Focusing on clathrin-mediated endocytosis

Abstract

Investigations into the mechanisms which regulate entry of integral membrane proteins, and associated ligands, into the cell through vesicular carriers (endocytosis) have greatly benefited from the application of live-cell imaging. Several excellent recent reviews have detailed specific aspects of endocytosis, such as entry of particular cargo, or the different routes of internalization. The aim of the present review is to highlight how advances in live-cell fluorescence microscopy have affected the study of clathrin-mediated endocytosis. The last decade has seen a tremendous increase in the development and dissemination of methods for imaging endocytosis in live cells, and this has been followed by a dramatic shift in the way this critical cellular pathway is studied and understood. The present review begins with a description of the technical advances which have permitted new types of experiment to be performed, as well as potential pitfalls of these new technologies. Subsequently, advances in the understanding of three key endocytic proteins will be addressed: clathrin, dynamin and AP-2 (adaptor protein 2). Although great strides have clearly been made in these areas in recent years, as is often the case, each answer has bred numerous questions. Furthermore, several examples are highlighted where, because of seemingly minor differences in experimental systems, what appear at first to be very similar studies have, at times, yielded vastly differing results and conclusions. Thus this is an exceedingly exciting time to study endocytosis, and this area serves as a clear demonstration of the power of applying live-cell imaging to answer fundamental biological questions.

Drosophila endocytic neoplastic tumor suppressor genes regulate Sav/Wts/Hpo signaling and the c-Jun N-terminal kinase pathway

“…Unlike Notch and JAK-STAT signaling, which are only activated by later blocks in the endolysosomal pathway (e.g., ept and vps25), we find that Yki activity is elevated by manipulating genes at each step in the endolysosomal pathway including AP-2σ, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization (reviewed in ref. 25). Moreover, we show that Yki …”

Advanced Optical Imaging of Endocytosis

“…Matters were complicated further when later data suggested that a single CCP could give rise to multiple vesicles (Rappoport, 2008). Single particle analysis of TIRF data showed that some CCV were initiated de novo -that is, the assembly of a single CCP resulted in complete disappearance of clathrin-associated signal into an internalized vesicle.…”

“…However, other CCPs were seen to separate into sub-structures, only a portion of which were seen to internalize, while other CCPs were seen to merge into larger structures (often termed clathrin coated plaques). As an example, Figure 1 shows TIRF images and analysis representing a single event of the latter type, taken from (Rappoport, 2008). Close inspection of the indicated point spread function from a single sub-diffraction sized CCP shows a broadening and eventual separation into two distinct features.…”

The emergence of multiple particle tracking in intracellular trafficking of nanomedicines

“…Reprinted with kind permission from Champion and Mitragotri (2006) 200 nm), which are pinched off from the plasma membrane by a small GTPase, dynamin (Pucadyil and Schmid 2009). Clathrin-coated vesicles lose their clathrin coat upon internalization and fuse with early endosomes, where they are sorted to late endosomes/lysosomes (pH 5.0-6.0), to the trans-Golgi network, or to recycling endosomes to be transported back to plasma membrane (Rappoport 2008). Contrary to phagocytosis, it is difficult to describe a thorough and consistent profile of nanoparticles uptake for CME.…”