InteractiveFly: GeneBrief

didum: Biological Overview | References

Gene name - dilute class unconventional myosin

Synonyms - MyosinV, MyoV

Cytological map position - 43C7-43D1

Function - cytoskeletal motor protein

Keywords - cytoskeleton, morphogenic vesicular secretory traffic, rhodopsin transport

Symbol - didum

FlyBase ID: FBgn0261397

Genetic map position - 2R: 3,387,660..3,396,130 [+]

Classification -

Cellular location - cytoplasmic

NCBI link:

didum orthologs: Biolitmine
Recent literature
Ji, H. H., Zhang, H. M., Shen, M., Yao, L. L. and Li, X. D. (2015). The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11. Biochem J [Epub ahead of print]. PubMed ID: 25940004
Myosin-5 (DmM5; Didum) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. This study overexpressed DmM5 in Sf9 cells and investigated its regulation using purified proteins. The actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid and dRab11 respectively. GTP-bound dRab11, but not Lightoid, significantly activates DmM5 actin-activated ATPase activity. Moreover, Gln1689 in the GTD was identified as the critical residue for the interaction with dRab11. Based on those results, it is proposed that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx.
Nishimura, Y., Shi, S., Zhang, F., Liu, R., Takagi, Y., Bershadsky, A. D., Viasnoff, V. and Sellers, J. R. (2021). The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily. J Cell Sci. PubMed ID: 33589498
The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formins from different species. This study found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. The effect of SMIFH2 was further checked on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and SMIFH2 was found to inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. The inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 than for the inhibition of retrograde flow and stress fiber contraction in cells. It was also found that SMIFH2 inhibits several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficient than inhibition of formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions.
Zhang, N., Zhou, S. J., Ji, H. H. and Li, X. D. (2022). Effects of the IQ1 motif of Drosophila myosin-5 on the calcium interaction of calmodulin. Cell Calcium 103: 102549. PubMed ID: 35144093
In Drosophila compound eyes, myosin-5 (DmMyo5) plays a key role in organelle transportation, including transporting pigment granules from the distal end to the proximal end of the photoreceptor cells to regulate the amount of light reaching the photosensitive membrane organelle rhabdomere. It is generally accepted that, upon exposure to light, the dark-adapted compound eyes produce a rapid rise of free Ca(2+) concentration, which in turn activates DmMyo5 to transport pigment granules. Considering the dynamic and compartmentation of Ca(2+) signaling in photoreceptor cells during light exposure, it is necessary to understand the kinetics of Ca(2+) interaction with DmMyo5. This study investigated the interaction of Ca(2+) with Drosophila calmodulin (CaM) in complex with the IQ1 of DmMyo5 using steady-state and kinetic approaches. These results show that IQ1 binding substantially increases the Ca(2+) affinity of CaM and decreases the dissociation rate of Ca(2+) from CaM. In addition, it was found that Mlc-C, the light chain associated with the IQ2 of DmMyo5, has little effect on the Ca(2+) kinetics of CaM in IQ1. It is proposed that, by decreasing the Ca(2+) dissociation rate from CaM, IQ1 delays the deactivation of DmMyo5 after Ca(2+) transition, thereby prolonging the DmMyo5-driven transportation of pigment granules.


Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and it was asked if myosin V (Didum) and the Drosophila Rab11 interacting protein, dRip11 (lethal (1) G0003), also participate in secretory transport. Reduction of either protein impairs rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin. MyoV-reduced photoreceptors also develop ectopic rhabdomeres inappropriately located in basolateral membrane, indicating a role for MyoV in photoreceptor polarity. Binary yeast two hybrids and in vitro protein-protein interaction predict a ternary complex assembled by independent dRip11 and MyoV binding to Rab11. It is proposed that this complex delivers morphogenic secretory traffic along polarized actin filaments of the subcortical terminal web to the exocytic plasma membrane target, the rhabdomere base. A protein trio conserved across eukaryotes thus mediates normal, in vivo sensory neuron morphogenesis (Li, 2007).

Across eukaryotes, a protein trio comprising a Rab protein, a member of the family of small GTPases that regulate exchange between membrane compartments, a myosin motor, notably myosin V (MyoV), and a linker/adaptor protein, powers organelle motility and polarized secretion (Hammer, 2002; Deneka, 2003; Seabra, 2004). For example, HeLa and MDCK cells recycle endocytosed cell surface receptors through a recycling endosome, the return leg mediated by Rab11 together with MyoV and the Rab11 adaptor/linker protein, family interacting protein 2 (FIP2) (Hales, 2002). Drosophila photoreceptors are typical polarized epithelial cells and morphogenesis of their photosensory membrane organelles, rhabdomeres, is driven by a late-pupal surge of secretory traffic that greatly expands the apical plasma membrane in a column of closely packed, rhodopsin-rich photosensitive microvilli. It was recently found that Rab11 mediates membrane transport to developing rhabdomeres (Satoh, 2005), prompting an investigation to see if Drosophila MyoV (Bonafe, 1998; MacIver, 1998) and dRip11, and Drosophila FIP2 (Prekeris, 2000) also participate in morphogenic secretory transport (Li, 2007).

Numerous observations link MyoV to polarized membrane transport (Reck-Peterson, 2000). Budding yeast lacking essential MyoV, Myo2p, accumulate cytoplasmic post-Golgi secretory vesicles; secretion continues in mutants, but is not correctly targeted to the growing bud (Johnston, 1991). Melanocytes of mouse dilute mutants lacking MyoVa fail to properly localize melanosome pigment organelles to the actin-rich cell periphery; expression of a MyoVa C-terminal fragment (MyoVa-CT) that displaces endogenous MyoVa from melanosomes mimics MyoVa loss (Wu, 1998). Expression of MyoVa-CT similarly inhibits Xenopus melanosome motility (Rogers, 1999) and HeLa cell transferrin receptor recycling (Lapierre, 2001; Hales, 2002; Rodriguez, 2002). Notably, in polarized MDCK cells, MyoVb-CT selectively disrupts Rab11-dependent apical, but not basolateral, membrane recycling (Lapierre, 2001) (Li, 2007).

Parallel loss-of-function phenotypes suggest MyoV and Rab11 cooperate in membrane transport. Loss of either activity inhibits recycling of CXCR2 chemokine and M4 muscarinic acetylcholine receptors (Volpicelli, 2002; Fan, 2003; Fan, 2004). Similarly, MyoV or Rab11 reduction prevents biogenesis of apical cannicular membranes in polarized hepatocytes (Wakabayashi, 2005) and decreases glutamate receptor 1 (GluR1) subunit delivery to developing synapses of hippocampal cells in culture (Lise, 2006) (Li, 2007).

Direct interaction between rabbit Rab11a and MyoVb is detected in yeast two-hybrid screens (Lapierre, 2001), and deletion of MyoVb-CT's Rab11 binding sequence neutralizes its dominant-negative impact on GluR1 delivery in hippocampal neurons, suggesting MyoVb binds Rab11 in GluR1 trafficking (Lise, 2006). Genetic interaction between Saccharomyces cerevisiae Myo2p and Sec4p mutants (Schott, 1999) is consistent with direct or close cooperation (Li, 2007).

In addition to MyoV, Rab11 interacts with Rab11-FIPs at a signature Rab11 binding domain (RBD) (Prekeris, 2003). Class I FIPs contain a C2 domain that targets recycling vesicles to the plasma membrane, and truncated FIPs lacking the C2 domain inhibit receptor recycling. Drosophila encodes a single class I FIP, dRip11 (Prekeris, 2000), but its function has not been reported (Li, 2007 and references therein).

The Drosophila genome includes a single MyoV gene, myoV (didum) (Bonafe, 1998; MacIver, 1998). Drosophila embryos receive substantial maternal MyoV and the protein is ubiquitously expressed throughout development, including the adult retina, where it localizes to the base of the rhabdomere (Mermall, 2005). Mutants lacking MyoV show strong developmental delays and substantial late larval lethality. Surprisingly, rare homozygous mutant escapers showed normal embryogenesis and cellular architecture, suggesting MyoV is dispensable for the wide range of membrane trafficking that supports normal development (Mermall, 2005). Actin staining of myoV mutant eyes showed apparently normal rhabdomeres and adult mutants were normally phototaxic, suggesting that MyoV does not play an obvious role in rhabdomere development or photoreception (Li, 2007 and references therein).

This paper investigated the role of MyoV and dRip11 in the polarized membrane transport that builds Drosophila rhabdomeres. Both were found to be essential. In MyoV mutants, rhodopsin 1 (Rh1) is not delivered to the growing rhabdomere, but instead accumulates in photoreceptor cytoplasm; rhodopsin-bearing vesicles, and the Rab11 and dRip11 they carry, do not approach the rhabdomere base. dRip11 loss similarly impairs secretory transport, delocalizing MyoV and Rab11 and promoting cytoplasmic Rh1. MyoV mutant photoreceptors also develop supernumerary rhabdomeres ectopically positioned within basolateral plasma membrane, suggesting MyoV-mediated transport suppresses formation of inappropriate rhabdomere primordia. Drosophila photoreceptors harness an evolutionarily conserved protein trio to deliver polarized apical membrane traffic in cellular morphogenesis (Li, 2007).

Drosophila photoreceptors, like many polarized epithelial cells, greatly amplify their apical membranes during terminal differentiation via targeted membrane delivery. This study shows that a protein trio (Rab11, dRip11, and MyoV) mediates this morphogenic secretory traffic. MyoV normally concentrates at the base and its loss causes three notable phenotypes of compromised apical transport: Rab11 and dRip11 delocalize from the base, Rh1 accumulates in photoreceptor cytoplasm, and ectopic rhabdomeres are formed. dRip11, the sole Drosophila class I Rab-FIP (Prekeris, 2003), is also required for normal Rh1 transport; its loss delocalizes Rab11 and MyoV. Together with the demonstration that Rab11 is essential for photoreceptor secretory traffic (Satoh, 2005), it is proposed MyoV pulls post-Golgi secretory vesicles, marked for rhabdomere delivery by Rab11 and dRip11, through an exclusionary subcortical cytoskeletal web along polarized microfilaments leading directly to the exocytic targeting patch at the rhabdomere base (Li, 2007).

Cytoplasm adjacent to the rhabdomere base is permeated by a dense microfilament brush, the rhabdomere terminal web (RTW), which extends from the rhabdomere base deep into photoreceptor cytoplasm (Arikawa, 1990; Chang, 2000). Microfilaments are poorly preserved in chemically fixed tissue, but distinct 'RTW cytoplasm' is manifest as organelle-poor cytoplasm behind the rhabdomere. RTW cytoplasm excludes even ribosomes, whose absence contributes to the light, clear appearance of RTW cytoplasm. Biosynthetic ER and Golgi are distributed the length of the cell, in close proximity to the RTW's cytoplasmic terminus (Li, 2007).

The rhabdomere base differentiates in mid-pupal photoreceptors as the photoreceptor apical membrane resolves to a central Moesin-rich rhabdomere primordium surrounded by a Crumbs-rich supporting domain. Once founded, the rhabdomere base organizes the RTW and receives morphogenic traffic. The stalk accepts little traffic, focusing exocytosis to the rhabdomere. The stalk links the rhabdomere to the retina's junctional network and projects it into an apical lumen, the IRS, aligned to the eye's optical axis (Li, 2007).

Membrane transport in light-adapted late pupal photoreceptors is dynamic, with biosynthetic and endocytic traffic reflected in numerous, complex membrane compartments. Post-Golgi secretory traffic is carried in tubular vesicles, approximately 100 nm across; endocytosed membrane gathers in multivesicular bodies. Complex membrane forms are common at the rhabdomere base, likely a consequence of extensive membrane fusion (Li, 2007).

Confocal immunofluorescence localizes Rab11 to puncta throughout photoreceptor cytoplasm, with a prominent concentration at the rhabdomere base . dRip11 immunolocalization resembled Rab11, with cytoplasmic puncta and localization at the rhabdomere base. Note that Rab11 and dRip11 lie within RTW cytoplasm, overlapping the actin brush extending from the rhabdomere's curving base. MyoV concentrates across the rhabdomere base of late pupal photoreceptors, often appearing strongest at the sides. Like Rab11 and dRip11, MyoV staining is strongly within RTW cytoplasm. Cytoplasmic MyoV is lightly diffuse with scattered brighter puncta (Li, 2007).

The RTW's role as both a barrier and a carrier for morphogenic traffic is an instance of a general theme of a dynamic regulatory role of the subcortical cytoskeleton in secretion. Myosin S1 decoration shows RTW filaments are oriented with plus-ends at the membrane, a correct orientation for MyoV-based secretory transport (Arikawa, 1990), and disruption of the actin cytoskeleton prevents the morphogenic traffic that rebuilds crab rhabdomeres at dusk. The RTW's strong polarization and anchorage to a secretory targeting patch resembles the polarized actin cables that mediate budding yeast secretory traffic (Li, 2007 and references therein).

Absorptive and secretory epithelial cell specialists often regulate apical membrane activity by dynamic, Rab11-dependent exchange of plasma membrane with recycling endosomes. For example, gastric parietal cells meet demand for additional acid secretion by Rab11-, Rab11-FIP2-, and MyoV-dependent delivery of additional H+/K+ ATPase pumps to the cell surface from a recycling endosome (Duman, 1999; Hales, 2001; Lapierre, 2001). Like GPCRs generally, Drosophila Rh1 is endocytosed upon stimulation but appears to be degraded rather than recycled back to the rhabdomere. Drosophila photoreceptor Rab11-dependent transport appears to be principally devoted to delivery of newly synthesized cargo from the TGN to the plasma membrane, a conserved Rab11 activity (Chen, 1998) now seen to further parallel recycling transport. Ectopic rhabdomeres in hypomorphs suggest MyoV normally suppresses the establishment of inappropriate rhabdomere primordia; once-founded ectopic rhabdomeres develop in concert with principal rhabdomeres, presumably drawing from the same secretory traffic. It is speculated that MyoV normally drives traffic to the differentiating rhabdomere primordium and that positive feedback driven by the incorporation of morphogenic determinants, perhaps proteins that anchor and promote RTW development, gives the original, 'true' apical membrane an overwhelming growth advantage, starving weak, inappropriate sites. MyoV reduction might diminish this advantage, allowing ectopic foci to capture sufficient morphogenic traffic to assemble a rhabdomere patch (Li, 2007).

The observation that MyoV is required for normal rhabdomere development differs from Mermall's report of normal rhabdomeres in MyoVQ1052st mutant eyes (Mermall, 2005). However, long ribbons of the principal rhabdomeres dominate phalloidin-stained longitudinal sections, and ectopic rhabdomeres, often patches a few microns across, are not prominent. Mermall's supplementary Fig. 1 L, a tangential section, shows actin-bright profiles apart from the principal rhabdomeres - potentially ectopic rhabdomeres. Massive biosynthetic traffic in late pupal photoreceptors sensitizes cells to compromise of efficient, accurate transport and accumulation of cytoplasmic Rh1 reflects an inability of transport to keep pace with biosynthesis (Li, 2007).

dRip11 loss inhibits secretory transport and misolcalizes Rab11 and MyoV. It is suggested that dRip11 couples two broad streams of membrane transport, Rab11- and MyoV-dependent activities, to drive morphogenic secretory traffic. The results are consistent with previously demonstrated roles for FIPs as contributors to membrane targeting (Meyers, 2002; Lindsay, 2004), and as scaffolds for the growing Rab11 effector ensemble (Pooley, 2006). Similar to chromaffin cells, where MyoV only partially overlaps with secretory vesicles (Rose, 2003), MyoV and Rab11 only partially overlap in developing photoreceptors, and it is likely MyoV transports multiple and changing cargoes (Li, 2007).

Rab11 participates in both constitutive and Ca2+-regulated secretion (Khvotchev, 2003), and both cargo binding and Ca2+ regulate MyoV activity (Krementsov, 2004; Li, 2005; Thirumurugan, 2006). Rhabdomere morphogenesis utilizes constitutive exocytosis, with substantial rhabdomere growth before Rh1 expression and photoresponse Ca2+ influx. Rhabdomeres likewise develop normally in the dark, indicating light-dependent Ca2+ elevation is not required for MyoV morphogenic transport. It is proposed that dRip11, in proximity to MyoV via their mutual binding to Rab11 on post-Golgi secretory vesicles, interprets or conveys non-Ca2+-stimulated MyoV activation, promoting developmental MyoV secretory transport (Li, 2007).

Crumbs regulates rhodopsin transport by interacting with and stabilizing myosin V

The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. This paper shows that myosin V (MyoV) coimmunoprecipitates with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. It is concluded that Crb protects against degeneration by interacting with and stabilizing MyoV, thereby ensuring correct Rh1 trafficking. The data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas (Pocha, 2011).

The role of the Crb complex in polarity is well studied, but the mechanism behind its ability to prevent light-dependent retinal degeneration is poorly understood. Some insight into the latter came from studies reporting that feeding flies a vitamin A (carotenoid)-depleted medium prevented the light-dependent degeneration of crb, sdt, and DLin7 mutant PRCs. These data suggested that degeneration in Crb complex mutants involves Rh1; however, the molecular mechanism behind this remained unknown. This study provides the missing link by showing that the Crb complex interacts with MyoV, an unconventional myosin, which has an established role in the transport of Rh1 to the rhabdomere. MyoV levels are reduced by ~90% in crb mutant retinas, which can be largely rescued by inhibition of the proteasome, and Rh1 transport is defective in crb mutant PRCs. Therefore, it is proposed that the Crb complex protects against light-dependent degeneration by interacting with and maintaining MyoV levels, thereby ensuring proper Rh1 transport to the rhabdomere (Pocha, 2011).

Blocking proteasome activity also allowed assessment of the localization of MyoV in the absence of Crb. Apical localization of MyoV was observed; however, rather than adopting the WT localization that spans the entire rhabdomere base, the rescued MyoV was seen in large clumps, which only partially covered the base of the rhabdomere. The steady-state WT localization of MyoV reflects its role in transporting Rh1 from the cell body to the rhabdomere base. Therefore, these large accumulations may suggest that some level of MyoV degradation is also important for maintaining efficient transport by the total pool of MyoV. Thus, the levels of MyoV and its ability to transport Rh1 to the rhabdomere base may depend on external cues (e.g., light), which alter the balance between stabilization and degradation (Pocha, 2011).

IPs of both Sdt and DPatJ contain the respective other members of the Crb complex, and together with these, MyoV is precipitated specifically. The strong reduction in MyoV protein seen in crb mutant photoreceptors raises the question of whether stability of MyoV is dependent on Crb itself or on the integrity of the Crb complex. As loss of Crb results in the loss of Sdt and the delocalization of DPatJ and DLin7, the data obtained using crb11A22 mutants can be used to analyze the role of the Crb complex. Data obtained from crb8F105 mutants, however, show that the integrity of the Crb complex is not required for the crb-dependent stabilization of MyoV. This was further supported by experiments in S2R+ cells that showed Crb alone, in the absence of Sdt, can recruit MyoV-GFP to the plasma membrane, suggesting that the interaction observed between Crb and MyoV is not mediated by any of the other core components of the Crb complex. As the interaction was detected by IP, the possibility remains that the interaction between Crb and MyoV is mediated by another still unknown protein (Pocha, 2011).

Interestingly, loss of MyoV in crb11A22 mutants cannot be overcome by overexpression of a MyoV transgene expressed under the control of an exogenous system, the UAS/Gal4 system. This demonstrates that Crb is required to maintain MyoV stability posttranscriptionally. This was investigated further by inhibiting proteasomal degradation, and a marked increase of MyoV staining was observed in crb mutant photoreceptors compared with controls. These findings support the conclusion that the interaction between Crb and MyoV is stabilizing the latter by protecting it from degradation by the proteasome (Pocha, 2011).

crb is known to have two main functions in the eye, one during development of the retina to ensure correct morphogenesis of the PRCs and the other to prevent degeneration of the adult eye in constant light. This study shows that MyoV does not show a polarized distribution at early pupal stages nor is its localization perturbed by loss of Crb in early stages, the time at which morphogenetic defects in crb mutants start, suggesting that the interaction between Crb and MyoV is not required for proper morphogenesis to occur. This is supported by reports that MyoV-null mutant adults display only mild morphological defects, which are distinct from those observed in crb mutants (Pocha, 2011).

The finding that MyoV fails to start accumulating apically in crb mutant cells during late pupal stages after Rh1 expression starts corroborates the conclusion that the Crb-MyoV interaction is required for the second role of Crb in the retina, preventing light-dependent degeneration. It is also plausible that the steady-state localization of MyoV seen in the adult is largely the result of its role in Rh1 transport to the rhabdomere, as MyoV is seen evenly distributed throughout the cell before Rh1 expression starts. This assumption is supported by published data showing that the localization of MyoV in the adult is light dependent and therefore reflects the status of Rh1 activation and transport. Fittingly, the apical accumulation of MyoV at later pupal stages coincides with increased MyoV staining and increased colocalization of MyoV with Crb (Pocha, 2011).

The effect that loss of Crb has on Rh1 was tested, and it was demonstrated that in normal 12-h light/12-h dark conditions, defects in Rh1 staining are seen only in old flies. This is suggestive of a subtle defect in Rh1 transport that is only visible at steady state if allowed to accumulate over time or if the system is under stress (i.e., constant light). As it was reported that only minimal MyoV activity is required for proper Rh1 localization, it is probable that the remaining 10% of MyoV seen in crb mutants is sufficient for Rh1 transport in young flies, but, over time, the effect of this deficiency accumulates, resulting in the retention of Rh1-positive punctae in the cell body. Together with the results from the Rh1 pulse-chase assay, it is concluded that in crb mutant tissue, Rh1 transport to the rhabdomere is delayed and that the cumulative effect of this delayed transport leads to the accumulation of Rh1 within the cell body, which is associated with a gradual deterioration of the rhabdomeres (Pocha, 2011).

Previous findings have shown that Crb-mediated protection against light-dependent retinal degeneration is not solely dependent on the ability of Crb to assemble and integrate into the Crb complex. Photoreceptors of crb8F105 mutants that express a Crb protein lacking the Sdt-interacting ERLI motif do not undergo light-dependent degeneration. This observation is in agreement with the findings presented in this study that MyoV is retained in Crb8F105 mutant photoreceptors. In addition, overexpression of a Crb transgene encoding the transmembrane and intracellular domains was not able to rescue the light-dependent degeneration observed in crb11A22 mutants. Interestingly, this membrane-tethered intracellular domain-encoding transgene does rescue the morphogenetic defects observed in both crb8F105 and crb11A22 mutants. Therefore, the two roles of Crb in the retina photoreceptor morphogenesis and maintenance — appear to occur through distinct mechanisms. Correct morphogenesis seems to necessitate the assembly of the Crb complex through the Crb ERLI motif. In contrast, Crb-mediated protection against light-dependent degeneration and stabilization of MyoV does not require an intact Crb complex. How do these finding correlate with reports of light-dependent retinal degeneration in other members of the Crb complex? It is proposed that in sdt and DPatJ mutants, it is the concomitant loss of Crb that is responsible for the degeneration phenotype rather than the loss of an intact Crb complex itself (Pocha, 2011).

The absence of endogenous Crb and Sdt from cultured Schneider S2R+ cells made them particularly useful to identify the regions of Crb required for its interaction with MyoV that were determined to include the membrane-spanning and first 14 amino acids of the cytoplasmic domain. However, the readout for this interaction — the recruitment of MyoV-GFP to the plasma membrane — may not reflect the purpose of this interaction in vivo, particularly considering the highly polarized and functionally specialized nature of PRC. Indeed, the finding that the majority of the residual MyoV in crb mutant photoreceptors localizes to the rhabdomere base suggests that in photoreceptors, the role of the Crb-MyoV interaction is primarily to stabilize MyoV and not to recruit it to the rhabdomere base. In addition, the ability of Crb lacking the extracellular domain to recruit MyoV to the plasma membrane of S2R+ cells but not to rescue light-dependent degeneration suggests that S2R+ cells lack many qualities (morphology, protein expression, and functionality) of PRCs. Considering the requirement of the extracellular domain, it is possible that in the context of a light-sensing photoreceptor, Crb responds to a stimulus that is transmitted via the extracellular domain, which then initiates the interaction with and/or the stabilization of MyoV. This hypothesis is an intriguing one, as the only function of Crb that requires its extracellular domain is its role in preventing light-dependent degeneration, and, to date, no known partner of the extracellular domain has been identified (Pocha, 2011).

A model is proposed in which the interaction between Crb and MyoV stabilizes the latter, maintaining a complete Rh1 transport cycle. In crb mutants, this cycle is slowed down at the MyoV-dependent stage of delivery to the rhabdomere. Whereas in normal light/dark conditions the effect of this is minimal, upon exposure to constant light, Rh1 accumulates in the cell body, suggesting that the rate of removal from the rhabdomere (as a result of constant activation) exceeds the rate of delivery to the rhabdomere. As previously discussed, photoreceptors are extremely sensitive to perturbations in the phototransduction cascade, and it has been well documented that mutations that affect the synthesis, delivery, and recycling of Rh1 lead to degeneration. Together with previously published data showing the rescue of Crb-dependent retinal degeneration in the absence of vitamin A, this strongly supports a model that the accumulation of Rh1 in the cell body as a result of a deficiency of Rh1 transport in crb mutants leads to degeneration (Pocha, 2011).

These data provide a molecular mechanism for the light-dependent degeneration observed in crb mutant animals. Recent findings that myoVIIa mutant mice display light-dependent degeneration as a result of defects in rod protein translocation (Peng, 2011) suggest that the efficient transport of opsins by myosins is crucial to prevent degeneration across species. Therefore, it will be intriguing to see whether the mechanism we identified here is conserved and whether human photoreceptors from patients with CRB1 mutations also display reduced myosin levels and delays in Rh1 transport (Pocha, 2011).

Calcium-activated Myosin V closes the Drosophila pupil

Approximately 40 years ago, an elegant automatic-gain control was revealed in compound eye photoreceptors: In bright light, an assembly of small pigment granules migrates to the cytoplasmic face of the photosensitive membrane organelle, the rhabdomere, where they attenuate waveguide propagation along the rhabdomere. This migration results in a 'longitudinal pupil' that reduces rhodopsin exposure by a factor of 0.8 log units. Light-induced elevation of cytosolic free Ca2+ triggers the migration of pigment granules, and pigment granules fail to migrate in a mutant deficient in photoactivated TRP calcium channels. However, the mechanism that moves photoreceptor pigment granules remains elusive. Are the granules actively pulled toward the rhabdomere upon light, or are they instead actively pulled into the cytoplasm in the absence of light? This study shows that Ca2+-activated Myosin V (MyoV) pulls pigment granules to the rhabdomere. Thus, one of MyoV's several functions is also as a sensory-adaptation motor. In vitro, Ca2+ both activates and inhibits MyoV motility; in vivo, its role is undetermined. This first demonstration of an in vivo role for Ca2+ in MyoV activity shows that in Drosophila photoreceptors, Ca2+ stimulates MyoV motility (Satoh, 2008).

Photoreceptor pigment-granule migration was assayed by observing an optical phenomenon called the deep pseudopupil (DPP). Upon illumination, the Drosophila DPP typically changes from dark red to bright green with a time constant as small as 2 s. In dark-adapted Canton-S wild-type eyes, the reflectance of the DPP is low; incident light is effectively channeled into the rhabdomeres and propagates there until it is absorbed by the visual pigment or by the pigment proximal to the rhabdomeres. Within seconds after the light is turned on, DPP reflectance rapidly increases, as pigment granules in the photoreceptor cytoplasm migrate to the rhabdomere base. The reflecting pigment granules cause a gold-green crescent in the light-adapted DPP. Note that although the pigment granules do not enter the rhabdomere, some of the light they reflect propagates distally along the rhabdomere, back to the source, seen as a dimmer central glow within the brighter crescent at the rhabdomere base. The pupil remains closed in the presence of continued strong light (Satoh, 2008).

In a strong MyoV loss-of-function mutant, MyoVKG04384, the pupil does not close, even after prolonged, bright illumination. Transgenic MyoV expression rescues MyoVKG04384 pupil closure. In fact, even when adapted to the dark, the pupil of transgenic animals appears partially closed, probably reflecting MyoV overexpression (Satoh, 2008).

Electron microscopy was used to identify pigment granule position in dark- and light-adapted wild-type and MyoV mutant photoreceptors. To localize pigment granules in dark-adapted photoreceptors, eyes were fixed by using infrared illumination and image-intensifying eyepieces. Cytoplasm adjacent to the rhabdomere excludes most organelles and appears relatively clear in electron micrographs. This cytoplasmic domain is dominated by the rhabdomere terminal web (RTW), a dense array of parallel, polarized terminal web microfilaments that emanate brush-like from the rhabdomere base and extend deep into photoreceptor cytoplasm; RTW microfilaments are oriented with their plus ends, toward which MyoV motility is directed, to the rhabdomere base. RTW cytoplasm is manifest as relatively clear cytoplasm from which organelles are excluded. In Musca photoreceptors and in Drosophila trp mutant photoreceptors pigment granules of dark-adapted wild-type photoreceptors stand off from the rhabdomere base; they are separated from it by RTW cytoplasm and are located along the back, or minus side, of the RTW. By 2 min after the room's lights come on, most pigment granules have moved through RTW cytoplasm to the rhabdomere base. In dark-adapted MyoVKG04384 photoreceptors, pigment granules still line up mostly along the back of the RTW; few migrate in response to illumination. MyoV is thus essential for pigment granule migration (Satoh, 2008).

Confocal immunofluorescence microscopy was used to ask whether MyoV localizes to pigment granules and migrates upon illumination. Because Lightoid (Ltd), a Rab-related protein, marks pigment granules, dark-adapted and illuminated photoreceptors were double stained with anti-MyoV and anti-Ltd antibodies. In dark-adapted photoreceptors, MyoV and Ltd localized prominently along a line at the back of RTW cytoplasm, consistent with the electron microscopic localization of pigment granules. It is notable that this line persists in w1118 mutants that lack the ommochrome pigment granules, suggesting that pigment granules per se are not necessary for MyoV and Ltd localization. Ltd is also prominent in large vesicles, appearing as bright circles, in pigment cells that surround photoreceptors. Both MyoV and Ltd immunofluorescence in wild-type eyes are similar, but dimmer, presumably as a result of fluorescence quenching by the dense ommochrome pigment. Upon illumination, MyoV and Ltd move to the rhabdomere base. It is thus concluded that pigment granules, Ltd, and MyoV migrate together upon Cai increase (Satoh, 2008).

In Ltd-null, ltd1 mutants, MyoV's characteristic localization is lost: MyoV is diffuse throughout the cytoplasm of both dark-adapted and illuminated photoreceptors. The formation of pigment granules in ltd1 mutants is impaired, and there are few ommochrome granules in ltd1 photoreceptors. However, decreased pigment granule number cannot account for MyoV delocalization because MyoV localizes normally in w1118 photoreceptors, which contain Ltd but not pigment granules. Thus, Ltd is essential for MyoV's localization in dark-adapted photoreceptors and its light-dependent translocation to the rhabdomere base (Satoh, 2008).

To evaluate potential interactions between Ltd and MyoV, binary yeast two-hybrid assays were used. A strong interaction was found between Ltd's Rab domain and the MyoV tail (amino acids 922-1800). A medium length of MyoV tail (amino acids 1063-1800) showed weak interaction, but the shortest tail fragment (amino acids 1383-1800) did not support colony growth. Direct protein-protein interaction is supported by coimmunoprecipitation of Ltd's Rab domain and a long fragment of MyoV tail from an in vitro transcription and translation system (Satoh, 2008).

Vertebrate MyoV binds CaM, and Drosophila MyoV binds CaM and Myosin light chain-cytoplasmic (Mlc-c) when the proteins are coexpressed in cells (Toth, 2005). Consequently, CaM and Mlc-c localization were investigated in dark-adapted and illuminated photoreceptors. CaM localized along the back of the RTW cytoplasm in dark-adapted photoreceptors. Lighter, diffuse anti-CaM staining, not overlapping with MyoV, was also seen in rhabdomeres and cytoplasm. Upon illumination, CaM and MyoV cotranslocate to the rhabdomere base. Both CaM and Mlc-c coimmunoprecipitate with Myc-tagged MyoV C-terminal. Thus, both CaM and Mlc-c are photoreceptor MyoV light chains (Satoh, 2008).

The impact of CaM loss on MyoV localization was examined. Although CaM-null flies are embryonic lethal, heteroallelic cam339/cam352 flies expressing less than 10% wild-type CaM are viable. CaM staining is greatly reduced in these animals, and MyoV localization is abnormal. In both light and dark conditions, some MyoV localizes to rhabdomere tips, whereas it is otherwise diffuse in the cytoplasm. CaM is thus essential for normal MyoV distribution. Together, these results suggest that CaM binds MyoV and, in response to illumination, they move together across the RTW (Satoh, 2008).

DPP reflectance was examined in cam339/cam352 flies. Similar to what is seen in MyoV mutants, the pupil does not close in these flies, even after bright, continuous illumination. In electron micrographs of dark-adapted cam mutants, pigment granules are positioned normally. In illuminated cam339/cam352 photoreceptors, pigment granules still line up along the back of the RTW; pigment granule translocation is completely abolished. CaM is thus essential for the migration of pigment granules (Satoh, 2008).

It is important to efficiently integrate MyoV motility with the specific cellular task at hand. Upon illumination, Drosophila phototransduction entrains rises of Cai, from 160 nM in the dark to 10 μM in bright light; pigment-granule migration is triggered at 1 μM Ca2+, a level attained at luminances above 0.3 cd/m2, which is roughly comparable to the threshold of human color vision. As in the pupil, Ca2+ elevation stimulates purified chick brain MyoV ATPase activity 90-fold over baseline activity, and there is a sharp activation threshold at 1 μM Ca2+. In vitro, low Ca2+ unfolds and activates MyoV, whereas higher Ca2+ dissociates CaM from MyoV lever arms and inhibits motility; addition of exogenous CaM restores motility (Krementsov, 2004; Nguyen, 2005; Lu, 2006). Results of this study suggest that the endogenous pool of CaM in fly photoreceptors suffices to preserve motility in the presence of elevated Ca2+ (Satoh, 2008).

Cargo binding to the MyoV globular tail in vitro also promotes MyoV unfolding and activation (Krementsov, 2004; Li, 2004; Wang, 2004; Liu, 2006; Thirumurugan, 2006), raising the question of whether vesicle-bound MyoV in vivo must be constitutively active. Results described in this study suggest this may not be necessary; in the dark, pigment granules localize to the back of the RTW, indicating cargo-bound MyoV activation additionally requires Ca2+ signaling. Ltd linkage of pigment granules might allow an extended MyoV form that is inactive at resting Cai (Satoh, 2008).

MyoV frequently partners with Rab family proteins to mediate organelle transport. In Drosophila photoreceptors, distinct Rab family members harness MyoV to distinct activities. In differentiating photoreceptors Rab11 partners with MyoV to deliver post-Golgi biosynthetic traffic to the rapidly expanding sensory membrane (Li, 2007). In mature photoreceptors, Ltd couples MyoV to pigment granules, employing it as a motor of sensory adaptation (Satoh, 2008).

MyoV positions melanosomes within melanocytes, delivers recycling and biosynthetic vesicles to the plasma membrane, propels endosomal traffic in neurons, and carries vacuoles into budding yeast daughter cells. Loss of MyoV-dependent motility causes human Griscelli disease, whose patients suffer hypopigmentation and neurological and immunological defects (OMIM 214450). Mutant mice lacking MyoV are hypopigmented and fail to deliver Ca2+-regulating endosomes to synaptic spines; they suffer severe, often fatal seizures. The Drosophila pupil is a genetically and molecularly tractable in vivo assay for this vital cellular motor (Satoh, 2008).

Competition between kinesin-1 and myosin-V defines Drosophila posterior determination

Local accumulation of oskar (osk) mRNA in the Drosophila oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for osk mRNA posterior localization. This study used Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for osk mRNA. Posterior localization of osk/Staufen was shown to be determined by competition between kinesin-1 and myosin-V. While kinesin-1 removes osk/Staufen from the cortex along microtubules, myosin-V anchors osk/Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of Drosophila oocytes is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density (Lu, 2020).

It is well established that kinesin-1 is essential for localization of osk/Staufen particles at the posterior pole of the Drosophila oocyte. However, it remained unclear how the compact posterior cap is anchored and retained over time. Cortical F-actin remodeling and Myosin-V, as well as the Arp1 subunit of the dynactin complex, have been all implicated in the osk/Staufen cortical localization. This study combined genetic and optogenetic tools to demonstrate that direct competition between two motors, kinesin-1 and myosin-V, ensures the posterior anchorage of osk/Staufen. Notably, it was demonstrated that the outcome of the competition is primarily determined by the density of cortical microtubules. High microtubule density at the anterior and lateral cortex favors kinesin-driven osk/Staufen cortical exclusion, while low microtubule density at the posterior pole favors myosin-driven cortical retention. Therefore, the kinesin-myosin competition and cortical microtubule density together determine the initial accumulation of osk mRNA at the posterior pole (Lu, 2020).

The cortical exclusion model was first proposed after uniform cortical localization of osk mRNA was observed in the kinesin-null oocytes. In agreement with this model, this study shows that constitutively active kinesin-1 causes osk/Staufen mislocalization in the cytoplasm of the oocyte, whereas reducing microtubule density at the lateral cortex leads to ectopic accumulation of Staufen at the cortex. These data support the model that kinesin-driven cortical exclusion along cortically-attached microtubules plays an essential role in restricting osk/Staufen to the posterior pole (Lu, 2020).

Previously, several groups have proposed that kinesin-1 transports osk/Staufen particles along slightly biased cortical microtubules, resulting in net movement of osk/Staufen from the anterior side to the posterior pole in stage 8-9 oocytes. In fact, kinesin-driven cortical exclusion and kinesin-driven transport towards the posterior pole are not mutually exclusive; they describe the same event of osk/Staufen movement. Within the oocyte, cortical microtubules are anchored to the cortex by their minus-ends while their plus-ends face towards the cytoplasm. Due to the anterior-posterior gradient of cortical microtubule density, more microtubule plus ends are oriented towards the posterior pole. Thus, kinesin-1-driven transport along microtubules is a prerequisite for kinesin-1-driven cortical exclusion. Cortical exclusion of osk/Staufen by kinesin-1 results in biased transport of osk/Staufen towards the posterior pole (Lu, 2020).

This kinesin-myosin competition model is suggested by genetic interaction data from a previous study. Specifically, increasing KHC dosage enhances osk/Staufen mislocalization phenotypes in myosin-V loss-of-function mutants, while reducing KHC dosage by half partially suppresses myosin loss-of-function phenotypes. Furthermore, double MyoV and Khc mutant clones have diffuse cytoplasmic localization of osk mRNA, compared to uniform cortical localization of osk mRNA in Khc single mutant clones. These data strongly imply that in the absence of kinesin-1, myosin-V promiscuously anchors osk/Staufen everywhere in the cortex. This study manipulated the activity of either kinesin-1 or myosin-V and found that proper balance between the activities of these two motors is critical for correct osk/Staufen localization, supporting the model in which kinesin-myosin competition is key to the correct posterior determination in the Drosophila oocyte (Lu, 2020).

The competition between kinesin-1 and myosin-V described in this study is not the first example of such a mechanism for cargo transport and localization. For instance, myosin-V opposes microtubule-dependent transport and provides a dynamic anchor for melanosomes, peroxisomes , recycling endosomes, mitochondria, and synaptic vesicles at sites of local accumulation of F-actin. At these sites, the abundance of F-actin tracks provides an upper hand for myosin-V to win the tug-of-wars over microtubule motors. Kinesin-myosin competition appears to be an evolutionarily conserved mechanism to allow flexible refinement and/or error correction, as motors constantly undergo reversible binding and releasing activity on cytoskeletal filaments (Lu, 2020).

In the oocytes, the machinery responsible for osk/Staufen localization contains the same basic building blocks; however, unlike the other systems, the outcome of the competition is not determined by actin filament density, as F-actin density is uniform along the oocyte cortex. Instead, the outcome of this competition is decided by abundance of microtubule tracks. Higher microtubule density at the anterior and lateral cortex favors kinesin-mediated cortical removal of osk/Staufen, while scarcity of microtubule tracks at the posterior pole favors myosin-V-dependent anchoring. To confirm this model, optogenetic tools were used to recruit a microtubule-depolymerizing kinesin, kinesin-13/Klp10A, to actin cortex, and thus locally modulate cortical microtubule density. Locally decreasing cortical microtubule density causes ectopic accumulation of Staufen at the cortex. The loss of microtubules prevents kinesin-driven cortical exclusion, which allows myosin-V to win the competition and form a patch of cortically localized Staufen. This recruitment of Staufen is reversible and repeatable, indicating this kinesin-myosin competition is continuous, and the outcome of this never-ending battle is decided by the local microtubule concentration (Lu, 2020).

Competition between kinesin-1 and myosin-V is sufficient for initial anchoring at the posterior pole Previously, synthetic motor domains of a plus-end motor, kinesin-1, and a minus-end motor, kinesin-14/Nod, were used to label overall microtubule polarity in Drosophila oocytes and neurons (Kin:βGal and Nod:βGal). As myosin-V is essential for osk/Staufen localization, in this study two synthetic motor constructs (KHC576 and MyoVHMM) were expressed in the oocyte, and their dimerization was induced using a rapalog-dependent dimerization system. Dimerized motors accumulate at the posterior pole, highly resembling the osk/Staufen localization. This posterior accumulation is dependent on the anterior-posterior microtubule gradient; dimerized motors fail to localize at the posterior pole after microtubule depolymerization. Together, using dimerized synthetic motors, this study demonstrated that direct competition (without any cargo binding) between a microtubule motor, kinesin-1, and an actin motor, myosin-V, is sufficient for initial posterior localization in a Drosophila oocyte (Lu, 2020).

In summary, this study has elucidated the anchorage mechanism for initial posterior localization of osk/Staufen during mid-oogenesis. Kinesin-1 competes with myosin-V to control osk/Staufen localization. The outcome of this kinesin-myosin competition is primarily determined by cortical microtubule density. Higher microtubule density at anterior-lateral cortex allows kinesin-1 to win and cortically exclude osk/Staufen, while lower microtubule density at posterior pole favors myosin-V-mediated anchorage at the cortex. Together, two cytoskeletal components (microtubules and F-actin) and two molecular motors (kinesin-1 and myosin-V) govern the posterior determination for future Drosophila embryos (Lu, 2020).

Lu, W., Lakonishok, M., Liu, R., Billington, N., Rich, A., Glotzer, M., Sellers, J. R. and Gelfand, V. I. (2020). Competition between kinesin-1 and myosin-V defines Drosophila posterior determination. Elife 9. PubMed ID: 32057294 z

Myosin V from Drosophila reveals diversity of motor mechanisms within the myosin V family

Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. The schematic structure of a DmV molecule demonstrates that the structural domain architecture of DmV is identical to that of vertebrate myosin V. Drosophila myosin V is represented by a single heavy chain gene which does not cluster with any of the three human isoforms (Va, b, and c) in a phylogenetic analysis. Rather, it is a distinct entity whose evolution diverged probably from the common ancestor of vertebrate myosin V; however, no major divergence was found in the well conserved regions of the motor domain. The mRNA of DmV is abundant in the fly through all developmental stages. In cultured cells, DmV seems to participate in vesicle transport just as other myosin V does. Myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va, and it is a nonprocessive, ensemble motor. Steady-state and transient kinetic measurements on single-headed constructs reveal that a single Drosophila myosin V molecule spends most of its mechanochemical cycle time detached from actin, therefore it has to function in processive units that comprise several molecules. Accordingly, in in vitro motility assays, double-headed Drosophila myosin V requires high surface concentrations to exhibit a continuous translocation of actin filaments. Comparison between vertebrate and fly myosin V demonstrates that the well preserved function of myosin V motors in cytoplasmic transport can be accomplished by markedly different underlying mechanisms (Toth, 2005; Full text of article).

Functions of Didem orthologs in other species

Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va. This study shows that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into Purkinje neuron spines. Specifically, myosin-Va accumulates at the ER tip as the organelle moves into spines, and hydrolysis of ATP by myosin-Va is required for spine ER targeting. Moreover, myosin-Va is responsible for almost all of the spine ER insertion events. Finally, attenuation of the ability of myosin-Va to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, this paper has established that an actin-based motor moves ER within animal cells, and have uncovered the mechanism for ER localization to Purkinje neuron spines, a prerequisite for synaptic plasticity (Wagner, 2011).

Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization

Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin V, Myo2, binds the vacuole-specific adaptor Vac17 to transport the vacuole from the mother cell to the bud. This study shows that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 that recruits the E3 ligase Dma1 to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, it was found that the termination of peroxisome transport also requires Dma1. It is predicted that this is a general mechanism that detaches myosin V from select cargoes (Yau, 2014).

Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. This study reports how Spir actin nucleators (see Drosophila Spire) and myosin V (see Drosophila Didum) motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. Crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 (see Drosophila Rab11) was determined, and it was shown that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes (Pylypenko, 2016).

Actin/Myosin-V- and activity-dependent inter-synaptic vesicle exchange in central neurons

Vesicle sharing between synaptic boutons is an important component of the recycling process that synapses employ to maintain vesicle pools. However, the mechanisms supporting and regulating vesicle transport during the inter-synaptic exchange remain poorly understood. Using nanometer-resolution tracking of individual synaptic vesicles and advanced computational algorithms, this study found that long-distance axonal transport of synaptic vesicles between hippocampal boutons is partially mediated by the actin network, with myosin V (see Drosophila Didum) as the primary actin-dependent motor that drives this vesicle transport. Furthermore, it was found that vesicle exit from the synapse to the axon and long-distance vesicle transport are both rapidly and dynamically regulated by activity. These findings were corroborated with two complementary modeling approaches of vesicle exit, which closely reproduced experimental observations. These findings uncover the roles of actin and myosin V in supporting the inter-synaptic vesicle exchange and reveal that this process is dynamically modulated in an activity-dependent manner (Gramlich, 2017).


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Biological Overview

date revised: 12 September 2022

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

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