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 ; Borner ); and various cell free assays to reconstitute and mechanistically probe the uncoating reaction (Rothman and Schmid, 1986), CME (Smythe ; Carter ; 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).

Sandra L. Schmid

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 ). 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 ). 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.

THE WATCHMAKERS' QUESTION: WHY IS THE DYNAMIC BEHAVIOR OF CCPs SO HETEROGENEOUS?

CCPs vary in size and in lifetimes; that is, some of the “stars” are dim and blink-out rapidly, whereas others are much brighter and remain for longer times. Dim and short-lived species might represent failed attempts to generate CCVs (Loerke ). But even the productive CCPs have lifetimes that range from 30 to >120 s. What distinguishes a failed from a productive CCP; is it a stochastic or directed event? If the latter, what cellular machinery monitors these criteria? What is the temporal hierarchy of molecular events leading to productive CCPs? Are all CCPs the same or are we viewing biology in action: operating with levels of specificity not previously appreciated?

Given the diversity of cargo molecules taken up through CME, is it possible that different classes of cargo confer different behaviors on the CCPs that carry them? Are CCPs more like taxis than busses? In the former scenario, signaling receptors (i.e., passengers) might control the rate at which they are internalized to prolong signaling events at the cell surface, determine what other cargo might be included to create specialized CCVs, or both. These and other receptors, together with the growing list of cytoplasmic adaptors (Traub, 2009), could play a programming role in determining the distinct dynamic and targeting behaviors of CCPs and thereby effect cellular behavior and homeostasis.

THE COSMOLOGISTS' QUESTION: WHAT ROLE DOES CME PLAY IN THE BROADER CONTEXTS OF ORGANISMAL DEVELOPMENT, PHYSIOLOGY, AND HOMEOSTASIS?

The plasma membrane serves as a physical barrier that separates the intracellular milieu from the harsh extracellular environment and as a conduit for communication among cells and between cells and their environment. Thus, endocytic CCVs carry both physical and informational traffic. Over the next 50 years, we will need to understand how the spatial and temporal regulation of CME functions to control and interpret this information load and to modulate cellular responses. This understanding will be especially relevant for CME of adhesion complexes, signaling receptors, mechanotransducers, morphogen sensors, and polarity markers that determine more complex cellular behaviors. These questions must ultimately be addressed at the organismal level. The watchmaker's work toward understanding the mechanisms of CME can guide the cosmologist. Such understanding can help to design and characterize mutations in components of the endocytic machinery that precisely alter specific properties of CME. Then, using ever more sophisticated approaches for genetic manipulation, these hypomorphic alleles can be introduced into animal models to systematically explore the role of CME in higher order cellular and organismal functions.

In summary, during the first 50 years we have begun to explore the CME universe. Guided and inspired by these twinkling stars, the next challenge is to understand how CME contributes to the development, physiology, and robust homeostasis of multicellular organisms. Once we gain the watchmaker's level of understanding of these cosmic questions, then we will know how to treat the many human diseases that result from small defects in this critical cellular process.