| Clathrin, a scaffolding protein that polymerizes onto membranes, transiently forms the outer coat of a specific subset of intracellular vesicles. Despite significant discoveries into its composition, structure, and biophysical properties, detailed information regarding clathrin's specific function has remained elusive. Most attempts to clarify clathrin's role in membrane traffic involved techniques to completely ablate clathrin's function and abundance. However, alterations in compensatory processes may confound results to approaches where clathrin function is assayed long after the initiation of experimental procedures to perturb clathrin.; In order to probe clathrin's function in living cells, we utilized a variety of techniques to specifically alter clathrin function combined with biophysical assays for cell function. First, we developed a novel inhibitor of clathrin function by generating a novel clathrin light chain allele that is specifically sensitive to the cell-permeant, small molecule FK1012-A that disrupts clathrin's normal assembly-disassembly cycle. Chemical inhibition of clathrin function in this manner is achieved rapidly (<2 hr) and is reversible. This novel tool illustrates that clathrin is the dominant cellular pathway for internalization of clathrin-dependent ligands in fibroblasts, although a substantial proportion of these ligands (∼20%) gain entrance to the cell via alternative pathways. Furthermore, cross-linking this clathrin allele in neurons essentially stops endocytosis at the synapse, which indicates that clathrin-mediated endocytosis is the predominant mode of synaptic vesicle retrieval.; Second, we targeted clathrin for destruction with siRNA in a manner that creates a heterogeneous reduction of clathrin abundance within a population of fibroblasts. The variable reduction in clathrin indicates that clathrin-dependent processes, including internalization of transferrin, are highly cooperative (apparent Hill coefficient, n > 6). Furthermore, this steep cooperativity indicates that clathrin is poised for tight regulatory control that may be used for physiological regulation.; Finally, we analyzed a genetic knockout of phosphatidylinositol phosphate kinase type Igamma, the principle enzyme at the synapse governing the creation of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which is presumed to orchestrate a variety of cellular functions, including clathrin-dependent vesicle retrieval. We conclude that PI(4,5)P2 serves a vital role in regulating many steps of vesicle trafficking at the synapse, including the speed of endocytosis and the kinetics of vesicle recycling. |
