Proteomics approach to study the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins

Abstract

Myosin VI is a molecular motor implicated in many processes, and it likely associates with a variety of cargoes that specify its functions. Although it is critical to Drosophila development, little is known about its cellular roles. To reveal its involvement in specific pathways, we sought to identify the binding partners of Drosophila myosin VI. We used affinity chromatography and mass spectrometry to discover interacting proteins, which we tested for direct binding. Using this approach, we found that the microtubuleassociated protein Cornetto bound myosin VI, and we demonstrated a role for both in secretion of the lipidated morphogen Hedgehog. We also identified a number of other binding proteins, and further characterization of their interactions with myosin VI will advance our understanding of the roles of these complexes in cellular and developmental processes. Thus, our method has provided us the means to gain valuable insight into the multifaceted roles of a motor protein in vivo.unconventional myosins | cytoskeleton | trafficking M yosins comprise a superfamily of actin-based motor proteins involved in a variety of processes (1). On the molecular scale, significant progress has been made in recent years toward understanding their mechanical properties (2, 3). On the organismal scale, it is also clear that they are important for the development and function of tissues and organs (4-6). Yet for many of these proteins, there is little data explaining their roles on the cellular level. As functions of motor proteins are thought to be determined by the types of cargoes they transport (7), an important step in furthering our understanding of motors' functions is identifying the proteins and organelles with which they interact (8).Toward this goal, we have focused on myosin VI, which participates in a wide range of processes such as vesicle trafficking and maintenance of stereocilia for mammalian hearing (9). Its broad utilization in higher organisms is perhaps due to its unique directionality toward the minus ends of actin filaments, unlike all other studied myosins (reviewed in ref. 10 and others). Although yeast two-hybrid screens and other approaches have made progress toward identifying some myosin VI cargoes (11), less is known about the molecular details of how myosin VI complexes function in vivo. Such information is particularly lacking about this protein in Drosophila, despite its significance in fly development.Depletion of Drosophila myosin VI (Jaguar; referred to as M6 throughout) protein levels, expression of a dominant negative M6 truncation, or injection of a function-blocking M6 antibody produces a variety of phenotypes that depend on the stage and tissue targeted (12). These data have revealed roles for M6 in pseudocleavage furrow formation in the syncytium (13), dorsal closure later in embryogenesis (14), and spermatogenesis in the adult male (15), among other processes (16).Though its importance is evident, it is unclear what M6 contributes as a motor protein to developmental events, because...

Myosin VI and its cargo adaptors – linking endocytosis and autophagy

“…1). In mammalian cells and also in Drosophila a substantial number of direct binding partners have been identified Finan et al, 2011). However, additional transient or low-affinity myosin VI cargo adaptor molecules as well as larger protein complexes may be identified in the future, by using in situ crosslinking or spatially-restricted enzymatic tagging in living cells or tissues (Rhee et al, 2013).…”

Multifunctional myosin VI has a multitude of cargoes

“…The PNAS study by Finan et al (4) now fills this gap in our knowledge and provides us with important information on the cargoes of Drosophila myosin VI. They used an affinity chromatography/mass spectrometry approach to identify the Drosophila myosin VI cargo-binding proteins.…”

“…The RRL motif is conserved between mammalian and Drosophila myosin VIs, whereas the WWY motif is changed to LWY in the Drosophila variant. Interestingly, the Drosophila homolog of GIPC called Kermit, which is likely to bind via the conserved RRL motif, is recognized as a myosin VI binding partner by Finan et al (4). However, the Drosophila homolog of Dab2 was not identified (Fig.…”

“…1), including Miranda (a protein already known to bind to myosin VI), Cornetto, Kermit (the ortholog of mammalian GIPC), and a number of presumed Golgi proteins and proteins similar to those in mammalian cells involved in vesicle trafficking. Focusing on one of the unique interacting proteins, Cornetto-a protein with no reported function-Finan et al (4) show that it binds to Drosophila myosin VI with a relatively weak affinity (in the micromolar range). This weak affinity is to be expected, because it is now recognized that in the cell, multicomponent motor complexes are believed to initially exist as weak transient interactions that are assembled, reinforced, and processed for transport around the cell to their destinations, where they are induced to rapidly disassemble.…”

“…Although the kinetic properties of the motor domains of the myosins are fine-tuned for their cellular roles, it is the identity of their cargobinding proteins that determines their precise cellular functions. In PNAS, Finan et al (4) use a biochemical interaction screen to identify the cargo-binding partners of myosin VI/Jaguar in Drosophila to further our understanding of the cellular roles of this motor protein.…”