To study at which developmental stages Lis1 is expressed, developmental Northern analysis was performed and Lis1 mRNA was found to be present throughout the Drosophila life cycle with high levels in the ovary. Four major transcripts of Lis1 are detected in all developmental stages. From an ovarian cDNA library, cDNAs were isolated corresponding to each of the mRNAs; sequence analysis determined that they share the same coding region and 5' untranslated region. Thus the difference in size is likely to arise from the usage of alternative polyadenylation sites or alternative splicing in the 3' untranslated region. Mouse antiserum raised against the N-terminal 164 amino acids of Lis1 recognizes a single band at 45 kDa in extracts from every developmental stage of flies, as indicated by Western analysis. Unfortunately, the antiserum fails to recognize the native protein in whole-mount ovaries or embryos (Liu, 1999). Nevertheless, Lis1 is reported to be strongly expressed in the central nervous system in Drosophila (Liu, 1999).

To further define which cell types express Lis1 in the ovary, whole-mount mRNA in situ hybridization was performed using a Lis1 antisense probe. Lis1 transcripts are detected in all germline and somatic cells in the germarium except the terminal filament cells and in the egg chambers. From stage 4 to stage 7 egg chambers, LIS1 RNA accumulates in the oocytes. Only low levels of LIS1 RNA are detected in stage 8 and 9 egg chambers. The RNA was strongly expressed in nurse cells of stage 10 egg chambers, indicating that Lis1 may function in early embryogenesis. Indeed, LIS1 RNA is uniformly distributed in early embryos before the onset of zygotic gene expression, indicating maternal derivation. The results from Northern analysis and mRNA in situ hybridization experiments suggest that Lis1 may well be required throughout Drosophila development (Liu, 1999).

During oogenesis LIS1 mRNA is expressed primarily in germ-line cells. Low levels of LIS1 mRNA can be detected in the germarium in regions 1 and 2 and in both nurse cells and the oocyte in stage 2-4 egg chambers. LIS1 message is enriched in the oocyte during stages 5-7; however, in stage 8-10 egg chambers LIS1 mRNA accumulates at much higher levels in nurse cells. In embryos LIS1 transcript is most abundant at precellular blastoderm stages, suggesting the presence of a maternal contribution. LIS1 mRNA is ubiquitously distributed in early embryos; however, the levels decline after the onset of gastrulation. Following germ-band retraction it is enriched in the developing central nervous system. In third-instar larvae, low levels of LIS1 transcript can be detected in the brain hemispheres and imaginal discs (Lei, 2000).

Regulation of dynein localization and centrosome positioning by Lis-1 and asunder during Drosophila spermatogenesis.

Dynein, a microtubule motor complex, plays crucial roles in cell-cycle progression in many systems. The LIS1 accessory protein directly binds dynein, although its precise role in regulating dynein remains unclear. Mutation of human LIS1 causes lissencephaly, a developmental brain disorder. To gain insight into the in vivo functions of LIS1, a male-sterile allele of the Drosophila homolog of human LIS1 was characterized. Centrosomes do not properly detach from the cell cortex at the onset of meiosis in most Lis-1 spermatocytes; centrosomes that do break cortical associations fail to attach to the nucleus. In Lis-1 spermatids, loss of attachments between the nucleus, basal body and mitochondria were observed. The localization pattern of LIS-1 protein throughout Drosophila spermatogenesis mirrors that of dynein. Dynein recruitment to the nuclear surface and spindle poles was shown to be severely reduced in Lis-1 male germ cells. It is proposed that Lis-1 spermatogenesis phenotypes are due to loss of dynein regulation, since similar phenotypes were observed in flies null for Tctex-1, a dynein light chain. asunder (asun) was previously identified as another regulator of dynein localization and centrosome positioning during Drosophila spermatogenesis (Anderson, 2009). It is now reported that Lis-1 is a strong dominant enhancer of asun and that localization of LIS-1 in male germ cells is ASUN dependent. Drosophila LIS-1 and ASUN colocalize and coimmunoprecipitate from transfected cells, suggesting that they function within a common complex. A model is presented in which Lis-1 and asun cooperate to regulate dynein localization and centrosome positioning during Drosophila spermatogenesis (Sitaram, 2012).

Analysis of a hypomorphic, male-sterile allele of Lis-1 revealed that Lis-1 plays essential roles during Drosophila spermatogenesis. The data suggest that loss of dynein function is the root cause of the defects that were observe in Lis-1k11702 testes, as mutation of the dynein light chain gene tctex-1 (Dynein light chain 90F) phenocopies mutation of Lis-1. Based on their overlapping phenotypes in male germ cells, genetic interaction, colocalization and co-immunoprecipitation, a model is presented in which Lis-1 and asun cooperate to regulate dynein localization during spermatogenesis (Sitaram, 2012).

These observations suggest that centrosomes of Lis-1 spermatocytes remain attached to the cell cortex and fail to migrate to the nuclear surface at entry into meiotic prophase. The phenotype of persistent cortical centrosomes during meiotic divisions has been characterized in abnormal spindles and nudE testes, and the presence of cortical centrosomes has been noted in Lis-1k11702 metaphase spermatocytes in studies of nudE mutants. Dynein-dynactin and LIS-1 localize to the cell periphery in lower eukaryotes and cultured mammalian cells, as well as to the posterior cortex of Drosophila oocytes. However, no enrichment of dynein-dynactin or LIS-1 at the cortex of Drosophila spermatocytes has been detected. Cortical dynein has been implicated in regulation of mitotic spindle orientation in several systems, although the mechanism is not clear. These data suggest that dynein and LIS-1 are required in spermatocytes to release centrosomes from the cortex prior to meiotic entry (Sitaram, 2012).

Lis-1 spermatocytes exhibit free centrosomes, albeit at a much lower frequency than the phenotype of cortical centrosomes. Detachment of centrosomes from the cortex of primary spermatocytes is an earlier step in male meiosis than reassociation of the centrosomes with the nuclear surface at G2/M; hence, a failure of centrosomes to detach from the cortex is likely to mask a subsequent failure of nucleus-centrosome coupling. LIS-1 colocalizes with dynein-dynactin at the nuclear surface, and localization of dynein-dynactin to this site is severely impaired in Lis-1 spermatocytes and spermatids. Dynein-dynactin anchored at the nuclear surface has previously been implicated in mediating interactions between the nucleus and centrosomes during both mitotic and meiotic cell cycles. It is proposed that defects in nucleus-centrosome coupling in Lis-1 spermatocytes stem from disruption in localization of dynein-dynactin to the nuclear surface (Sitaram, 2012).

Previous studies in other systems concerning the role of LIS1 in dynein-dynactin recruitment to the nuclear surface have yielded conflicting results. In C. elegans embryos, dynein-dynactin was reported to localize normally to this site in the absence of Lis-1. In mammalian neural stem cells, however, Lis1 was shown to be required for recruitment of dynein to the nuclear surface at prophase entry. Similarly, it was observed that severe reduction of perinuclear dynein-dynactin in Drosophila Lis-1 spermatocytes at meiotic onset, suggesting that Lis-1 is required for this process. Conversely, normal levels of Drosophila LIS-1 were found at the nuclear surface of tctex-1 spermatocytes; thus, dynein-dynactin does not appear to be reciprocally required for LIS-1 recruitment to this site. This finding of reduced levels of dynein heavy chain on the nuclear surface of tctex-1 spermatocytes suggest that Tctex-1 light chain plays a specific role in localizing dynein complexes to the nuclear surface; alternatively, complex integrity may be compromised in tctex-1 mutants (Sitaram, 2012).

Previously reported has been the finding that asun regulates dynein localization during Drosophila spermatogenesis (Anderson, 2009). The characterization of the hypomorphic Lis-1k11702 allele and the null asun93 allele during Drosophila male meiosis reveals overlapping but distinct phenotypes. Lis-1k11702 spermatocytes exhibit two classes of centrosome positioning defects: cortical (major phenotype) and free centrosomes (minor phenotype). By contrast, although most asun93 spermatocytes have free centrosomes, they do not share with Lis-1k11702 spermatocytes the phenotype of cortical centrosomes. These observations suggest that the role of asun in spermatocytes is limited to events at the nuclear surface, whereas Lis-1 additionally regulates cortical events. asun93 spermatocytes undergo severe prophase arrest, possibly owing to failure of astral microtubules of free centrosomes to promote nuclear envelope breakdown. In Lis-1k11702 spermatocytes, however, meiosis apparently progresses on schedule despite cortical positioning of centrosomes. The high percentage of asun93 spermatids with increased numbers of variably sized nuclei, probably a consequence of cytokinesis and chromosome segregation defects, are also absent in Lis-1k11702 testes. These observations suggest that spindle formation and normal progression through male meiosis require centrosomes to be anchored, either to the nuclear surface or the cortex (Sitaram, 2012).

Hypomorphic Lis-1k11702 and null asun93 round spermatids also show similarities and differences in their phenotypes. Both genes are required for recruitment of dynein-dynactin to the nuclear surface; this pool of dynein probably mediates nucleus-basal body and nucleus-Nebenkern attachments, which are defective in both mutants. Genes encoding Spag4 (a SUN protein), Yuri Gagarin (a coiled-coil protein) and GLD2 [a poly(A) polymerase] are required for nucleus-basal body coupling in spermatids, although it is not known whether they interact with ASUN or LIS-1 in this process. The current studies suggest that Lis-1, but not asun, is required for proper Nebenkern shaping and Nebenkern-basal body association; these functions might be mediated by dynein/microtubules acting at the Nebenkern surface. Nebenkerne are generated through fusion of mitochondria following Drosophila male meiosis. Two Nebenkerne bodies are occasionally present in Lis-1 and tctex-1 spermatids, implicating dynein in regulation of mitochondrial aggregation at this stage. Together, these observations suggest that the role of asun in spermatids is limited to events at the nuclear surface, whereas Lis-1 plays additional roles in regulating Nebenkerne (Sitaram, 2012).

Based on the studies of hypomorphic Lis-1k11702 and null asun93 mutant testes, a model is proposed in which LIS-1 is required for several dynein-mediated processes during Drosophila spermatogenesis, and ASUN is required for the subset of these processes that involve the nuclear surface. Both LIS-1 and ASUN promote recruitment of dynein-dynactin to the nuclear surface of spermatocytes and spermatids. The strong genetic interaction that was observe between Lis-1 and asun suggests that they cooperate in regulating dynein localization during spermatogenesis; the finding that LIS-1 accumulation on the nuclear surface is lost in asun male germ cells provides further support for this notion. The observed colocalization and coimmunoprecipitation of LIS-1 and ASUN suggest that they function within a shared complex to promote dynein-dynactin recruitment to the nuclear surface. Not interaction between Drosophila LIS-1 and ASUN proteins was detected by in vitro binding or yeast two-hybrid assays, suggesting that their association may be mediated by another protein(s) rather than being direct. Future studies on the nature of the ASUN-LIS-1 interaction should help elucidate the mechanism by which dynein-dynactin localizes to the nuclear surface during spermatogenesis (Sitaram, 2012).

Several proteins that promote dynein recruitment and centrosomal tethering to the nuclear surface have been identified. In C. elegans embryos, the KASH-domain protein ZYG-12, which localizes to the outer nuclear membrane and binds the inner nuclear membrane protein SUN-1, is required for these events. Another KASH-domain protein, Syne/Nesprin-1/2 (see Drosophila Nesprin), works in concert with SUN-1/2 to mediate nucleus-centrosome interactions during mammalian neuronal migration. Two additional pathways required for dynein recruitment to the nuclear surface at prophase have recently been identified in cultured mammalian cells. BicD2 binds dynein and anchors it to the nuclear envelope via its interaction with a nuclear pore complex protein, RanBP2. Similarly, CENP-F and NudE/EL act as a bridge between dynein and Nup133. It has not yet been determined whether mammalian LIS1 and ASUN function within these pathways or whether they act via a parallel mechanism to promote dynein recruitment to the nuclear surface (Sitaram, 2012).

The finding that a single copy of Lis-1k11702 can drastically decrease the size of asunf02815 testes suggests potential roles for Lis- 1 and asun in regulating division of male germline stem cells of Drosophila, as loss of cell proliferation can lead to reduction of testes size. Interestingly, Lis-1 has been reported to regulate germline stem cell renewal in Drosophila ovaries. Orientation of the cleavage plane during male germline stem cell division requires proper migration of centrosomes along the nuclear surface, and misorientation of the plane can lead to stem cell loss. Given the importance of Lis-1 and asun in mediating nucleus-centrosome coupling in Drosophila spermatocytes, it is possible that these genes also cooperate to regulate centrosomes during stem cell divisions in testes. In humans, the LIS1 gene is dose sensitive during brain development, as the disorder lissencephaly results from deletion or mutation of a single copy. Lis-1 spermatogenesis phenotypes reported in this study were observed in flies homozygous for a hypomorphic Lis-1 allele; flies carrying one copy of this allele displayed many of the same phenotypes but to a lesser degree. These findings suggest that precise regulation of LIS- 1 protein levels is essential for normal development in Drosophila. A requirement for Lis1 during spermatogenesis is conserved in mammals. Deletion of a testis-specific splicing variant of Lis1 in mice blocks spermiogenesis and prevents spermatid differentiation. LIS1 and dynein were shown to partially colocalize around wild-type spermatid nuclei, but dynein localization in Lis1 testes was not assessed. It remains to be determined if the functions of LIS1 in mammalian spermatogenesis are mediated through dynein and if the ASUN homolog regulates LIS1 localization in this system (Sitaram, 2012).

Effects of Mutation or Deletion

To reveal its function, mutations were generated in the Lis1 gene. Based on the observation that Lis1 is expressed throughout development and that a null Lis1 mutation in the mouse is lethal, it was expected that Lis1 should be an essential gene also in Drosophila. Lis1 homozygous embryos hatch normally and the first instar larvae look indistinguishable from their heterozygous siblings. At the L2 stage, the development of the homozygotes is clearly retarded and they die 5-6 days later without phenotypically entering the L3 stage. The survival of Lis1 mutants through embryonic and early larval development is probably due to the contribution of maternal wild-type Lis1 protein, as directly demonstrated by Western analysis. When homozygous larvae were picked at L1 stage or early L2 stage, wild-type Lis1 protein was clearly detectable in the extract. But Lis1 protein is reduced or not detectable when the extracts are made from homozygotes close to death (Liu, 1999).

The expression pattern of Lis1 during oogenesis and early embryogenesis suggests that Lis1 may have important functions in the ovary and embryo. To investigate such a possibility, mosaic egg chambers whose germline lacks wild-type Lis1 were produced using the FLP/DFS technique. The germline phenotype in clones of all four Lis1 mutants were similar and failed to produce any eggs. Egg chambers specifically lacking Lis1 in germline cells and control egg chambers retaining Lis1 in both germline and follicle cells were compared. Control egg chambers invariably contain 15 nurse cells and one oocyte. Lis1 egg chambers often contain fewer than 16 cells and usually lack an oocyte. All the cells in the mutant Lis1 cysts appear to develop as nurse cells, as indicated by their polyploid nuclei. Lis mutation results in a 59-fold reduction in the frequency of oocyte formation. As a result of synchronized cystocyte division, there are either 1, 2, 4, 8 or 16 cells in developing wild-type cysts. However, in Lis1 mutant cysts, the cell numbers range consecutively from 1 to 16, indicating that the synchrony of germline cell divisions is disrupted in the absence of Lis1 germline function (Liu, 1999).

Quantitative differences in the number of cells in the Lis1 mutant cysts were observed, dependent on when the germline clones were induced. Fewer cells in the mutant cysts were observed the earlier the induction. When clones were induced at embryonic or first instar larval stages, the average nurse cell number in Lis1 egg chambers was 7.2. The average cell number increased to 11.9 with egg chambers containing mostly even numbers of nurse cells, when the induction was done at the third instar larval stage. The frequency of mutant clone formation was also increased significantly with late induction. However, the frequency of oocyte differentiation was similar in early and late induction. These results suggest that oocyte differentiation is more sensitive to the perturbation of Lis1 function than is cystocyte division. The weaker phenotype observed with later induction may be a reflection of the perdurance of wild-type Lis1 protein in germline stem cells. The relatively low frequency of clones resulting from early induction may be due to the death and growth arrest of germline stem cells or cystoblasts. Lis1 protein level in these cells is expected to be lower in early induced clones than in those induced later (Liu, 1999).

Mosaic egg chambers that contain wild-type germline cells surrounded by only mutant follicle cells produce normal germline cysts of 15 nurse cells and one oocyte. Thus, Lis1 function is not required in the follicle cells for normal cyst formation. These results clearly demonstrate that Lis1 is required in the germline but not in somatic follicle cells to regulate cystocyte division and oocyte differentiation (Liu, 1999).

The cyst defects observed in Lis1 mutants are similar to those of hts and alphalpha spectrin mutants. The hts and alpha spectrin genes encode membrane skeletal proteins that are components of the fusome, and mutations in either gene abolish fusome formation. The absence of a fusome is thought to block the rapid and synchronous cystocyte divisions in both mutants. The morphology of fusomes was examined in cysts mutant for Lis1 by double-staining the egg chambers with anti-beta-galactosidase antibody and monoclonal anti-Hts antibody 1B1. In a wild-type germarium, a spherical fusome, also called spectrosome (ss), is found in germline stem cells. The fusome (f) grows from a spherical fusome in the cystoblast into a polarized branched structure in 2-, 3-, 8- and 16-cell cysts (Liu, 1999). Several major defects in Lis1 mutant cysts have been observed. (1) Some mutant cystoblasts fail to divide, while their fusomes continue to grow to a larger sphere and their nuclei sometimes become polyploid, suggesting that Lis1 is required for cystoblast division (2) When all germline cells in a germarium are mutant for Lis1, the number of developing cysts is dramatically reduced compared with a wild-type germarium, suggesting that the division of germline stem cells is also retarded in Lis1 mutants. (3) Branched fusomes in the Lis1 mutant cysts are often thinner and fragmented as compared to the intact, smoothly branched fusomes in wild-type cysts. The DNA staining of these mutant cysts indicates that the nuclear appearance of most cystocytes is normal, although some cystocytes appear arrested in cell division and the nuclei become prematurely polyploid. (4) Finally, fusomes in more posterior regions of the germarium are sometimes absent and, in all these cysts, the nuclei are often polyploid or apoptotic as determined by DNA staining. Most likely, these cysts are growth-arrested based on their location in the more posterior region of the germarium, eventually degenerating the fusomes (Liu, 1999).

Sometimes, cysts with apparently normal fusomes are also detected. However, they probably do not function normally, since virtually no mutant egg chambers with a normal complement of 15 nurse cells and one oocyte were observed. It is concluded that the fusome is defective in the absence of Lis1 protein. The wide spectrum of fusome phenotypes may result from the different levels of perdurance of Lis1 protein in germline stem cells after the clone is induced (Liu, 1999).

Lis1mutant egg chambers have packaging defects About 5% of the mutant cysts are compound egg chambers that contain a wild-type cyst and some mutant cells. These hybrid cysts are almost always found in association with a mutant cyst. This condition occurred even in egg chambers that were surrounded by wild-type follicle cells. There were usually fewer than 16 combined Lis1 cells in the two adjacent mutant cysts. The mutant cells in the compound egg chamber are not transient clones generated during cystocyte division because the size of the mutant nuclei is the same in the two neighboring mutant egg chambers and differs in size from the wild-type nurse cells in the same egg chamber. Also, the wild-type cyst in the hybrid egg chamber contains a full complement of 15 nurse cells and one oocyte (Liu, 1999).

The likely explanation for this packaging defect is that the Lis1 cysts are unstable and break into smaller groups of interconnected cells during or shortly after the cystocyte divisions. It is possible that Lis1 is required for normal functions of the cyst cytoskeleton, important for maintaining the integrity of a cyst. Alternatively, the formation of compound egg chambers is due to mis-signaling between the Lis1 germline cells and the wild-type follicle or wild-type germline cells (Liu, 1999).

Animals homozygous for EMS-induced DLis1 alleles isolated in a screen, as well as the P-element mutant DLis113209, are late larval or early pupal lethals. However, flies bearing these alleles in trans to the weak DLis111702 allele are viable, but male and female sterile. Eggs laid by such transheterozygous females show defects in the location and morphology of the dorsal appendages. Dorsal appendages are paired paddle-shaped structures that arise from the dorsal-anterior side of the eggshell and are used by the embryo for respiration. Defects in dorsal-ventral as well as anterior-posterior patterning during oogenesis are often associated with alterations in the morphology or placement of the dorsal appendages (Lei, 2000).

Eggs laid by females carrying a copy of DLis111702 in trans to three different EMS alleles were examined. Based on the severity of the dorsal appendage phenotype the mutant eggs were placed in five categories. Class 1 comprises eggs with apparently normal dorsal appendages, in both their structure and their position. In Class 2 eggs the appendages are located closer than normal but remained separate. A third class consists of eggs in which the dorsal appendages are partially fused. In Class 4 eggs the dorsal appendages are fused along their entire length to form a single structure. In a small fraction of the eggs laid by DLis1 mutant mothers, the fused dorsal appendages are severely reduced in size and displaced posteriorly. These eggs constitute Class 5. All three DLis1 alleles tested in trans to DLis111702 show similar defects, although the severity of the phenotype varies in an allele-specific manner. In all cases, greater than 68% of the eggs show fused dorsal appendages typical of Classes 4 and 5. By comparison, females carrying the EMS alleles or DLis111702 in trans to wildtype lay morphologically normal eggs (Lei, 2000).

In normal development, the follicle cells provide spatial cues that are required to establish the dorsal-ventral axis of the embryo. Thus many mutations that alter eggshell polarity also perturb patterning of the embryo. In order to determine whether reduction in DLis1 activity results in ventralization of the embryos, DLis13.1.2/DLis111702 transheterozygous females were crossed to wild-type males and the development of the eggs were monitored under halocarbon oil. Fewer than 20% of the eggs laid undergo cellularization and gastrulation. Of these, about half (10% of the total) show obvious signs of ventralization. During early gastrulation the cephalic furrow that normally occupies a lateral position is displaced dorsally, indicating that the dorsal cells have acquired a more ventral identity. DLis1 mutants also show defects in germ-band extension consistent with a shift in cell fate along the dorsal-ventral axis. Germ-band extension is delayed in mutant embryos with respect to wildtype and does not extend as far anteriorly. At later stages, the germ-band fails to extend completely and instead invaginates into the interior of the embryo. However, the majority of these embryos fail to complete embryogenesis. This was confirmed by examining cuticle preparations of egg lays from DLis1 mutant mothers. Only a small fraction of the embryos differentiate a cuticle, all of which are wild-type in appearance (Lei, 2000).

The defects observed in DLis1 transheterozygotes are similar to those caused by reduction in EGF signaling. Genetic interactions between DLis1 and components of the EGF signaling pathway, such as grk, torpedo (top), and cornichon (cni), were tested. A strong enhancement of the eggshell phenotype is observed in flies double heterozygous for DLis111702 and the strong grkHK36 allele. About 68% of the eggs laid by such females showed completely fused dorsal appendages typical of Classes 4 and 5. Similar phenotypes have been observed with other Lis1 alleles in trans to grkHK36. Flies double heterozygous for DLis1 and topCO, a strong loss-of-function mutation in the Egf receptor, also display a significant increase in the frequency of fused dorsal appendages in comparison to the control females. In contrast, only weak interactions have been detected between DLis1 and the cniAR and cniAA alleles (Lei, 2000.

The ventralized chorion phenotype resulting from reduction in Lis1 activity, as well as the genetic interactions observed between Lis1 and components of the Egf signaling pathway, raised the possibility that Lis1 is required for localization of the Grk ligand. In order to test this, the distribution of GRK mRNA was examined in ovaries from females transheterozygous for DLis111702 and different Lis1 alleles. In wild-type egg chambers from stages 1-7, GRK transcript is localized in a crescent between the oocyte nucleus and the posterior of the oocyte. At stage 8, the microtubule cytoskeleton undergoes a reorientation and the oocyte nucleus migrates to the anterior. Consequently GRK mRNA accumulates transiently along the anterior margin of the oocyte and from late stage 8 onward is tightly localized around the dorsal anterior side of the oocyte nucleus. In Lis1 mutant ovaries, no abnormalities are observed in GRK mRNA localization during stages 1-7. However, from stage 8 onward GRK message is mislocalized in 10%-20% of mutant egg chambers, depending on the strength of the allelic combination. Most frequently, the transcript is less tightly confined to the anterior cortex compared to wildtype. In about 5% of the egg chambers, GRK mRNA is detected in the middle of the oocyte rather than at the anterior, in association with an abnormally positioned oocyte nucleus. Ovaries from Lis1 trans-heterozygotes were also stained with polyclonal antisera against Grk protein to determine the extent to which localization of the ligand was perturbed. Consistent with the effects on GRK transcript, Grk protein distribution is essentially normal in early egg chambers. However, from stage 8 onward, Grk protein appears to be less tightly localized or mislocalized in Lis1 mutant egg chambers. In addition, the level of staining is lower than in comparably staged wild-type oocytes (Lei, 2000).

The preceding results indicate that the altered distribution of GRK mRNA could be a consequence of the mislocalization of the oocyte nucleus. Therefore the position of the germinal vesicle was directly examined in Lis1 mutant egg chambers. A P-element enhancer trap line that drives beta-galactosidase expression in the nuclei of germ-line cells was crossed into a Lis1 mutant background. Ovaries from DLis111702/DLis13.1.2 flies were dissected and lacZ expression in the oocyte nucleus was visualized by activity staining. In ~8% of stage 9-10 egg chambers, the nucleus was misplaced and located approximately midway along the anterior-posterior axis of the oocyte. The frequency of nuclear mislocalization correlates well with the most severe fused dorsal appendage phenotype and the instances of mislocalization of GRK mRNA to the center of the oocyte. An additional 13% of the egg chambers showed less severe but detectable displacement of the oocyte nucleus. Similar effects were observed in eggs from females of the genotype DLis111702/DLis18.25.3 and DLis111702 /DLis111.4.13 (Lei, 2000).

The mislocalization of GRK mRNA in Lis1 transheterozygotes could result from defects in the machinery involved in general transcript localization during oogenesis. In this event one might expect that other localized mRNAs would be aberrantly distributed in Lis1 ovaries. To test this idea the distributions of Bicoid and Oskar, two well-characterized transcripts that are normally localized to opposite ends of the mature oocyte, were examined. In wild-type stage 8-10 egg chambers, BCD mRNA is confined to the anterior margin of the oocyte, while OSK mRNA is present at the posterior pole of the oocyte. No difference in the abundance or localization of either OSK or BCD transcripts was observed in ovaries from Lis1 transheterozygotes (Lei, 2000).

In order to determine whether loss of Lis1 activity affects some global aspect of the microtubule cytoskeleton the distribution of a protein containing the motor domain of the plus-end-directed microtubule motor kinesin fused to beta-galactosidase (Kin:betagal) was examined. Kin:betagal protein accumulates at the plus ends of microtubules in the oocyte as well as in neurons and columnar epithelial cells, and its localization has been used as a sensitive assay for the polarity and integrity of the microtubule network. In wild-type egg chambers Kin:betagal is transiently localized to the posterior of the oocyte during stages 8-9. This localization is lost at stage 10, with the initiation of cytoplasmic streaming in the oocyte. Stage 9 egg chambers from DLis111702/DLis13.1.2 females were examined and Kin:betagal was found tightly focused to the posterior in a majority of the egg chambers (92%). In the remaining cases the fusion protein was present at the posterior of the oocyte but less tightly localized. In comparison, Kin:betagal was found at the posterior in 100% of wild-type stage 9 egg chambers examined, suggesting that the microtubule cytoskeleton and kinesin activity are largely unaffected in DLis111702/DLis13.1.2 egg chambers (Lei, 2000).

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 DLis18.25.3/DLis111702 ;Gl1/+ adults. Thus the genetic interactions between Dhc64C, Gl and Lis1 are consistent with a functional relationship between these genes (Lei, 2000).

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/Dynactin in spindle assembly and mitotic checkpoint control

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. This study generated and/or characterized mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, an improved time-lapse video microscopy technique was developed that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, GFP-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: initially promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both C. elegans and mammals. It is concluded that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release (Siller, 2005).

This study shows that both Lis1 and Gl are enriched on centrosomes/spindle poles in wild type neuroblasts, and Lis1/Gl are required for centrosome separation in prophase neuroblasts. A role for centrosome separation has been reported for dynein in Drosophila embryos, dynein in mammalian cells, and dynein/dynactin/Lis1 in C. elegans blastomeres. However, the exact mechanism by which they promote centrosome separation is unclear. One proposed model suggests that dynein may promote centrosome separation by generating pulling forces on astral MTs attached to the cortex or cytoplasmic structures. Alternatively, dynein associated with the nuclear envelope may exert pulling forces on astral MTs to promote centrosome separation. No GFP-Lis1 was detected on the nuclear envelope or at the neuroblast cortex, although it is possible that high cytoplasmic levels mask low levels of Lis1/dynactin at these sites. Thus, it remains unclear how Lis1/Gl promotes centrosome separation in neuroblasts. Centrosome separation is not completely blocked in Lis1 or Gl mutant neuroblasts, either due to residual amounts of maternal protein or due to the presence of a Lis1/dynactin/dynein-independent pathway. Interestingly, cortical non-muscle myosin II has been shown to contribute to centrosome separation in some cell types, raising the possibility that Lis1/dynactin/dynein and myosin II play partially redundant roles in neuroblast centrosome separation (Siller, 2005).

These observations further support a role for centrosomal/spindle pole-associated Lis1/Gl in spindle assembly, spindle pole focusing, and centrosome attachment in prometaphase and metaphase neuroblasts. Detachment of centrosomes from the spindle has been observed in dynein mutants in Drosophila, and in mammalian cells with reduced dynein or dynactin function. These findings show that Lis1 and dynactin act as cofactors for dynein-dependent focusing of spindle poles and attachment of spindle MTs minusends to centrosomes. In vertebrate cells dynein/dynactin is thought to contribute to focusing of spindle poles and attaching MT-minus ends to centrosomes by transporting pericentriolar proteins and MT-binding proteins, such as NuMA, to centrosomes. Although no clear NuMA orthologue is encoded in the Drosophila genome, a dynein/dynactin/Lis1 complex may contribute to spindle pole focusing by concentrating other MT cross-linking proteins with NuMA-like function at spindle MT minus ends (Siller, 2005).

Gl and Lis1 mutant neuroblasts occasionally form multipolar spindles and have more than two centrosome-like Centrosomin/gamma-tubulin structures. Due to the lack of Drosophila centriolar markers it was not possible to determine whether these extra centrosomelike structures contained centrioles. Multipolar spindles have also been observed in mammalian cells overexpressing Lis1 protein or in which Lis1 function was reduced. Time-lapse analysis of Lis1 mutant neuroblasts reveal occasional co-segregation of both centrosomes into the neuroblast as a consequence of incomplete centrosome separation and centrosome detachment from the spindle. Such a mis-segregation event may be followed by duplication of both centrosomes during the next cell cycle leading to supernumerary centrosomes. Alternatively, extra centrosomes in Lis1 and Gl mutant neuroblasts may be due to uncoupling of centrosome duplication from the cell cycle or centrosome fragmentation (Siller, 2005).

Time-lapse imaging experiments show that loss of Lis1/Gl in neuroblasts results in extension of both prometaphase and metaphase. Prometaphase in Lis1 mutant neuroblasts is characterized by delayed congression of chromosomes to the equatorial plate: this is likely to be largely due to inefficient kinetochore capturing as an indirect result of spindle assembly defects. Importantly, in Lis1 mutant neuroblasts congression of all chromosomes into a tight metaphase plate eventually occurs, suggesting that Lis1/Gl are not absolutely critical for MT/kinetochore attachment per se (Siller, 2005).

In addition, severe delays were observed in metaphase-to-anaphase transition. A few of these neuroblasts showed individual chromosomes that were transiently lost from and recongressed to the metaphase plate. Thus, consistent with findings in mammalian cells, Lis1 appears to play some role in maintaining stable chromosome alignment in metaphase neuroblasts. However, in contrast to mammalian studies, it was found that loss of Lis1 function causes delays in metaphase-to-anaphase transition even when all chromosomes stay aligned in a tight metaphase plate. Thus, mitotic checkpoint activity remains high even after apparent bipolar kinetochore attachment. Two defects appear to contribute to prolonged checkpoint activity in Lis1 mutant metaphase neuroblasts: reduced inter-kinetochore tension and failure to transport checkpoint proteins (e.g., Rod) off kinetochores. Reduced inter-kinetochore tension may be due to lack of Lis1/dynactin on kinetochores or on spindle pole/MTs (which may affect forces acting on kinetochore pairs as a consequence of altered spindle morphology or MT dynamics). Defects in Rod checkpoint protein transport off kinetochores can be explained as a direct consequence of depletion of kinetochore-associated Lis1/dynactin/dynein motor complex, which in wild type cells is loaded with Rod at kinetochores. However, previous studies have indicated that Rod and Zw10 are removed from kinetochores in response to inter-kinetochore tension not MT-attachment. Therefore, in addition to its direct role as a 'carrier', Lis1/dynactin/dynein may also play an indirect role in modulating Rod transport by generating the inter-kinetochore tension required to trigger initiation of Rod streaming (Siller, 2005).

In summary, the data is consistent with and extends a model recently proposed for dynein function in checkpoint protein transport in Drosophila and mammalian cells. According to this model a Lis1/dynactin/dyneinRod/Zw10 complex, pre-assembled on unattached kinetochores, is critical for timely anaphase onset by promoting poleward streaming of checkpoint proteins away from kinetochores after correct kinetochore-MT attachment has occurred. The data demonstrate that in Drosophila, the Lis1 protein is an obligate component in this process. Although the Lis1-binding proteins NudE/Nudel have been implicated in facilitating dynein-dependent checkpoint protein transport, it remains to be directly tested whether Lis1 has a similar function in mammalian cells (Siller, 2005).

What is the link between Rod/Zw10 and Mad2 in mitotic checkpoint function? Two recent studies demonstrate that the Rod/Zw10 complex is required for efficient recruitment of Mad2 to unattached kinetochores in mammalian cells and Drosophila neuroblasts, and that Mad2 and Rod colocalize during poleward transport along kMTs in Drosophila neuroblasts. Although a physical link between the Rod/Zw10 complex and Mad2 has not been discovered, an attractive model is that Rod/Zw10 links Mad2 to the Lis1/dynactin/dynein complex during poleward checkpoint protein transport (Siller, 2005).

Epistasis of Lis1/dynactin localization at kinetochores Lis1/dynactin localization is regulated differently in worm and mammalian cells. In mammalian cells, dynactin is required for Lis1 kinetochore association, but Lis1 is not required for dynactin localization. Whereas in C. elegans, Lis1 localizes to kinetochores independently of dynactin. Surprisingly, a third mechanism is found in Drosophila neuroblasts, where Lis1 and dynactin (Gl) are co-dependent for their localization to kinetochores. In neuroblasts, Lis1 may have a 'structural' role in recruiting dynein/dynactin to the kinetochore, in addition to stimulating dynein/dynactin activity. Thus, despite the conservation of the physical interaction between Lis1/dynein/dynactin, subcellular localization of these proteins can be regulated differently in various organisms (Siller, 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).

New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye.

The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Genetic screens have been performed using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, this study has analyzed the DGC modifiers' function in the developing Drosophila brain and eye. Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, the function of previously screened components and their genetic interaction with the DGC in this tissue were determined. This study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. It was determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. These data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton (Marrone, 2011).

The roles that Dys and Dg play in disease have been apparent for some time since their disruption or misregulation has been closely linked to various MDs. Dg depletion results in congenital muscular dystrophy-like brain malformations associated with layering defects and aberrant neuron migration. These defects arise due to extracellular matrix protein affinity problems that influence neuronal communication and result in learning and memory defects. Similar to brain layer formation, the migration of R1-R6 growth cones into the lamina occurs in a similar manner where glia cells that migrate from progenitor regions into the lamina provide a termination cue to innervating axons. In Drosophila Dys and Dg are expressed in the CNS, PNS and visual system and both proteins are required for proper photoreceptor axon guidance and rhabdomere elongation. This work has identified novel components implicated in the process of eye-neuron development. Moreover, it was found that Nrk, Mbl, Cam and Capt genetically interact with Dys and/or Dg in visual system establishment (Marrone, 2011).

The proteins Mbl, Capt, Cam, Robo, Lis1 and Nrk have been shown previously to be associated with the nervous system, and this study has additionally found that mutations in chif, SP2353, CG34400 and vimar cause abnormal photoreceptor axon pathfinding and/or differentiation phenotypes. Lis1 has been shown to bind microtubules in the growth cone, and the human Lis1 homologue is important for neuronal migration and when mutated causes Lissencephaly, a severe neuronal migration defect characterized by a smooth cerebral surface, mental retardation and seizures. This study has found that Lis1RNAi/GMR-Gal4 mutants have abnormally formed lamina plexuses, shortened rhabdomeres, and retinal vacuoles. Chif has been shown to regulate gene expression during egg shell development and is related to a DNA replication protein in yeast. The human ortholog for SP2353 (AGRN) is involved in congenital MD development. Drosophila SP2353 is a novel agrin-like protein that contains Laminin G domains, which makes it a potential new extracellular binding partner for Dg. CG34400 encodes for a protein homologues to human DFNB31 (Deafness, autosomal recessive 31) that causes congenital hearing impairment in DFNB31 deficient people and mouse whirlin, that causes deafness in the whirler mouse. Hearing loss has been as well demonstrated in association with various forms of muscular dystrophy. Vimar has been shown to regulate mitochondrial function via an increase in citrate synthase activity (Marrone, 2011).

Mbl is a Drosophila homologue of the human gene MBNL1. Mutations of this gene cause myotonic dystrophy and are associated with the RNA toxicity of CUG expansion diseases protein. This study shows that Mbl deficiency results in similar phenotypes to Dys and Dg loss of function, and to specifically interact with Dys in axon projections which is in accord with the Dys specific interaction seen in muscle. Dys has multiple isoforms, and the variability of DMD patients to have mental impairment has been linked in part to small Dys isoform mutations, which leads to speculation that Dys is a target for Mbl mediated splicing (Marrone, 2011).

Interestingly, Mbl isoforms have been demonstrated to regulate splicing of α-actinin, which belongs to the spectrin gene superfamily that also includes dystrophins. α-actinin and Capt, the Drosophila homologue of Cyclase-associated protein (CAP) are actin-binding proteins in the growth cone. Capt was first identified in yeast and is highly conserved throughout eukaryotic evolution. The main known function of Capt is to act in the process of actin recycling by working in conjunction with Actin Depolymerization Factor (ADF a.k.a. Cofilin) to help displace Cofilin from G-actin during depolymerization. It has already been reported that ADF/Cofilin has a role in retinal elongation. The actin cytoskeleton is a major internal structure that defines the morphology of neurons, and Capt has already been shown to be required to maintain PNS neuronal dendrite homeostasis in Drosophila via kinesin-mediated transport. Additionally, Capt has been found to lead to excessive actin filament polymerization in the eye disc and to cause premature differentiation of photoreceptors. The rate of axon projection is much slower than the rate of microtubule polymerization during axonal growth, implying that depolymerization/polymerization of actin is important during pathfinding. This study has also shown that Capt interacts with Dys and is necessary for proper projection of photoreceptor axons in the developing brain, and when absent, eyes develop with abnormal rhabdomeres. Furthermore, captRNAi mutants exhibit overgrowth of photoreceptor axons, and it is believed that a possible explanation for this is improper turnover of actin (Marrone, 2011).

Importantly, proteins that can be regulated by Ca2+ to organize actin filament bundles and to promote filament turnover include α-actinin and (ADF)/Cofilin, respectively. Cam functions as an intracellular Ca2+ sensor, and when Ca2+-Cam was selectively disrupted in a subset of neurons in Drosophila embryos, stalls in axon extension and errors in growth cone guidance resulted. Actin turnover is highly regulated by Ca2+ levels, and many proteins are Ca2+-mediated to regulate motility and axon guidance. The results and those from prior studies suggest that Cam is a major functional player of Ca2+ regulation in growth cones. Since it was shown here that mutations in Cam and capt have similar phenotypes in photoreceptor axon pathfinding and rhabdomere development, it is postulated that actin dynamics is the link between these two proteins and the phenotypes described here. Due to the importance of Cam for actin dynamics, its interaction with both Dg and Dys suggests that the DGC coordinates the actin cytoskeleton in the developing eye (Marrone, 2011).

The last gene identified in this work is Nrk. Recently various kinases, channels and other enzymes have been shown to associate with the DGC, although only a few of these interactions have been confirmed in vivo. Since Nrk is a component found to interact with Dys in photoreceptor axon pathfinding, it is most likely that it functions as a receptor to sense guidance cues rather than as a molecule affecting actin cytoskeletal rearrangement. The data here hint that Dg and Nrk could be two receptors integral to transferring signals important for neuronal layering (Marrone, 2011).

It is concluded that dynamic rearrangement of the actin cytoskeleton is crucial for the central and peripheral nervous system establishment, which depends on proper neuron migration and differentiation. This process requires not only the cell autonomous regulation of neuron motility, but also the interaction between the migrating cell and its underlying substrate. This interaction is often dependent on the signaling transduced via the ECM. The DGC and other factors are believed to be mediators of actin dynamics in growing axons and during neuronal cell morphogenesis, and this study found components that interact with Dys and/or Dg in both of these activities (see The DGC coordinates actin cytoskeleton remodeling). Additionally, disruption in gene expression of these components results in the same phenotypes seen with Dys and Dg mutants in the developing and adult eye. The data lead to the conclusion that the DGC is involved in signaling to cause cytoskeletal rearrangement and actin turnover in growth cones. Since many cases of muscular dystrophies are associated with mental retardation, it is believed that it is important to understand the role of the DGC in axon migration because understanding of this process could aid in finding an adequate therapy for this aspect of the disease's physiology. Since the human brain continues to develop well after gestation, and evidence shows that nerves maintain plasticity throughout an individual's lifespan, therapies could be devised that reverse these defects after birth (Marrone, 2011).

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. This study examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function (Folker, 2012).

This study has used the Drosophila musculature to investigate the mechanisms that control muscle size and intracellular organization. Muscle length is regulated independently of the number of fusion events and demonstrate that perturbations that affect embryonic muscle length correlate with decreased larval muscle size and poor muscle function. Additionally, it was found that intracellular organization, specifically myonuclear positioning, is essential for muscle function. Moreover, it was shown that the length and intracellular organization of the myofiber are mechanistically independent. Although a number of factors link both processes, this study identified factors that contribute solely to muscle length or myonuclear position and demonstrates that each fature can be independently manipulate (Folker, 2012).

Dynein regulates both muscle length and myonuclear positioning. Some Dynein-interacting proteins, such as Dlc90F and Pins, are necessary for both processes. That these factors regulate both muscle length and myonuclear positioning suggests that specific aspects of muscle growth and the positioning of myonuclei can indeed be coordinated. However, Lis1 affects muscle length specifically, whereas CLIP-190 and Glued specifically affect myonuclear position. Within the contexts of muscle length and myonuclear position CLIP-190 and Lis1 do not genetically interact, illustrating that, although linked, the two processes are mechanistically distinct (Folker, 2012).

Identification of CLIP-190 and Lis1 as regulators of Dynein is not novel. CLIP-190 and Lis1 are known to interact with Dynein both physically and functionally, and they usually cooperate toward a single goal. This study provides the first example of CLIP-190 and Lis1 serving completely independent, Dynein-dependent functions (Folker, 2012).

Mechanistically, the data suggest that Dynein function, with respect to the regulation of muscle length and myonuclear positioning, is specified by Lis1 and CLIP-190 downstream of its localization. That Dynein localization to the myofiber pole is important is illustrated by pins (raps193) mutants in which Dynein does not accumulate at the myofiber pole, and defects were observed in both muscle length and myonuclear positioning. This suggests that Pins recruits and stabilizes Dynein at the cortex as it does during mitosis. Lis1 also affects Dynein localization, but in Lis1 mutants Dynein is localized to the muscle pole and is tightly restricted to the pole compared with controls. This is interpreted to mean that Lis1 is necessary for retrograde trafficking of Dynein away from the myofiber pole. It is hypothesized that, in the absence of Dynein retrograde trafficking, cellular components accumulate at the extending muscle end, thus inhibiting the trafficking of factors necessary for further directed growth. Thus, when Dynein is incapable of moving cargo away from the myofiber pole, muscles are shorter than in control embryos. Indeed, a similar correlation between retrograde trafficking and directed growth was recently reported during mechanosensory bristle growth and axonal transport. Interestingly, it was recently reported that decreased retrograde transport of Dynein results in longer processes in both fibroblasts and neurons in culture (Folker, 2012).

This contradictory finding raises several interesting questions. Is there an opposing pathway in muscle or is another aspect of muscle size increased? For example, does the length of the muscle impact its volume? Complications due to muscle contraction in the live embryo make such analysis difficult, however. Likewise, working in vivo on a dynamically changing tissue located 30-150 microm below the surface of the developing embryo presents challenges for imaging the rapid cellular processes that underlie motor activity, microtubule organization, organelle positioning and cell size. Nevertheless, the link between different aspects of cell size and their relationships to each other and to motor activity are being explored (Folker, 2012).

It is not clear why LT muscle growth/length is more sensitive to Dynein activity than the VL muscles in the embryo. A simple explanation is that the maternally loaded Dynein persists longer in the VL muscles than in the LT muscles. Additionally there might be a physical explanation. Although a cluster of potential tendon cells for the VL muscles exist, they are clustered at the segment border. Therefore, the size of the hemisegments, and thus the distance from segment border to segment border, determines muscle length. Conversely, the cluster of potential tendon cells for the LT muscles might be more broadly dispersed and the location of the muscle pole at the time of tendon specification determines the length of the muscle. Under this hypothesis, inefficient extension of the LT muscles would result in the muscles being shorter during tendon specification/maturation and therefore shorter throughout embryonic development. Alternatively, differences in guidance/signaling systems could explain why the LT muscles, but not the VL muscles, are shorter when Dynein activity is compromised. Different signaling mechanisms are employed by these different muscle types. For example, Derailed (Drl) plays a crucial role in the ability of LT muscles to recognize their target. Perhaps, altered trafficking of Drl or another factor in the signaling pathway causes slight, but significant, changes in LT muscle length (Folker, 2012).

It is interesting that the LT muscles are smaller in Dhc64C, Dlc90F, pins and Lis1 mutant embryos, but that other muscles are unaffected, whereas in larvae all muscles appear to be smaller. During the larval stages, muscles remain stably attached to tendon cells and grow through insulin signaling/Foxo- and dMyc-dependent pathways. The observations are intrepreted to mean that Dynein and its regulatory proteins Dlc90F, Pins and Lis1 contribute to insulin receptor-mediated muscle growth. Indeed, it would not be surprising to find that Dynein-dependent cellular trafficking is essential to that signaling pathway (Folker, 2012).

CLIP-190 does not dramatically affect Dynein localization, but it does affect microtubule organization, which, in turn, affects nuclear positioning. CLIP-190 mutant embryos have fewer microtubules at the myofiber pole, suggesting that, similar to its functions in other systems, the role of CLIP-190 is to stabilize microtubule-cortex interactions, which Dynein then uses to move nuclei towards the myofiber pole. The specification of Dynein function, downstream of its localization, is novel. Although phospho-regulation has been shown to alter Dynein function during mitosis and the possibility for competitive regulation of Dynein has been suggested, this is the first example in which Dynein at a single location has its activity modified through interactions with unique binding partners. The ability to specify Dynein function without dramatically altering its localization is likely to be an important factor during development when temporal constraints are high (Folker, 2012).

With regards to physiology, the small, but significant, changes in myonuclear positioning and muscle size seen in the embryo continue throughout larval development and are associated with impaired muscle function. Additionally, that the same defects and impairments are found in larvae that were depleted of Dynein, Lis1 or CLIP-190 specifically in the muscle shows that the effects are, at least in part, muscle specific (Folker, 2012).

Many muscle myopathies are characterized by smaller myofibers and mispositioned nuclei. However, it is unclear whether these pathologies are linked and which of these defects are paramount in causing the muscle weakness associated with these myopathies. This study has shown that in Drosophila these two processes are linked via the requirement for Dynein activity at the muscle pole. It was further shown that these two processes are mechanistically distinct, yet that both are necessary for muscle function. Together, these data suggest that therapeutics aimed at improving the functional capacity of diseased muscles must counteract effects on both muscle size and myonuclear positioning. This highlights Drosophila as an ideal model system with which to identify the genes and mechanisms required for distinct aspects of muscle morphogenesis and to shed light on key features of muscle disease (Folker, 2012).

Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth

Seamless tubes form intracellularly without cell-cell or autocellular junctions (see Labarsky, 2003). Such tubes have been described across phyla, but remain mysterious despite their simple architecture. In Drosophila, seamless tubes are found within tracheal terminal cells, which have dozens of branched protrusions extending hundreds of micrometres. This study has found that mutations in multiple components of the dynein motor complex block seamless tube growth, raising the possibility that the lumenal membrane forms through minus-end-directed transport of apical membrane components along microtubules. Growth of seamless tubes is polarized along the proximodistal axis by Rab35 and its apical membrane-localized GAP, Whacked. Strikingly, loss of whacked (or constitutive activation of Rab35) leads to tube overgrowth at terminal cell branch tips, whereas overexpression of Whacked (or dominant-negative Rab35) causes formation of ectopic tubes surrounding the terminal cell nucleus. Thus, vesicle trafficking has key roles in making and shaping seamless tubes (Schottenfeld-Roames, 2013).

Three tube types -- multicellular, autocellular and seamless -- are found in the Drosophila trachea. Most tracheal cells contribute to multicellular tubes or make themselves into unicellular tubes by wrapping around a lumenal space and forming autocellular adherens junctions, but two specialized tracheal cell types, fusion cells and terminal cells, make 'seamless' tubes. How seamless tubes are made and how they are shaped are largely unknown. One hypothesis holds that seamless tubes are built by 'cell hollowing', in which vesicles traffic to the centre of the cell and fuse to form an internal tube of apical membrane, whereas an alternative model proposes that apical membrane is extended internally from the site of intercellular adhesion. In both models, transport of apical membrane would probably play a key role. As terminal cells make seamless tubes continuously during larval life, they serve as an especially sensitive model system in which to dissect the genetic program (Schottenfeld-Roames, 2013).

Tracheal cells are initially organized into epithelial sacs with their apical surface facing the sac lumen. During tubulogenesis, γ-tubulin becomes localized to the lumenal membrane of each tracheal cell, generating microtubule networks oriented with minus ends towards the apical membrane. Terminal and fusion cells are first selected as tip cells that undergo a partial epithelial-to-mesenchymal transition and initiate branching morphogenesis: they lose all but one or two cell-cell contacts and become migratory. Branchless-FGF signalling induces a subpopulation of tip cells to differentiate as terminal cells. During larval life, terminal cells ramify on tissues spread across several hundred micrometres, with branching patterns that reflect local hypoxia. A single seamless tube forms within each branched extension of the terminal cell (Schottenfeld-Roames, 2013).

How trafficking contributes to seamless tube morphogenesis is unknown. Despite clues that vesicle transport plays a role in the genesis of seamless tubes, the tube morphogenesis genes remain elusive. This study characterized the cytoskeletal polarity of larval terminal cells, shows that a minus-end-directed microtubule motor complex is required for seamless tube growth, and characterizes mutations in whacked (wkd) that uncouple seamless tube growth from the normal spatial cues. Sequence analysis indicates that wkd encodes a RabGAP, and it was shown that Rab35 is the essential target of Wkd, and that together, Wkd and Rab35 can polarize the growth of seamless tubes (Schottenfeld-Roames, 2013).

Apical-basal polarity and cytoskeletal organization was examined in mature larval terminal cells. The lumenal membrane was decorated by puncta of Crumbs, a definitive apical membrane marker. Actin filaments were found enriched in three distinct subcellular domains: surrounding seamless tubes, decorating filopodia and outlining short stretches of basolateral membrane. The microtubule cytoskeleton also seemed polarized, with γ-tubulin lining the seamless tubes and enriched at tube tips. These data are consistent with tracheal studies in the embryo. EB1::GFP analyses of growing (plus-end) microtubules demonstrated that some are oriented towards the soma and others towards branch tips. Stable acetylated microtubules ran parallel to the tubes and extended beyond the lumen at branch tips where they may template tube growth. Consistent with such a role, microtubule-tract-associated fragments of apical membrane were observed distal to the blind ends of the seamless tubes. Filopodia extended past the stable microtubules as expected. These data indicate that mature terminal cells maintain the polarity and organization described for embryonic terminal cells. On the basis of γ-tubulin localization, it is inferred that a subset of microtubules is nucleated at the apical membrane, and that apically targeted transport along such microtubules would require minus-end motor proteins. Indeed, homozygous mutant Lissencephaly-1. As γ-tubulin lines the entire apical membrane, growth through minus-end-directed transport might be expected to occur all along the length of seamless tubes, and indeed, a pulse of CD8::GFP (transmembrane protein tagged with GFP) synthesis uniformly labelled the apical membrane as it first became detectable (Schottenfeld-Roames, 2013).

The cytoplasmic dynein motor complex drives minus-end-directed transport of intracellular vesicles in many cell types; to test for its requirement in seamless tube formation, terminal cells were examined mutant for any of four dynein motor complex genes: Dynein heavy chain 64C (Dhc64C), Dynein light intermediate chain (dlic), Dynactin p150 (Glued) and Lis-1. Mutant terminal cells showed a cell autonomous requirement for these genes. Mutant terminal cells had thin cytoplasmic branches that lacked air-filling, and antibody staining revealed that seamless tubes did not extend into these branches although acetylated microtubules often did. It was also noted that formation of filopodia at branch tips is disrupted in dynein motor complex mutants, which may account for the decreased number of branches in mutant terminal cells. Ectopic seamless tubes that were not air-filled were detected near the nucleus, as described below. Interestingly, discontinuous apical membrane fragments (similar to those in Lis-1 embryos) were found in terminal branches lacking seamless tubes, and were associated with microtubule tracts. Whereas γ-tubulin was enriched on truncated tubes and on these presumptive seamless tube intermediates, diffuse γ-tubulin staining was detected throughout the mutant cells, indicating that assembly of apical membrane is required to establish or maintain γ-tubulin localization. Likewise, Crumbs seemed reduced and aberrantly localized. Reduced levels of acetylated microtubule staining in these cells may reflect loss of apical γ-tubulin. Importantly, these data show that stable microtubules extend through cellular projections that lack seamless tubes. Thus, without minus-end-directed transport, stable microtubules are insufficient to promote seamless tube formation, but stable cellular projections are formed and maintained in the absence of seamless tubes (Schottenfeld-Roames, 2013).

In contrast to these defects in seamless tube generation, mutations in wkd confer overly exuberant tube growth. Examination of wkd terminal cell tips revealed a 'U-turn'; phenotype in which seamless tubes executed a series of 180 degree turns - the possibility is entertained that branch retraction, similar to that observed in talin mutants, could contribute to the U-turn defect (Schottenfeld-Roames, 2013).

Homozygous wkd animals survived until pharate adult stages, and, other than the seamless tube defects, had normal tracheal tubes at the third larval instar. Mosaic analysis revealed a terminal cell autonomous requirement for wkd. Mutant clones in multicellular tubes, and in unicellular tubes that lumenize by making autocellular adherens junctions, were of normal morphology. Strikingly, fusion cells, which also form seamless tubes, were unaffected by loss of wkd (Schottenfeld-Roames, 2013).

To determine the molecular nature of wkd, a positional cloning approach was taken. Mapping techniques defined a candidate gene interval of ~ 75 kilobases (kb). Focused was placed on CG5344 as it encodes a protein containing a TBC (Tre2/Bub2/Cdc16) domain characteristic of Rab GTPase-activating proteins, and hence was likely to participate in vesicular trafficking, a process that could lie at the heart of seamless tube formation. A single nucleotide change was identified that resulted in mis-sense (PC24) and non-sense (220) mutations in the CG5344 coding sequence. Pan-tracheal knockdown of wkd by RNA-mediated interference (RNAi) caused terminal cell-specific U-turn defects (other defects characteristic of the ethyl methanesulfonate (EMS)-induced alleles of wkd were detected at a low frequency). A genomic rescue construct for CG5344 rescued wkd mutants, confirming gene identity. On the basis of these results, it is concluded that wkd is CG5344 and that it probably regulates vesicular trafficking during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

To determine the Rab target(s) of Wkd regulation, whether tracheal expression of constitutively active 'GTP-locked'; Rab isoforms (henceforth, RabCA) might phenocopy wkd was investigated. RabCA for 31 of the 33 Drosophila Rabs were tested individually in the tracheal system. Rab35CA alone conferred terminal-cell-specific U-turns defects (Schottenfeld-Roames, 2013).

To evaluate Wkd overexpression, UAS-wkd was expressed in wild-type animals in a pan-tracheal pattern. Excess Wkd caused formation of ectopic seamless tubes surrounding the terminal cell nucleus. At higher levels of expression, small spheres of apical membrane were found adjacent to the nucleus and less abundantly at more distal sites. Consistent with Wkd regulation of vesicle trafficking by modulation of Rab35, expression of a dominant-negative Rab35 (henceforth, Rab35DN) caused formation of ectopic proximal tubules (Schottenfeld-Roames, 2013).

Attempts were made to determine whether Rab35 was the essential target of Wkd GAP activity. Wkd primary structure is equally conserved in three human RabGAPs. All three act as Rab35GAPs, although each has been proposed to have additional targets. To further determine if Wkd acts as a Rab35GAP, whether Rab35DN could suppress wkd mutants was examined; tracheal-specific expression of Rab35DN strongly suppressed the 'U-turn'; defects of wkd-null animals and, surprisingly, also rescued the lethality of wkd. Since mutant Rab35 isoforms phenocopy wkd gain and loss of function, Rab35DN bypasses the requirement for wkd and human Wkd orthologues are Rab35GAPs, it is concluded that the critical function of Wkd is as a GAP for Drosophila Rab35 (Schottenfeld-Roames, 2013).

In other systems Rab35 is implicated in polarized membrane addition to plasma membrane compartments - for example, immune synapse, cytokinetic furrow and so on - or, in actin regulation. A role for actin in fusion cell seamless tube formation has been proposed, so whether Wkd and Rab35 act by modulation of the terminal cell actin cytoskeleton was examined. As the actin-bundling protein Fascin (Drosophila singed) was recently identified biochemically as a Rab35 effector, a role of singed in terminal cell tubes was examined, but found no evidence was found for one. Furthermore, overexpression of Wkd, or of Rab35DN, did not significantly alter the terminal cell actin cytoskeleton, leading to the conclusion that actin regulation is not a primary function of Wkd/Rab35 during seamless tube morphogenesis (Schottenfeld-Roames, 2013).

The alternative model -- that Rab35 acts in polarized membrane addition -- was found to be attractive, because extra Rab35-GTP activity promoted seamless tube growth at branch tips whereas depletion of Rab35-GTP promoted tube growth at the cell soma. To test this model, advantage was taken of the information that expression of an activated Breathless-FGFR (lambdaBtl in terminal cells induces robust growth of ectopic seamless tubes surrounding the nucleus; whether growth of the ectopic tubes could be redirected from the soma to the branch tips was investigated by eliminating wkd. The activated FGFR phenotype was not altered in wkd heterozygotes, but in wkd mutant animals (or wkd-RNAi animals) the site of ectopic seamless tube growth was strikingly different. In some cells, extra tubes were found throughout the cell - in the soma and at branch tip - whereas in others extra tubes were present only at the branch tip. Thus, the position of seamless tube growth is dependent on Wkd activity, although Wkd itself is not essential for tube formation. These data provide evidence against branch retraction (as occurs in talin mutants) as the mechanism for generating a U-turn phenotype, because branch retraction would not redirect ectopic tube growth (Schottenfeld-Roames, 2013).

To better understand how Wkd and Rab35 determined the site of seamless tube growth, their subcellular distribution was examined. Pan-tracheal expression of mKate2-tagged Wkd (Wkd::mKate2) rescued wkd-null animals. The steady-state subcellular localization of Wkd::mKate2 was restricted to the lumenal membrane with higher accumulation at the growing tips of seamless tubes. At lower levels, cytoplasmic puncta of Wkd::mKate2 were noted that could reflect vesicular localization, as well as labeling of filopodia. It was found that YFP::Rab35 was distributed in a diffuse pattern throughout the terminal cell cytoplasm with some apical enrichment, and notable localization to filopodia. Substantial co-localization of Wkd::mKate2with YFP::Rab35 was found at the apical membrane, in cytoplasmic puncta, and in filopodia. Among endosomal Rabs, Rab35 seemed uniquely abundant within filopodia, and showed the greatest overlap with Wkd at the apical membrane. Substantial overlap was noted between Wkd/Rab35 and acetylated microtubules, including at positions distal to the blind end of seamless tubes. The enrichment of Wkd along seamless tubes indicates that Rab35 functions in an apical membrane trafficking event, leading to the speculation that recycling endosomes at filopodia might be targeted to the growing seamless tube by minus-end motor transport (Schottenfeld-Roames, 2013).

In a similar vein, it is speculated that vesicles might be transported from the soma towards branch tips in a process regulated by Wkd and Rab35. Disruption of such transport might explain why overexpression of Wkd leads to ectopic seamless tube growth in the soma. Whether Wkd::mKate2 localization was compromised in dynein motor complex mutants was examined. As these cells have branches that lack apical membrane/seamless tubes, disruption in the localization pattern of Wkd was anticipated, but it was wondered whether co-localization with acetylated tubulin would be intact, indicative of a microtubule association independent of dynein motor transport. It was found that Wkd::mKate2 is broadly distributed throughout the cytoplasm of dynein motor complex mutants, and does not show enrichment on acetylated microtubule tracts; indeed, substantial basal enrichment was detected of Wkd::mKate2. If Wkd/Rab35-dependent trafficking of apical vesicles was dynein motor complex dependent, ectopic seamless tubes would be expected in the soma of dynein motor complex mutants, similar to those seen with Wkd overexpression or expression of Rab35DN. In fact, such ectopic tubes were consistently found in the dynein motor complex mutants, consistent with dynein-dependent trafficking of Rab35 vesicles. It cannot be ruled out that these defects are due to dynein-dependent processes unrelated to Wkd and Rab35; however, whether the ectopic tubes could be redirected distally by expression of Rab35CA, or elimination of Wkd, was examined. The motor complex ectopic tube phenotype could not be altered, indicating that the phenotype does not arise as an indirect consequence of altered Wkd localization or Rab35 activity (Schottenfeld-Roames, 2013).

The roles of RabGAP proteins have started to become clear only in recent years. Historically, it has been difficult to determine which Rab proteins are substrates of specific RabGAPs. Tests of in vitro GAP activity produced conflicting results, and in some cases did not seem indicative of in vivo function. Indeed, the specificity of Carabin (also known as Wkd orthologue TBC1D10C) has been controversial: it was first shown to act as a RasGAP, whereas later studies indicate a Rab35-specific GAP activity. The in vivo genetic data for wkd, together with recent studies characterizing the function of all three human Wkd-like TBC protein, make a compelling case that this family of proteins acts as GAPs for Rab35. Furthermore, this study establishes a role for classical vesicle trafficking proteins in seamless tube growth. As seamless tubes, but not multicellular or autocellular tracheal tubes, are affected by mutations in wkd and Rab35, this study also establishes an in vivo cell-type-specific requirement for trafficking genes in tube morphogenesis (Schottenfeld-Roames, 2013).

It is concluded that Wkd and Rab35 regulate polarized growth of seamless tubes, and it is speculated that Wkd and Rab35 direct transport of apical membrane vesicles to the distal tip of terminal cell branches (when equilibrium is shifted towards active Rab35-GTP), or to a central location adjacent to the terminal cell nucleus (when equilibrium is shifted towards inactive Rab35-GDP). Analogous to its previously described roles in targeting vesicles to the immune synapse in T cells, the cytokinetic furrow in Drosophila S2 cells and the neuromuscular junction in motor neurons, Rab35 would promote transport of vesicles from a recycling endosome compartment to the apical membrane. It is further speculated that Breathless-FGFR activation at branch tips may couple terminal cell branching with seamless tube growth within that new branch (Schottenfeld-Roames, 2013).


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Lissencephaly-1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 October 2014

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