Highly cooperative control of endocytosis by clathrin.
Clathrin assembles into a dynamic two-dimensional lattice on the plasma membrane where it plays a critical role in endocytosis. To probe the regulation of this process, we used siRNA against clathrin, in combination with single cell assays for transferrin uptake as well as total internal reflection microscopy, to examine how endocytic rates and membrane dynamics depend upon cellular clathrin concentration ([Clathrin]). We find that endocytosis is tightly controlled by [Clathrin] over a very narrow dynamic range such that small changes in [Clathrin] can lead to large changes in endocytic rates, indicative of a highly cooperative process (apparent Hill coefficient, n > 6). The number of clathrin assemblies at the cell surface was invariant over a wide range of [Clathrin]; however, both the amount of clathrin in each assembly and the subsequent membrane dynamics were steeply dependent on [Clathrin]. Thus clathrin controls the structural dynamics of membrane internalization via a strongly cooperative process. We used this analysis to show that one important regulator of endocytosis, the actin cytoskeleton, acts noncompetitively as a modulator of clathrin function.
Moskowitz
Howard S
HS
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021, USA.
Yokoyama
Charles T
CT
Ryan
Timothy A
TA
D000199:Actins / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
D000199:Actins; D002966:Clathrin; D034741:RNA, Small Interfering; D014168:Transferrin