Gene name - jaguar
Synonyms - myosin 95F; myosin VI
Cytological map position - 95F6
Function - motor protein
Symbol - jar
FlyBase ID: FBgn0011225
Genetic map position - 3-
Classification - myosin VI
Cellular location - cytoplasmic
|Recent literature||Yoong, L. F., Lim, H. K., Tran, H., Lackner, S., Zheng, Z., Hong, P. and Moore, A. W. (2020). Atypical myosin tunes dendrite arbor subdivision. Neuron. PubMed ID: 32155441
Dendrite arbor pattern determines the functional characteristics of a neuron. It is founded on primary branch structure, defined through cell intrinsic and transcription-factor-encoded mechanisms. Developing arbors have extensive acentrosomal microtubule dynamics, and this study reports an unexpected role for the atypical actin motor Myo6 in creating primary branch structure by specifying the position, polarity, and targeting of these events. In vivo time-lapse imaging was carried out of Drosophila adult sensory neuron differentiation, integrating machine-learning-based quantification of arbor patterning with molecular-level tracking of cytoskeletal remodeling. This revealed that Myo6 and the transcription factor Knot regulate transient surges of microtubule polymerization at dendrite tips; they drive retrograde extension of an actin filament array that specifies anterograde microtubule polymerization and guides these microtubules to subdivide the tip into multiple branches. Primary branches delineate functional compartments; this tunable branching mechanism is key to define and diversify dendrite arbor compartmentalization.
|Dye, N. A., Popovic, M., Iyer, K. V., Fuhrmann, J., Piscitello-Gomez, R., Eaton, S. and Julicher, F. (2021). Self-organized patterning of cell morphology via mechanosensitive feedback. Elife 10. PubMed ID: 33769281
Tissue organization is often characterized by specific patterns of cell morphology. How such patterns emerge in developing tissues is a fundamental open question. This study investigated the emergence of tissue-scale patterns of cell shape and mechanical tissue stress in the Drosophila wing imaginal disc during larval development. Using quantitative analysis of the cellular dynamics, a pattern of radially oriented cell rearrangements was revealee that is coupled to the buildup of tangential cell elongation. Developing a laser ablation method, tissue stresses were mapped, and key parameters of tissue mechanics were extract. A continuum theory is presented showing that this pattern of cell morphology and tissue stress can arise via self-organization of a mechanical feedback that couples cell polarity to active cell rearrangements. The predictions of this model are supported by knockdown of MyoVI, a component of mechanosensitive feedback. This work reveals a mechanism for the emergence of cellular patterns in morphogenesis.
Jaguar (Jar), an unconvential myosin (see The Myosin Home Page for information about the different families of myosins) is the founding member of class VI myosins (Kellerman, 1992). Mammalian myosin VI moves toward the pointed (minus) end of actin in vitro (Wells, 1999). This unusual directional movement requires a region within the motor core domain (Homma, 2001) that is highly conserved, indicating that all myosin VI proteins are pointed end-directed motors (Rodriguez, 2000). Drosophila Myosin VI has been implicated in the movement of vesicles and particles, for instance during cellularization of the embryonic syncytial blastoderm (Mermall, 1994; Mermall, 1995). Myosin VI is associated with clathrin-coated vesicles and has been implicated in facilitating endocytosis (Buss, 2001). Another function of Jar is to stabilize DE-cadherin (Shotgun) and Armadillo at adherens junctions in the ovaries (Geisbrecht, 2002). Myosin VI/Jar has also been implicated in the regulation of actin dynamics during sperm individualization (Hicks, 1999; Rogat, 2002). Jar has also been identified as an essential motor protein for asymmetric division of neuroblasts by virtue of its requirement for basal protein targeting and spindle orientation (Petritsch, 2002 and references therein).
Asymmetric cell divisions generate cellular diversity. In Drosophila, embryonic neuroblasts target cell fate determinants basally, rotate their spindles by 90° to align with the apical-basal axis, and divide asymmetrically in a stem-cell-like fashion. In this process, apically localized Bazooka recruits Inscuteable and other proteins to form an apical complex, which then specifies spindle orientation and basal localization of the cell fate determinants and their adapter proteins such as Miranda. Miranda localization requires Jaguar. In jar null mutant embryos, Miranda is delocalized and the spindle is misoriented, but the Inscuteable crescent remains apical. Miranda directly binds to Jar, raising the possibility that Miranda, and the complex it assembles, including Prospero, Staufen, and prospero mRNA, may be one of the principle cargos for Jar in its role in asymmetric protein localization and spindle positioning in neuroblasts (Petritsch, 2002).
Based upon its amino acid sequence the structure of mammalian myosin VI has been predicted to be composed of a head domain, a coiled-coil domain, and a globular tail domain. The head domain is itself divided into a globular motor domain and a neck domain containing a light chain binding region. The sequence at the neck region contains a single IQ motif that is implicated as a calmodulin or myosin light chain binding consensus motif as found in a variety of calmodulin-binding proteins and myosins. The coiled-coil domain is present at the C-terminal side of the neck region, so it is predicted that myosin VI is a two-headed myosin. Finally, the globular tail domain is hypothesized to be a targeting domain that determines the cellular binding counterpart (Yoshimura, 2001 and references therein).
There are two unique inserts in the head domain, one at the surface in the upper 50-kDa domain and the other at the junction between the converter domain and the IQ domain. Until quite recently, all myosin motors were characterized as moving toward the barbed end of actin filaments. Since unconventional myosins play a role in translocating cellular organelles along actin cables, a myosin having opposite moving directionality would be expected to have unique cellular functions. Of the 18 different classes of myosins, it was found that only myosin VI moves toward the minus end of F-actin filaments (Wells, 1999). While the mechanism underlying the reverse movement of myosin VI is unclear, the unique large insertion in the myosin VI head domain between the motor domain and the light chain binding domain (lever arm) has led to the idea that this alters the angle of the lever arm switch movement, thus changing the direction of motility (Yoshimura, 2001 and references therein).
The importance of light chains in the regulation of myosin motor function is seen in both vertebrate smooth muscle/nonmuscle myosin and invertebrate myosin, in which the phosphorylation of the regulatory light chain and Ca2+ binding to the essential light chain, respectively, trigger the activation of motor activity (Yoshimura, 2001 and references therein).
The role of the IQ motif and bound calmodulin (see Drosophila Calmodulin) serving as a regulatory component of unconventional myosins was first studied for mammalian myosins I. For both brush border myosin I and myosin I, high Ca2+ inhibits motor activity due to its binding to the calmodulin light chain. Myosin VI undergoes dual regulation by phosphorylation and Ca2+ binding to calmodulin light chain. Myosin VI contains a potential phosphorylation site in a loop near the tip of the head. The phosphorylation of myosin VI significantly facilitates the actin-translocating activity of myosin VI. In contrast, Ca2+ diminishes the actin-translocating activity of myosin VI although the actin-activated ATPase activity is not affected by Ca2+. Calmodulin is not dissociated from the heavy chain at high Ca2+, suggesting that a conformational change of calmodulin upon Ca2+ binding, but not its physical dissociation, determines the inhibition of the motility activity. It is anticipated that myosin VI has a processive nature in its motor function, although more direct evidence is required to determine the processivity of myosin VI (Yoshimura, 2001).
Neuroblasts divide asymmetrically after delamination from Drosophila embryonic epithelium to generate a neuroblast and a smaller ganglion mother cell, by first forming an apical complex, which then targets cell fate determinants basally and reorients the mitotic spindle (Jan, 2001). This complex recruits Inscuteable, Partner of Inscuteable (Pins), and the G protein subunit Galphai to form an apical crescent at prophase. In metaphase, cell fate determinants and their respective adapters Numb/PON, prospero mRNA/Staufen, and Prospero/Miranda form a basal crescent. Miranda is required for basal localization of Prospero, Staufen, and prospero mRNA, and binds not only Prospero and Staufen but also Inscuteable. Basal but not apical crescent formation requires the cortically localized Lethal giant larvae (Lgl) and Discs large (Dlg). Just how the apical complex directs basal crescent formation is not known. Because Miranda/Prospero and Staufen/prospero mRNA are transiently localized to the apical cortex before forming a basal crescent, conceivably Miranda is transported from the apical to the basal cortex by a motor, thereby mediating basal targeting of Prospero, Staufen, and prospero mRNA (Petritsch, 2002 and references therein).
What could be the motor for basal protein targeting? Lgl interacts with myosin II (Strand, 1994), and the lgl mutant phenotype is suppressed by a loss-of-function mutation of the myosin II gene zipper (zip). However, zip mutants exhibit no alterations in cell fate determinant localization or spindle orientation. Thus, motors other than Zip are probably involved in basal protein transport. To search for such motor proteins, Miranda-containing complexes were isolated from Drosophila embryos and two associated proteins were identified, myosin II/Zipper and myosin VI/Jaguar. Various methods were used to reduce jar activity and these treatments were found to disrupt Miranda localization and proper spindle orientation, but not the apical complex formation. As opposed to zip mutations, reducing jar activity enhances the basal protein transport defects in lgl mutants. Jar binds Miranda directly, and partially overlaps with Miranda in the distribution in neuroblasts, suggesting that Miranda and the complex it assembles including Prospero, Staufen, and prospero mRNA may be one of the cargos for Jar (Petritsch, 2002).
It thus appears likely that the hitherto unknown motor protein(s) that mediates basal protein targeting may form a complex with Miranda. This possibility was tested and it was shown that Jar not only binds Miranda directly but is in a complex with Miranda in vivo. Also in a complex with Miranda is Zipper, a myosin II, which negatively regulates basal transport of Miranda. Jar exhibits a dynamic, punctate distribution concentrating at the basal side of the dividing neuroblast and partially overlapping when the Miranda basal crescent forms. Importantly, three independent ways of reducing Jar activity in neuroblasts all resulted in mislocalization of Miranda. In addition, a reduction or loss of Jar function compromised spindle orientation. However, the apical complex does not depend on Jar activity, suggesting that Jar acts downstream of or in parallel to the apical complex to ensure proper basal protein localization. These studies have therefore identified Jar as a myosin that targets basal proteins and aligns the mitotic spindle along the apical-basal axis in neuroblasts. The involvement of barbed end-directed myosins in asymmetric cell division has been demonstrated in budding yeast. In Drosophila neuroblasts, Jaguar, a pointed end-directed myosin motor, regulates asymmetric protein localization and spindle positioning (Petritsch, 2002).
It is intriguing that the same myosin VI may coordinate mitotic spindle alignment with the basal crescent of cell fate determinants. In an attempt to pursue this possibility further, an in vivo association of Jar with the microtubule-associated protein dEB1 has been demonstrated as well as an association of Jar with the microtubule-associated protein D-CLIP-190. However, loss-of-function mutations of dEB1 and D-CLIP-190 are not available. Reducing the levels of dEB1 by injection of double-stranded RNA (RNAi) results in a mild spindle orientation phenotype in epithelial cells, either due to functional redundancy -- there are four predicted dEB1 genes found in the database -- or due to a strong maternal contribution of dEB1. These technical difficulties have hampered attempts to examine the function of these genes in spindle orientation in neuroblasts (Petritsch, 2002).
It is not yet clear how Jar mediates basal targeting of Miranda. Identification of Jar as an essential motor protein for asymmetric division of neuroblasts raises the following questions for future studies: how might Jar interpret the apical-basal polarity set by the apical complex and target basal proteins? Does Jar organize actin cables along the apical-basal axis or simply move along preexisting actin filaments? Besides the pointed end-directed myosin VI, the barbed end-directed myosin II, Zip, is also associated with Miranda in vivo. Whereas both Jar and Lgl are required for basal protein targeting, loss of zipper function suppresses the lgl mutant phenotype. Given that Lgl and Jar synergize to control basal transport, Zip and Jar might have antagonistic activities in basal protein targeting. Genetic interactions between zipper and jar could not be easily assessed, due to overall abnormal morphology of the double mutant embryos at late stages. Whereas both Jar and Zip interact with Miranda in vivo, Zip is not detectable in Jar-containing complexes immunoprecipitated from embryo extracts, raising the possibility that Jar and Zip might compete for binding to Miranda (Petritsch, 2002).
Jaguar is required for spermatogenesis in Drosophila. Partial loss of function mutations in jaguar result in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. Jaguar is a component of the IC whose function is essential for individualization. In wild-type testes, Jaguar localizes to the leading edge of the IC. Two independent mutations in jar reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of Jar causes male sterility as a result of defects in spermatid individualization. Germ line transformation with jar cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. It is proposed that 95F myosin is a motor that participates in membrane reorganization during individualization (Hicks, 1999).
Individualization proceeds from the head region of each spermatid to the tail. The cystic bulge, a spindle-shaped enlargement of the cyst, is the region in which individualization is occurring. Within the bulge the individualized portion is devoid of cytoplasmic organelles (except the major and minor mitochondrial derivatives and the axoneme) and contains structures termed investment cones. A single investment cone, filled with fine fibrils ~60 Å in diameter, surrounds each spermatid. Golgi reside at the individualization boundary, and beyond that are dense concentrations of ribosomes and organelles. As the cystic bulge progresses toward the caudal end of the spermatids, it increases in size because of the accumulation of organelles. Eventually the excluded membrane and organelles are pinched off at the caudal end in the waste bag structure. After individualization, each sperm is contained in its own membrane, and only a remnant of the axonemal sheath remains next to the condensed mitochondria (Hicks, 1999 and references therein).
Jaguar is not required for the initial assembly of actin into the IC, because in mutant animals actin initially assembles normally. After actin is recruited to the complex but before any movement, Jaguar localizes in particulate structures along the length of the nucleus. These particulate structures are reminiscent of Jaguar localization in other tissues (Kellerman, 1992; Mermall, 1995; Lantz, 1998) and may represent vesicular or organellar cargo. In the absence of Jaguar, actin still forms investment cones, but the alignment of cones within each cyst is disrupted compared with wild type. It is not clear from these results whether this misalignment is a result of an initial defect in positioning of the nuclei or whether the complex begins to move in the absence of Jaguar, becoming disorganized as it progresses and dragging nuclei out of alignment. However, because spermatid elongation and nuclear condensation appear unaffected in mutant testes, it is unlikely that Jaguar plays a direct role in nuclear positioning (Hicks, 1999).
After assembly of the complex, the IC progresses down the cyst, maintaining the alignment between investment cones of neighboring spermatids. Jaguar becomes tightly associated with the base of the advancing investment cone, and this distribution is maintained as the IC progresses down the cyst to the waste bag. In the absence of Jaguar, either the IC never advances away from the nuclei, or its component cones advance out of register, resulting in a disrupted IC. This suggests that Jaguar is involved in IC movement. Based on the known functions for other myosins there are three possible models for the role of Jaguar during individualization: (1) a force-producing motor, which moves the IC; (2) an actin cross-linking structural protein, and (3) a motor involved in short-range movements, which are important for membrane reorganization (Hicks, 1999).
The simplest model for the role of Jaguar is that it provides the force that moves the IC along the length of the axoneme. If this is the case, when Jaguar is absent the investment cones should not move away from their assembly site adjacent to the nuclei. In fact, investment cones were observed that had translocated away from the nucleus in the mutant; this observation makes this model unlikely. In addition, in this model, Jaguar would require some type of 'track' on which to translocate down the spermatid. There is no obvious actin track preceding the IC. The microtubules that constitute the spermatid tail axoneme might form a track for movement, but it seems likely that a myosin would only be able to use these microtubules to move actin filaments and membrane in collaboration with microtubule-binding proteins and microtubule motors (Hicks, 1999).
In other tissues, such as nervous system and posterior of the early embryo, Jaguar colocalizes with CLIP-190, the Drosophila homolog of the vertebrate vesicle linker protein CLIP 170 (Lantz, 1998). Both CLIP-170 and CLIP-190 bind microtubules in vitro. To determine whether Jaguar might interact with microtubules by way of CLIP-190, the localization of CLIP-190 was examined in testes. CLIP-190 does not colocalized with Jaguar. It is hypothesized that if Jaguar produces force for movement during IC progression it does not do so by binding indirectly to microtubules by way of CLIP-190. This does not exclude the possibility that there is another as yet undiscovered CLIP family member or other microtubule-associated protein in Drosophila that could interact with Jaguar in this process (Hicks, 1999).
A second model for the role of Jaguar suggests that Jaguar might stabilize the actin structure as Jaguar moves. In this case, actin would assemble, and ICs would begin to move. However, because Jaguar is absent in jar mutants, they would rapidly disassemble, leading to few progressed ICs. The data show that in fact there is no substantial change in the proportion of ICs that have progressed. This model also does not easily explain why ICs and their associated nuclei are initially out of register in the mutant (Hicks, 1999).
The third hypothesis, that Jaguar might function to transport components important for individualization to the site of membrane growth, is favored. Progression of the investment cone down the spermatid bundle reorganizes the previously shared membrane so that each sperm is enclosed by a distinct membrane. This membrane remodeling is likely to involve an organized mechanism for delivery and incorporation of recycled and/or newly synthesized membrane and cytoskeletal components. It is hypothesized that Jaguar functions to recruit vesicles from the apical side of the IC (ahead of individualization machinery) to the site of membrane remodeling -- the leading edge of the investment cone. Jaguar would travel along cortical actin filaments toward the plasma membrane and release vesicles at the site of membrane remodeling. This model would explain the accumulation of Jaguar in the band at the base of the leading edge of the investment cones. In this model, Jaguar would not serve as the motor that generates force for IC movement down the spermatogenic cyst. Instead it is postulated that the force that moves the IC is actin polymerization. This model is reminiscent of the mechanism of actin-based movement of Listeria monocytogenes (Hicks, 1999).
Subsequent studies have clarified the role of Myosin VI in individualization. Myosin VI colocalizes with and is required for the accumulation of the actin polymerization regulatory proteins, cortactin and arp2/3 complex, on actin structures that mediate membrane remodeling during spermatogenesis. In addition, dynamin/Shibire localizes to these actin structures and when dynamin and myosin VI function are both impaired, major defects in actin structures are observed. It is concluded that during spermatogenesis myosin VI and dynamin function in parallel pathways that regulate actin dynamics and that cortactin and arp2/3 complex may be important for these functions. Regions of myosin VI accumulation are proposed as sites where actin assembly is coupled to membrane dynamics (Rogat, 2002).
During individualization, myosin VI localizes to a complex of filamentous actin, the individualization complex, responsible for remodeling the syncytial membrane around a bundle of 64 spermatids. The actin complex initially assembles around spermatid nuclei and then progresses away from the nuclei and down the tails of the spermatids, extruding cytoplasm and remodeling membrane as it moves (Fabrizio, 1998). Initially, myosin VI accumulates in a particulate fashion as they assemble around the nuclei, but just as the actin individualization complex initiates movement away from the nuclei, myosin VI concentrates at the front of each actin cone. As the complex progresses down the length of the spermatid tails, myosin VI further concentrates into a tight band at the front of the actin cones. Myosin VI is also diffusely localized in front of the actin cones in association with the membrane and cytoplasm of the cystic bulge (Rogat, 2002).
The site at which myosin VI concentrates is the junction between a moving actin structure and a zone of active membrane remodeling. This location places myosin VI in an ideal position to link sites of remodeling to actin dynamics. Therefore, the localization of proteins that have been implicated in membrane/actin coordination was examined. One such protein is the actin-binding protein cortactin. Cortactin is thought to link membrane signaling proteins to actin dynamics by virtue of is ability to associate with both actin polymerization components and membrane-associated kinases (Rogat, 2002).
The distribution of cortactin was examined in individualizing spermatids with anti-Drosophila cortactin antibodies. Like myosin VI, cortactin concentrated at the front of actin cones, and double labeling of spermatids with cortactin and myosin VI antibodies shows that they colocalize at the front of each actin cone. Cortactin is also present on the cyst membrane. The distribution of cortactin in individualization complexes indicates that the fronts of the actin cones are sites where actin polymerization might be coupled with membrane dynamics. Myosin VI colocalization with cortactin at these sites suggests myosin VI may also be involved in these dynamics (Rogat, 2002).
To further demonstrate that the fronts of the actin cones are sites of regulated actin assembly, the distributions of the arp2/3 complex and capping protein were examined in individualizing spermatids. The arp2/3 complex is a complex of seven proteins that binds actin filaments and nucleates new actin filament assembly. Capping protein is a barbed-end actin-binding protein with a known role in regulating actin polymerization at sites where the arp2/3 complex promotes assembly. It is also concentrated in regions of dynamic actin assembly in many cell types. In individualizing spermatids, the arp2/3 complex, as demonstrated by arp3 and ARPC2/p34 staining, and capping protein, as demonstrated by capping protein β staining, concentrated at the front of actin cones. In both cases, staining was also visible generally through the cytoplasm of the cyst and along the actin cones. Double labeling experiments have shown that myosin VI colocalizes with concentrated arp3 and capping protein at the front of actin cones. The accumulation of proteins involved in actin polymerization at the front of the actin cones supports the idea that the zone where myosin VI concentrates is a zone of active actin assembly (Rogat, 2002).
Myosin VI is required for the proper distribution of cortactin and the arp2/3 complex on individualization complexes. The colocalization of cortactin, arp2/3 complex and myosin VI on individualization complexes prompted an examination of cortactin and arp2/3 complex distribution on individualization complexes in jaguar mutants. Cortactin can be detected on actin individualization complexes in jaguar mutants (jar1). However, its distribution is not normal. Cortactin is not concentrated at the front of the actin cones. Instead, it is weakly present uniformly along the cones. The complexes have a disrupted morphology and reduced actin staining, as is typically observed for progressed actin cones in jaguar mutants. When early individualization complexes were examined in jaguar mutants, no early complexes showed any concentration of cortactin at the front of cones. By contrast, in wild-type spermatids, some early individualization complexes had cortactin concentrated at the front and others did not. When doubly stained for myosin VI, those complexes with concentrated myosin VI also showed concentrated cortactin. It is concluded that myosin VI is required for the proper asymmetrical distribution of cortactin on actin cones (Rogat, 2002).
Defects in arp2/3 complex localization were observed in jaguar mutants. Arp3 does not concentrate at the front of actin cones, either early or on progressed complexes, in jaguar mutants. In addition, there appears to be a higher level of arp3 staining in the cytoplasm of the cysts in jaguar mutants in comparison to wild-type cysts. This may be because arp3 cannot concentrate on the actin cones and, instead, accumulates in the cytoplasm. Like arp3, ARPC2/p34 concentration at the front of actin cones is abolished in jaguar mutants. Therefore, like cortactin, asymmetric distribution of the arp2/3 complex on the actin cones is dependent on myosin VI function. These findings support a role for Jaguar in regulating actin dynamics by participating in the localization of cortactin and arp2/3 complex at the front of the individualization complex (Rogat, 2002).
The close coupling between actin assembly and membrane remodeling during individualization prompted an examination of proteins involved in membrane dynamics to determine if Jaguar works with these proteins. Dynamin, encoded by the Drosophila shibire gene, is a large GTPase with known roles in promoting the fission of clathrin-coated pits into clathrin-coated vesicles during endocytosis. Dynamin binds cortactin and regulates actin dynamics at membrane sites of actin assembly. Thus, dynamin is a likely molecule to function in individualization complexes (Rogat, 2002 and references therein).
Dynamin distribution was examined in individualizing spermatids; it localizes along the length of the actin cones in a distribution most similar to actin. As expected from the distribution of dynamin on actin cones, myosin VI concentrates at the front of dynamin-stained cones. Dynamin localization along the actin cones suggests that this region might either be an area of high endocytic membrane trafficking or a region where actin dynamics are regulated by dynamin. Furthermore, the close juxtaposition of myosin VI and dynamin suggests that they might participate in the same process during individualization (Rogat, 2002).
To gain a clearer understanding of dynamin's function in the actin cones, the distribution was examined of two proteins known to interact with dynamin in vertebrates, but which appear to function in different pathways in Drosophila. alpha-Adaptin is the alpha subunit of the AP-2 adaptor complex, which is known to bind clathrin and function in early endocytosis. alpha-Adaptin is also required for endocytosis in Drosophila. Amphiphysin, however, is not required for endocytosis in Drosophila. Amphiphysin can influence filamentous actin localization and has been implicated in membrane morphogenesis and organization in Drosophila (Rogat, 2002 and references therein).
alpha-Adaptin is neither concentrated on actin cones nor at the front of the cones, as indicated by anti-Drosophila alpha-adaptin antibodies. Instead, it localizes in a particulate fashion throughout the cystic bulge ahead of the actin cones. By contrast, Drosophila Amphiphysin antibodies localize to actin cones in a manner similar to dynamin. However, unlike dynamin, Amphiphysin also concentrates at the front of cones in a manner similar to Jaguar, Cortactin and the arp2/3 complex. Thus, Amphiphysin's distribution is intermediate between dynamin and cortactin/myosin VI. Since Amphiphysin localizes to the actin cones whereas alpha-Adaptin does not, it is concluded that dynamin on the actin cones participates in a non-endocytic function. It is hypothesized that Amphiphysin function is related to actin dynamics or organization on the basis of the Amphiphysin localization (Rogat, 2002).
These results suggest that myosin VI regulates the dynamics of actin structures during individualization. This idea is supported by these observations: (1) myosin VI colocalizes with the actin polymerization regulatory proteins cortactin, arp2/3 complex and capping protein; (2) cortactin and arp2/3 complex distribution are disrupted when myosin VI function is reduced; and (3) actin individualization complexes disassemble when both myosin VI and dynamin function are compromised. This is the first demonstration of a genetic interaction between myosin VI and dynamin. In addition, the observation that myosin VI can influence the localization of actin regulatory proteins is novel (Rogat, 2002).
A model is proposed in which myosin VI acts in a structural capacity in the individualization complex to regulate actin dynamics at sites of active membrane remodeling. Myosin VI might participate in actin dynamics solely by its influence on Cortactin and arp2/3 complex localization. Cortactin can bind both actin and the arp2/3 complex. Cortactin also enhances actin polymerization and stabilizes actin filaments during arp2/3-complex-dependent actin polymerization through its actin-binding activity. Myosin VI could help localize or maintain the localization of arp2/3 complex and cortactin at the fronts of the actin cones. The localization of these components at the front of the cones would then facilitate actin assembly. This localized actin assembly would drive actin cone movement in a manner similar to the leading-edge protrusion or Listeria motility. In the absence of myosin VI, the fronts of the cones would lose assembly sites and thus actin cones would depolymerize (Rogat, 2002).
Alternatively, myosin VI might directly stabilize actin at the front of the actin cones. Since myosin VI has a coiled-coil domain that is thought to mediate its dimerization, myosin VI itself could crosslink actin filaments and provide a stabilizing force to newly generated actin filaments. Loss of myosin VI function in this scenario would result in destabilization of actin filaments and the actin-binding proteins associated with those filaments such as arp2/3 complex and cortactin. Thus, in this case, loss of arp2/3 complex and cortactin concentration would be secondary effects owing to loss of actin filaments at the front of actin cones. At this point, the results do not distinguish between a direct or indirect effect of myosin VI on actin assembly/stability (Rogat, 2002).
In addition to a role for myosin VI in localizing actin assembly proteins, it is speculated that myosin VI might be important for coupling actin assembly sites to regions where the membrane is remodeled at the front of individualization complexes. Membrane lies in close proximity to actin along the length of the cone, including the front of the cone, and vesicles, organelles and ribosomes are excluded from this region. The close proximity of the actin cones to the plasma membrane suggests they are tightly linked. The front of the actin cones is also the region where concentrated myosin VI and concentrated sites of actin-assembly-regulating proteins are seen. Immediately in front of the cones there is an abrupt transition to the syncytial cytoplasm in the cystic bulge, a region of large vesicles, organelles and ribosomes. Membrane remodeling takes place at the junction between the front of the actin cones and the syncytial cytoplasm. During individualization complex progression, the connection between membrane and actin must be maintained, otherwise membrane remodeling would not progress synchronously with movements of the actin complex. Myosin VI, perhaps through its effects on Cortactin localization, might be part of the structure that links actin and membrane as the individualization complex moves down the spermatids (Rogat, 2002).
Moreover, the detection of Cortactin at the front of actin cones where myosin VI accumulates indicates that this region is not only a site of active actin assembly but also a region of coupling between membrane and the actin cytoskeleton. Cortactin is thought to act as a link between membrane-associated kinases and actin assembly since it associates with proteins such as Src, Syk and the arp2/3 complex and concentrates in zones of membrane/actin linkage such as lamellipodia (Rogat, 2002).
Myosin VI may be carrying out similar functions in other cellular contexts. The suggestion that myosin VI facilitates actin assembly through effects on the arp2/3 complex and cortactin and couples actin dynamics to membrane remodeling through either a direct or cortactin-dependent mechanism may be applicable to myosin VI function in other systems. For example, vertebrate myosin VI has been proposed to function in endocytosis. During endocytosis, plasma membranes undergo dynamic morphological change in order to invaginate and form a budding vesicle. It has been proposed that F-actin networks help deform membrane during coated-vesicle formation and that F-actin polymerization helps propel endocytic vesicles away from the plasma membrane. In these scenarios actin assembly is required and membrane topology changes must be coordinated with actin assembly. Given such requirements it is not surprising that cortactin has been suggested to function in endocytosis. It is speculated that myosin VI's role in this process may be to link actin assembly sites containing arp2/3 complex and cortactin to sites of membrane dynamics involving dynamin (Rogat, 2002).
It is also tempting to consider that myosin VI might facilitate actin assembly and its coupling with membrane reorganization in other cellular contexts. Examples include membrane invagination in syncytial blastoderm embryos (Mermall, 1995), membrane ruffling in EGF stimulated cells (Buss, 1998) and stereocilia morphogenesis (Self, 1999) in developing hair cells (Rogat, 2002).
It is unclear if the ability of myosin VI to translocate along actin filaments is required for actin cone movement or function. In particular, it is not known whether the minus-end movement of myosin VI is important. The orientation of actin filaments in individualization complexes has not been visualized directly, although the capping protein and arp2/3 complex localization are consistent with the barbed ends of actin filaments at the front of actin cones oriented away from the spermatid nuclei. If this is true, then pointed-end motility would lead to myosin VI walking away from the advancing edge. This seems inconsistent with maintaining a high concentration of myosin VI at the front of the cones. Until the orientation of actin filaments is known, the mobility of membrane and myosin VI observed, and the dynamics of actin in the actin cones determined, it is premature to formulate specific models for the involvement of myosin VI's actin-based motility in this process. However, the studies of spermatid individualization are providing insight into the molecules required for actin cone assembly and movement, as well as myosin VI's specific role in this process (Rogat, 2002).
As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, molecular analysis of a 140-kD ATP-sensitive actin-binding protein has been undertaken. This protein represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29%-33% identical (60%-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain an approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, the new gene has been named 95F myosin heavy chain (95F MHC) (Kellerman, 1992).
date revised: 10 July 2021
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