Mechanisms and Functions of Early Endocytosis

Mechanisms of clathrindependent endocytosis Although clathrin-mediated internalization has been investigated for many years, new technologies keep providing us with novel insights into its underlying mechanisms, with an unprecedented scale down to molecular details regarding the structure and dynamics of the proteins involved. The plasticity of clathrin-mediated internalization was well illustrated by the fact that this pathway is used not only to traffi c extracellular molecules or cellular proteins, but also to mediate entry of certain toxins, viruses, and bacteria. The issue of lifetimes of clathrincoated pits (CCPs) and vesicles (CCVs) has remained controversial, as values reported in the literature range from seconds to minutes. Sandra Schmid (The Scripps Research Institute) described quantitative computational analyses to track the dynamics of CCP/CCV formation on the plasma membrane. In this way, three kinetically distinct populations of CCPs could be distinguished, two shortlived (earlyand late-abortive with lifetimes in the range of seconds) and one long-lived productive population stable for over one minute. Interestingly, cargo appears to increase a number of productive, long-lived CCPs/CCVs without affecting their lifetimes, which can in turn be regulated by the activity of dynamin. Morphological heterogeneity of CCVs was emphasized by Tomas Kirchhausen (Harvard Medical School). Cryoelectron tomography of individual CCVs revealed a broad range of patterns used to organize a clathrin lattice, with asymmetrically located membrane vesicles buried inside the shell ( Cheng et al., 2007 ). Moreover, high-resolution imaging of live cells based on total internal refl ection fl uorescence technology indicates that AP-2 adaptor proteins are also localized nonsymmetrically within an individual CCV. This may result from an initially restricted localization of adaptors, as they are captured during the nucleation and early phases of coated pit assembly, while retaining the adaptors concentrated at the place of their original recruitment at the time of vesicle pinching and CCV formation. Clathrin-mediated endocytosis serves some specialized functions in various tissues, including the nervous system. Knockout (KO) studies in mice, reported by Pietro De Camilli (Yale University School of Medicine), demonstrated that dynamin-1 appeared not to be essential for the biogenesis and endocytic recycling of synaptic vesicles ( Ferguson et al., 2007 ), although studies of dynamin mutants in cultured cells would have predicted a crucial role for this protein in vivo. The role of dynamin-1 in synaptic vesicle endocytosis is activity dependent and becomes evident during strong stimulation of neurons. The morphology of KO nerve terminals was visualized by EM tomography followed by tridimensional reconstruction. Such synapses are fi lled with clusters of clathrincoat components, forming tubular networks capped by clathrin-coated pits that open to the plasma membrane.