Search for Myosin I Receptor

University Of Texas Southwestern Medical Center, Dallas TX

Investigators

Abstract

Myosin I-beta is a ubiquitously expressed and relatively abundant member of the superfamily of so-called unconventional myosins. Unlike the 'conventional' muscle type myosins (myosin II), these myosins do not form bipolar filaments, but rather are believed to interact with specific subcellular targets by virtue of their diverse cargo-binding tail domains. The precise functions of unconventional myosins are not yet known, but their importance is evident from the recent discoveries that myosin VII is involved in subcellular movements of membrane-bound vesicles and that mutations in myosins VI and VII result in deafness and visual impairment. Myosin I-beta is highly enriched in plasma membranes, as determined by immunofluorescence microscopy and subcellular fractionation. More specifically, it associates with detergent-insoluble glycolipid-rich membranes (DIGs), which are also enriched in growth factor receptors, G proteins, and other signaling molecules. Therefore, it is tempting to speculate that myosin I-beta is a motor that translocates these membrane 'rafts' along the cortical actin cytoskeleton. To test this possibility it will be important to verify that myosin I-beta actually docks with an authentic component of membrane rafts. Therefore, a major goal of this project is to identify, characterize, and localize a myosin I-beta receptor. Whether or not this receptor is located on raft domains, this information will greatly help to elucidate the function of myosin I-beta by pointing to the cargo to which it is targeted. Another goal of the project is to determine how the interaction of myosin I-beta with membranes is regulated. Myosin I-beta exists in both cytosolic and membrane-bound states. The hypothesis to be tested here is that its association with the membrane is not constitutive, but rather is subject to regulation by phosphorylation and calmodulin-binding. In addition, the possibility that membrane-binding and specific targeting to DIGs is enhanced by myosin palmitoylation will be examined. Finally, two approaches to interfere with myosin I-beta function in cells will be developed: expression of dominant negative forms of either myosin I-beta or its receptor; and deletion of the myosin I-beta gene (or the receptor gene) in avian DT40 cells. These tools will allow the testing of the working hypothesis that the myosin is involved in membrane protein translocation, and will be useful in determining the actual function of myosin I-beta should the hypothesis prove to be incorrect.