The expression profile of the 95F MHC gene (Jaguar) is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein (Kellerman, 1992).
Drosophila 95F is an unconventional myosin and the original member of class VI, which includes a homolog found in pig kidney. Some unconventional myosins have been suggested as mediators of some types of intracellular transport, but there is little direct evidence for this function. Transport of cytoplasmic particles have been observed in live Drosophila embryos in three dimensions using computational optical sectioning microscopy. Evidence is presented that this transport is actin-based, ATP-dependent and catalysed by the 95F myosin. This is the first direct observation of transport catalysed by an unconventional myosin in living cells (Mermall, 1994).
The 95F myosin, a class VI unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three-dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions led to aberrant nuclear divisions and to the nuclear defects observed. 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form (Mermall, 1995).
The density of Jaguar particles in neuroblasts is highest in prophase and metaphase, coinciding with Miranda translocalization and spindle rotation. Jar particles accumulate preferentially to the basal half in 45% of metaphase neuroblasts, whereas they are more homogeneously distributed in the rest. In telophase, Jar particles are inherited by the ganglion mother cell preferentially but are also seen in the neuroblast. The transient accumulation of Jar particles on the basal side in metaphase neuroblasts is consistent with its involvement in the transport of Miranda to the basal pole (Petritsch, 2002).
Jaguar is present in most if not all tissues throughout the lifetime of the fly. However, its level of expression in particular tissues is regulated, such that a subset of tissues express higher levels of protein at certain stages of embryonic development. The overall immunostaining pattern of CLIP-190, a Jaguar interacting protein, and Jaguar in stage 14-16 embryos is quite similar. When viewed at low magnification, both proteins are enriched in the anterior structures of the head, the posterior spiracles, and the ventral nerve cord (Lantz, 1998).
The most striking example of their colocalization is the enrichment of both proteins in the central nervous system (CNS) located on the ventral side of the embryo. The cell bodies of the central nervous system form a broad, flat, compact region that surrounds the ladder-like connectives (longitudinal tracts) and commissures (transversal fibers that cross the ventral midline between the connectives). The connectives and commissures are formed by axons from multiple neurons that are in large bundles as they run anterior, posterior, and across the midline to innervate their targets. Axons that leave the CNS do so at regular intervals, in a segmentally repeated manner. Both Jaguar and CLIP-190 are enriched in the axonal processes that make up the connectives and commissures relative to overall staining levels in the embryo and in the nerve cell bodies. This enrichment is also apparent in processes that exit the CNS. In contrast, the two are differentially enriched in what appear to be glial cells in different positions. Jaguar, but not CLIP-190, is enriched in the midline glial cells that serve as guide cells for the formation of the commissures. Conversely, CLIP-190 is enriched in several cells that lie at the extreme periphery of the CNS in a segmentally repeated pattern that correlates with where the axons exit the CNS. At this position are glial cells that guide the axons as they exit the CNS. Thus, whereas in some cells or parts of cells the two proteins colocalize, in others they do not (Lantz, 1998).
At higher magnification, the staining of the axons is nonuniform. Strikingly, the two proteins are present in the same particulate structures, possibly vesicles, in the neuronal processes. The anterior and posterior commissures stain for Jaguar as well as the longitudinal fibers that connect them. Within individual axons, punctate staining is observed with Jar antibodies. There is a one to one correspondence between the punctate spots that contain Jaguar and those that contain CLIP-190. The punctate Jaguar/CLIP-190 structures observed are distinct from the distribution of a uniformly distributed protein in axons of neurons of the CNS. Because the Jaguar/CLIP-190 punctate structures are present in the axonal processes of neurons, it seems likely that they are vesicular or organellar structures (Lantz, 1998).
Because subcellular localization is difficult to discern in whole embryos, Jaguar and CLIP-190 have been localized in primary cultures of embryonic cells. These cultures contain several different cell types, including myoblasts, which eventually fuse to form myotubes with sarcomeric structures, neurons that elaborate extensive processes, and hemocytes, a macrophage-like cell that has a flat, spread morphology. Cultures made from gastrulating embryos were fixed and stained with antibodies specific for the two proteins. Neurons show Jaguar and CLIP-190 particles like those seen in intact embryos. Colocalization in hemocytes is quite striking. There are a number of large inclusions in the cytoplasm, possibly vesicles or other organelles, that stain brightly for both proteins. In myoblasts, cytoplasmic organelles are seen in which both proteins are present. Whereas Jaguar staining appears to be primarily or exclusively confined to these organelles, CLIP-190 is clearly present in other areas of the cell. Particularly prominent is the CLIP-190 staining in the region of the nucleus. Jaguar is not enriched in this area. It is concluded that Jaguar and CLIP-190 are colocalized in a subset of organelles in several cell types (Lantz, 1998).
One of the most surprising features of the localization pattern of CLIP-190 is its distribution before migration of the nuclei to the cortex in precellularization embryos. In unfertilized eggs and during nuclear cycles 1-8 of embryogenesis, CLIP-190 protein is present at the posterior pole. Consistent with the association of the two proteins, Jaguar is also enriched at the posterior pole. Because of the high level of protein present in the cortex of the embryo, the enrichment of Jaguar at the posterior pole is not as striking as that of CLIP-190 protein; however, a higher level of protein is consistently observed at the posterior than in the adjacent cortex (Lantz, 1998).
The localization of CLIP-190 and Jaguar at the posterior of this large syncytial cell permits a test to see whether the localization of these two proteins is dependent on the actin and microtubule cytoskeletons. As associated proteins, one might expect them to behave similarly in response to disruption of the cytoskeleton. Therefore, early embryos were treated with cytochalasin D to disrupt the actin cytoskeleton or colchicine to disrupt microtubules. When embryos were treated with cytochalasin D, the actin cytoskeleton appeared to be substantially depolymerized. In addition, the nuclei were not distributed normally in treated embryos; this is expected if the cytochalasin D depolymerization was effective because the axial expansion or spreading of nuclei is an actin-dependent process. When such embryos were stained with CLIP-190 or Jaguar antibodies, the localization of these two proteins at the posterior pole was substantially disrupted. Most cytochalasin D-treated embryos had little or no CLIP-190 or Jaguar present at the posterior pole. A low level of CLIP-190 and Jaguar may remain in some treated embryos because cytochalasin D does not depolymerize all actin filaments in some cases. From these results it is concluded that the posterior localization of both CLIP-190 and Jaguar is dependent on the actin cytoskeleton (Lantz, 1998).
Embryos were also treated with colchicine to assess the effect of depolymerizing the microtubule cytoskeleton on localization at the posterior pole of CLIP-190 and Jaguar. Microtubules appeared to be substantially depolymerized by colchicine treatment. The distribution of Jaguar appeared to be unaffected by disrupting the microtubule cytoskeleton. CLIP-190 also appears unaffected, although at higher concentrations of colchicine (20 µg/ml), the protein does appear somewhat more diffuse. Thus, the posterior localization of CLIP-190 and Jaguar does not appear to be dependent on the microtubule cytoskeleton. Embryos were also treated with both cytochalasin D and colchicine. Because this treatment did not completely abolish localization in the majority of embryos, it is concluded that CLIP-190 is not substantially more affected when compared to treatment with cytochalasin D alone (Lantz, 1998).
Drosophila unconventional myosin VI, encoded by jaguar, is required for both imaginal disc and egg chamber morphogenesis. During oogenesis, Jaguar is expressed in migrating follicle cells, including the border cells, which migrate between the nurse cells to lie at the anterior of the oocyte; the columnar cells that migrate over the oocyte; the centripetal cells that migrate between the oocyte and nurse cells; and the dorsal-anterior follicle cells, which migrate to secrete the chorionic appendages (Deng, 1999).
In a screen for enhancer-trap Gal4 lines that show reporter gene expression in subsets of follicle cells line C865 was identified as of potential interest. It shows reporter gene expression in anterior follicle cells and migrating border cells during stages 7-11 of oogenesis. C865 is homozygous viable and fertile. DNA flanking the P[Gal4] insert was isolated by plasmid rescue, and used to isolate surrounding genomic DNA. Sequence analysis of these clones showed that the P[Gal4] insertion site is located at the 5' end of the Myosin heavy chain at 95F. Sequence analysis of the 'plasmid rescued' 3' flanking genomic DNA fragment shows that P[Gal4] is inserted 80 bp 3' of the transcription initiation site of the Mhc95F gene. The first exon of jaguar is 334 bp and untranslated, and this is followed by a large intron of approximately 8 kb (Deng, 1999).
The distribution pattern of jaguar mRNA during oogenesis was studied by whole-mount in situ hybridization. There are very high levels of expression of jaguar in the nurse cells from stages 9-13; this could reflect a role for myosin 95F in nurse cell cytoplasmic dumping. jaguar transcripts are present in the anterior follicle cells prior to their delamination and migration at stages 7-8 through the nurse cells to lie at the anterior of the oocyte. During stages 9-10, expression is also detected in these border cells during and after their migration through the nurse cell cluster (Deng, 1999).
Expression in these two groups of follicle cells is similar to that of the reporter gene in the original P[Gal4] line, C865. In addition to these two subsets of follicle cells, expression is found in other follicle cells that undergo morphogenetic movement during stages 9-13 of oogenesis. These include the follicle cells that retract from the nurse cell cluster to cover the oocyte at stage 9. To show these it is necessary to focus on the surface of the oocyte which makes the oocyte itself out of focus. The centripetal follicle cells at stage 10b can be seen as a strong band of staining next to the heavily stained nurse cells, and the dorsal-anterior follicle cells during stages 10b to 12. The expression domain in dorsal-anterior follicle cells shifts rapidly. Initially it appears as two curved lines over the stage-10b oocyte; this changes to a two-circle pattern at stage 12. The two to three rows of dorsal-anterior follicle cells stained during stage 10b appear to be at the leading edge of the follicle cell epithelium that undergoes dorsal-anterior migration. Particulate staining was observed at specific stages. High power magnification shows it as spots of staining in the nucleus representing the sites of transcription. This is most common for large genes which will take some time to transcribe and reflects active new synthesis of transcripts. In summary, all types of follicle cell migration during stages 9-13 are coincident with jaguar expression. This observation, along with the report that strong expression of Jaguar is detected in three of the four rows of dorsal-lateral epidermal cells which were at the leading edge of the epithelial sheet involved in dorsal closure (Kellerman, 1992), suggests that myosin 95F is likely to be involved in morphogenetic movements of epithelial cell sheets. During stage 10B, Mhc95F expression is also detected in a small group of posterior follicle cells. The function related to expression in these follicle cells is unclear (Deng, 1999).
In order to examine if the myosin 95F protein is expressed in the same pattern as its RNA during oogenesis, monoclonal antibody 3C7 raised against this myosin was used to label ovaries (Kellerman, 1992). Myosin VI is observed in the nurse cells at high levels until stage 6 of oogenesis and after this weak staining is observed. Small amounts of protein are also observed in the oocyte but not specifically localized. Myosin 95F is present in the anterior follicle cells around stage 7 prior to their migration towards the oocyte. Strong staining in the border cells is also detected at stages 9-10. During stage 10A, myosin VI is found in all columnar cells covering the oocyte with stronger staining in the cells lying most anterior in the oocyte, which will migrate to form the centripetal cells. At stage 10B, when centripetal cells are migrating to cover the anterior end of the oocyte, myosin 95F is predominantly localized in these cells while the staining in the columnar cells is fading. The staining remains strongest in the leading centripetal cells, which form a circle to squeeze into the frontier of the nurse cell cluster and the oocyte. During stages 12-13, when dorsal anterior follicle cells are migrating towards the anterior to synthesize the dorsal appendages, myosin 95F is strongly expressed in these cells but not in other follicle cells over the oocyte. All these observations show that the myosin 95F protein is expressed in a similar pattern to its RNA in the migrating follicle cells. Myosin VI protein is seen in the posterior follicle cells until stage 13 at least (Deng, 1999).
The nurse cells have much lower protein levels in relation to the follicle cells. This difference in RNA and protein staining in the nurse cells is likely to reflect the fact that stored mRNAs are made for transfer to the oocyte, and less protein is translated for the functions of myosin VI in the germline. This is a common observation for germline expressed genes. Since nonmuscle myosin II is also expressed in migrating follicle cells in a pattern similar to that of myosin 95F expression, a double labelling of both myosins was carried out. It was found that in most stages, both myosins are present in the same follicle cells. At stage 10A, both myosins are expressed in the border cells and columnar cells. However, their subcellular distribution seems to be different; it appears that they are not entirely colocalized within each cell, with the myosin 95F being more dominant in the cytoplasm close to the oocyte (Deng, 1999).
jaguar, is required for both imaginal disc and egg chamber morphogenesis. jaguar function during development has been studied using a targeted gene silencing technique, combining the Gal4-UAS targeted expression system and the antisense RNA technique. Antibody staining shows that the expression of jaguar is greatly decreased in follicle cells when antisense jaguar RNA is expressed. Interfering with expression of jaguar at various developmental stages frequently results in lethality. During metamorphosis it results in adult flies with malformed legs and wings, indicating that jaguar is essential for imaginal disc morphogenesis. During oogenesis, abnormal follicle cell shapes and aberrant follicle cell migrations are observed when antisense jaguar is expressed in follicle cells during stages 9 to 10, suggesting that Drosophila jaguar is required for follicle cell epithelial morphogenesis (Deng, 1999).
To examine the functional requirement of Jar in asymmetric division of neuroblasts, a jar zygotic null mutant, jar322, was generated by imprecise excision of the P element in the jar1 mutant allele, which carries a P[lacW, ry+] insertion in the first intron of the jar gene (Hicks, 1999). Single-embryo PCR has revealed that the jar322 mutation deletes the entire jar coding region and at least the first exon of the adjacent gene CG5706 for a putative phenylalanine-tRNA ligase, leaving intact the other adjacent gene of jar, CG13610, and the first gene downstream of CG5706, CG31138. Whereas the jar1 allele significantly reduces Jar levels only in the testis and leads to male sterility (Hicks, 1999), the jar zygotic null mutant jar322 animals die as first/early second instar larvae. At stage 16, 30% of metaphase neuroblasts have Miranda mislocalized in patches around the cortex and the cytoplasm, whereas Inscuteable is properly localized to an apical crescent in 95% of the embryonic neuroblasts with mislocalized Miranda. Thus, Jar acts downstream of, or in parallel to, the apical complex to control basal protein localization. These mutant embryos also exhibited improperly oriented spindles; 21% of the spindles examined were misoriented by 80°-90°, compared to 2% in jar322 heterozygotes. Maternal jar mRNA and Jar protein persists in zygotic jar322 homozygous embryos until stage 16, and Jar protein is significantly reduced thereafter. Attempts to induce maternal germline clones homozygous for jar322 yielded no surviving embryos, probably due to an essential role of Jar during oogenesis (Deng, 1999). In the first instar larvae of homozygous jar322 mutants devoid of maternal Jar protein, Miranda is mislocalized to the cytoplasm in metaphase neuroblasts. Hence, the residual maternal Jar in zygotic null jar322 mutant embryos may be sufficient for Miranda localization to the cortex though not for its basal targeting. A total loss of Jar, however, leaves Miranda within the cytoplasm in a punctate form (Petritsch, 2002).
To reduce both maternal and zygotic jar mRNA, double-stranded RNA (RNAi) was injected into wild-type embryos, reducing Jar levels at an earlier stage than in the jar322 mutant (stage 14). This jar RNAi treatment resulted in improper Miranda localization in about 50% of neuroblasts with Miranda most often detected in cortical and cytoplasmic patches (45%) and rarely in a mispositioned crescent, as well as randomized spindles (45%) with over 30% misoriented by 80°-90° (Petritsch, 2002).
In another approach to interfering with Jar function, a truncated form of Jar (DeltaATP), which lacks the ATP binding domain, was expressed. Zygotic expression of this putative dominant-negative myosin VI driven by neuralized-Gal4 caused mislocalization of Miranda to patches at the cortex and in the cytoplasm and misorientation of spindles in neuroblasts. Maternal expression of the truncated Jar with V32A-Gal4 driving the UAS line (UAS-deltaATP-jar) resulted in similar but earlier defects in basal but not apical protein localization, with 25% of neuroblasts showing Miranda mislocalization and spindle misorientation by 80°-90° in stage 9 embryos. Thus, three independent lines of evidence have been obtained for an essential role of Jar in basal Miranda localization (Petritsch, 2002).
Having found a requirement for jar in basal protein targeting, it was of interest to enquire whether jar interacts with other genes known to be involved in basal targeting. The tumor suppressor protein Lgl is involved in the localization of basal but not apical proteins. In lgl loss-of-function mutant (lgl4), Miranda is distributed all around the cortex, becomes associated with the spindle, and is also present in the cytoplasm at metaphase. In contrast, in lgl4; jar322 double mutants, Miranda is mostly in the cytoplasm with a diffuse distribution similar to that in jar322 mutants, but evident earlier in development, at stage 14, and with higher penetrance. The apically localized Inscuteable is not affected in lgl4. Thus, unlike myosin II Zipper, Jar appears to act synergistically with Lgl, downstream of or in parallel with apical crescent formation, to localize Miranda (Petritsch, 2002).
The temperature-sensitive mutation in Drosophila dynamin, shibire (shi1), was used to determine if dynamin function is required for individualization of spermatids during spermatogenesis. This mutation is in the GTPase domain of dynamin and is thought to block the GTPase cycle of dynamin, thereby acting as a functional null at the non-permissive temperature. Exposure of the dynamin mutant flies to the non-permissive temperature for up to 6 hours did not have any obvious effect on actin in actin cones or organization of individualization complexes. After dynamin inactivation, myosin VI and cortactin localization and accumulation at the front of actin cones was not significantly affected either, although myosin VI accumulates abnormally elsewhere in individualizing cysts. Individualization complexes moved normally when dynamin mutants were shifted to the non-permissive temperature as judged by observations in real time using cysts cultured in vitro. Thus, dynamin inactivation alone does not have a strong effect on individualization complexes. However, striking defects were observed when the temperature-sensitive dynamin mutation was placed into the background of a hypomorphic myosin VI mutation (jar1). When these shibire;jaguar double mutants were exposed to the non-permissive temperature to inactivate dynamin function, the total number of individualization complexes per testis was dramatically reduced. In addition, most of the individualization complexes that could be observed stained very weakly with phalloidin in comparison with those in flies bearing only the shibire or jaguar mutations. It is concluded that actin assembly or stability is dramatically reduced in the shibire;jaguar double mutant. Therefore, it is suggested that myosin VI and dynamin function in parallel pathways and that each pathway contributes to the regulation of actin structures in individualizing spermatids (Rogat, 2002).
The defects in shibire;jaguar double mutants were quantified to demonstrate the severity of the effect on actin structures. In these experiments shibire;jaguar double mutants, shibire single mutants, jaguar single mutants and wild-type flies (OreR) were exposed to the non-permissive temperature for different times then fixed and the testes were stained with phalloidin to visualize actin. The total number of early individualization complexes (complexes that are just assembling or that have just begun to move) were quantitated and the proportion of individualization complexes that stained strongly or weakly with phalloidin was determined. In wild-type flies there are typically 13 early individualization complexes per testis, of which most stain strongly with phalloidin. These numbers are not significantly changed even after 6 hours at the non-permissive temperature. Similar numbers were observed for the shibire mutants even after 6 hours at the non-permissive temperature. Therefore, exposing flies to as much as 6 hours of dynamin inactivation does not, obviously, affect actin structures (Rogat, 2002).
In jaguar single mutants there was a slight reduction in the total number of early actin complexes relative to wild-type and shibire mutant flies at all time points tested. Approximately 40% of the early complexes stained weakly with phalloidin. In jaguar mutants, actin complexes form, but as complexes begin to progress they fall apart and apparently lose actin filaments. This results in fewer total actin complexes in jaguar mutant testes and accounts for the larger proportion of weakly staining complexes. Thus, absence of myosin VI alone has a slight effect on actin assembly and/or stability in early individualization complexes (Rogat, 2002).
In comparison with wild-type controls and the single mutants, the shibire;jaguar double mutant at the non-permissive temperature displays a large reduction in the total number of early actin complexes per testis. Moreover, in the double mutant a large proportion of the actin complexes that remained stained weakly with phalloidin (indicating that those few complexes remaining had greatly reduced F-actin levels). By contrast, when the shibire;jaguar double mutant was exposed to the non-permissive temperature for only 2 minutes, the total number of actin complexes per testis and the number of weakly staining complexes were similar to the jaguar single mutant. This demonstrates that shibire;jaguar double mutants at the permissive temperature are similar to jaguar single mutants, in that actin cones can assemble. Only after inactivation of dynamin are filamentous actin structures disrupted (Rogat, 2002).
Since continuous actin assembly is often required to maintain stable actin structures, the loss of individualization complexes in the double mutant could be due to a disruption of actin filament assembly. Alternatively or in addition, the loss of actin filaments could be due to a direct disruption of actin filament stability. Therefore, it is concluded that myosin VI and dynamin function in pathways that regulate actin assembly or stability in individualizing spermatids. It is also concluded that, although dynamin activity is not required for actin cone formation, it plays a redundant role with myosin VI in regulating actin dynamics (Rogat, 2002).
Myosin VI is an unconventional Myosin that has been implicated in vesicle transport and membrane trafficking. Lethal mutants of Myosin VI have been isolated; these flies lack protein once maternal supplies have been utilised during embryogenesis. Dorsal closure, where there is a ring of Myosin VI at the edge of the migrating epithelial sheet, is often abnormal. The sheet of migrating cells is irregular, rather than a smooth epithelium and cells begin to detach. Some embryos hatch into larvae, containing detached cells loose in the haemolymph. Myosin VI is crucial for correct cell morphology and maintenance of adhesive cellular contacts within epithelial cell layers (Millo, 2004).
Previous studies showed that in Drosophila, Myosin VI plays a role in cell migration during oogenesis, actin dynamics during sperm individualisation, membrane synthesis during cellularization and spindle orientation in the metaphase neuroblast. Evidence is provided that Myosin VI is necessary for cell-cell adhesion and maintaining cell rigidity during dorsal closure. Disruption in Myosin VI function causes folding-in of the migrating epithelial tissue and rupture of the tissue, which suggests that Myosin VI is necessary not only for the cell-cell adhesion but also for keeping the cells rigid as they migrate (Millo, 2004).
Lethal mutants were isolated in the jar gene of Drosophila that encodes Myosin VI. The resulting embryos die late in embryogenesis failing to complete dorsal closure or as early larvae. The expression of fluorescent tagged Myosin VI in the mutant embryos rescued the lethal phenotype of embryos indicating that the lethality is caused due to the jaguar mutation (Millo, 2004).
Genetic and molecular analysis reveal that the two mutants have been disrupted in the sequence: AGTATACT. In jarR235 this sequence has been changed and in jarR39 the sequence is deleted. This is a palindromic sequence and as such could potentially regulate the transcription of jar. In Drosophila palindromic sequences located within the promoter region are often necessary for accurate transcription.The palindromic region might be a specific binding site for transcription factors (Millo, 2004).
Not all the embryos failed to complete dorsal closure and some hatched as larvae. RTPCR results show that in homozygous mutant embryos (stage 14 onwards) Myosin VI transcripts are still observed. However, in the homozygous mutant larvae, the expression level of Myosin VI transcripts is lower than OrR (these results are consistent with the Western Blot and antibody localization results). These results suggest that survival throughout embryogenesis is due largely to the use of maternal Myosin VI (Millo, 2004).
In the three mutants: jarmmw14, jarR39 and jarR235 the 5′ end of the mRNA was truncated, however the truncated region in jarR39 and jarR235 is bigger than that observed in jarmmw14 (1027 bp compared with 517 bp). This could explain why jarmmw14 mutants survive until adulthood while the jarR39 and jarR235 mutants die during embryogenesis and the first larval instar (Millo, 2004).
Analysis of the null mutant jar322 shows that it affects the embryonic neuroblasts but there is no mention of any defects in cell shape in the lateral epidermis, or of some embryos failing in dorsal closure. However, the same dominant negative Myosin VI that causes mislocalisation of Miranda and misorientation of the spindle in neuroblasts is shown to disrupt the cell attachment in the amnioserosa and the leading edge epidermis during dorsal closure. Therefore, Myosin VI is necessary for both neuroblast morphogenesis and dorsal closure during embryogenesis. It is surprising to find that the null mutant jar322 lives until the early stages of second instar, while the homozygous mutants jarR39 and jarR235 die during embryogenesis and during first instar. Since no protein was detected in the mutants jarR39 and jarR235, the severe effect of the mutants might be expected to be the same as in a null mutant (Millo, 2004).
Depletion and disruption in Myosin VI function causes detachment of cells and folding-in of the migrating epidermis during dorsal closure. In en-Gal4/ΔATP-jar embryos these affects are dramatic; if the cells expressing ΔATP-jar are not as robust as the wild type they might not be able to bear the multiple forces applied to them during dorsal closure, which could lead to the rupture of the tissue caused initially by the detachment of cells (Millo, 2004).
Cells that express ΔATP-jar and mutant cells have an aberrant organisation of the actin filaments in the cell cortex at the leading edge, and the cells lose their elongated shape. This is the area that was found to fold-in in many embryos. The connection between the change in cell shape and the disorganisation of the cytoskeleton could suggest that the cytoskeleton is necessary for maintaining cell rigidity. This suggests that Myosin VI preserves cell shape by participating in the synthesis or the patterning of the actin filaments during the migration of the epithelial cells. This is consistent with previous studies showing the role of Myosin VI in actin dynamics in the individualisation complex in the testes (Millo, 2004).
The creation of multiple wounds by laser beams in embryos did not prevent their quick recovery. Disruption caused by Myosin VI prevents recovery of the ripped tissues, probably due to the loss of cell adhesion properties. This suggests a potentially important role for Myosin VI in wound healing (Millo, 2004).
The failure in dorsal closure corresponds well with antisense experiments during development which led to failures in all the cell migrations of follicle cells during oogenesis. In each case Myosin VI is needed for the correct movement of a sheet or tight cluster of follicle cells, and in each case the cells lost their shape, therefore Myosin VI might also be needed for the preservation of cell shapes as they migrate. It was also observed that the follicle cell layer is disorganised and does not remain as a unicellular sheet. The phenotype is similar in the lethal embryos, where cells detach from sheets and are found lose in the haemolymph and the cells in the leading edge lose their tight organisation. The reduced expression and mislocalisation of DE-cadherin in the follicle cells and the lateral epidermis following the disruption in Myosin VI expression and function support the assumption that Myosin VI might play similar roles in the organisation of the two tissues (Millo, 2004).
During spermatogenesis in Drosophila, male sterile jar mutants fail in sperm individualisation, an event which requires separation of sperm by membranes. It is possible that Myosin VI also plays a role in the remodelling of cell membranes at the leading edge and that this is crucial for the adhesive properties of the cells during dorsal closure. The presence of Myosin VI in the filopodia and lamellipodia in epithelial cells at the leading edge could be necessary for the remodelling of the cell membrane (Millo, 2004).
It seems that Myosin VI is crucial for maintaining epithelial sheets and keeping cells in the correct spatial relationship to one another. This could be achieved by Myosin VI anchoring cells to the basement membranes that surround epithelial sheets, or perhaps if Myosin VI is absent from the apical surface of cells, cell polarity is not correctly defined and cell contacts are thus lost (Millo, 2004).
It is necessary to identify additional potential cargoes carried by the Myosin VI tail by protein-protein interaction experiments in vitro. Although the jar mutants are lethal, the abnormal phenotypes they cause at later developmental stages can still be investigated producing clones of mutant cells failing to express Myosin VI at various times and in various tissues (Millo, 2004).
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date revised: 20 July 2005
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