The Dynamics of Clathrin Coats in Living Cells Measured by Advanced Fluorescence Microscopy

Abstract

Clathrin coated pits and coated vesicles are responsible for receptor-mediated endocytosis of a wide variety of ligands that bind at the cell surface (Anderson et al., 1977; Goldstein et al., 1985). Clathrin coated pits are also involved in the sorting of receptors to be transported to intracellular acidic compartments such as the Mannose-6-phosphate receptor rich pre-lysosomal compartment (Lemansky et al., 1987), the secretory granules in pancreatic endocrine insulin-secreting cells (Orci et al., 1987), or the ACTH-containing vesicles in anterior pituitary cells (Tooze and Tooze, 1986). Clathrin triskelions are composed of three heavy (180 kD) and three light (30 kD) chains. They can assemble in vitro at low pH to form clathrin baskets, the geometry of which has been extensively studied (Crowther and Pearse, 1981; Harrison and Kirchhausen, 1983; Blank and Brodsky, 1986). Clathrin triskelions are associated with adaptins (Pearse, 1988; Ahle et al., 1988; Ahle and Ungewickell, 1989), which are forming multi-units complexes specific for either plasma membrane or Golgi-derived vesicles (Robinson and Pearse, 1986; Robinson, 1987, 1989). In coated vesicles, the adaptins are presumably located between the external shell formed by clathrin triskelions and the cytoplasmic domains of transmembrane proteins with which they might interact (Vigers et al., 1986). It is generally accepted that in addition to clathrin, adaptins are required at the level of the plasma membrane or of Golgi derived membranes to form coated pits that bud into coated vesicles. The coat is then removed presumably by an uncoating ATPase characterized in vitro (Schlossman et al., 1984; Brael et al., 1984) which is identical to the hsc 70 heat shock protein (Rothman and Schmid, 1986).