Studying the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins [electronic resource]

Molecular motors utilize energy from ATP hydrolysis to perform mechanical work, and examples include the myosins, a superfamily of proteins that utilizes actin filaments as a track for processive movement. There are many classes of myosins, and they share common elements necessary to regulate actin binding and force generation based on the identity of the nucleotide bound. Their diversity is most evident in their C-terminal tails; in the case of the unconventional myosins that do not form bipolar thick filaments, these regions mediate their associations with specific binding partners. Interactions between myosins and cognate adaptors allow for their recruitment onto cargoes, and it is these cargoes that define the functions of myosins in a cellular environment. Numerous studies have suggested developmental and cellular roles for myosins, but in general, few binding proteins have been discovered for most family members. In the case of mammalian myosin VI, yeast two-hybrid screening has revealed several adaptors that link this motor to vesicles, endosomes, and the Golgi, where it participates in a number of trafficking events. In contrast, very little is known about the proteins that bind to Drosophila myosin VI, despite the necessity of this protein for many essential processes during fly development. To better understand the specific pathways to which myosin VI contributes in flies, we set out to identify its cargoes. We used a combination of affinity chromatography and mass spectrometry in a proteomics-based screen to discover candidates, given the many advantages of this method over yeast two-hybrid and other approaches. Upon obtaining data indicating that over 1000 proteins could potentially associate with myosin VI, we were next charged with determining which interactions were specific and direct, and we chose to screen through select candidates with an in vitro assay. We identified a number of novel cargoes for myosin VI, including those associated with the Golgi, protein trafficking, microtubules, and others of unknown function. Next, we attempted to perform another screen to identify binding partners that might mediate the function of myosin VI in dorsal closure during embryogenesis. Because this motor is very concentrated at the leading edge of cells during this process, we developed an antibody to myosin VI for immunolocalization studies. We then examined embryos mutant for or depleted of myosin VI cargoes and assessed any effect on myosin VI localization. Although some showed slight differences in the staining pattern, we chose a different assay to test for a shared function between myosin VI and one of its novel cargoes. After commencing this project, we became aware of data indicating that myosin VI and Cornetto, one of the novel binding proteins we discovered, participate in Hedgehog secretion. Without further information about the assay used or the results obtained, we used cell culture to validate the original screen data and found that both proteins are indeed required for the timely secretion of exogenously expressed Hedgehog (Hh) from S2R+ cells. We then began examining embryos for defects associated with reduced Hedgehog secretion and found that a small percentage of flies expressing Cornetto RNAi or a dominant negative myosin VI truncation in Hh-producing cells indeed have the types of segmentation problems found in mild hedgehog mutants. Thus, we found a shared function for myosin VI and one of its binding proteins, which not only validates our screen data but also provides important information not previously available about their roles in development. In a second project, I turned my attention to another function for myosin VI to analyze its binding protein CG3529, which is orthologous to Tom1 family proteins involved in membrane trafficking. Based on information available about orthologs, I reasoned that CG3529 might mediate the involvement of myosin VI in asymmetric Notch signaling during pupal development, a role identified for this motor in another large-scale RNAi screen. Despite finding no evidence linking CG3529 to asymmetric trafficking of Notch in the dorsal epidermis as I had hypothesized, it was apparent that CG3529 depletion does affect mechanosensory bristle length, which had been previously noted for myosin VI as well. I then performed experiments to help determine by what pathway these proteins contributed to this process, but the data I collected did not indicate a shared function or binding interactions between CG3529 and the proteins to which its orthologs had been linked. Although it is still not clear what the function of this myosin VI adaptor is, I suspect that it is involved in protein trafficking at or near the endoplasmic reticulum, given that CG3529 appears to localize to this compartment. Beyond the proteins whose functions we were able to address, we obtained many other candidates that could potentially link myosin VI to a variety of cellular compartments and signaling pathways. The data presented here should be useful in future work to analyze the roles of myosin VI in specific systems upon utilization of information available about its novel binding proteins.

Keywords: Biochemistry
Advisor: Spudich James A.
Krasnow Mark
Pfeffer Suzanne advisor. Thesis advisor