Cooperative force generation by actin assembly and myosin-I during endocytosis
Clathrin-mediated endocytosis (CME) is a fundamental cellular membrane trafficking process. The process generates nascent cytoplasmic vesicles from the plasma membrane through a molecular pathway that brings about membrane invagination and scission. Over the last 2 decades, extensive studies in the budding yeast Saccharomyces cerevisiae have identified many of the molecules involved in the CME pathway. More than 60 proteins localize to CME sites in distinct spatial and temporal patterns. Initially, the earliest arriving proteins mark the presumptive CME site on the plasma membrane and initiate the assembly of an endocytic coat complex. The timing of this stage of CME is quite variable (< 30 s to > 4 min.). Eventually, additional coat proteins are recruited and the site transitions into a fast, regular phase (~30 s). Later arriving coat proteins recruit actin assembly factors, triggering a burst of actin assembly that invaginates the membrane facilitating scission. Despite the known identities of scores of proteins involved in CME, the molecular mechanisms of the process are incompletely understood. My dissertation work provides important mechanistic details into the molecular mechanism of force generation for membrane invagination and the molecular mechanisms governing the transition from the early, variably-times phase of CME into the later, more regular phase.
The actin cytoskeleton can generate force on membranes either through its assembly, its associated myosin motors, or both. How actin assembly and myosin motor activity are coordinated during processes that require both modes of force generation was unclear. Most recent studies of CME treat the process as an actin assembly-based force generation process. However, in budding yeast, type I myosins (Myo3 or Myo5) are also required. In Chapter 2 of this dissertation, I demonstrate that membrane binding by the type I myosin Myo5 constitutes a critical membrane anchor for actin assembly at endocytic sites, facilitating actin-assembly based force generation. In Chapter 3, I demonstrate that the Myo5 motor itself is also capable of generating appreciable force, indicating that actin assembly can be assisted by myosin activity to bring about membrane morphogenesis.
After initiation, CME sites enter a variably-timed early phase before maturing through a transition point into a phase of more regular kinetics. A previous study from our lab demonstrated that the presence of CME cargo is necessary for efficient maturation through the transition point. In Chapter 4 of this dissertation, I demonstrate that the presence of cargo is also sufficient to drive accelerated maturation through the transition point. In addition to providing insight into the mechanisms of CME progression, this observation also explains the longstanding observation that late CME markers are polarized towards sites of cell growth. Since the majority of secretory traffic is directed towards sites of cell growth, endocytic cargos are likely to be concentrated there, accelerating maturation of CME sites into the regular late phase of the process.