Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

The actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral-facial-digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits, and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein cep164. Myosin VI depletion in non-tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be causally linked to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.

Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single molecule motility shows thatsaturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable to constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and “stop-and-go” motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers result in actin remodeling and foci formation. In contrast, Dab2 MIRmyosinVI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Formation of Aberrant Myotubes by Myoblasts Lacking Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast Adhesion and Fusion

We have previously postulated that unconventional myosin VI (MVI) could be involved in myoblast differentiation. Here, we addressed the mechanism(s) of its involvement using primary myoblast culture derived from the hindlimb muscles of Snell’s waltzer mice, the natural MVI knockouts (MVI-KO). We observed that MVI-KO myotubes were formed faster than control heterozygous myoblasts (MVI-WT), with a three-fold increase in the number of myosac-like myotubes with centrally positioned nuclei. There were also changes in the levels of the myogenic transcription factors Pax7, MyoD and myogenin. This was accompanied by changes in the actin cytoskeleton and adhesive structure organization. We observed significant decreases in the levels of proteins involved in focal contact formation, such as talin and focal adhesion kinase (FAK). Interestingly, the levels of proteins involved in intercellular communication, M-cadherin and drebrin, were also affected. Furthermore, time-dependent alterations in the levels of the key proteins for myoblast membrane fusion, myomaker and myomerger, without effect on their cellular localization, were observed. Our data indicate that in the absence of MVI, the mechanisms controlling cytoskeleton organization, as well as myoblast adhesion and fusion, are dysregulated, leading to the formation of aberrant myotubes.

Loss of myosin VI expression affects acrosome/acroplaxome complex morphology during mouse spermiogenesis†

During spermiogenesis in mammals, actin filaments and a variety of actin-binding proteins are involved in the formation and function of highly specialized testis-specific structures. Actin-based motor proteins, such as myosin Va and VIIa, play a key role in this complex process of spermatid transformation into mature sperm. We have previously demonstrated that myosin VI (MYO6) is also expressed in mouse testes. It is present in actin-rich structures important for spermatid development, including one of the earliest events in spermiogenesis—acrosome formation. Here, we demonstrate using immunofluorescence, cytochemical, and ultrastructural approaches that MYO6 is involved in maintaining the structural integrity of these specialized actin-rich structures during acrosome biogenesis in mouse. We show that MYO6 together with its binding partner TOM1/L2 is present at/around the spermatid Golgi complex and the nascent acrosome. Depletion of MYO6 in Snell’s waltzer mice causes structural disruptions of the Golgi complex and affects the acrosomal granule positioning within the developing acrosome. In summary, our results suggest that MYO6 plays an anchoring role during the acrosome biogenesis mainly by tethering of different cargo/membranes to highly specialized actin-related structures.