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).
In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue - the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen Kermit, previously implicated in G protein and PCP signaling, was unvcovered as a novel binding partner of Go. Through pull-down and genetic interaction studies, it was found that Kermit interacts with Go and another PCP component Vang (Strabismis), known to undergo intracellular relocalization during PCP establishment. It was further demonstrated that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI (Jaguar). The results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment (Lin, 2013).
At the top of the signaling hierarchy in PCP lies a G protein-coupled receptor Fz. The heterotrimeric Go protein emerged as an immediate transducer of Fz in Drosophila as well as other organisms. One of the mediators of Go signaling in PCP is the endocytic GTPase Rab5 required for the proper Fz internalization and relocalization. During PCP establishment, Fz concentrates at the distal apical position of wing epithelia. This study describes identification of Kermit as another transducer of Go in PCP. kermit downregulation suppresses the Gαo-overexpression phenotypes, and Gαo and kermit co-overexpression results in a prominent synergism in PCP malformations (Lin, 2013).
Kermit and its mammalian homolog GIPC, through their PDZ domain, are known to interact with a number of proteins in various organisms. Observations in Xenopus and mice indicated that Kermit/GIPC could interact with members of the Fz and RGS protein families -- Fz3, Fz7, and RGS19 (De Vries, 1998; Tan, 2001). Since Go also binds Fz and RGS proteins, it was hypothesized that a quaternary complex consisting of Fz, Go, Kermit, and RGS19 could form in Drosophila PCP, with Kermit as a potential organizer of these interactions. However, Drosophila Kermit was found not interact with Fz. Similarly, no binding between Kermit and the Drosophila RGS19 homolog could be seen. Thus Kermit is unlikely to act as a scaffold in Fz-Go signaling, and another mode of action of Kermit in transducing Go signal exists in PCP (Lin, 2013).
In a recent study using mouse genetics and cellular assays, a role of GIPC1 in regulating Vangl2 (a murine homolog of Drosophila Vang) intracellular trafficking has been revealed (Giese, 2012). In Drosophila PCP, Vang relocalizes to the site opposite to Fz at the proximal apical tip of wing epithelia. This study provides genetic evidence placing Vang downstream from Kermit in Drosophila PCP, suggesting that the Kermit-Vang connection is conserved from insects to mammals (Lin, 2013).
kermit expression is strongly upregulated in the developing wing during PCP establishment, and kermit overexpression induces strong PCP phenotypes (Djiane, 2010). In Xenopus, both up- and down-regulation of kermit lead to defective Fz3-dependent neural crest induction. It is thus surprising that Drosophila kermit loss-of-function alleles were homozygous viable and did not reveal PCP phenotypes. It is proposed that Kermit may regulate Drosophila PCP redundantly with some other PDZ domain-containing proteins, such as Scribble or Patj, which genetically interact with PCP components but on their own also produce only mild phenotypes; of those Scribble has been shown to interact with Vang both in Drosophila and mammals. In general, up to 75% of genes Drosophila are estimated to be phenotypically silent in loss-of-function due to redundancy, and the significance of gain-of-function analysis in discovery of novel important pathway components has been highlighted in a recent large-scale Drosophila-based assay. Kermit, based on the presented overexpression and genetic interaction studies, can thus be considered as an important regulator of Drosophila PCP (Lin, 2013).
A genetic and physical interaction between Kermit and the unconventional actin-based motor MyoVI has been described. This study confirmed that the dominant UAS-kermit PCP phenotypes critically depend on the MyoVI activity. MyoVI has been previously shown to mediate removal of endocytic vesicles away from the cell's periphery. The excessive activity of Kermit or MyoVI may thus result in removal of Vang-containing vesicles from the apical membrane, contributing to mislocalization of Vang and appearance of the PCP defects. In contrast, microtubule-based transport along the apical microtubule cables, polarized below the apical plasma membrane in wing epithelia, mediates the correct relocalizations of Fz and Vang in PCP. It is probable that a competition between the actin-based and microtubule-based motors may exist for the endocytic vesicles containing PCP components, and that excessive Kermit activity unbalances this competition in favor of the actin-based transport. Thus whether reduction in the levels of the microtubule-based transport system would further aggravate the dominant UAS-kermit PCP phenotypes was tested. And indeed, reduction in either the minus end-directed motor dynein or the plus end-directed motor kinesin significantly enhances the UAS-kermit effects (Lin, 2013).
The following model is proposed to collectively explain the results. It is speculated that endocytic vesicles containing PCP components can be transported in a planar manner, along the microtubule meshwork underlying the apical plasma membrane -- the mode of transport required for the proper apical relocalizations of these components. Alternatively, the vesicles can be trapped by the actin cables and transported away from the apical membrane, removing them from the active pool of PCP components. In the case of Vang, the choice between these decisions is regulated by the Kermit protein, which favors the actin-based transport (Lin, 2013).
These findings and model shed new light on the mechanisms of complex inter-regulations ensuring the robust epithelial polarization, likely conserved across the metazoans (Lin, 2013).
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).
Myosin VI is an actin-based motor that has been implicated in many cellular processes. Studies in vertebrates have demonstrated that animals lacking this ubiquitously expressed myosin are viable. However in Drosophila, myosin VI loss of function has been thought to be lethal. This study shows that complete loss of myosin VI is not lethal in flies and that the previously reported lethality of the null mutation (jar322) is most likely due to deletion of a neighboring gene. Maternally provided myosin VI does not account for the survival of myosin VI null animals. Mutant animals are recovered at a lower than expected Mendelian frequency, suggesting that myosin VI participates in processes which contribute to normal development, but its participation is not essential (Morrison, 2008).
Previous experiments suggested that in Drosophila, myosin VI loss of function was lethal. However, the studies reported here using genetic null animals demonstrate conclusively that myosin VI loss of function is not lethal in flies and that the lethality attributed previously to loss of myosin VI function in jar322 deletion must be caused by loss of the neighboring gene. Interestingly, these results are consistent with results in other species that loss of myosin VI function is not lethal. Despite the fact that animals completely lacking myosin VI can survive, they are not obtained at the expected frequency. This partially penetrant lethality is attributable to myosin VI loss of function, because this defect can be rescued by ubiquitous expression of a myosin VI cDNA transgene. The same transgene fails to rescue the lethality of the jar322 homozygotes and jar322/Df(3R)S87-4 transheterozygotes. Thus, the lethality observed in these animals is not associated with myosin VI loss of function (Morrison, 2008).
In addition to observations reported in this study, an ENU mutagenesis screen for new myosin VI mutants, in which both for lethal and male sterile alleles were screened, lends additional support to the idea that myosin VI loss of function is not lethal. No myosin VI lethal mutations were obtained, even though this screen was successful in identifying three new male sterile myosin VI alleles (Morrison, 2008).
Because of the phenotypes previously observed using other loss-of-function techniques and other myosin VI alleles, it was surprising that the null mutant animals were viable. The phenotypes attributed to myosin VI loss of function in other mutant alleles, including jar322 homozygous mutants, and defects generated by other loss of function techniques, such as antibody injection and antisense expression, must be reassessed in the jar322/Df(3R)S87-5 background. In neuroblasts, both expression of dominant negative fragments and lack of myosin VI (jar322 homozygotes) resulted in a partially penetrant defect in basal determinant localization, but perhaps such a defect is not severe enough to cause lethality. During early embryonic development, function-blocking antibody injection prevented metaphase pseudocleavage furrow formation, leading to massive defects in nuclear division. This severe phenotype would be expected to cause embryonic lethality. However, loss of myosin VI does not cause embryonic lethality. Perhaps the furrow defects caused by antibody injection can be explained by the sequestration of myosin VI-associated proteins, rather than as a direct effect of inhibiting myosin VI function. When myosin VI is absent (in null embryos), the myosin VI-associated proteins may still be able to function in furrow formation. Transgenic antisense expression caused severe defects in epithelial integrity and morphogenesis at a number of times in development and these animals were not viable. It is unclear why antisense expression causes more severe defects than the null mutation, but one explanation is off-target effects of the antisense. Possibly, impairment of epithelial integrity and defects in morphogenesis due to lack of myosin VI could contribute to the partially penetrant lethality observe. However, the processes in question cannot be completely blocked by loss of myosin VI function, since 40% of the null animals survive and these survivors have no obvious defects. The significant differences in phenotype of this null mutation compared to those observed using other functional manipulations reinforces the importance of careful genetic analysis of loss-of-function alleles in understanding in vivo functions (Morrison, 2008).
Myosin VI, encoded by jaguar (jar) in Drosophila, is a unique member of the myosin superfamily of actin-based motor proteins. Myosin VI is the only myosin known to move towards the minus or pointed ends of actin filaments. Although Myosin VI has been implicated in numerous cellular processes as both an anchor and a transporter, little is known about the role of Myosin VI in the nervous system. Previous studies recovered jar in a screen for genes that modify neuromuscular junction (NMJ) development and this study reports on the genetic analysis of Myosin VI in synaptic development and function using loss of function jar alleles. The experiments on Drosophila third instar larvae revealed decreased locomotor activity, a decrease in NMJ length, a reduction in synaptic bouton number, and altered synaptic vesicle localization in jar mutants. Furthermore, studies of synaptic transmission revealed alterations in both basal synaptic transmission and short-term plasticity at the jar mutant neuromuscular synapse. Altogether these findings indicate that Myosin VI is important for proper synaptic function and morphology. Myosin VI may be functioning as an anchor to tether vesicles to the bouton periphery and, thereby, participating in the regulation of synaptic vesicle mobilization during synaptic transmission (Kisiel, 2011).
Although Myosin VI function in the vesicle cycle has been implicated in mammalian cells, this report provides the first evidence that Myosin VI is important for maintaining normal peripheral vesicle localization at the bouton. In Drosophila, there are four types of boutons, which are the sites of neurotransmitter release at the NMJ, and they differ in their morphological and chemical properties. Of interest for this study were the largest synaptic boutons found at type I axon terminals, which are present at all NMJs of mature larvae. Visualization of synaptotagmin staining using confocal imaging revealed a mislocalization of synaptic vesicles in jar mutant boutons. An increasing number of jar mutant boutons, corresponding to the severity of Myosin VI loss of function, were found to exhibit diffuse staining over the entire bouton area as opposed to the doughnut-shaped staining pattern present in control boutons. Bouton centre occupancy has previously been observed at Drosophila NMJs of larvae lacking Synapsin, a phosphoprotein that reversibly associates with vesicles, using FM1-43 loading under low frequencies. EM analysis confirmed that in synapsin knockouts there was a spread of vesicles into the bouton centre, accompanied by a reduction in the size of the reserve pool. Thus, synapsin is thought to function in maintaining the peripheral distribution of vesicles in Ib boutons. Likewise, the unexpected diffuse synaptotagmin staining of jar mutant boutons suggests Myosin VI participates in restricting vesicles to the bouton periphery. It is possible that Myosin VI is functioning as a regulator of the actin cytoskeleton at the synapse. Mutant studies have revealed that the presynaptic actin cytoskeleton is required for proper synaptic morphogenesis. Myosin VI has already been shown to function in regulating the actin cytoskeleton during the process of spermatid individualization, by acting either as a structural cross-linker or as an anchor at the front edge of the actin cone, and during nuclear divisions in the syncytial blastoderm. However, live imaging of actin dynamics at the synaptic boutons revealed no major defects in the actin cytoskeleton at jar loss of function mutant nerve terminals (Kisiel, 2011).
To assess whether the morphological defects in vesicle localization observed at jar mutant synapses impact synaptic transmission, electrophysiological assays with different stimulation paradigms were used to recruit vesicles from different functional pools. The data add to the knowledge of this protein's physiological role at synapses. Myosin VI mutant mouse hippocampal neurons exhibit defects in the internalization of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, responsible for fast glutamatergic transmission, suggesting Myosin VI normally plays a role in AMPAR endocytosis. In addition, basal synaptic transmission is reduced in Myosin VI deficient mouse hippocampal slices compared to wild-type controls. Electrophysiological experiments also indicate that Myosin VI mediates glutamate release induced by brain-derived neurotrophic factor, which is known to modulate synaptic transmission and plasticity in the mammalian central and peripheral nervous system (Kisiel, 2011).
This study is the first to show that Myosin VI's role in synaptic transmission involves mobilization of vesicles from different functional pools, indicating that Myosin VI is important for synaptic plasticity. At the Drosophila NMJ, three pools of vesicles with differential release properties have been identified using FM1-43 staining loaded by various stimulation protocols. The immediately releasable pool (IRP), representing approximately 1% of all vesicles at the NMJ, consists of vesicles docked and primed at active zones for immediate release and experiences rapid depletion within a few stimuli. The readily releasable pool (RRP), making up 14% to 19% of all vesicles at the NMJ, is mobilized by moderate stimulation of ≤3 Hz and maintains exo/endocytosis at these stimulation frequencies. The reserve pool (RP) represents the vast majority of vesicles, 80% to 90%, and is mobilized upon depletion of the RRP. Recruitment from the RP occurs with high frequency stimulation of ≥10 Hz. Spontaneous release was reduced in the most severe jar loss of function mutants. Evoked response at 1 Hz stimulation was also reduced in the jar maternal null mutant. Although less severe jar mutants exhibited a significant decrease in bouton number, they did not experience an accompanying reduction in evoked potential amplitude at low frequency stimulation, suggesting that other homeostatic mechanisms are important for maintaining synaptic strength. The impaired synaptic response in the jar maternal null mutant may be due to a reduction in the probability of RRP vesicle release or in RRP size. If Myosin VI functions to anchor synaptic vesicles, it may act on the RRP to ensure vesicles are localized in manner that makes them readily available for release. Thus, in jar maternal null mutants the reduction in EJP amplitude may occur because a significant number of vesicles were displaced from areas of higher probability release. Alternately, RRP pool size may be reduced at jar mutant synapses (Kisiel, 2011).
Different synaptic vesicle pool properties, such as rate of recruitment of the RP in response to high frequency stimuli, may translate to changes in short-term synaptic plasticity. The increase in EJP amplitude observed at 10 Hz stimulation in 1 mM Ca2+ saline may be attributable to enhanced mobilization of the RP for jar322/Df(3R)crb87-5 NMJs. Filamentous actin has been implicated in RP mobilization as cytochalasin D, an inhibitor of actin polymerization, has been shown to reduce RP dynamics. This suggests translocation from the RP to the RRP may be mediated by an actin-based myosin motor protein. If Myosin VI functions as a synaptic vesicle tether to regulate recruitment from the RP pool, RP vesicles would be more readily mobilized and transitioned into the RRP upon high frequency stimulation in jar loss of function mutants. Consistent with the idea that RP vesicles were more rapidly incorporated into the RRP, a greater initial depression is observed at jar322/Df(3R)crb87-5 mutant synapses during high frequency stimulation in 10 mM Ca2+ saline corresponding to the depletion of vesicles at high calcium concentrations. Taken together, the data suggest that Myosin VI mediates synaptic transmission and short-term plasticity by regulating the mobilization of synaptic vesicles from different functional pools. In mammalian cells, Myosin VI has been implicated as a mediator of vesicle endocyctosis and has been shown to transport uncoated vesicles through the actin-rich periphery to the early endosome. The current experiments, however, indicate that endocytosis is not likely affected at jar mutant synapses. Typically, endocytotic mutants are unable to maintain synaptic transmission in response to high frequency stimulation, whereas the Myosin VI loss of function mutants exhibited enhanced EJP amplitude observed at 10 Hz stimulation in 1 mM Ca2+ saline. Additional experiments are required to confirm that Myosin VI is functioning as a vesicle tether. Fluorescence recovery after photobleaching analysis can be used to examine the effect of Myosin VI on synaptic vesicle mobility. If Myosin VI is functioning as a vesicle tether, synaptic vesicle mobility is expected to be increased in jar mutants compared to controls (Kisiel, 2011).
In summary, the present work shows that Myosin VI is important for proper synaptic morphology and physiology at the Drosophila NMJ. Myosin VI function in peripheral vesicle localization at the bouton may underlie its contribution to basal synaptic transmission and expression of synaptic plasticity. Future work will address the mechanism by which Myosin VI performs its roles at the synapse, whether as a vesicle tether or by some other involvement in vesicle trafficking (Kisiel, 2011).
At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. This study investigated vesicle dynamics in Myosin VI mutants of Drosophila. In Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling FM dye loading was used, and loss of function alleles of jar was compared with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching. This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. Mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility were observed when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton (Kisiel, 2014. PubMed ID: 25062032).
Altman, D., Sweeney, H. L. and Spudich, J. A. (2004). The mechanism of Myosin VI translocation and its load-induced anchoring. Cell 116: 737-749. 15006355
Avraham, K. B., Hasson, T., Steel, K. P., Kingsley, D. M., Russell, L. B., Mooseker, M., Copeland, N. G., and Jenkins, N .A. (1995). The mouse Snell's waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells. Nat. Genet. 11: 369-375. 7493015
Bahloul, A., Chevreux, G., Wells, A. L., Martin, D., Nolt, J., Yang, Z., Chen, L.-Q., Potier, N., Van Dorsselaer, A. and Rosenfeld, S. et al. (2004). The unique insert in myosin VI is a structural calcium-calmodulin binding site. Proc Natl Acad Sci. 101(14): 4787-92. 15037754
Breckler, J., Au, K., Cheng, J., Hasson, T. and Burnside. B. (2000). Novel myosin VI isoform is abundantly expressed in retina. Exp. Eye Res. 70(1): 121-34. 10644428
Buss, F., Kendrick-Jones, J., Lionne, C., Knight, A. E., Cote, G. P. and Luzio, J. P. (1998). The localization of myosin VI at the Golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation. J. Cell Biol. 143: 1535-1545. 9852149
Buss, F., Arden, S. D., Lindsay, M., Luzio, J. P. and Kendrick-Jone,s J. (2001). Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis. EMBO J. 20: 3676-3684. 11447109
Buss, F., Luzio, J.P. and Kendrick-Jones, J. (2002). Myosin VI, an actin motor for membrane traffic and cell migration. Traffic 3: 851-858. 12453148
De La Cruz, E. M., Ostap, E. M. and Sweeney. H. L. (2001). Kinetic mechanism and regulation of myosin VI. J. Biol. Chem. 276(34): 32373-81. 11423557
Deng, W., Leaper, K. and Bownes, M. (1999). A targeted gene silencing technique shows that Drosophila myosin VI is required for egg chamber and imaginal disc morphogenesis. J. Cell Sci. 112 (Pt 21): 3677-90. 10523504
De Vries, L., Lou, X., Zhao, G., Zheng, B. and Farquhar, M. G. (1998). GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP. Proc Natl Acad Sci U S A 95: 12340-12345. PubMed ID: 9770488
Djiane, A. and Mlodzik, M. (2010). The Drosophila GIPC homologue can modulate myosin based processes and planar cell polarity but is not essential for development. PLoS One 5: e11228. PubMed ID: 20574526
Fabrizio, J. J., Hime, G., Lemmon, S. K. and Bazinet, C. (1998). Genetic dissection of sperm individualization in Drosophila melanogaster. Development 125: 1833-1843. 9550716
Geisbrecht, E. R. and Montell, D. J. (2002). Myosin VI is required for E-cadherin-mediated border cell migration. Nat. Cell Biol. 4: 616-620. 12134162
Giese, A. P., Ezan, J., Wang, L., Lasvaux, L., Lembo, F., Mazzocco, C., Richard, E., Reboul, J., Borg, J. P., Kelley, M. W., Sans, N., Brigande, J. and Montcouquiol, M. (2012). Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear. Development 139: 3775-3785. PubMed ID: 22991442
Hasson, T., Gillespie, P. G., Garcia, J. A., MacDonald, R. B., Zhao, Y., Yee, A. G., Mooseker, M. S., and Corey, D. P. (1997). Unconventional myosin in inner-ear sensory epithelia. J. Cell Biol. 137: 1287-1307. 9182663
Hicks, J. L., Deng, W. M., Rogat, A. D., Miller, K. G. and Bownes, M. (1999). Class VI unconventional myosin is required for spermatogenesis in Drosophila. Mol. Biol. Cell, 10: 4341-4353. 10588662
Homma, K., Yoshimura, M., Saito, J., Ikebe, R. and Ikebe, M. (2001). The core of the motor domain determines the direction of myosin movement. Nature 412: 831-834. 11518969
Inoue, A., Sato, O., Homma, K. and Ikebe, M. (2002). DOC-2/DAB2 is the binding partner of myosin VI. Biochem. Biophys. Res. Commun. 292(2): 300-7. 11906161
Jan, Y. N. and Jan, L. Y. (2001). Asymmetric cell division in the Drosophila nervous system. Nat. Rev. Neurosci. 2: 772-779. 11715054
Joshi, M. K., Moran, S., Beckingham, K. M. and MacKenzie, K. R. (2012). Structure of androcam supports specialized interactions with myosin VI. Proc Natl Acad Sci U S A 109: 13290-13295. PubMed ID: 22851764
Kelleher, J. F., et al. (2000). Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis. Curr. Biol. 10(23): 1489-96. 11114515
Kellerman, K. A. and Miller, K. G. (1992). An unconventional myosin heavy chain gene from Drosophila melanogaster J. Cell Biol. 119: 823-834. 1429838
Kisiel, M., Majumdar, D., Campbell, S. and Stewart, B. A. (2011). Myosin VI contributes to synaptic transmission and development at the Drosophila neuromuscular junction. BMC Neurosci. 12: 65. PubMed Citation: 21745401
Kisiel, M., McKenzie, K. and Stewart, B. (2014). Localization and mobility of synaptic vesicles in Myosin VI mutants of Drosophila. PLoS One 9: e102988. PubMed ID: 25062032
Lantz, V. A., and Miller, K. G. (1998). A class VI unconventional myosin is associated with a homologue of a microtubule-binding protein, cytoplasmic linker protein-170, in neurons and at the posterior pole of Drosophila embryos. J. Cell Biol. 140: 897-910. 9472041
Laplante, C. and Nilson, L. A. (2006). Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila. Development 133(16): 3255-64. PubMed Citation: 16854971
Lin, C. and Katanaev, V. L. (2013). Kermit interacts with gαo, vang, and motor proteins in Drosophila planar cell polarity. PLoS One 8: e76885. PubMed ID: 24204696
Lin, H. P., et al. (2007). Cell adhesion molecule Echinoid associates with unconventional myosin VI/Jaguar motor to regulate cell morphology during dorsal closure in Drosophila. Dev. Biol. 311(2): 423-33. PubMed Citation: 17936269
Mermall, V., McNally, J. G. and Miller, K. G. (1994). Transport of cytoplasmic particles catalysed by an unconventional myosin in living Drosophila embryos. Nature 369: 560-562. 8202156
Mermall, V. and Miller, K. G. (1995). The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm. J. Cell Biol. 129: 1575-1588. 7790355
Millo, H., Leaper, K., Lazou, V. and Bownes, M. (2004). Myosin VI plays a role in cell-cell adhesion during epithelial morphogenesis. Mech. Dev. 121(11): 1335-51. 15454264
Morris, S. M., et al. (2002). Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton. Traffic 3(5): 331-41. 11967127
Morrison, J. K. and Miller, K. G. (2008). Genetic characterization of the Drosophila jaguar322 mutant reveals that complete myosin VI loss of function is not lethal. Genetics 179(1): 711-6. PubMed Citation: 18493084
Mukherjea, M., Ali, M. Y., Kikuti, C., Safer, D., Yang, Z., Sirkia, H., Ropars, V., Houdusse, A., Warshaw, D. M. and Sweeney, H. L. (2014). Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles. Cell Rep 8: 1522-1532. PubMed ID: 25159143
Nishikawa, S., et al. (2002). Class VI myosin moves processively along actin filaments backward with large steps. Biochem. Biophys. Res. Commun. 290(1): 311-7. 11779171
Park, H., et al. (2006). Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering. Mol. Cell 21: 331-336. PubMed Citation: 16455488
Petritsch, C. Tavosanis, G., Turck, C. W. Jan, L. Y. and Jan, Y. N. (2002). The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts. Developmental Cell 4: 273-281. 12586070
Rock, R. S., et al. (2001). Myosin VI is a processive motor with a large step size. Proc. Natl. Acad. Sci. 98(24): 13655-9. 11707568
Rodriguez, O. C. and Cheney, R. E. (2000). A new direction for myosin. Trends Cell Biol. 10: 307-311. 10884682
Rogat, A. D. and Miller, K. G. (2002). A role for myosin VI in actin dynamics at sites of membrane remodeling during Drosophila spermatogenesis. J. Cell Sci. 115: 4855-4865. 12432073
Self, T., et al. (1999). Role of myosin VI in the differentiation of cochlear hair cells. Dev. Biol. 214(2): 331-41. 10525338
Suter, D. M., Espindola, F. S., Lin, C. H., Forscher, P. and Mooseker, M. S. (2000). Localization of unconventional myosins V and VI in neuronal growth cones. J. Neurobiol. 42(3): 370-82. 10645976
Tan, C., Deardorff, M. A., Saint-Jeannet, J. P., Yang, J., Arzoumanian, A. and Klein, P. S. (2001). Kermit, a frizzled interacting protein, regulates frizzled 3 signaling in neural crest development. Development 128: 3665-3674. PubMed ID: 11585793
Warner, C. L., et al. (2003). Loss of myosin VI reduces secretion and the size of the Golgi in fibroblasts from Snell's waltzer mice. EMBO J. 22(3): 569-79. 12554657
Wells, A. L., Lin. A. W., Chen. L. Q., Safer. D., Cain, S. M., Hasson, T., Carragher, B. O., Milligan, R. A. and Sweeney H. L. (1999). Myosin VI is an actin-based motor that moves backwards. Nature 401: 505-508. 10519557
Yoshimura, M., et al. (2001). Dual regulation of mammalian Myosin VI motor function. J. Biol. Chem. 276: 39600-39607. 11517222
Yu, C., et al. (2009). Myosin VI undergoes cargo-mediated dimerization. Cell 138(3): 537-48. PubMed Citation: 19665975
date revised: 5 February 2015
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