Expression of a mutant dynamin protein in cells was equally effective in attenuating endocytosis with or without the GFP tag. Thus, these combined observations suggest that the GFP tag does not interfere

with the distribution or function of the dynamin to which it is attached. Initial observations suggesting that clathrin-based endocytosis might occur at concentrated Cobimetinib nmr sites came from live mammalian cells that express GFP-tagged clathrin light chain. The formation of coated pits appeared to be restricted to discrete domains of the PM20, 25, 26 that liberate several clathrin-coated vesicles over short times. Because these spots moved in temporal and spatial synchrony at the surface of cells treated with detergents, it was suggested that these sites are interconnected and positioned by an actin cytoskeletal network that might also act to sequester coat-forming components. We have found that

these sites in cultured hepatocytes are much more extensive than originally reported, represent exceptionally large (2-10 μm) tubuloreticular structures that may form hundreds of nascent vesicles, and are dependent on dynamin function. Thus, it appears that hepatocytes, like neurons, form specialized endocytic domains for the large-scale production of clathrin-coated vesicles. This sequestration and organization at predefined platforms in the hepatocyte is likely to increase endocytic

efficiency substantially, as is well known ABT-263 in vivo to occur at the neuronal synapse. As depicted by the illustration in Fig. 7, the generation of endocytic vesicles is markedly increased 上海皓元医药股份有限公司 at hotspots (15-20/min) in comparison to the conventional internalization of clathrin-coated pits (<1/min) by providing a site for large-scale vesiculation of the PM. The location of these platforms is likely to be dictated by the enrichment of specific lipids into microdomains and are highly dependent on actin and actin-binding proteins that recruit and stabilize many components of the endocytic machinery, from clathrin and dynamin to endophilin and intersectin, to name just a few. In comparison, the formation of a single clathrin vesicle from an isolated site would require the time-consuming process of a sequential recruitment and assembly of many proteins from the cytosol. One might conclude that clathrin hot spots have been observed for some time from early electron micrographs taken by Palade, Porter, and others. For example, high-magnification images of hepatocytes in situ show the PM decorated with individual clathrin-coated pits and vesicles at different stages of maturation. Most striking is that within very small domains of the cell surface (<1.5 μm2) reside 7-8 clathrin-coated pits.