Review

Authors: P Stahl, A L Schwartz

Topics: Animals; Biological Transport; Clathrin; Coated Pits, Cell-Membrane; Endocytosis; Endosomes; Humans; Microscopy, Electron; Microscopy, Electron, Scanning; Organoids

PubMed: 2869052
DOI: 10.1172/JCI112359

Authors: Emanuele Cocucci, Raphaël Gaudin, Tom Kirchhausen...

Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.

Topics: Animals; Base Sequence; Cattle; Cell Line, Tumor; Clathrin; Clathrin-Coated Vesicles; Coated Pits, Cell-Membrane; Dynamin II; Dynamins; Endocytosis; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Humans; Microscopy, Fluorescence; Models, Molecular; Molecular Sequence Data; Protein Multimerization; Protein Transport; RNA, Small Interfering; Signal Transduction

PubMed: 25232009
DOI: 10.1091/mbc.E14-07-1240

Review

Authors: T Wileman, C Harding, P Stahl...

Topics: Adenosine Triphosphate; Amines; Animals; Anions; Biological Transport; Coated Pits, Cell-Membrane; Endocytosis; Endosomes; Kinetics; Ligands; Models, Biological; Protons; Receptors, Drug

PubMed: 2867759
DOI: 10.1042/bj2320001

Review

Authors: Margaret S Robinson

The purification of coated vesicles and the discovery of clathrin by Barbara Pearse in 1975 was a landmark in cell biology. Over the past 40 years, work from many labs has uncovered the molecular details of clathrin and its associated proteins, including how they assemble into a coated vesicle and how they select cargo. Unexpected connections have been found with signalling, development, neuronal transmission, infection, immunity and genetic disorders. But there are still a number of unanswered questions, including how clathrin-mediated trafficking is regulated and how the machinery evolved.

Topics: Animals; Cell Biology; Clathrin; Clathrin-Coated Vesicles; Coated Pits, Cell-Membrane; History, 20th Century; History, 21st Century; Humans; Models, Molecular; Protein Conformation; Protein Transport

PubMed: 26403691
DOI: 10.1111/tra.12335

Review

Authors: Zhiming Chen, Sandra L Schmid

Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.

Topics: Adaptor Protein Complex 2; Cell Membrane; Clathrin; Clathrin-Coated Vesicles; Coated Pits, Cell-Membrane; Endocytosis; Humans

PubMed: 32770195
DOI: 10.1083/jcb.202005126

Review

Authors: Keita Soda, Shuta Ishibe

PURPOSE OF REVIEW

In this review, we discuss the role of endocytosis, a fundamental process internalizing molecules from the plasma membrane, and its critical importance in podocyte biology.

RECENT FINDINGS

Endocytic clathrin and nonclathrin-coated pits have been visualized in podocytes using electron microscopy, but the functional biological relevance has not been well defined. Recent evidence suggests that loss of key clathrin endocytic regulatory apparatus, such as dynamin, synaptojanin 1 or endophilin, in genetic mouse models of disease results in severe proteinuria and foot process effacement. In addition, several genes implicated in human nephrotic syndrome directly or indirectly associate with these endocytic proteins, thus creating a protein network that is linked in actin dynamics, signalling and endocytosis.

SUMMARY

This review summarizes our current understanding of membrane trafficking specifically in podocytes, thus giving further novel insights into the molecular mechanisms and pathogenesis of nephrotic syndrome.

Topics: Actin Cytoskeleton; Animals; Clathrin-Coated Vesicles; Coated Pits, Cell-Membrane; Endocytosis; Humans; Nephrotic Syndrome; Podocytes; Protein Transport; Proteinuria; Signal Transduction

PubMed: 23703394
DOI: 10.1097/MNH.0b013e3283624820

Review

Authors: Joshua Z Rappoport, Sandford M Simon, Alexandre Benmerah...

Most knowledge of clathrin-mediated endocytosis has been gained by biochemical fractionation and in vitro assays. Recently, the study of endocytosis has extended into the living cell. The tracking of individual clathrin-coated pits and vesicles (CCPs and CCVs) has provided new insight into understanding the dynamic nature of CCPs. The use of total internal reflection fluorescence microscopy (TIR-FM), also termed evanescent field microscopy, has enabled the direct observation of events occurring within a restricted area of the cell adjacent to and including the adherent plasma membrane. TIR-FM is now actively being pursued in the study of endocytic processes. The direct observation of CCP-associated proteins including clathrin itself, dynamin and, most recently, AP-2 has considerably challenged old models, confirming some points but raising very interesting new questions.

Topics: Adaptor Protein Complex 2; Animals; Clathrin; Coated Pits, Cell-Membrane; Endocytosis; Humans; Microscopy, Fluorescence; RNA, Small Interfering

PubMed: 15086782
DOI: 10.1111/j.1398-9219.2004.00187.x

Authors: Aubrey V Weigel, Michael M Tamkun, Diego Krapf...

Clathrin-mediated endocytosis takes place through the recruitment of cargo molecules into a growing clathrin-coated pit (CCP). Despite the importance of this process to all mammalian cells, little is yet known about the interaction dynamics between cargo and CCPs. These interactions are difficult to study because CCPs display a large degree of lifetime heterogeneity and the interactions with cargo molecules are time dependent. We use single-molecule total internal reflection fluorescence microscopy, in combination with automatic detection and tracking algorithms, to directly visualize the recruitment of individual voltage-gated potassium channels into forming CCPs in living cells. We observe association and dissociation of individual channels with a CCP and, occasionally, their internalization. Contrary to widespread ideas, cargo often escapes from a pit before abortive CCP termination or endocytic vesicle production. Thus, the binding times of cargo molecules associating to CCPs are much shorter than the overall endocytic process. By measuring tens of thousands of capturing events, we build the distribution of capture times and the times that cargo remains confined to a CCP. An analytical stochastic model is developed and compared with the measured distributions. Due to the dynamic nature of the pit, the model is non-Markovian and it displays long-tail power law statistics. The measured distributions and model predictions are in excellent agreement over more than five orders of magnitude. Our findings identify one source of the large heterogeneities in CCP dynamics and provide a mechanism for the anomalous diffusion of proteins in the plasma membrane.

Topics: Coated Pits, Cell-Membrane; Endocytosis; Green Fluorescent Proteins; HEK293 Cells; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Models, Biological; Molecular Imaging; Potassium Channels; Protein Binding; Time Factors

PubMed: 24218552
DOI: 10.1073/pnas.1315202110

Authors: F Santini, J H Keen

The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we studied the relationship between activation of IgE-bound high affinity Fc receptors (FCepsilonRI) and coated pit formation. Because these receptors are rapidly internalized via clathrin-coated pits only when cross-linked by the binding of multivalent antigens, we were able to separate activation from internalization by using an immobilized antigen. The FCepsilonRIs, initially uniformly distributed over the cell surface. relocalized and aggregated on the antigen-exposed membrane. The process was specific for the antigen, and temperature- and time-dependent. This stimulation initiated a cascade of cellular responses typical of FCepsilonRI signaling including membrane ruffling, cytoskeletal rearrangements, and, in the presence of Ca2+, exocytosis. Despite these responses, no change in coated pit disposition or in the distribution of clathrin and assembly protein AP2 was detected, as monitored by immunoblotting and by quantitative (vertical sectioning) confocal microscopy analysis of immunofluorescently stained cells. Specifically, there was no decrease in the density of clathrin-coated pits in regions of the cell membrane not in contact with the antigen, and there was no apparent increase in clathrin-coated pits in proximity to stimulated FCepsilonRI receptors as would have been expected if the receptors were inducing formation of new pits by active recruitment. These results indicate that de novo formation of clathrin-coated pits is not a prerequisite for rapid internalization or a direct response to stimulation of FCepsilonRI receptors. Therefore, increases in coated pits reported to occur in response to activation of some signaling receptors must be consequences of the signal transduction processes, rather than strictly serving the purpose of the internalization of the receptors.

Topics: Adaptor Protein Complex 2; Adaptor Protein Complex alpha Subunits; Adaptor Proteins, Vesicular Transport; Animals; Clathrin; Coated Pits, Cell-Membrane; Dinitrophenols; Endocytosis; Golgi Apparatus; Immunoglobulin E; Immunologic Capping; Leukemia, Basophilic, Acute; Membrane Proteins; Microscopy, Confocal; Microscopy, Electron, Scanning; Neoplasm Proteins; Rats; Receptors, IgE; Serum Albumin, Bovine; Transferrin; Tumor Cells, Cultured

PubMed: 8601582
DOI: 10.1083/jcb.132.6.1025

Review

Authors: Tom Kirchhausen, David Owen, Stephen C Harrison...

Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation.

Topics: Actins; Animals; Auxilins; Biological Transport; Cell Membrane; Clathrin; Coated Pits, Cell-Membrane; Dynamins; Humans

PubMed: 24789820
DOI: 10.1101/cshperspect.a016725