Clathrin-mediated endocytosis is a conserved pathway in eukaryotic cells for internalizing external macromolecules, membrane lipids, and cell surface receptors. Perturbation of this process is associated with multiple types of cancer and endocytic internalization is used by multiple viruses during infection. Therefore, understanding the underlying molecular mechanisms will improve our understanding of multiple disease processes besides providing fundamental insights into an essential part of cell biology. The yeast Saccharomyces cerevisiae is an ideal model for endocytosis because it is easily manipulated experimentally. Actin polymerization is critically required for endocytic internalization. Interestingly, the class I myosins Myo3p and Myo5p (Myo3/5) are essential for this inward movement in yeast and metazoan cells. Deletion of these proteins or mutation of the motor domain prevents internalization of either actin or endocytic vesicles. This is surprising because actin polymerization in the absence of myosin is sufficient to propel intracellular movement of pathogens like lysteria and propel membrane projections. Therefore, we propose to determine how Myo3/5 contributes to endocytosis.
Specific Aims : The motor domain and the tail homology domain 1 (TH1) of Myo3/5 contribute to endocytosis by unknown mechanisms. Therefore, we propose the following specific aims:
Aim I. To understand how the Myo3/5 motor domain contributes to endocytosis;
Aim II. To determine the roles of the TH1 domain of Myo3/5. Study Design:
Aim 1. We hypothesize that the motor domain both enhances the force generated by actin polymerization by transiently binding actin filaments and directly pushes on the actin network to drive internalization. To test this hypothesis, we propose to generate a series of motor domain mutations that will alter the translocation of actin using strategies that have been validated in multiple other myosins. These mutations will cause the translocation distance to increase, decrease, or become reversed while preserving the ability of the motor to transiently bind actin and hydrolyze ATP. This strategy will allow us to independently evaluate whether Myo3/5 contribute to internalization by transiently binding actin filaments, directly pushing on actin filaments, or both mechanisms. Discriminating between these two models has not been possible previously because previous motor mutations disrupted both the ability to translocate and the ability to transiently bind actin.
Aim II. We hypothesize that the TH1 domain contributes to localization of Myo3/5 by binding to acidic phospholipids in the inner leaflet of the plasma membrane. To test this hypothesis, we will also evaluate whether Myo3/5 bind to acidic phospholipids in vitro through the TH1 domain. We will also determine how TH1 domain mutations affect the endocytosis phenotype in live cells. Whether the TH1 domain functions by binding acidic phospholipids or by an unanticipated mechanism, our assays will allow us to fully characterize the phenotype and generate new models if appropriate.
Endocytosis is normal process used by all cells to internalize particles and recycle the cell membrane. This process impacts human health in that it is hijacked by multiple viral pathogens to infect cells, and perturbation of this process can result in cancer. Therefore, understanding the underlying mechanisms of endocytosis will benefit multiple distinct areas of human health research.
|Lewellyn, Eric B; Pedersen, Ross T A; Hong, Jessica et al. (2015) An Engineered Minimal WASP-Myosin Fusion Protein Reveals Essential Functions for Endocytosis. Dev Cell 35:281-94|
|Cortesio, Christa L; Lewellyn, Eric B; Drubin, David G (2015) Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2. Mol Biol Cell 26:1509-22|