Unconventional myosin proteins of the MyTH-FERM superclass are involved in intrafilopodial trafficking, are thought to be mediators of membrane-cytoskeleton interactions, and are linked to several forms of deafness in mammals. This study shows that the Drosophila myosin XV homolog, Sisyphus (Myo10A), is expressed at high levels in leading edge cells and their cellular protrusions during the morphogenetic process of dorsal closure. Sisyphus is required for the correct alignment of cells on opposing sides of the fusing epithelial sheets, as well as for adhesion of the cells during the final zippering/fusion phase. Several putative Sisyphus cargos have been identifed, including DE-cadherin (also known as Shotgun) and the microtubule-linked proteins Katanin-60, EB1, Milton and aPKC. These cargos bind to the Sisyphus FERM domain, and their binding is in some cases mutually exclusive. These data suggest a mechanism for Sisyphus in which it maintains a balance between actin and microtubule cytoskeleton components, thereby contributing to cytoskeletal cross-talk necessary for regulating filopodial dynamics during dorsal closure (Liu, 2008).

Epithelial movements underlie fundamental physiological processes including embryonic morphogenesis, wound healing and cancer metastasis. During dorsal closure (DC), a morphogenetic event that occurs late in Drosophila embryogenesis, two lateral sheets of epithelial cells move towards one another over a dorsally exposed region of extraembryonic tissue and fuse together at the midline. During this process, the dorsal-most face of leading edge (LE) epithelial cells exhibit dynamic cellular protrusions, lamellipodia and filopodia, required initially for sensing their environment and finding their appropriate counterpart on the opposing epithelial sheet. Subsequently, the opposing protrusions adhere to one another, facilitating the formation of transient cell-cell contacts as the epithelial sheets zipper together, followed by permanent cell-adhesion structures. Filopodia contain a core of organized bundled actin filaments, oriented with their barbed (plus) ends towards the tip. Filopodia continuously assemble and disassemble, with growth occurring via de novo actin nucleation and polymerization locally at their tips. Although the dynamic rearrangements of cells and filopodia during DC are mainly attributed to actin dynamics, a recent report has described a role for microtubules in epithelial zippering, the final step of DC (Liu, 2008).

The coordination of environmental sensing, cell-cell recognition and adhesion mediated by LE cell protrusions must require orchestrated movements of structural, adhesive and regulatory molecules within filopodia. Unconventional myosins have recently been implicated in the movement of such cellular molecules/machineries within filopodia. Unconventional myosins are actin-based motor proteins that can be subdivided into at least 18 distinct classes (I-XVIII) based on their motor and tail domain structural and functional characteristics. One subset, the `MyTH-FERM' unconventional myosins, includes classes VII, X, XII and XV that share structurally conserved features in their tails: MyTH4 (myosin tail homology 4) domains that bind to microtubules (Weber, 2004) and FERM (band 4.1, ezrin, radixin, moesin) domains that are involved in cargo binding (Sousa, 2005; Sheetz, 1999). The tail regions are thought to determine where the myosins are located and what cargos they transport (Liu, 2008).

Mutations in MyTH-FERM unconventional myosins result in disorganized stereocilia leading to deafness and vestibular dysfunction in humans and mice: myosin VIIa is responsible for human Usher Syndrome type IB and the mouse shaker 1 mutation, whereas myosin XV is linked to human non-syndromic deafness, DFNB3, and the mouse shaker 2 mutation (Gibson, 1995; Weil, 1995; Liang, 1999; Libby, 2000). In Dictyostelium, Myosin VIIa (MVII) localizes to filopodial tips and is required for the formation of filopodia and cell attachment (Titus, 1999; Tuxworth, 2001). In mammalian cells, Myosin X (Myo10) moves bidirectionally within filopodia and accumulates at filopodial tips (Berg, 2002; Bohil, 2006). Ectopic expression of Myo10 is sufficient to direct assembly of filopodia in cells lacking them. The Myo10 FERM domain has been shown to bind β-integrin (Zhang, 2004) and transport it to filopodial tips where it is needed for proper filopodial extension and substratum adherence (Liu, 2008).

Drosophila has three MyTH-FERM myosin homologs: myosin VIIa, VIIb and XV (Tzolovsky, 2002). Functional data has only been reported for crinkled (myosin VIIa), mutants of which are semi-lethal with escaper adults exhibiting defects in actin-rich structures such as bristles and hairs (Kiehart, 2004), and deafness due to disruption of scolopidia auditory organ integrity required for transducing auditory signals (Todi, 2005; Liu, 2008 and references therein).

This study shows that the Drosophila myosin XV homolog, which is here named Sisyphus (Syph), is required for proper DC where it traffics sensory, cytoskeletal, and adhesion cargos within LE cells and their filopodial protrusions (Liu, 2008).

Dorsal closure is a complex morphogenetic process that is dependent on cell protrusions that are highly dynamic, requiring F-actin filaments and microtubules present in the structures to grow and shrink very rapidly. Transport of proteins to and within these protrusions and the leading edge is essential for the environmental sensing, cell-cell recognition and adhesion events that take place during DC. This paper presents the first characterization of the Drosophila myosin XV homolog, Sisyphus, and shows that it is required for DC during at least two key steps: for proper epithelial alignment, and for zippering and fusion of the two epithelial sheets. Live imaging of Syph shows that this motor protein accumulates at the leading edge during DC, whereas mapping and RNAi studies demonstrate interactions with its cargo proteins, consistent with a role as a transport protein. The results also suggest a possible role for Syph in the coordination of the actin and MT networks required for the dynamic protrusions during DC (Liu, 2008).

The MyoX MyTH-FERM myosin has been proposed to play a structural role by facilitating actin polymerization at filopodia tips by pushing the plasma membrane away from the growing actin filament barbed ends to create a space for actin monomer addition (cf. Sousa, 2005). Although Syph is present at filopodium ends and could perform a similar role for actin or MT assembly, unlike MyoX, it is not preferentially found at tips. Instead, Syph moves bi-directionally within LE cells and their protrusions. Reduction of Syph by RNAi disrupts filopodia formation, and this probably contributes to the segment mismatching and zippering/fusion phenotypes observed in syph-deficient embryos. Dictyostelium cells mutant for the myosin-VII MyTH-FERM protein have also been shown to exhibit loss of filopodia and adhesion defects (Titus, 1999; Tuxworth, 2001). How filopodial formation is disturbed in syph-deficient embryos remains to be answered, though improper distribution of cargo proteins required for proper filopodial dynamics and integrity of the leading edge is a strong possibility. Another interesting and nonexclusive model is that disruption of the actin-microtubule network caused by Syph knockdown in turn disrupts filopodial formation and/or integrity of the leading edge. Additional studies will be required to differentiate between these two possibilities (Liu, 2008).

Mapping data shows that Syph appears to play a key role in transporting structural, adhesive and regulatory molecules within filopodia via its C-terminal FERM domain. This is consistent with studies in mammals that show that, in addition to the motor domain, the FERM domain of MyoXV is critical for development of stereocilia required for normal hearing and balance (Anderson, 2000). One Syph cargo that binds to the FERM domain of Syph was identified as DE-cadherin. Cadherin is required at filopodium ends where it forms transient cell-cell contacts, followed by more permanent cell adhesion ones. syph-deficient embryos are defective in epithelial fusion during DC, presumably due to the failure of cadherin to correctly accumulate at the dorsal-most edge of LE cells to mediate fusion and adhesion. Interestingly, this 'failure to close' phenotype is reminiscent of that observed in Rac GTPase mutants proposed to interfere with the contact-inhibition machinery (Woolner, 2005). These results may have clinical relevance, since mutations in the X, VIIA and XV classes of MyTH-FERM unconventional myosins have been shown to cause deafness and aberrant morphology of stereocilia in inner-ear hair cells. Interestingly, in addition to MyoVIIa alleles, mapping of recessive mutations for the most common form of hereditary deaf-blindness in humans, Usher syndrome, has identified alleles of cadherin-23 and protocadherin-15. The MyoVIIa MyTH-FERM myosin has also been shown to interact with cadherin complexes through the vezatin adaptor protein (Kussel-Andermann, 2000). Together, these observations suggest that proper distribution and localization of cadherin is essential for normal stereocilia formation or maintenance and may be a conserved task among MyTH-FERM unconventional myosins (Liu, 2008).

In addition, the binding site for Syph on DE-cadherin was mapped to a C-terminal 40aa fragment of the cad^ICD. The Drosophila genome contains 17 cadherin proteins in addition to DE-cadherin; three of these show conservation within this C-terminal 40aa motif. Interestingly, this region of DE-cadherin has not been previously assigned a function, but shows conservation with over 15 human cadherins, and thus may define a novel unconventional myosin-binding domain by which cadherins are tethered and transported (Liu, 2008).

A remarkable recent finding is the presence of and requirement for MTs in filopodia, as well as in the final stages of zippering during fly DC (Jankovics, 2006; Schober, 2007). Although unconventional myosins have been generally considered as actin-based motor proteins, members of the MyTH-FERM class were recently shown to bind to and travel along MTs via their MyTH4 domains (Weber, 2004). Thus, in addition to trafficking on actin, Syph could traffic on MTs through its two MyTH4 domains. Dynamic MTs have been proposed to work by regulating local concentrations of the cadherin needed to establish and maintain cell-cell contacts, or by delivering actin-organizing proteins to filopodia tips. Thus, Syph could use MTs both as a transport substrate, and be involved in their dynamic assembly/disassembly (Liu, 2008).

Another appealing possibility is that Syph may play a role in coordinating the two cytoskeletons. Several of the putative cargos identified for Syph are MT-associated proteins: α-tubulin, Katanin-60, Milt and EB1 are all MT components or binding partners. Transport of the MT subunit, α-tubulin, and the MT-severing protein, Katanin-60, suggest a role for Syph in the assembly and disassembly of MTs, whereas the plus end MT-binding protein EB1 suggests a role in stabilization and regulation. A recent study has shown that ectopic expression of another Drosophila MT-severing protein, spastin, resulted in delayed epithelial hole closure [taking about 9 hours instead of 3 hours to reaching completion (Jankovics, 2006)]. The similar phenotype that was observe in syph-deficient embryos suggests that Syph modulates MT cytoskeleton regulation by transporting cargo proteins essential for its regulation (Liu, 2008).

Identification of cadherin and several MT-linked proteins as Syph cargos and the mutually exclusive - and perhaps competitive - binding for these cargos on the Syph FERM domain, leads to a proposal that one role of Syph is to coordinate the actin and MT networks during filopodial dynamics through differential cargo transport. Consistent with this possibility, it was found that both α-tubulin and actin overexpression rescues the Syph 'failure to close' phenotype, and in the case of actin, the delayed closure phenotype as well. These results suggest that the actin network can partially compensate for disruption of the MT one, and that Syph, in addition to serving as a delivery system, may play a role in the regulation of actin and MT cytoskeleton cross-talk during processes such as DC. The finding that the binding sites for putative cargo proteins are mutually exclusive implies that Syph itself must be regulated by proteins that help it 'choose' particular proteins to transport. Future studies aimed at uncovering and deciphering the rules governing the choice of cargo and transport substrate for unconventional myosin motors are likely to provide exciting new insight into the coordinate regulation of and cross-talk between the actin and MT cytoskeletons during highly orchestrated morphogenetic events such as DC (Liu, 2008).