proposed to act as an adaptor between bcd mRNA and the Dynein motor. Swa itself localizes to the anterior cortex of stage-10 oocytes, and this localization requires the coiled-coil domain, suggesting that polar transport of Swa and its cargo, bcd mRNA, occurs in a Dynein-dependent manner. The observation that bcd mRNA is delocalized in oocytes overexpressing p50 Dynamitin (Dmn), a component of the Dynactin complex provides further support for this suggestion (Januschke, 2002).

Surprisingly, in khc mutant oocytes, bcd mRNA is not tightly concentrated to the anterior cortex but is diffusely spread out in a wide cortical ring that expands toward the posterior. Thus, correct bcd localization depends not only on minus end-directed, but also on plus end-directed, motors. Kinesin I might be directly involved in anchoring of bcd mRNA to the anterior cortex. Alternatively, Kinesin I might be required for efficient Dynein-dependent transport of bcd. The observation that Dynein is mislocalized in khc mutant oocytes supports the latter hypothesis. Dhc fails to accumulate at the posterior pole of khc mutant oocytes and instead is enriched at the anterior cortex. Thus, Kinesin I appears to be necessary to relocate Dynein to the posterior pole after it has moved together with its cargo to the anterior pole. This would allow for renewed rounds of cargo loading and transport to the anterior cortex. Without sustained posterior-to-anterior transport, the bcd mRNA/adaptor complexes might become delocalized by diffusion. This scenario indicates that sustained transport could be an alternative to an independent anchorage step (Januschke, 2002).

In contrast to what was detected with bcd, no dual motor requirement has been detected for osk localization to the posterior, which is Kinesin I dependent. osk mRNA is clearly localized and translated when Dynein function is impaired. However, several features of the phenotypes produced by Dynamitin overexpression suggest that Dynein function is not completely abolished. Thus, it cannot be strictly ruled out that low levels of Dynein are required for efficient osk transport to the posterior pole. Indeed, the mechanism of osk localization is more complex than that of bcd localization. osk localization occurs through a series of distinct steps first to the anterior, then to the middle, and finally to the posterior pole of the oocyte (Januschke, 2002).

The correct positioning of the oocyte nucleus requires two different anchoring processes: one to the lateral cortex and a second to the anterior cortex. The former might be a prerequisite for nuclear movement from the posterior to the anterior pole. The latter occurs after completion of the movement. While Kinesin I appears to be dispensable for nuclear movement, the role of Dynein remains to be clarified. The Dynein-Dynactin complex is essential for nuclear migration in many cell types, from yeast to vertebrates. Overexpression of Dmn, though, seems not to interfere with correct positioning of the nucleus at early stages. Since nuclear migration is targeted toward MT minus ends, like bcd mRNA localization, it is assumed that it requires Dynein and it is suggested that, in the Dmn overexpression experiment, residual Dynein function is present at early stages, which allows nuclear migration (Januschke, 2002).

While the question of migration needs further analysis, both motors are clearly required for nuclear anchorage. Impairment of Dynein leads to nuclear detachment from both the anterior and lateral cortex. Kinesin I, however, is only required for maintaining the nucleus at the anterior cortex. The fact that both anchoring processes fail when Dmn is overexpressed indicates that Dynein fulfils a complex function in nuclear positioning. Two Dynein-Dynactin pools are present in the oocyte: the posterior pool, which is microtubule dependent and maintained by Kinesin I-dependent transport, and the perinuclear pool, whose maintenance is independent of MTs and MT motor activity. The perinuclear Dynein-Dynactin pool appears to be involved in organizing a MT cage around the nucleus, and this cage is likely to be necessary for the attachment of the nucleus to the lateral cortex. The mislocalization of the nucleus in Kinesin mutants might be explained in a similar way as the mislocalization of bcd mRNA. Cortical Dynein activity might be required during some stage after the nucleus has reached the anterior pole. During this period what appears to be anchorage would be the result of sustained minus end-directed movement. To maintain this movement, Dynein is supplied from the posterior pool, which constantly has to be replenished by Kinesin-dependent transport (Januschke, 2002).

The nuclear MT scaffold might not only be important for nuclear migration and nuclear anchoring. It harbors centrosomal components such as Centrin, which probably contribute to the formation of the MT scaffold but might also influence the MT network of the entire oocyte. Due to these properties, the nucleus is likely to have a central role in polarizing the Drosophila oocyte. During migration, it might contribute to the overall anterior-posterior repolarization of the oocyte MT network, which is required to establish the anterior and posterior cortical domains. After migration, MTs emanating from the asymmetrically positioned nucleus are likely to polarize the transport of grk mRNA and Grk protein, which establishes the anterodorsal cortical domain (Januschke, 2002).

grk mRNA is produced by both the nurse cells and the oocyte nucleus. After nuclear migration, grk mRNA accumulates briefly along the anterior margin of the oocyte, before it concentrates in a perinuclear position. The anterior localization of grk is not affected when Dynein function is reduced or if Kinesin I function is completely abolished. However, both motors are required to transport grk to the nucleus. It is suggested that grk mRNA is transported toward the minus ends of MTs, which emanate from the nucleus. This would explain the Dynein requirement for grk transport to the nucleus. The role of Kinesin I in anterodorsal grk transport might again reflect the need to retrieve the Dynein motors for renewed cargo loading, as suggested for bcd and the oocyte nucleus (Januschke, 2002).

This model has to assume, however, that Dynein-Dynactin complexes carrying different cargos can distinguish between distinct populations of MTs: Dynein-Dynactin complexes loaded with bcd mRNA should be transported to and remain at anterior cortex, while those loaded with grk mRNA should be subject to a second transport step toward the nucleus. Deletions within the grk 3'UTR allow anterior localization of grk mRNA but prevent its transport to the nucleus. This suggests that specific factors distinguish anterior and anterodorsal transport of grk. The heterogeneous nuclear RNA binding protein (hnRNP) Squid plays a central role in this process. It regulates both grk localization and translation and binds directly to the grk 3'UTR. Squid protein, like grk, appears to be transiently localized along the anterior cortex during the transition from stage 7 to stage 8 (Januschke, 2002).

grk mRNA, though mislocalized, is frequently translated when Kinesin I or Dynein motor activities are impaired. Since grk mRNA is found around the anterior cortex in those cases, Grk secretion should occur around the entire circumference of the oocyte instead of being restricted to the dorsal side. Secreted Grk induces dorsal follicle cell fates. Thus, ectopic secretion should lead to the formation of dorsalized eggs as in squid and fs(1)K10 mutants in which grk mRNA is also mislocalized. However, impaired MT motor activity leads to ventralized eggs and thus to reduced Grk signaling. An analysis of Grk distribution in oocytes shows that, in contrast to wild-type or squid and fs(1)K10, Grk protein is not closely associated with grk mRNA and fails to reach the plasma membrane. Thus, polar transport of Grk protein and exocytosis requires Dynein and Kinesin I activity. This is not surprising, since both motors have been shown to be involved in Golgi dynamics in higher eukaryotes and it has been shown that vesicular trafficking from the Golgi to the plasma membrane requires Kinesin activity (Januschke, 2002).

Interestingly, no requirement has been detected for the two motors in earlier Grk signaling, which induces posterior follicle cells and prevents the formation of a second micropyle at the posterior pole. In the case of Dynamitin overexpression, this might be due once more to residual levels of Dynein function. In the case of Kinesin I, it is assumed that Grk secretion is only impaired, but not entirely blocked. The phenotypic series of grk mutations suggests that minute amounts of secreted Grk are sufficient to induce posterior follicle cells (Januschke, 2002).

Opposite polarity motors can interact with the same cargo in two fundamentally different ways. They can function in an opposition mode, like Myosin V and Kinesin II in the migration of Xenopus melanophores, or in a coordination mode, like Dynein and Kinesin in the motion of lipid droplets in the Drosophila embryo. In the opposition mode, the two motors produce opposing forces on a single cargo. Inactivation of the minus end-directed motor leads to a delocalization of the cargo to the plus end, whereas inactivation of the plus end-directed motor leads to a delocalization of the cargo to the minus end. In the coordination mode, the motors are not competing with each other. For example, when plus end motors are active, minus end motors are turned off, and vice versa. Inactivation of either of the two motors leads to the delocalization of the cargo to the same side. According to this scheme, Dynein and Kinesin I act in a coordination mode during transport of bcd and grk mRNAs, and the same might be true for their role in the positioning of the oocyte nucleus. The observation that Dynein accumulation at the posterior pole depends on Kinesin I suggests that both motors are associated with the same vesicle or macromolecular complex, as has been proposed for axonal transport in Drosophila. Dynein has to be inactive during Kinesin I-dependent transport to the posterior pole but then has to be activated again for renewed cargo loading and transport to the anterior cortex. If this recycling model holds true for the described polar transport processes in the oocyte, it will be challenging to find those factors that regulate motor activity and cargo loading in successive transport cycles (Januschke, 2002).

The cytoplasmic Dynein and Kinesin motors have interdependent roles in patterning the Drosophila oocyte

Despite their opposing polarity, Dynein and kinesin motors may cooperate in vivo. In Drosophila circumstantial evidence suggests that dynein acts in the localization of determinants and signaling factors during oogenesis. However, the pleiotropic requirement for dynein throughout development has made it difficult to establish its specific role. Dynein function in the oocyte has been examined by disrupting motor activity through temporally restricted expression of the dynactin subunit, dynamitin. The results indicate that dynein is required for several processes that impact patterning; such processes include localization of bicoid (bcd) and gurken (grk) mRNAs and anchoring of the oocyte nucleus to the cell cortex. Surprisingly, dynein function is sensitive to reduction in kinesin levels, and germ line clones lacking kinesin show defects in dorsal follicle cell fate, grk mRNA localization, and nuclear attachment that are similar to those resulting from the loss of dynein. Significantly, dynein and dynactin localization is perturbed in these animals. Conversely, kinesin localization also depends on dynein activity. It is concluded that dynein is required for nuclear anchoring and localization of cellular determinants during oogenesis. Strikingly, mutations in the kinesin motor also disrupt these processes and perturb dynein and dynactin localization. These results indicate that the activity of the two motors is interdependent and suggest a model in which kinesin affects patterning indirectly through its role in the localization and recycling of dynein (Duncan, 2002).

In order to investigate the effects of targeted disruption of dynein activity, heat shock-inducible (hsDmn) and Gal4-responsive (UAS-Dmn) transgenic lines were created. The hsDmn transgene permits tight temporal control of misexpression, whereas UAS-Dmn allows spatially restricted transcription when coupled with the appropriate Gal4 drivers. After mapping the insert position, hsDmn flies were examined for the ability to induce Dmn expression by probing immunoblots with polyclonal antisera against the Drosophila protein. A single band migrating at 45 kDa, close to the predicted size of the endogenous protein, was detected in untreated control flies. This band was present at approximately 5- to 10-fold higher levels in animals that had been heat shocked for 60 min. A time course of induction shows that elevated Dmn levels are present 15 min after heat shock and persist for at least 6 hr. To test whether Dmn overexpression perturbs the stability or localization (or both) of the dynein/dynactin complex in vivo, egg chambers were stained with antisera against Dmn, Gl (the largest subunit of dynactin), and the dynein intermediate chain (Cdic). In the wild-type, Dmn preferentially accumulates in the oocyte during early oogenesis and shows both perinuclear and cortical staining through stage 8. By stage 9 Dmn is enriched in a crescent at the posterior cortex as well as in lateral regions. Overall, this distribution mimics that of Gl and the dynein intermediate and heavy chains. Within 60 min of hsDmn induction, high levels of Dmn were detected throughout the oocyte, nurse cells and follicle cells. In contrast, Gl and Cdic staining was undetectable in stage 9/10 oocytes and was strongly reduced at earlier stages, demonstrating that Dmn overexpression disrupts the localization of the dynein/dynactin complex (Duncan, 2002).

One explanation for the strong genetic interaction observed between kinesin and Dmn overexpression could be that kinesin is required to transport dynein toward microtubule plus ends. This would allow individual dynein complexes to be reused for multiple rounds of minus end-directed motion. A reduction in kinesin levels may compromise this recycling and decrease the pool of available dynein; it would thus affect dynein's ability to translocate cargo toward microtubule minus ends. This model provides a mechanistic basis for why processes that involve dynein, such as nuclear attachment and grk RNA localization, could be severely impacted in oocytes lacking Khc. It is also consistent with the observation that in Khc mutant egg chambers the dynein/dynactin complex is not localized to the lateral cortex of the oocyte after stage 8. Significantly, localization of grk transcript and protein are relatively unaffected prior to stage 8, when defects in dynein/dynactin localization first become apparent (Duncan, 2002).

In this context, it is interesting that the distribution of Khc in the oocyte resembles that of dynein and dynactin components; i.e., it is enriched at the cortex and the perinuclear region, where microtubule minus ends are expected to be most abundant. Such a pattern is consistent with a role for kinesin in recycling dynein from the cortex, similar to its proposed function in transporting osk mRNA, but raises the paradoxical question of how kinesin localization is established. After hsDmn induction cortical staining for Khc is reduced, suggesting that Khc localization is in turn dependent on dynein activity. Transport of kinesin to the cortex could occur as a result of a direct physical interaction between the two motors. Alternatively, kinesin and dynein could bind common cargoes or adaptor proteins; this would be analogous to the situation in the embryo, where dynein and a so-far-unidentified plus-end motor both associate with individual lipid droplets. Transport of the particles and the associated motors could occur in either direction if the activity of the opposite polarity motors is appropriately regulated. Interaction with a common intermediate anchored to the posterior cortex could also explain why kinesin, dynein, and dynactin colocalize in this region. The recent finding that dynein-associated structures move rapidly along microtubules in both directions in Dictyostelium suggests that motor recycling may be a common mechanism for enhancing optimal utilization of a limited pool of these mechanochemical enzymes (Duncan, 2002).

The results indicate a role for dynein in grk transcript localization. The fact that grk mRNA cannot be detected in late-stage oocytes 1-6 hr after Dmn induction argues that dynein could be required for both the transport and anchoring of grk message. When microtubules are depolymerized, grk mRNA forms aggregates on the oocyte nuclear lamina, suggesting that this represents a site where it is anchored. It might therefore be expected that if dynein functions exclusively in transport, inhibition of its activity would cause an increase in the perinuclear concentration of grk mRNA. Furthermore, transcripts that were already at the cortex should not have been disrupted. In oocytes assayed 1 or 6 hr after Dmn induction, grk message was absent from the nuclear periphery and the cortex, irrespective of where the nucleus was positioned. However, 12 hr after Dmn induction, grk mRNA localization to the nucleus had partially recovered. Interestingly, when the oocyte nucleus was incorrectly positioned along the A/P axis but remained cortically attached, grk transcript was also detected at the cortex. This argues that nuclear position and proximity to the cortex are primary determinants of grk localization. It is notable that grk message is insensitive to Dmn overexpresssion and the absence of kinesin in earlier-stage egg chambers, when it may be transported by a diffusion-based mechanism and is known to accumulate even in the absence of microtubules or microfilaments. Localization of bcd message at the anterior of the oocyte is also highly susceptible to Dmn misexpression. Although the results cannot distinguish between inhibition of transport or anchoring of the mRNA, other data argue that dynein is likely to be involved in both of these aspects. Resolution of this issue may require direct observation of RNA localization in live egg chambers after hsDmn induction (Duncan, 2002).

In contrast to the dramatic effect of Dmn overexpression on grk and bcd transcripts, osk mRNA distribution is altered in a more subtle fashion. The increased level of osk mRNA in the cytoplasm after Dmn overexpression is consistent with the proposal that osk transcript binds to cortex throughout the oocyte and that kinesin transports it toward the interior in the anterior and lateral regions. Accordingly, dynein may contribute to osk localization by transporting transcripts toward the cortex or maintaining them there (Duncan, 2002).

The requirement for dynein activity in positioning the oocyte nucleus at the anterior cortex could reflect a role in nuclear anchoring alone or in both nuclear migration and anchoring. Misplaced nuclei are found in stage 10 egg chambers dissected 1 hr after heat shock even though nuclear migration would have occurred 13-25 hr earlier (at stage 7/8). This clearly shows that reduction of dynein activity disrupts nuclear anchoring through a mechanism that is still unclear. One possibility is that sustained activity of perinuclear dynein (acting on microtubules oriented with minus ends toward the cortex) is required to maintain nuclear position. Alternatively, cortically localized dynein may have to be continually active to keep the nucleus 'reeled in' through a subset of microtubules that have the opposite orientation. In either case, the nucleus would be predicted to fall away from the cortex in the absence of dynein activity. With respect to nuclear migration, there is considerable evidence that dynein motors power such a process in fungi. The data do not permit a firm conclusion as to whether dynein also performs this role in the oocyte. Although severely ventralized eggs were obtained 40 hr after hsDmn expression, suggesting a failure of nuclear migration, this could also result from defects in anchoring after migration because of perdurance of excess Dmn (Duncan, 2002).

Compared to oocytes in which dynein activity has been disrupted, those lacking kinesin show a higher frequency of nuclear-positioning defects. One explanation could be that kinesin function is completely abolished in Khc null clones, whereas residual dynein activity remains after hsDmn induction. Alternatively, it is conceivable that kinesin is the primary motor involved in nuclear positioning and that dynein plays an accessory role. In either event, the similarity in nuclear localization defects is consistent with a model in which the function of the two motors is linked. Dynein- and kinesin-related motors also act cooperatively to bring about nuclear migration in S. cerevisiae. Deletion of either of the kinesin-related proteins Kip2p and Kip3p or the dynein heavy chain results in nuclear migration defects. Epistatic analysis suggests that Kip2p acts cooperatively with dynein, whereas Kip3p may affect nuclear migration through an independent pathway involving Kar9p. Similarly, in Aspergillus, where nuclear migration is primarily thought to be dynein mediated, it has recently been shown that kinesin mutations affect nuclear movement and distribution in the hyphae. It is concluded that both dynein and kinesin are required for nuclear anchoring and localization of cellular determinants during oogenesis. The subcellular localization of dynein and dynactin is perturbed in kinesin mutants, and kinesin distribution is affected by Dmn misexpression. The interdependence of the two motors suggests a model in which kinesin affects patterning by localizing and recycling dynein and thus maximizing its utilization (Duncan, 2002 and references therein).

Effects of Mutation or Deletion

The microtubule motor cytoplasmic dynein has been implicated in a variety of intracellular transport processes. The previously identified and characterized Drosophila gene Dhc64C encodes a cytoplasmic dynein heavy chain. To investigate the function of the cytoplasmic dynein motor, a mutational analysis of the Dhc64C dynein gene was initiated. A small deletion that removes the chromosomal region containing the heavy chain gene was used to isolate EMS-induced lethal mutations that define at least eight essential genes in the region. Germline transformation with a Dhc64C transgene rescues 16 mutant alleles in the single complementation group that identifies the dynein heavy chain gene. All 16 alleles are hemizygous lethal, which demonstrates that the cytoplasmic dynein heavy chain gene Dhc64C is essential for Drosophila development. Furthermore, failure to recover somatic clones of cells homozygous for a Dhc64C mutation indicates that cytoplasmic dynein function is required for cell viability in several Drosophila tissues. The intragenic complementation of dynein alleles reveals multiple mutant phenotypes including male and/or female sterility, bristle defects, and defects in eye development (Gepner, 1996).

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. Biochemical, cytological, and genetic evidence is presented that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. Similar to the vertebrate homologs in dynactin, the Glued polypeptides are components of a 20S complex. Biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In an analysis of the Glued polypeptides encoded by the dominant mutation, Glued, a truncated polypeptide was identified that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act are shown to act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes (McGrail, 1995).

During animal development, cellular differentiation is often preceded by an asymmetric cell division whose polarity is determined by the orientation of the mitotic spindle. In Drosophila the oocyte differentiates in a 16-cell syncytium that arises from a cystoblast that undergoes 4 synchronous divisions with incomplete cytokinesis. During these divisions, spindle orientation is highly ordered and is thought to impart a polarity to the cyst that is necessary for the subsequent differentiation of the oocyte. Using mutations in the Drosophila cytoplasmic dynein heavy chain gene Dhc64C, it has been shown that cytoplasmic dynein is required at two stages of oogenesis. Early in oogenesis, dynein mutations disrupt spindle orientation in dividing cysts and block oocyte determination. The localization of dynein in mitotic cysts suggests spindle orientation is mediated by the microtubule motor cytoplasmic dynein. Later in oogenesis, dynein function is necessary for proper differentiation, but does not appear to participate in morphogen localization within the oocyte. These results provide evidence for a novel developmental role for the cytoplasmic dynein motor in cellular determination and differentiation (McGrail, 1997).

Cytoplasmic dynein is a multisubunit minus-end-directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. Recessive lethal mutations in the dynein heavy chain gene, Dhc64C, were used to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immunofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo (Robinson, 1999).

Recessive lethal alleles of the dynein heavy chain gene, Dhc64C, were identified that exhibit intragenic complementation and reveal mitotic phenotypes during the rapid divisions of the syncytial embryo. The nonlethal Dhc64C^6-8/Dhc64C^6-6 combination of mutations results in fully viable transheterozygous adult females that produce eggs that are endowed with strictly mutant dynein heavy chain. The rapid rounds of nuclear divisions that follow fertilization of these mutant embryos are compromised by the defective dynein and result in maternal effect lethality with greater than 94% of the embryos dying. The nature of the lesions within the dynein heavy chain mutations, Dhc64C^6-8 and Dhc64C^6-6, are not known. However, the presence of a wild-type dynein heavy chain transgene rescues the mitotic defects, as well as maternal effect lethality, demonstrating that the phenotype is specific to a loss in dynein function. Importantly, the mitotic defects that were uncovered are not unique to the syncytial nature of early nuclear divisions. Similar defects occur in the larval neuroblasts of late-lethal alleles of the dynein heavy chain (Robinson, 1999).

Within the mutant syncytium, nuclear cycles proceed and repeatedly show defects in specific centrosome behaviors and spindle morphogenesis at each nuclear cycle. This progression of the nuclear cycles and the repetitive occurrence of centrosome misbehavior and aberrant multipolar spindle formation is consistent with the defects being a primary consequence of dynein dysfunction. In this regard, it is suggested that the combination of hypomorphic heavy chain alleles provides a means to specifically attenuate dynein function in order to investigate its mitotic function in early syncytial embryos. Strong loss of function alleles or null mutations in the dynein heavy chain are cell lethal and prohibit such analysis. This result contrasts with findings in other genetically tractable systems, such as yeast, in which it has been shown that dynein is not an essential gene. The results demonstrate that the mitotic function(s) of cytoplasmic dynein are essential in Drosophila (Robinson, 1999).

Analysis of centrosome behavior in vivo within the dynein mutant embryos occasionally reveals centrosomes detaching from the nuclear envelope. In time-lapse movies some centrosomes leave the envelope never to return, while other centrosomes detach briefly and then moved back to the nucleus and reattach. These events are never seen in wild-type embryos and provide evidence for a novel function for dynein in maintaining the association of centrosomes with the nuclear envelope. The reattachment of centrosomes, as well as the low penetrance of the detached centrosome phenotype, is consistent with the prediction that the hypomorphic dynein gene products are only partially compromised for nuclear attachment. One interpretation of these phenotypes is that dynein is associated with the nuclear envelope, where it acts as a minus-end motor to draw in centrosomal microtubules and secure the centrosomes to the nuclear membrane. Alternatively, or in addition, dynein may reside in the centrosome and act to stabilize the attachment of nucleated microtubules that are themselves required for nuclear attachment. In this case, loss of dynein function may increase the frequency of microtubule release from centrosomes and thus weaken nuclear attachment. Evidence for active dynein complex associated with the nuclear envelope has been reported in vitro in Xenopus extracts. In Drosophila, cytoplasmic dynein is localized to the oocyte nucleus, where it might power nuclear migration. However, in embryos, dynein is present on the mitotic spindle and appears concentrated at spindle poles, but no accumulation on nuclear envelopes has yet been detected. In mammalian cell lines, dynein is localized to kinetochores, centrosomes, and at the nuclear periphery (Robinson, 1999 and references).

Most centrosomes observed in mutant embryos retained their nuclear attachments, but exhibit defects in migration along the nuclear membrane during prophase. Time-lapse analysis in living embryos demonstrates that dynein is required for the initial separation of centrosomes along the nuclear envelope and is distinct from the function of antagonistic motors that maintain the separation of centrosomes once initial separation is complete. There is an observed failure of the duplicated centrosomes to fully migrate along the nuclear envelope to a position 180° apart from one another before nuclear envelope breakdown occurs. The centrosome migration defect is consistent with results from antibody knockout experiments performed in a vertebrate cell culture system. The predominant defect in centrosome migration can be viewed as an intermediate phenotype, the consequence of only partial loss of dynein function. The 'detached-centrosome' phenotype might occur when the same dynein-based mechanism is further compromised. However, whether dynein function in centrosome attachment and centrosome migration are mechanistically related remains to be determined. Indeed, recent studies show that centrosome migration in Xenopus extracts depends upon the activity of the plus-end directed kinesin-like protein Xklp2. Xklp2 is a member of the BimC class of conserved kinesin-like proteins which likely play similar roles in several different eukaryotes. Furthermore, the localization of a COOH-terminal fragment of Xklp2 to the minus-ends of spindle and astral microtubules requires the activity of cytoplasmic dynein. Mutations in dynein may affect centrosome separation by reducing or preventing the normal accumulation of BimC class motor proteins to astral and spindle microtubules. An opposing category of models for dynein involvement in centrosome separation predicts that force production occurs within the cortical cytoplasm and acts to pull on centrosomal microtubules. While such a model readily explains the centrosome migration defect, this mechanism does not account for the observed detachment of centrosomes (Robinson, 1999 and references).

In dynein mutant embryos, the release of centrosomes from spindle poles was observed, as well as 'loosely attached' centrosomes, where the distance between a centrosome and the associated metaphase spindle pole is significantly greater than in wild-type. Furthermore, the morphology of the spindle poles which lose a centrosome is affected in a manner consistent with current models of dynein function in spindle morphogenesis. In mutant embryos the detachment of a centrosome from bipolar spindles results in the partial collapse of the affected pole. In some cases, the blunt-ended pole becomes refocused, suggesting that either a residual function of the mutant dynein or an additional minus-end motor can rescue the spindle pole. Loss of a centrosome from multipolar spindles also results in collapse of the affected pole. In living embryos the maintenance of a focused spindle pole requires dynein and the stabilizing influence of a centrosome. This result is not contingent upon the syncytial environment of the embryo since a similar requirement for centrosomes in the organization of spindle poles is apparent in Drosophila larval neuroblasts (Robinson, 1999).

In vivo time-lapsed analysis provides a temporal understanding of the relationship between centrosome behavior and spindle morphogenesis, and reveals another novel aspect to the dynein mutant phenotype. Nuclei that undergo incomplete centrosome migration are predisposed to suffer further defects in spindle assembly, which frequently lead to multipolar spindle configurations. As a further consequence, the size of interphase nuclei is variable and suggests a significant defect in karyokinesis. Although dynein is likely to be present on Drosophila kinetochores, as it is in other organisms, evidence for a direct role for dynein in chromatid congression or segregation is lacking. The alignment of chromosomes at metaphase is apparently normal in mutant embryos and the interpretation that aberrant nuclear size results predominantly from abnormal chromatid segregation on multipolar spindles is favored, rather than a direct effect on kinetochore-mediated chromatid movement (Robinson, 1999).

How does a loss in dynein function and defective centrosome behavior lead to multipolar spindle assembly? The regular spacing between metaphase spindles in the Drosophila syncytium is dependent upon centrosome positions on the nuclear envelope before M phase. One interesting possibility is that organization of the cortical cytoskeleton and the pseudocleavage furrow acts to help isolate nuclei from one another during late nuclear division cycles and is disrupted by mispositioned centrosomes. In this case, loss of spindle autonomy and the formation of multipolar spindle configurations is an indirect effect of the role of dynein in centrosome positioning. However, mutations known to disrupt the cortical cytoskeleton and pseudocleavage furrows are reported to promote spindle fusions during late nuclear cycles when nuclear density is high. For dynein mutant embryos, spindle fusions are detected during the earliest rounds of division before cortical migration and when nuclear density is quite low (Robinson, 1999 and references).

Alternatively, a reduction in dynein function in mitotic spindles and/or the syncytial cytoplasm may allow spindle or centrosomal microtubule bundles to interact inappropriately with neighboring arrays. Measurements of an increase in spindle girth in the mutant embryos is consistent with a reduced organization of microtubules within mutant spindles. In spite of the reduced affinity of centrosomes for both nuclear envelopes and spindle poles in the dynein mutants, centrosomes retain a strong capacity to organize spindle poles. Ectopic spindle pole formation has been observed on neighboring mitotic arrays by both free centrosomes and spindle-associated centrosomes. The ectopic poles form by splitting off bundles of microtubules from the adjacent spindle, rather than by nucleation of microtubule bundles from the errant centrosome toward the adjacent spindle. Subsequently, the formation of interspindle microtubule bundles and the fusion of neighboring spindles may result from the action of other motor activities (Robinson, 1999). For example, the separation of spindles during late nuclear cycles in Drosophila embryos has been shown to require the function of KLP61F (Sharp, 1999).

In summary, evidence has been provided that cytoplasmic dynein is required for the attachment of centrosomes to prophase nuclei. Time-lapsed analysis has further demonstrated in living embryos the role of dynein in the initial migration of centrosomes along the nuclear envelope before spindle assembly, as well as in the attachment of centrosomes to spindle poles. The inappropriate behaviors of centrosomes that result from the reduction in dynein function disrupt spindle morphogenesis. These results show that dynein function in centrosome attachments is essential for autonomous spindle function, and the global spatial organization of early development in the Drosophila embryo (Sharp, 1999).

Centrosome migration into the Drosophila oocyte is independent of BicD and egl, and of the organization of the microtubule cytoskeleton

During early Drosophila oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte, and accumulates oocyte-specific proteins and the centrosomes from the other 15 cells. The microtubule cytoskeleton and the centrosomes follow the same stepwise restriction to one cell as other oocyte markers. Surprisingly, the centrosomes still localize to one cell after colcemid treatment, and in BicD and egl mutants, which abolish the localization of all other oocyte markers and the polarization of the microtubule cytoskeleton. In contrast, the centrosomes fail to migrate in cysts mutant for Dynein heavy chain 64C, which disrupts the fusome. Thus, centrosome migration is independent of the organization of the microtubule cytoskeleton, and seems to depend instead on the polarity of the fusome (Bolivar, 2001).

Since BicD and egl abolish the polarization of the MT cytoskeleton in the cyst, the only candidate for a polarized structure that could direct centrosome migration in these mutants is the fusome. In fact, the cells that accumulate the centrosomes of egl and BicD mutant cysts possess the largest portion of the degenerating fusome. This observation demonstrates that, as in wild type, the asymmetry established during fusome morphogenesis persists until the stages when the centrosomes migrate in BicD and egl cysts. In order to test a direct role for the fusome in centrosome movement, the behavior of the centrosomes was analyzed in a mutant that affects the integrity of the fusome. Germline clones of null alleles of Dynein heavy chain 64C divide correctly and produce cysts that show a very similar phenotype to BicD and egl. These mutant cysts contain 16 nurse cells in which oocyte cytoplasmic markers do not accumulate in a single cell. In addition to this phenotype, a null allele of Dhc64C affects the integrity of the fusome. Dhc64C mutant cysts possess a normal-looking fusome in region 1 and early in region 2a. However, the fusome of older region-2a cysts shows a fragmented appearance. Interestingly, the centrosomes of these cysts fail to migrate to a single cell, strongly suggesting that centrosome migration requires an intact fusome (Bolivar, 2001).

Dhc64C is necessary for the localization of centrosomes and cytoplasmic markers to the oocyte. It was then investigated if Dhc64C is also required for the restriction of meiosis to the oocyte and the distribution of the synaptonemal complex was analysed in Dhc64C germline clones. Like BicD mutants, Dhc64C is required for the formation of the SC. Thus, lack of function of dynein blocks the three asymmetries present in region-3 oocytes, suggesting that the restriction of meiosis to the oocyte, the organization of a polarized MT centered in this cell, and the migration of centrosomes to the oocyte, depend upon the polarization of the fusome (Bolivar, 2001).

Since the MT cytoskeleton seems to be polarized toward the oocyte prior to the migration of the centrosomes, this suggests that most of the centrosomes of region-2 cysts might have lost their MT nucleating properties. These post-mitotic centrosomes thus would act differently from their region-1 counterparts, which retain the ability to grow microtubules, at least during the mitotic divisions of the cyst. A test was performed to see whether the molecular composition of post-mitotic centrosomes is different to mitotic ones. The distribution of Centrosomin (Cnn), a marker for the active centrosomes of mitotic cells, was examined. Cnn, like gamma-tubulin, is present in region-1 centrosomes. In contrast, Cnn is absent or barely detectable in region-2 and -3 cysts. This change in composition of centrosomes depends upon the activity of egl, since in egl mutant cysts Cnn reappears in post-mitotic region-2b centrosomes and by region 3 they possess a noticeable staining with the alpha-cnn antibody. Although no explanation for this difference is available, it suggests that the correct determination of the oocyte among the cells of the cyst affects the composition of the germline centrosomes (Bolivar, 2001).

Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning

To understand the role of Bicaudal-D in the establisment of oocyte polarity, a screen was carried out for genetic enhancers of Bic-D mutation. This screen identified the mutation E415 as a dominant genetic enhancers of Bic-D. The mutation E415 maps to a genetic locus that codes Drosophila Lissencephaly-1. Mutations in the human Lissencephaly-1 (Lis-1) gene cause Miller-Dieker syndrome. This syndrome is characterized by a smoothened brain surface and disorganized cortical layers, resulting from failed neuronal migration during brain development. Homologs of Lis-1 have also been identified in the filamentous fungus Aspergillus nidulans and in Saccharomyces cerevisiae, and both function with the microtubule minus-end-directed motor dynein/dynactin in nuclear migration (Swan, 1999).

Patterning of the Drosophila embryo depends on the correct localization of patterning determinants within the oocyte, beginning in mid-oogenesis. Starting in stage 8, specific mRNAs begin to accumulate either at the posterior end of the oocyte or along the anterior cortex; this pattern is dependent on Bic-D and microtubules. The distribution of anteriorly localized factors (orb, egl, nos and bcd mRNAs and Orb protein) and posteriorly localized factors (osk mRNA and Osk protein) were studied in E415 ovaries. All of these factors show disruptions in their normal subcellular localization within the oocyte. These effects on localization could be due to effects on transport, anchoring or stability of transcripts in the mutant (Swan, 1999).

In stage 7 of oogenesis, the oocyte nucleus migrates from the posterior to the future dorsal-anterior corner of the oocyte. In about half of stage-9 and stage-10 E415/E415 mutant egg chambers, the oocyte nucleus is positioned incorrectly. This could be due to a failure in nuclear migration or anchoring. Since the oocyte nucleus takes up almost the entire oocyte in stage-8 and earlier mutant egg chambers, it is not possible to determine whether nuclear positioning is affected before stage 9. Bic-D is also required for positioning of the oocyte nucleus2, and therefore E415 could function in nuclear positioning through the Bic-D-Egl complex. Consistent with this view, Bic-D and Egl proteins accumulate at high levels in a region between the oocyte cortex and nucleus: this localization is abolished in E415 mutants. Microtubules are also required for oocyte nuclear positioning; whereas microtubule organization in E415 homozygous mutants appears to be unaffected in early oogenesis, some alterations in microtubules are observed after stage 7. In wild-type oocytes, microtubules appear to be concentrated at the anterior cortex, apparently reflecting their nucleation from anterior sites. Microtubules still appear to be organized in this way in E415 mutants, but are often less focused (Swan, 1999).

DLis-1 shares with the dynein heavy chain gene, Dhc, a requirement in oocyte determination, indicating that the dynein heavy chain may function like its fungal homologs in a pathway with dynein/dynactin. A specific allele of Dhc (Dhc^6-6) dominantly suppresses the rough-eye phenotype produced by a mutation in the dynactin component Glued (Gl¹ mutation) whereas a deficiency removing Dhc has no effect. This allele-specific interaction is evidence that Gl and Dhc may act in the same pathway. Similarly, Dhc^6-6 behaves as a strong dominant suppressor of the DLis-1^E415 homozygous phenotype, resulting in fertility, proper nuclear positioning and near normal oocyte growth. A deficiency of Dhc and the point mutation Dhc ^6-12 have no effect on the DLis-1^E415 phenotype. Therefore Dhc shows the same allele-specific interaction with DLis-1 as it does with Gl, indicating that DLis-1 may function in a genetic pathway with Dhc, and implicating dynein in nurse-cell-to-oocyte transport and nuclear positioning in the oocyte. Tests were also made for genetic interactions between Gl and DLis-1 and between Gl and Bic-D. The antimorphic Gl¹ mutation confers lethality in a DLis-1^E415 homozygote and in a Bic-D^PA66/Df(2L)TW119 background, indicating that both DLis-1 and Bic-D may function in the same essential process as dynactin (Swan, 1999).

To determine how DLis-1 could interact with dynein/dynactin, the localizations of DLis-1 and Dhc proteins were studied in wild-type oocytes and in oocytes with mutations in either gene. DLis-1 signal is concentrated along the cortex of wild-type oocytes from as early as stage 5 of oogenesis. This localization is not detectable in DLis-1 ^E415/DLis-1^E415 ovaries, indicating that the antiserum specifically recognizes DLis-1. To determine whether DLis-1 localization is dependent on dynein function, DLis-1 distribution was studied in Dhc^6-6/ Dhc^6-12 mutants. This hypomorphic allelic combination has no effect on the cortical accumulation of DLis-1 protein. DLis-1 localization was mapped in egg chambers treated with the microtubule-destabilizing drug colchicine. Under conditions that disrupt microtubules and dynein localization, cortical DLis-1 signal is still present, indicating that its cortical localization or maintenance does not depend on localized dynein or microtubules. In contrast, Dynein localization is dependent on DLis-1. In wild-type egg chambers, Dhc localizes to the presumptive oocyte in region 2 of the germarium and remains enriched in the oocyte throughout oogenesis. In DLis-1^E415 mutants, this specific accumulation is completely abolished. This strong effect of DLis-1^E415 on Dhc localization contrasts with its subtle effect on the accumulation of other oocyte-specific factors, indicating that DLis-1 may specifically regulate Dhc localization and that the localization of most oocyte-specific factors to the oocyte is independent of DLis-1 and Dhc function. Thus there appear to be two distinct microtubule-dependent nurse-cell-to-oocyte transport mechanisms at work during oogenesis. One mechanism involving Bic-D, DLis-1 and Dhc is involved in bringing oocyte determinants into the presumptive oocyte, and, subsequently, is responsible for oocyte growth in stages 1-7 of oogenesis. A second microtubule-based transport mechanism would function during these stages in the transport of oocyte-specific mRNAs and proteins into the oocyte, possibly using other dyneins. In wild-type egg chambers at stages 7-9, Dhc accumulates along the oocyte cortex and around the oocyte nucleus. Later in stage 9, Dhc accumulates mainly at the posterior and anterior oocyte margins. These aspects of Dhc localization are disrupted in DLis-1 mutants and, therefore, DLis-1 is necessary for most or all aspects of dynein localization within the female germ line (Swan, 1999).

Lis-1 and Bic-D function in nuclear migration in neurons. Given the role of Lis-1 homologs in fungi, it has been suggested that the requirement for human Lis-1 in neuronal migration could also reflect a role in nuclear migration. In the developing cerebral cortex and cerebellum, migrating neurons project out a cytoplasmic extension towards their target, and then their nucleus translocates along this extension. An analogous process occurs during neural development in Drosophila. In the third-instar eye imaginal disc, undifferentiated cells lie at the basal surface and extend processes apically. As photoreceptor cells differentiate posterior to the morphogenetic furrow, their nuclei translocate to the apical surface of the eye disc. In serial confocal sections nuclei start to appear ~4 µm below the apical surface and are no longer visible beyond 8 µm. In flies homozygous for a pupal-lethal allele of DLis-1, nuclei also start to appear 4 µm below the apical surface, but many nuclei are also found more basally. This phenotype is identical to that of mutants for Glued, suggesting that DLis-1 functions with dynein/dynactin in nuclear migration in these neural cells. Given that DLis-1 is 70% identical to the human Lis-1, these findings strongly support the possibility that the failure in neuronal migration in Miller-Dieker syndrome also results from a failure in dynein/dynactin-dependent nuclear migration (Swan, 1999).

As in the ovary, DLis-1 appears to function with Bic-D in nuclear migration during eye development. Bic-D mutant eye discs also exhibit a severe defect in nuclear positioning, with photoreceptor nuclei being found at all levels basal to 4 µm and, frequently, in the axons that project basally from these cells. The requirement for DLis-1 and Bic-D in nuclear positioning in the developing eye imaginal disc could reflect a role for these genes in microtubule organization. Microtubules in the eye disc extend longitudinally along the apical-basal axis, but the polarity of these microtubules is not known. To establish the orientation of these microtubules, third-instar eye imaginal discs were immunostained with antibodies to gamma-tubulin. In wild-type imaginal discs, gamma-tubulin is found at high levels along the apical cortex and within this region in a single strong focus about 2 µm below the apical cortex and 2 µm apical to the photoreceptor nuclei. In DLis-1K13209 mutants, gamma-tubulin still accumulates at the apical surface, indicating that microtubules are orientated normally within the mutant photoreceptors. However, the subapical focus of gamma-tubulin is more diffuse and is undetectable in many photoreceptors, indicating a requirement for DLis-1 in focusing microtubule minus ends. A similar effect has been noted in Glued mutants, suggesting that DLis-1 and dynein/dynactin also function in the same pathway in focusing microtubule minus ends in the eye. Interestingly, whereas Bic-D mutants consistently exhibit a more marked defect in nuclear positioning, localization of gamma-tubulin is normal in these mutants (Swan, 1999).

Several models for nuclear localization have been advanced. This analysis of DLis-1 allows for the proposal of a model for nuclear migration in the Drosophila oocyte in which a cortical protein (DLis-1) anchors microtubules via the minus-end-directed microtubule motor dynein: DLis-1 appears to function at the cortex, and its localization to the cortex is independent of microtubules and dynein. The dynein heavy chain, Dhc, also associates with the oocyte cortex and this localization requires DLis-1. Association of the nucleus with these microtubules would then allow it to be anchored to the cell cortex. The cortical localization of Dhc is also microtubule dependent, and this could indicate that dynein uses microtubules to reach the cortex. Bic-D-Egl could mediate the interaction between DLis-1 and dynein or the interaction between microtubules and the oocyte nucleus. As well as localizing to the oocyte cortex, Dhc also associates with the oocyte nucleus throughout oogenesis, indicating that it may function as well in linking the nucleus to microtubules (Swan, 1999).

The Drosophila Lissencephaly1 (DLis1) gene is required for nuclear migration

The minus-end-directed motor cytoplasmic dynein has been implicated in nuclear migration in several fungal systems. In addition, genetic epistasis data suggest that nudF, the Aspergillus homolog of Drosophila Lis1, acts upstream of dynein in mediating nuclear migration. Lis1 egg chambers were examined to determine whether the level or distribution of Dhc64C protein was affected. In wild-type ovaries high levels of Dhc64C can be detected in the oocyte from region 2 in the germarium onward. Later, in stage 9 egg chambers Dhc64C is enriched at the posterior of the oocyte and also outlines the oocyte nucleus. In Lis1 mutants Dhc64C localization appears unperturbed through early oogenesis. However, in stage 9 mutant egg chambers localization to the posterior of the oocyte is strongly reduced, suggesting that Lis1 activity is required for dynein localization or activity (Lei, 2000).

The dependence of dynein localization on Lis1 activity suggests a functional interaction between these two genes. A test was performed to see whether the Lis1 phenotype is affected by mutations in the Gl and Dhc64C genes that encode structural components of the dynein-dynactin microtubule motor. The Gl¹ allele causes a synthetic lethality with strong heteroallelic combinations of Lis1 mutations, while in combinations with weaker Lis1 alleles Gl¹ causes a reduction in viability. Adult escapers from the latter crosses generally do not survive for more than a few days. More interestingly, they display defects in eye morphology, bristles, and abdominal tergites. Flies carrying the dominant Gl¹ allele have small, rough eyes with irregularly positioned bristles. A reduction in Lis1 activity results in an enhancement of the Gl¹ eye phenotype. In addition, the scutellar bristles of escaper flies are reduced in size and frequently lost. Most adult escapers lack bristles on the ventral side of the abdomen, and in many animals abdominal tergites are completely absent in one or more segments. Lis1 mutants show similar but weaker interactions with Dhc64C alleles. A deficiency for Dhc64C was viable in combination with Lis1 mutants. However, the adults display defects in scutellar and abdominal bristle morphology resembling those seen in DLis1^8.25.3/DLis1¹¹⁷⁰² ;Gl¹/+ adults. Thus the genetic interactions between Dhc64C, Gl and Lis1 are consistent with a functional relationship between these genes (Lei, 2000).

Drosophila wingless and pair-rule transcripts localize apically by Dynein-mediated transport of RNA particles

Asymmetric mRNA localization targets proteins to their cytoplasmic site of function. The mechanism of apical localization of wingless and pair-rule transcripts in the Drosophila blastoderm embryo has been elucidated by directly visualizing intermediates along the entire path of transcript movement. After release from their site of transcription, mRNAs diffuse within the nucleus and are exported to all parts of the cytoplasm, regardless of their cytoplasmic destinations. Endogenous and injected apical RNAs assemble selectively into cytoplasmic particles that are transported apically along microtubules. Cytoplasmic dynein is required for correct localization of endogenous transcripts and apical movement of injected RNA particles. It is proposed that dynein-dependent movement of RNA particles is a widely deployed mechanism for mRNA localization (Wilkie, 2001).

To study the mechanism of apical localization, whether actin and/or MTs are necessary for localization of injected mRNA was tested by preinjecting cytoskeletal inhibitors 10 min before injecting the RNA. It was found that preinjection of Cytochalasin B, at concentrations that disrupt the organization of actin filaments, has no affect on Runt mRNA localization. However, a similar disruption of nuclear position has been observed in the cortical cytoplasm. In contrast, preinjection of colcemid, which destabilizes blastoderm MTs, disrupts runt, wingless, and fushi tarazu RNA localization almost entirely. It is concluded that an intact MT cytoskeleton is required for apical localization of injected RNA and that actin does not play a major role in the process. However, some minor role for actin in apical localization of RNA cannot be excluded (Wilkie, 2001).

Whether the localization of injected RNA occurs by minus end directed MT-dependent motor movement was tested by preinjecting embryos with antibodies against Drosophila cytoplasmic dynein heavy chain (dhc). Two independently raised monoclonal antibodies against dhc are each sufficient to inhibit RUN, FTZ, and WG mRNA apical localization in most, or all, embryos. Either one, the anti-dynein antibody or the colcemid injections, is sufficient to cause apical RNA to partly diffuse away from the site of injection in a similar manner to embryos injected with HB RNA alone. Injected apical RNA does not diffuse in the absence of anti-dynein antibodies or Colcemid preinjections. These results suggest that apical RNA is tethered to MTs by dynein and that dynein is required for the transport of RNA particles (Wilkie, 2001).

To further test the involvement of cytoplasmic dynein in apical transcript localization, RNA was injected into mutant cytoplasmic dynein heavy chain (Dhc64C) embryos. A marked reduction was found in the speed of movement of injected apical targeted RNAs in dynein mutants. Cytoplasmic dynein is essential for many cellular processes, so strong mutations in Dhc64C are homozygous lethal in Drosophila and cannot be studied at the blastoderm stage. Instead hypomorphic allelic combinations of Dhc64C, which are viable in trans due to intragenic complementation, were used. In two different allelic combinations of Dhc64C, injected RNA particles move at speeds 60% to 70% slower than they do in wild-type. Staining Dhc64C mutant embryos with anti-tubulin antibodies showsthat MT distribution is indistinguishable from wild-type, indicating that the reduced speed of localization is not due indirectly to a disruption of the MTs. Instead, the reduction in speed is likely to show a direct requirement for dynein in particle transport (Wilkie, 2001).

To test whether cytoplasmic dynein is also required for apical localization of endogenous transcripts, the effects of Dhc64C hypomorphic mutants and anti-dhc antibodies on the apical localization of endogenous FTZ transcripts was tested by in situ hybridization. As expected, hypomorphic Dhc64C mutants show no detectable effects on FTZ apical mRNA localization since injected RNA localizes correctly, but more slowly. In contrast, injection of anti-dhc antibody disrupts endogenous FTZ localization, leading to unlocalized stripes of ftz mRNA 20–30 min after injection. Given that FTZ mRNA has a half-life of 6 min in the blastoderm, the FTZ transcripts observed are likely to have been synthesized after the injection. It is concluded that endogenous apical mRNA localization is also dynein dependent (Wilkie, 2001).

Dynactin is a protein complex that is involved in coordinating the activities of cytoplasmic dynein, and is thought to be required for most forms of dynein-based transport. To test whether dynactin is also required for apical RNA localization, a large excess of p50/dynamitin is preinjected into embryos 10 min before injecting apically targeted RNA. p50/dynamitin causes a significant reduction in the speed of RNA particle movement. p50/dynamitin is a subunit of dynactin whose overexpression is a widely used method of disrupting the dynactin complex and demonstrating conclusively dynein-dependent motility. Dynactin is required for some cargo binding and for dynein processivity. It is concluded that apical transcript localization in the blastoderm embryo occurs by cytoplasmic dynein- and dynactin-mediated transport along MTs toward their minus ends (Wilkie, 2001).

It is thought that export and localization of apical mRNA in the blastoderm embryo can be divided into six distinct steps. (1) During or after completion of transcription and processing, transcripts are assembled into particles, which contain various hnRNPs and export factors, some of which may form part of the cytoplasmic localization machinery. (2) mRNA particles diffuse freely after release from the site of transcription and processing until they reach nuclear pore complexes (NPCs) on the nuclear periphery. (3) mRNA particles are exported through NPCs in all parts of the nuclear envelope. (4) The composition of the particles probably changes during export from the nucleus and in the cytoplasm to recruit dynein, dynactin, and associated proteins. (5) Particles attach to MTs and are actively transported to the apical cytoplasm. (6) Particle movement arrests in the apical cytoplasm, where they may associate with other particles and become anchored (Wilkie, 2001).

The first three steps of apical localization are thought to be common to most mRNAs, because they are essential universal processes in eukaryotic cells. However, the last three steps of the localization pathway are likely to vary among different kinds of transcripts, since the key determinant in sorting different mRNAs to their correct cytoplasmic destinations is presumably RNP particle composition in the cytoplasm. It is possible that some components required for cytoplasmic sorting are preassembled in the nucleus, as suggested by studies showing that the localization of injected FTZ mRNA depends on preincubation with the hnRNPA1 protein Squid. Indeed, a requirement for hnRNPs has also been shown for GRK mRNA localization in the oocyte, for myelin basic protein mRNA in rat oligodendrocytes, and for Vg1 transcripts in Xenopus oocytes. However, the data in this study show that injected protein-free apical RNA assembles in the cytoplasm into particles that localize correctly, arguing that all the factors needed to assemble competent localization particles can also be recruited in the cytoplasm (Wilkie, 2001).

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

The hypothesis was tested that amyloid precursor protein (APP) and its relatives function as vesicular receptor proteins for kinesin-I. Deletion of the Drosophila APP-like gene (Appl) or overexpression of human APP695 (an alternatively spliced version of APP) or APPL constructs causes axonal transport phenotypes similar to kinesin and dynein mutants. Genetic reduction of kinesin-I expression enhances while genetic reduction of dynein expression suppresses these phenotypes. Deletion of the C terminus of APP695 or APPL, including the kinesin binding region, disrupts axonal transport of APP695 and APPL and abolishes the organelle accumulation phenotype. Neuronal apoptosis was induced only by overexpression of constructs containing both the C-terminal and Ab regions of APP695. The possibility is discussed that axonal transport disruption may play a role in the neurodegenerative pathology of Alzheimer's disease (Gunawardena, 2001).

Although reducing the amount of kinesin-I to 50% of normal by deleting one of two copies of either the klc or khc gene ordinarily has no significant phenotype, such a reduction in an animal overexpressing APP proteins that contain the cytoplasmic C terminus is predicted to significantly enhance the axonal blockage phenotype. This behavior is expected, because if kinesin-I becomes limiting by virtue of binding excess APP C termini, then further reduction of kinesin-I by deleting one gene copy should dramatically enhance the axonal transport phenotype. To test this prediction, larvae were generated that overexpressed APPL or APP695 and that were also heterozygous for a null mutation, khc⁸, so that kinesin-I was reduced to 50% of normal. Although larvae overexpressing APPL or APP by themselves or reduced in KHC dosage alone have no striking organismal phenotype, larvae combining these two features exhibit a dramatic new neuromuscular phenotype. These larvae flip their tail and head upwards during crawling, rocking back and forth as they struggled to crawl. Their neurons also contained an enhanced number of organelle accumulations. The extent of accumulations in larvae expressing wild-type APP695 and APPL in the context of reduced KHC dosage was comparable to homozygotes for kinesin-I or dynein mutants and was similarly lethal. Quantitative analysis has revealed a statistically significant difference between siblings with a normal and reduced dose of KHC (Gunawardena, 2001).

To confirm the specificity of the genetic interactions observed between reduction in KHC and overexpression of APP695 or APPL, larvae heterozygous for a null mutation of klc [Df(3L)8ex94, which removes the entire kinesin light chain gene] were generated in combination with constructs expressing APPL and APP695. Neurons from these larvae contain an increased number of organelle jams relative to larvae with a normal dose of KLC. Quantitative analysis reveals a statistically significant difference between siblings containing a normal and reduced dose of KLC, although the extent of enhancement is not as dramatic as when the dosage of KHC is reduced. Although these larvae do not show a larval neuromuscular phenotype as dramatic as was observed when the dosage of KHC was reduced, these larvae show a clear posterior paralysis phenotype. This result is again consistent with a direct functional interaction of the C-terminal region of APP695 and APPL with kinesin-I (Gunawardena, 2001).

Although reducing the amount of cytoplasmic dynein to 50% of normal by deleting one of two copies of either dynein heavy chain (dhc) or dynein light chain (dlc) genes ordinarily has no significant phenotype, such a reduction in an animal overexpressing APP family members that contain the cytoplasmic C terminus is predicted to suppress significantly the severity of the axonal accumulation phenotype. The basis for this prediction is that dynein drives retrograde axonal transport, which is antagonistic to kinesin-I-mediated anterograde axonal transport. In addition, many vesicles or organelles that exhibit net anterograde movement experience periods of retrograde movement owing to the simultaneous presence of kinesins and dyneins on the same vesicle or organelle. Thus, vesicle stalling and axonal accumulations induced by APP are predicted to be ameliorated by dynein reduction by (1) reducing the rate at which vesicles and organelles moved by dynein are transported into regions that have stalled or accumulated vesicles caused by APP expression; or (2) reducing the contribution of dynein-driven movement to a vesicle experiencing stalling because of reduced kinesin-driven activity; this reduction should attenuate vesicle stalling by restoring the balance of movements toward normal (Gunawardena, 2001).

To test this prediction, larvae overexpressing APP695 or APPL were generated that were also heterozygous for either a deficiency of either dhc [Df(3L)GN24] or dlc (robl^k). Reduction of dynein suppresses the extent of organelle accumulations in APP695 or APPL transgenic lines. In addition, no significant larval crawling phenotype was observed in these animals. Surprisingly, reduction in dynein dosage also rescues the inviability of males overexpressing APP695. No effect is seen in the lines expressing a C-terminal deletion of APP695 or APPL. Thus, reduction in dosage of a retrograde motor protein appears to be sufficient to decrease organelle accumulations induced by APP or APPL expression (Gunawardena, 2001).

Whether larvae bearing heterozygous deletions of dynein components and the Appl gene in combination would exhibit abnormal axonal transport was investigated. In contrast to the situation with kinesin and APPL, female larvae with one copy (50% dose) of Appl and one copy of dhc or dlc do not have typical axonal accumulations. Intriguingly, male larvae bearing a deletion of the Appl gene, and hence lacking all APPL function, in combination with one copy of dhc or dlc also lack axonal accumulations. Thus, reduction in dynein levels appears to suppress axonal accumulations induced by loss of APPL (Gunawardena, 2001).

Genetic analysis in Drosophila strongly supports the hypothesis that mammalian APP and its homolog, APPL, have kinesin-I receptor functions in vivo. The genetic data and tests complement the in vitro biochemical evidence for a kinesin-I receptor function for APP. In these experiments, APP has been shown to form a complex with conventional kinesin by directly binding to KLC. Transport of APP depends upon kinesin-I and KLC in particular. In addition, the finding that, in Drosophila, APP695 can enter and be transported down axons to neuromuscular junctions and that this transport depends upon the cytoplasmic C terminus containing the proposed KLC binding region supports this view. In toto, these data strongly support the hypothesis that APP functions as a kinesin-I receptor. Perhaps APP bound to kinesin-I may be required for the axonal transport of a subset of cargoes, such as vesicles containing signaling or other molecules used at the synapse. Identifying these vesicles and their cargoes is an important next step (Gunawardena, 2001).

A surprising finding is the suppression of APP and APPL-induced organelle accumulations by genetic reduction of cytoplasmic dynein. Although further work is needed to define the mechanism of this suppression, one simple explanation comes from previous observations about the functionally antagonistic relationship of dynein and kinesin. A general observation is that kinesin and dynein are both present on many of the same axonal vesicles and organelles. Such vesicles and organelles often exhibit alternating anterograde (kinesin) and retrograde (dynein) movements, with net anterograde or retrograde movement resulting from a regulated bias in the balance of opposing movements along the microtubule. Thus, reduction of kinesin-I on non-APP vesicles caused by binding of kinesin-I to excess APP might cause vesicle stalling and organelle accumulations. Stalling of these vesicles and subsequent phenotypes might be rescued by reducing the antagonistic component of movement produced by dynein (Gunawardena, 2001).

The fusome and microtubules enrich, Par-1 in the oocyte, where it effects polarization in conjunction with Par-3, BicD, Egl, and Dynein

After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Par-1 is shown to become restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. baz is required for Par-1 relocalization within the oocyte and the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization (Vaccari, 2002).

During oocyte specification, localization of the determinants BicD, Egl, and Orb to the early oocyte relies on the asymmetric distribution of microtubules in the cyst, evident as a dense focus of MT in the oocyte. Depolymerization of the MT by colchicine abolishes the localization of BicD, Egl, and Orb and results in egg chambers with 16 nurse cells and no oocyte. It was therefore asked if the restriction of Par-1 to the oocyte during the transition from region 2a to region 2b is also MT dependent. Ovaries of flies fed with colchicine for 12 hr fail to localize Par-1 and Orb to the oocyte, indicating that Par-1 restriction to the oocyte is indeed MT dependent. This is in contrast to the localization of Par-1 to the fusome, which occurs independently of MT (Vaccari, 2002).

The distribution of Par-1 within the oocyte was further examined by focusing on the transition between regions 2b and 3, when par-1-dependent polarization of the oocyte occurs. In germarial region 2b, Par-1 is enriched anterior to the oocyte nucleus. In region 3, the protein is mainly detected at the posterior of the oocyte, where it remains. During this relocalization, Par-1 colocalizes completely with BicD in the germline. Because par-1 is required for BicD relocalization within the oocyte, the distribution of Par-1 was examined in BicD hypomorphs that allow differentiation of an oocyte. Par-1 is detected but mislocalized in an anterior dot within the BicD mutant oocytes. Hence, Par-1 and BicD are interdependent for their relocalization to the posterior of the oocyte region 3b (Vaccari, 2002).

To assess whether the MT cytoskeleton mediates relocalization of Par-1 and BicD within the oocyte, wild-type ovaries were dissected a short time after treatment with colchicine. A screen was carried out for region 3 egg chambers in which the focus of oocyte MT was destroyed. In these, BicD and Par-1 remain anterior to the oocyte nucleus, indicating that MTs are required for oocyte polarization (Vaccari, 2002).

The MT motor Dynein has been reported to influence development of the germline cyst. Loss-of-function mutants in dhc64C, encoding the heavy chain of the minus end-directed molecular motor Dynein, fail to develop an egg chamber because of mitotic failure in the germarium. However, hypomorphic dhc64C mutants develop an oocyte and 15 nurse cells. In a high percentage of such egg chambers, both Par-1 and BicD remain at the anterior of the oocyte in region 3. Hence, after its initial requirement in cyst formation, the minus end-directed motor Dynein is involved in the relocalization of Par-1 and BicD to the posterior of the oocyte (Vaccari, 2002).

Thus, impairment of the MT cytoskeleton and mutations in BicD and dhc64C affect Par-1 relocalization within the oocyte. Conversely, in par-1 mutants, the MT cytoskeleton is not focused in the oocyte, BicD fails to relocalize, and Dynein is not enriched in the oocyte. The mutual interdependence of these genes and the MT suggests that all these components cooperate to form a polarization complex in the oocyte. Interestingly, the N1 antibody begins to detect Par-1 only when its function is genetically required, suggesting that, in region 2, the kinase may undergo a change in conformation or in its association with other factors (Vaccari, 2002).

The presence and localization of Par-1 in the oocyte at the time of its determination and polarization complements the previously reported localization of Par-1 on the fusome prior to oocyte determination and establishes Par-1 as a unique oocyte marker, for at least two reasons. (1) Absence of any one of the oocyte determinants, BicD, Egl, or Orb, prevents the concentration of the two other determinants in this cell. In contrast, in the absence of Par-1, it is the relocalization of the determinants within the oocyte that is specifically affected. (2) BicD, Egl, and Orb are not present on the fusome, and the observed enrichment of these determining factors in the oocyte is the result of the enrichment of their RNAs in this cell during its specification. In contrast, no par-1 RNA is detected in the germline at such early stages. The idea that Par-1 is initially loaded on the fusome, where it perdures during the cyst divisions, and that it is later preferentially inherited by the oocyte, is favored. Taken together, the facts that par-1 mutants show no fusomal defects and that accumulation of Par-1 itself in the oocyte requires MT suggest that Par-1 does not affect the oocyte MT cytoskeleton from its fusomal location. It is proposed that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where it acts in conjunction with BicD, Egl, and Dynein to effect polarization (Vaccari, 2002).

Transport of germ plasm on astral microtubules directs germ cell development in Drosophila

In many organisms, germ cells are segregated from the soma through the inheritance of the specialized germ plasm, which contains mRNAs and proteins that specify germ cell fate and promote germline development. Whereas germ plasm assembly has been well characterized, mechanisms mediating germ plasm inheritance are poorly understood. In the Drosophila embryo, germ plasm is anchored to the posterior cortex, and nuclei that migrate into this region give rise to the germ cell progenitors, or pole cells. How the germ plasm interacts with these nuclei for pole cell induction and is selectively incorporated into the forming pole cells is not known. Live imaging of two conserved germ plasm components, nanos mRNA and Vasa protein, revealed that germ plasm segregation is a dynamic process involving active transport of germ plasm RNA-protein complexes coordinated with nuclear migration (see graphical abstract). Centrosomes accompanying posterior nuclei induce release of germ plasm from the cortex and recruit these components by dynein-dependent transport on centrosome-nucleated microtubules. As nuclei divide, continued transport on astral microtubules partitions germ plasm to daughter nuclei, leading to its segregation into pole cells. Disruption of these transport events prevents incorporation of germ plasm into pole cells and impairs germ cell development. These results indicate that active transport of germ plasm is essential for its inheritance and ensures the production of a discrete population of germ cell progenitors endowed with requisite factors for germline development. Transport on astral microtubules may provide a general mechanism for the segregation of cell fate determinants (Lerit, 2011).

This study has uncovered a dynamic mechanism for germ plasm inheritance involving release of germ plasm RNPs from the posterior cortical actin anchor coordinated with their dynein-dependent transport to centrosomes that are associated with posterior nuclei. Transport of these RNPs occurs primarily, if not exclusively, on astral microtubules throughout the mitotic cycle. The results suggest that directed transport of germ plasm components during pole bud formation ensures the production of a discrete population of germ cell progenitors and partitions factors required for germline development during subsequent divisions. Through this process, germline fate determinants are segregated away from somatic nuclei (Lerit, 2011).

Pole cell formation is highly sensitive to the dosage of germ plasm components, because mutations that reduce the accumulation of germ plasm at the posterior pole result in fewer pole cells. This study found that pole cell formation is similarly reduced when germ plasm transport is disrupted, as it is in Dhc mutants or in mutants that affect centrosome function. Although the molecular mechanism by which germ plasm promotes pole cell formation is unknown, the results suggest that directed transport of germ plasm components toward the small subset of nuclei that are the first to arrive at the posterior pole provides the requisite concentration of one or more factors necessary to impart germline fate and induce pole cell formation. In addition, because the first divisions of the nascent pole cells occur before budding is complete, the persistence of germ plasm transport toward centrosomes during these divisions would ensure that factors required for germline development, such as nos, are maintained within pole buds, segregated to daughter nuclei, and ultimately incorporated into the forming germ cells (Lerit, 2011).

Germ plasm produced ectopically in osk-bcd3′UTR (transgene used to generate embryos with germ plasm containing nos*GFP localized ectopically at the anterior of the embryo in addition to its normal localization at the posterior) and Khc mutant embryos is transported to nearby nuclei, indicating that nuclei are not predetermined to recruit germ plasm. Thus, the release of germ plasm from its actin-based anchor and the onset of germ plasm motility must be tightly coordinated with the arrival of nuclei at the posterior cortex to target germ plasm specifically to these nuclei and prevent the misspecification of cell fate. Egg activation triggers the release of bcd mRNA from the anterior cortex, probably through a generalized activation-dependent restructuring of the cortical actin cytoskeleton. This event does not release nos and Vas, however. Nor is germ plasm release scheduled by an intrinsic timing mechanism, as was shown in this study. Consistent with the observation that nos release is delayed at the anterior in osk-bcd3′UTR embryos, formation of ectopic germ cells at the anterior lags behind pole cell formation at the posterior in these animals. Moreover, centrosomes isolated from nuclei, either pharmacologically or genetically, are sufficient to trigger germ plasm release from the posterior. These data thus support a model whereby centrosomes and/or centrosome-nucleated microtubules associated with migrating nuclei trigger germ plasm release from the cortical anchor (Lerit, 2011).

Astral microtubules provide the tracks along which germ plasm RNPs travel upon their initial release from the cortex. During mitosis in the syncytial embryo, astral microtubules appear to secure the partitioning of germ plasm RNPs to daughter nuclei. The preferential association with astral microtubules may also prevent the dilution of inductive signals during asymmetric division events, when only one aster is proximal to the germ plasm. The apparent specificity for astral microtubules suggests that the RNP-motor complexes may include factors that recognize particular microtubule-associated proteins or modifications that distinguish these microtubules as preferred tracks (Lerit, 2011).

The observed dynein-dependent transport of nos during pole cell formation contrasts with its diffusion-based mode of localization during oogenesis. Given that dynein-dependent transport of bcd mRNA to the oocyte anterior is ongoing during late oogenesis, it is essential that nos be excluded from interaction with the dynein transport machinery. nos may reside in a dynein-associated transport complex that is inactive or incompatible with the various oocyte microtubule subpopulations. Alternatively, the composition of the nos RNP in the oocyte may simply preclude its association with the dynein motor complex. The observed cotransport of nos and Vas in the embryo suggests that nos becomes linked to dynein through its packaging into a complex with Vas and other germ plasm components. Whether germ plasm RNPs are coupled to dynein motors while they are anchored at the posterior or only after their release remains a subject for future investigation. A similar switch between motor-independent and motor-dependent modes of germ plasm mRNA translocation may occur in Xenopus, although the role of motors in Xenopus germ plasm inheritance is not yet clear (Lerit, 2011).

Recent in situ hybridization studies have now identified over 50 mRNAs that are localized at the posterior of the Drosophila embryo and incorporated into pole cells. Further characterization of a subset of these mRNAs showed that they accumulate near posterior nuclei, suggesting that they may be transported similarly to nos. Determining whether the different transcripts are cotransported will require the development of methods to simultaneously visualize multiple RNAs and germ plasm proteins. However, packaging of even subsets of RNAs together into germ plasm RNPs competent for dynein-mediated transport would greatly simplify the problem of partitioning a complex pool of transcripts to pole cells (Lerit, 2011).

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. This study characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. A novel localization of Ncd to the growing tips of C-MTs is reported, that is shown is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, a model is proposed on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis (Goshima, 2005; full text of article).

EB1 is a highly conserved microtubule plus end-tracking protein that binds various cargo proteins (e.g., APC [adenomatous polyposis coli protein]). Ncd also was recently observed to bind to an EB1 affinity column. It was therefore of interest investigate whether the plus end accumulation of Ncd-GFP is mediated by EB1. After EB1 depletion by RNAi in Ncd-GFP-NES cell line, no plus end accumulation of Ncd-GFP-NES was seen; instead the microtubules were evenly labeled with this protein. After EB1 RNAi, microtubules become less dynamic and frequently enter a pause state where they exhibit minimal growth or shrinkage. However, even the subset of growing microtubule never accumulated Ncd-GFP-NES at their tips (Goshima, 2005).

Next, an in vitro interaction between purified Ncd and EB1 was tested using a GST pull-down assay. It was found that the nonmotor 'tail' domain (aa 1-290) of Ncd can bind directly to the COOH terminus domain of EB1 (EB1-C; aa 208-278), albeit weakly. This binding was competed by addition of a fragment of human APC protein (2744-2843 aa) that binds to EB1's COOH-terminal domain, suggesting that the Ncd tail and APC bind to the same site on EB1. However, expression of the Ncd tail domain (1-290 aa) fused to GFP did not track along microtubule plus ends in vivo, suggesting that the motor domain may augment affinity for the microtubules and thereby aid plus end localization (Goshima, 2005).

No specific mutagenesis strategy was availabe for eliminating plus end tracking of Ncd while retaining its other critical mitotic activities such as microtubule cross-bridging. However, the consequences were tested of pole focusing and Ncd-GFP localization in mitosis after RNAi of EB1. Time-lapse imaging of mitotic EB1 RNAi cells showed no plus end microtubule enrichment, as expected from the observed interphase results. However, Ncd-GFP still strongly and dynamically (revealed by FRAP) localized to spindle MTs, indicating that K fiber binding does not require EB1. In this setting of EB1 RNAi where Ncd was mislocalized from microtubule plus ends but not the spindle, centrosome detachment and K fiber focusing was examined. Qualitative study of EB1 RNAi found both centrosome detachment and pole defocusing. When these phenotypes were examined quantitatively, it was found that the EB1 phenotype consists of pronounced K fiber defocusing and has less pronounced centrosome detachment, which is more similar to the Ncd RNAi than the dynein RNAi phenotype. Although not definitive proof because EB1 RNAi causes microtubule dynamics defects in addition to displacing Ncd from the microtubule plus end, this result is consistent with a functional link between EB1-dependent localization of Ncd to microtubule tips and K fiber coalescence (Goshima, 2005).

Based on live cell imaging and computer simulation analyses, it is proposed that the coalescence of spindle poles involves the following steps: (1) inter-K fiber cross-linking, (2) 'search and capture' of K fibers by the tip of growing C-MTs, and (3) K fiber transport on C-MTs. RNAi analysis of dynein and Ncd as well as live cell imaging of Ncd-GFP has provided insight into the roles of these minus end-directed motor proteins in these processes. By quantitatively comparing Ncd and dynein knockdown phenotypes in the same cell type, it was found that these Ncd and dynein have distinct but overlapping functions in the three steps of pole focusing (Goshima, 2005).

RNAi results suggest that Ncd plays a role in K fiber focusing through three mechanisms described above. Ncd's major role is likely to be in inter-K fiber cross-linking, as evidenced by the splaying of K fibers after Ncd RNAi and the prominent localization of Ncd-GFP to K fibers. This process most likely involves cross-linking of microtubules by Ncd's force generating motor domain and its positively charged 'tail' domain that also binds to microtubules independently of the motor. This process occurs in the absence of centrosomes and C-MTs, because acentrosomal spindles created by centrosomin RNAi also show severe K fiber unfocusing when Ncd is also knocked down by RNAi. It is also showm that the process of lateral K fiber interactions is highly dynamic, because K fibers are continually splaying and coalescing. Such observations are also consistent with FRAP measurements of Ncd-GFP, which show that these motors are associating and dissociating with K fibers on a rapid time scale and thus are not behaving as static cross-linkers. Albeit less efficient than dynein, Ncd also likely contributes to minus end-directed transport of K fibers along C-MTs, because the centrosome to K fiber distance is somewhat greater in the Ncd/Dhc64C double RNAi compared with Dhc64C alone. Finally, it is believed that Ncd at the tips of C-MTs may act to capture K fibers and facilitate subsequent minus end transport of the K fiber (Goshima, 2005).

Cytoplasmic dynein in S2 cells plays a dominant role in transporting K fibers along microtubules, as evidenced by finding that Dhc64C RNAi causes detachment of centrosomes from the minus ends of K fibers. Although secondary to Ncd, dynein also contributes to the focusing of the minus ends of K fibers. This role of dynein becomes particularly clear after Ncd depletion, since Ncd/Dhc64c double RNAi causes very severe splaying of K fibers. It is believed that this K fiber focusing effect also primarily involves dynein's role as a transporter of K fiber bundles along C-MTs, which causes the coalescence of most peripheral K fibers toward the centrosome as shown in computer simulations. However, dynein may have other roles in K fiber coalescence, such as potentially transporting and concentrating cross-linking proteins at minus ends of K fibers. Indeed, synthetic effects of kinesin-14 and dynein motors on pole focusing have been reported in centrosome-free spindles reconstituted in Xenopus egg extract (Goshima, 2005).

The molecular properties of kinesin-14/Ncd and cytoplasmic dynein are well designed to support the above proposed functions of these two motors in pole focusing. Cytoplasmic dynein is a fast, processive motor. Thus, small numbers of dyneins could rapidly transport K fibers along C-MTs. In contrast, Ncd is nonprocessive, slow motor that is not designed for cargo transport. Instead, its ability to bind two microtubules and its slow motor activity makes it an effective cross-bridger between microtubules in the spindle. These properties are likely to be advantageous for inter-K fiber cross-linking, as well as for crossbridging of C-MTs plus ends to K fiber. However, the rapid on-off rates of these cross-bridges, as shown by FRAP data, would still enable dynein to effectively transport the K fibers along the C-MTs (Goshima, 2005).

Phenotypic RNAi analyses may account for differences in the pole unfocusing phenotypes of Ncd or dynein depletions that have been described in the literature. Specifically, spindle architecture in the given system (e.g., the presence or absence of centrosome and differences in microtubule dynamics) may determine whether Ncd or dynein acts as the essential contributor to pole coalescence. For example, Ncd/kinesin-14 function, is particularly important for K fiber focusing, may become more crucial when centrosomes are detached or absent from the spindle. Consistent with this idea, the most dramatic pole unfocusing phenotypes for kinesin-14 mutations/depletions have been described in plant mitosis and animal meiosis, systems in which spindle assembly occurs through a centrosome-independent mechanism and in which interactions between C-MTs and K fibers are simply absent. However, dynein also is likely to play crucial roles in pole coalescence in some acentrosomal spindles, as shown convincingly in Xenopus extract system. In contrast, somatic animal cell mitosis utilizes centrosomes, and kinesin-14 is less important for pole focusing in such cells (e.g., Ncd is nonessential in fly development). Microtubule dynamics, specifically the relative number of K- to C-MTs in the bipolar spindle, also may alter the relative contribution of the two motors. For example, if C-MTs are very abundant, the high probability of close approximation of K- and C-MTs may enable dynein to easily link these two networks without any assistance from kinesin-14 motors at microtubule plus ends (Goshima, 2005).

An unexpected finding of this study is the microtubule plus end tracking of Ncd. Localization of a kinesin-14 motor protein to the plus end of interphase microtubules has been recently reported in plants. The accumulation of kinesin-14 at the plus end overlap zone in mitotic spindle has been shown but whether this localization reflects localization of plus ends of individual microtubules is not known. Nevertheless, this work does suggest that the plus end tracking in mitotic microtubules might be a broadly conserved feature of kinesin-14 motors. Yeast Kar3p also was shown to accumulate at the plus ends of microtubules at the shmoo, but it is more enriched on the depolymerizing microtubules, which is not observed for Ncd. Even though it was found that C-MTs are still dynamic after Ncd RNAi, it is possible that Ncd at the plus end also modulates microtubule dynamics, as does EB1 or other tip-localized proteins (Goshima, 2005).

The enhanced K fiber unfocusing in EB1 RNAi-treated cells, which displaces Ncd from plus ends but not K fibers, suggests that plus end tracking of Ncd may serve a function in pole focusing. It is proposed that plus end tracking of Ncd on newly nucleated C-MTs, as a 'capture factor,' facilitates their connection to K fibers, possibly using its second microtubule binding site located in its NH₂-terminal tail domain. This idea is analogous to a 'search and capture' model for how C-MTs find chromosomes. In this case, the tip-localized motor Ncd enables C-MTs to 'search' for and then 'capture' a second major microtubule network in the spindle, the K fibers. Ncd may generate a connection between K fiber and C-MTs temporary, and thereby facilitate the recruitment of minus end-directed transporter (primarily dynein but Ncd contributing as well) for the transport of K fibers. Additionally, Ncd at the plus end may act as a K fiber transporter once it binds, although this transport would be less efficient than that produced by fast and processive dynein motors. It is also noted that the simulations are two-dimensional and encounters between C-MTs and K fibers would become less likely in three dimensions, and one might expect the effect of a C-MT-mediated capture/transport mechanism to become more important under such circumstances (Goshima, 2005).

The microtubule plus end search-and-capture mechanism might apply to other aspects of metazoan cell division. For example, cross-linking interactions between antiparallel microtubules occurs at overlap zone of microtubules, and genetic study demonstrates that Ncd produces an inward force on antiparallel microtubules during early mitosis. Ncd at the tips of growing microtubules may act to capture microtubules that arise from the opposite pole. Another possible target of tip-localized Ncd may be free microtubules, which are either released from centrosomes or generated de novo in cytoplasm and are eventually incorporated into the spindle by a dynein-dependent transport process (Goshima, 2005).

Dynein-dynactin complex is essential for dendritic restriction of TM1-containing Drosophila Dscam

Many membrane proteins, including Drosophila Dscam, are enriched in dendrites or axons within neurons. However, little is known about how the differential distribution is established and maintained. Dscam isoforms carrying exon 17.1 (Dscam[TM1]) are largely restricted to dendrites, while Dscam isoforms with exon 17.2 (Dscam[TM2]) are enriched in axons. This study investigated the mechanisms underlying the dendritic targeting of Dscam[TM1]. Through forward genetic mosaic screens and by silencing specific genes via targeted RNAi, it was found that several genes, encoding various components of the dynein-dynactin complex, are required for restricting Dscam[TM1] to the mushroom body dendrites. In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl. Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites. The results of this study suggest multiple mechanisms of dendritic protein targeting. Notably, dynein-dynactin plays a role in excluding dendritic Dscam, but not Rdl, from axons by retrograde transport (Yang, 2008).

Multiple lines of evidence indicate that the dynein/dynactin complex has an important function in maintaining proper distribution of dendritic Dscam in MB neurons. First, mutations in three components (Lis1, Dmn and p24) of the dynein/dynactin complex were recovered based on mislocalization of dendritic Dscam through a MARCM-based genetic mosaic screen. Second, silencing other components of the complex with RNAi also resulted in mistargeting of dendritic Dscam to axons. Third, disrupting dynein/dynactin function with dominant-negative Glued reproduced the mislocalization phenotype. Further, newly synthesized Dscam[TM1] was preferentially targeted to dendrites. Interestingly, compromising dynein/dynactin function did not affect the targeting from cell bodies to dendrites but disrupted the continuous exclusion of dendritic Dscam from axons. Altogether, these findings show that dynein/dynactin normally acts to prevent Dscam[TM1] from entering axons by retrograde axonal transport (Yang, 2008).

Acute induction by TARGET, in which GAL4-dependent expression of UAS-transgene is acutely controlled by a temperature-sensitive GAL4 repressor, GAL80ts, revealed two mechanisms underlying the dendritic distribution of Dscam[TM1]. Newly synthesized Dscam[TM1] was largely excluded from axons, suggesting directed dendritic targeting and the involvement of selective transport in the dendritic distribution of Dscam[TM1]. Though dynein/dynactin is essential for restricting Dscam[TM1] to dendrites, knocking down dynein/dynactin function did not disrupt the directed dendritic targeting. This leads to the belief that dynein/dynactin is required for preventing dendritic Dscam from misdistributing into axons. When dynein/dynaction function was compromised, newly synthesized Dscam[TM1] remained consistently targeted to dendrites but later leaked into axons. Dendritic Dscam gradually filled the axons; and it took about six hours for Dscam[TM1] to reach the axon termini. This protracted process of mislocalization suggests that dendritic Dscam passively leaks into the axons, and that dynein/dynactin-mediated retrograde axonal transport normally acts to rapidly move leaked Dscam[TM1]-containing vesicles out of the axons. In summary, these phenomena not only demonstrate a dynein-dynactin-independent mechanism of selective transport that preferentially targets Dscam[TM1]-containing vesicles to dendrites, but also implicate the involvement of retrograde axonal transport in preventing accumulation of Dscam[TM1] in axons. These two independent mechanisms act together to ensure restriction of dendritic Dscam to the dendrites (Yang, 2008).

Although the dynein/dynactin complex is essential for maintaining dendritic distribution of Dscam[TM1], the results do not reveal whether mislocalized Dscam[TM1] is on the plasma membrane or in vesicles inside the cytoplasm. It is possible that dendritic Dscam passively leaks into axons either through membrane diffusion or mistargeting of vesicles. Since blocking endocytosis with temperature-sensitive shibire mutant showed no obvious effect on Dscam dendritic distribution, the model is favored that dynein/dynactin acts to prevent axonal accumulation of Dscam[TM1] by actively moving mistargeted Dscam[TM1]-containing vesicles out of axons by retrograde axonal transport (Yang, 2008).

Dscam[TM1]-containing cargos are primarily targeted to dendrites via a dynein/dynactin-independent process. In addition, they are effectively excluded from the axons by dynein/dynactin-mediated retrograde axonal transport. However, dynein/dynactin is not routinely needed for excluding dendritic proteins from the axons. Since no biological process can be carried out with absolute fidelity, it is conceivable that dendritic molecules of most kinds may accidentally leak into the axons. Some salvage mechanism(s) should exist for actively clearing mislocalized molecules to prevent any significant accumulation in the wrong places. One of the possibilities is that dynein/dynactin mediates retrograde axonal transport and can serve as a general mechanism for removing dendritic molecules out of axons. This hypothesis remains to be tested thoroughly. Nonetheless, blocking dynein/dynactin function did not affect the distribution of two other dendritic markers checked. Nod-β-gal is a reliable minus-end reporter of microtubules, and misdistribution of Nod-β-gal in MB axons has been shown in short stop mutant clones, in which microtubule polarity is perturbed. Absence of Nod-β-gal from the axons of dynein/dynactin mutant neurons demonstrates that the microtubules in axons remained uniformly polarized with minus ends pointing toward cell bodies, and rules out the possibility that dendritic Dscam became mislocalized due to abnormal microtubule organization. As to Rdl-HA, which, like Dscam[TM1], is a membrane protein, a lack of effect on its somatodendritic distribution indicates that dynein/dynactin is selectively involved in preventing dendritic Dscam from leaking into the axons. Diverse mechanisms may be utilized to efficiently clear different dendritic proteins in axons (Yang, 2008).

Regarding the mechanism(s) of selective transport, directed dendritic targeting apparently requires motor proteins that selectively move cargos toward the dendrites. Since dendrites, but not axons, carry microtubules with minus ends pointing away from cell bodies, potential candidates that underlie directed dendritic targeting include all minus-end-directed microtubule motors. Notably, dynein/dynactin is dispensable to the initial dendritic targeting of Dscam[TM1] or the continuous dendritic restriction of Rdl, arguing against any critical role for minus-end-directed dynein/dynactin in transporting cargos into the dendrites. Other microtubule motors that might support such directional movement include dendrite-specific plus-end-directed motors (e.g. KIF17 and KIF21B), though it remains mysterious how a plus-end-directed motor can be well restricted to dendrites. In theory, forward genetic mosaic screens will ultimately allow uncovering of the diverse mechanisms of dendritic protein targeting. Encouragingly, mutants have been obtained that exhibit different mislocalization phenotypes, further characterization of which should shed additional light on neuron polarity and its underlying cellular/molecular mechanisms. Notably, in DC-B9 mutant clones, mistargeted Dscam[TM1]::GFP existed abundantly in the MB peduncle, preferentially accumulated at the end of the peduncle, but never extended into the axon lobes. This intriguing phenotype suggests presence of distribution barriers not only in the beginning of axons but also at the junction between the proximal axon domain (peduncle) and the distal axon segment (lobe), and implies another possible mechanism for restricting Dscam[TM1] to the dendritic membrane (Yang, 2008).

Furthermore, the functional roles of each subunit of the dynein/dynactin complex have not been fully determined. Although several studies of the dynein light chains in mammalian cells indicate that dynein subunits can be functionally specialized, studies in Drosophila show that strong loss-of-function mutations in different dynein/dynactin subunits show extensive overlap in the resulting mutant phenotypes. The current data indicate that Lis1, Dmn, Glued, p24, p25, Dhc64C, Dhc62B, and Dlc90F all participate in the complete function of dynein/dynactin complex in maintaining dendritic distribution of Dscam. This result supports the idea that all the dynein/dynactin subunits work together to fulfill its diverse functions, and loss of any subunits may result in different degrees of similar dynein/dynactin-dysfunctional phenotypes (Yang, 2008).

With respect to Dscam targeting motifs, the cytoplasmic juxtamembrane domain of Dscam may dictate its TM-dependent subcellular localization. However, further structure-distribution analysis only allowed location of an axonal targeting motif to the cytoplasmic juxtamembrane region of TM2, leaving its dendritic targeting motif(s) still undetermined. In addition, using the same system it could not be determined whether any of the mutants recovered here also affects the axonal targeting of Dscam[TM2], since transgenic Dscam[TM2] becomes uniformly distributed upon overexpression following an analogous induction. The involvement of multiple mechanisms in targeting specific Dscams to specific neuronal domains further supports the notion that Dscam isoform compositions in the dendrites versus axons of the same neurons need to be independently regulated, elucidation of the physiological significance of which promises to shed new light on how the brain develops and operates (Yang, 2008).

In summary, this study has uncovered a scavenger mechanism for maintaining dendritic distribution of Dscam[TM1] and provide an in vivo model to study neuron polarity and differential protein targeting. On top of the many known functions of dynein/dynactin (including mitosis, vesicular transport, retrograde signaling, neuronal migration), dynein/dynactin helps restrict certain dendritic proteins to the somatodendritic domain of neurons by preventing them from spreading into the axons. Notably, multiple independent mechanisms act together to locate Dscam[TM1] to dendrites; and diverse mechanisms are utilized to target different dendritic proteins to the dendrites (Yang, 2008).

Dynein regulates epithelial polarity and the apical localization of stardust A mRNA

Intense investigation has identified an elaborate protein network controlling epithelial polarity. Although precise subcellular targeting of apical and basolateral determinants is required for epithelial architecture, little is known about how the individual determinant proteins become localized within the cell. Through a genetic screen for epithelial defects in the Drosophila follicle cells, it was found that the cytoplasmic Dynein motor is an essential regulator of apico-basal polarity. The data suggest that Dynein acts through the cytoplasmic scaffolding protein Stardust (Sdt) to localize the transmembrane protein Crumbs, in part through the apical targeting of specific sdt mRNA isoforms. The sdt mRNA localization signal maps to an alternatively spliced coding exon. Intriguingly, the presence or absence of this exon corresponds to a developmental switch in sdt mRNA localization in which apical transcripts are found only during early stages of epithelial development, while unlocalized transcripts predominate in mature epithelia. This work represents the first demonstration that Dynein is required for epithelial polarity and suggests that mRNA localization may have a functional role in the regulation of apico-basal organization. A unique mechanism is introduced in this study in which alternative splicing of a coding exon is used to control mRNA localization during development (Horne-Badovinac, 2008).

Dynein's role in MT-based apical transport has been studied in the epithelia of both mammals and flies, but an explicit link between Dynein and apico-basal polarity has not been found. One cause for this deficiency may be that Dynein is required for a number of essential cellular processes, making it difficult to study this motor under strong loss-of-function conditions. For instance, previous studies of Dynein function in Drosophila embryos have relied on combinations of hypomorphic Dhc alleles or injection of anti-Dhc antibodies which only partially block Dynein function. Notably, these manipulations have failed to produce epithelial polarity phenotypes. This study shows that strong loss of Dynein function in the FCs disrupts both molecular and morphological aspects of apico-basal polarity, providing the first direct evidence for the role of this motor in this key cell biological process. It is currently unclear why the FCs tolerate strong loss of Dynein function better than other tissues, but this property provides a unique opportunity to begin dissecting the many roles that Dynein is likely to play in epithelial organization (Horne-Badovinac, 2008).

How does Dynein regulate apico-basal polarity? Since Dynein is a minus-end directed MT motor and minus ends are apically oriented in epithelia, whether Dynein might ferry components of the Baz and/or the Crb complexes to their sites of action at the apical surface was investigated. Two pieces of data indicate that the Baz complex is not the primary Dynein cargo contributing to FC polarity. Although Baz is occasionally relocalized to the lateral FC surface in Dhc clones, most mutant cells display significant amounts of apical Baz. Furthermore, ECs in which the entire epithelium is mutant for baz or aPKC display multilayering at both EC poles, a phenotype that is distinct from the posterior multilayering in Dhc mutant epithelia. These observations are consistent with recent studies in the embryonic blastoderm, where it was shown that Dynein does play a role in Baz localization, but that it is not the only means by which this protein is targeted to the apical domain. Together, these data indicate that, while Dynein likely does play a role in the apical targeting of Baz in both the embryo and the FCs, Dynein's major contribution to FC apico-basal polarity corresponds to a different cargo (Horne-Badovinac, 2008).

The results favor a model in which Dynein works primarily through the Crb complex to influence epithelial polarity. When Dynein function is reduced in the FCs, Crb and Sdt disappear from the apical surface, even in Dhc cells that remain cuboidal. Moreover, the morphology of an egg chamber in which the entire epithelium is mutant for Dhc is quite similar to that seen for crb and sdt mutant egg chambers, as all three display multilayering predominantly in the posterior. A major challenge comes in deciphering which Crb complex components are specifically transported by Dynein. This study focused on the relationship between Dynein and Sdt because genetic interaction and molecular epistasis experiments indicate that loss of apical Sdt can account for many aspects of the Dhc polarity phenotype. The finding that the apical localization of sdt transcripts is Dynein-dependent suggests a mechanism by which Dynein could localize Sdt, in part, through the apical targeting and localized translation of its mRNA. sdt transcripts cannot be the only Crb complex component transported by Dynein, however, as the phenotype of sdt^EH681 mutant FCs does not recapitulate all aspects of the polarity phenotype of Dhc mutant clones. Interestingly, crb transcripts are also targeted to the apical FC cytoplasm in a Dynein-dependent manner, although at a later stage than sdt. This finding raises the possibility that, in addition to Baz, crb mRNA and/or Crb complex proteins represent other Dynein cargoes required for full epithelial polarization. Future work will be required to more finely dissect Dynein's complex contributions to the apical targeting of the Crb complex (Horne-Badovinac, 2008).

While investigating whether sdt mRNA was likely to be a primary Dynein cargo contributing to apico-basal polarity, the surprising discovery was made that the apical targeting of sdt transcripts is regulated through the alternative splicing of a coding exon, exon 3. Only two other genes are known in which transcript localization is regulated in this way. In both instances, however, the signal lies within the 3'UTR, so the splicing event does not affect protein structure. It is curious that alternative splicing of the sdt mRNA localization signal also deletes 433 amino acids from the protein. The role of the amino acids encoded by exon 3 in Sdt function is not yet known. These amino acids are not conserved in vertebrate Sdt homologs. Moreover, Sdt A and Sdt B bind the intracellular domain of Crb with equal efficiency in vitro and this work with the sdt^EH681 allele, as well as over-expression studies with the sdt A and sdt B transgenes indicate that both protein isoforms stabilize Crb in vivo. Although the possibility that exon 3 regulates both mRNA localization and protein function cannot be ruled out, together these observations suggest that the splicing of exon 3 may primarily regulate mRNA localization (Horne-Badovinac, 2008).

A potential role for Dynein-dependent apical targeting of sdt mRNA in epithelial polarity is supported by analysis of the relative contributions of the sdt A and sdt B isoforms. Although the lack of Sdt A in sdt^EH681 FC clones reduces Crb at the apical membrane, this deficit leads to relatively mild effects on other aspects of polarity. This apparent discrepancy can be explained by the extrinsic cue for apical identity that is provided to the FCs through their direct contact with the germline, which may compensate for reduced Crb complex function in this tissue. However, in the embryo, where no such cue is available, sdt^EH681 has a nearly null phenotype, which is rescued much more efficiently by sdt A than sdt B. Overall, these data suggest that the apical targeting of sdt transcripts may represent an important mechanism contributing to apico-basal polarity (Horne-Badovinac, 2008).

Interestingly, the apical targeting of sdt mRNA is developmentally regulated in both embryonic and adult epithelia. Specifically, it was shown that apically localized sdt transcripts are found only during early stages, while unlocalized transcripts predominate at later stages. Why is sdt transcript localization regulated in this way? It is tempting to speculate that apical transcripts are required primarily for the establishment of apico-basal polarity but not its maintenance. In reality, however, the functional distinction between the two phases of sdt mRNA localization is almost certainly more subtle. When apical sdt A transcripts are present, the epithelia are relatively immature; they tend to be proliferative, display incomplete junctional structures and have yet to adopt their final cell morphology. By contrast, when unlocalized sdt B transcripts predominate, the epithelia are more likely to be post-mitotic and highly differentiated. These observations raise the possibility that a concentrated pool of Sdt protein, generated by localized translation of apical transcripts, could function to stabilize Crb primarily during periods when cell polarity is labile, but that this dynamic regulatory mechanism would be dispensable in fully differentiated cells (Horne-Badovinac, 2008).

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