Clathrin-mediated Endocytosis: A Universe of New Questions

I’m a detail-oriented person, a watchmaker. I like to take things apart, put them back together, and understand exactly how they work. For most of the past 30 years, I have focused (some might say fixated) my attention on clathrin-coated vesicles (CCVs) and clathrin-mediated endocytosis (CME). CME, a major endocytic pathway, is involved in the ubiquitous uptake of nutrient–receptor complexes, membrane transporters, adhesion molecules, and signaling receptors, as well as in the recycling of synaptic vesicles—all of which play central roles in human health and disease. The role of coated vesicles in efficient endocytosis was first proposed in 1964 based on electron micrographs of yolk protein uptake in insect oocytes (Roth and Porter, 1964). CME involves the assembly of clathrin coats on the plasma membrane that concentrate receptors; invaginate; pinch off to form CCVs; and finally uncoat, releasing transport vesicles that fuse and deliver their contents to the endosomal system. To understand the mechanisms underlying CME, we and others have developed and used the now “classical” techniques of cell biology. These techniques include subcellular fractionation to purify coated vesicles (Pearse, 1975); biochemical fractionation coupled to proteomics to identify components of the CME machinery (Blondeau et al., 2004; Borner et al., 2006); and various cell free assays to reconstitute and mechanistically probe the uncoating reaction (Rothman and Schmid, 1986), CME (Smythe et al., 1992; Carter et al., 1993; Miwako and Schmid, 2005), and dynamin-catalyzed membrane fission (Pucadyil and Schmid, 2008). Consequently, during the almost 50 years since its discovery, we have learned a great deal about the molecular mechanisms underlying clathrin-mediated endocytosis (Conner and Schmid, 2003; Doherty and McMahon, 2009). In 1999, Jim Keen used the then newly developed green fluorescent protein (GFP) technology to tag the clathrin light chain and, for the first time, visualized CME in real time (Gaidarov et al., 1999). Subsequent imaging by total internal reflection fluorescence microscopy (which selectively illuminates an 100-nm-deep plane in the cytosol of adherent cells, thereby increasing signal-to-noise ratios) produced spectacular live-cell movies of CME. The GFP-clathrin appeared as small points of light that grew to varying intensities and then abruptly disappeared as the nascent CCVs quickly moved deeper into the cytosol and out of the zone of illumination (Merrifield et al., 2002). The dynamic clathrin-coated pits (CCPs) seen in these movies (see Supplemental Movie 1) are reminiscent of stars blinking in the night’s sky—and they have opened up a universe of new questions that will need to be addressed over the next 50 years. Some (those of a watchmaker) reflect the need for further understanding of the molecular basis for CME, whereas others (those of a cosmologist) seek to integrate CME with the bigger picture of organismal physiology in development, health, and disease.