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Clathrin mediated endocytosis (CME) is the main route of receptor internalization in mammalian cells and this well conserved mechanism has been intensively studied for over 40yrs. In the general or 'canonical' model of CME clathrin coated pits form stochastically at the plasma membrane and coated pit curvature develops as the coated pit grows through clathrin polymerization. However, the canonical model of CME does not explain the diversity of endocytically active clathrin coated structures (CCSs) found at the plasma membrane by both electron and light microscopy. In this review we examine the canonical model of CME, highlight discrepancies with published experimental data and suggest future avenues of exploration while paying particular attention to the relationship between clathrin coated pits, plaques, sites of adhesion and the formation of endocytic 'hotspots'.

An actin filament coat promotes cargo expulsion from large exocytosing vesicles, but the mechanisms of coat formation and force generation have been poorly characterized. Elegant imaging studies of the Drosophila melanogaster salivary gland now reveal how actin and myosin are recruited, and show that myosin II forms a contractile 'cage' that facilitates exocytosis.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

Nat. Cell Biol. 18, 142–144 (2016); published online 28 January 2016; corrected after print 4 February 2016 In the version of this News and Views article originally published, the author's affiliation should have read “Institute for Integrative Biology of the Cell, Bât. 34, Avenue de la Terrasse, 9198 Gif sur Yvette cedex, France”.

Simultaneous ion conductance and confocal microscopy in live cells reveal a new form of asymmetric clathrin-coated pit closure., Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin–enhanced green fluorescent protein (EGFP) and actin-binding protein–EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.

Dishevelled is a crucial effector upstream in the Wnt signalling pathway, but the molecular mechanism by which it transduces the Wnt signal remains elusive. Dishevelled is a cytoplasmic protein with a strong tendency to form puncta, which correlates with its potent activity in stimulating Wnt signal transduction when overexpressed. These puncta are thought to reflect cytoplasmic vesicles. However, we show here that the mammalian Dishevelled protein Dvl2 does not colocalise with known vesicle markers for clathrin-mediated or clathrin-independent endocytic pathways. Furthermore, Dvl2 puncta do not stain with lipid dyes, indicating that these puncta do not contain membranes. Instead, our evidence from live imaging by TIRF microscopy of Dvl2 tagged with green fluorescent protein (GFP-Dvl2) revealed that these puncta move in and out of the evanescent field near the plasma membrane in an undirected fashion, and that they can grow by collision and fusion. Furthermore, high-resolution confocal microscopy and photobleaching experiments indicate that the GFP-Dvl2 puncta are protein assemblies; there is a constant exchange of GFP-Dvl2 between puncta and a diffuse cytoplasmic pool, which, therefore, are in a dynamic equilibrium with each other. The same is true for the DIX domain of Dvl2 itself and also for Axin-GFP, which equilibrates between the punctate and cytosolic pools. Our evidence indicates that Dvl2 and Axin puncta are dynamic protein assemblies rather than cytoplasmic vesicles.

Visual and auditory information is encoded by sensory neurons that tonically release neurotransmitter at high rates. The synaptic ribbon is an essential organelle in nerve terminals of these neurons. Its precise function is unknown, but if the ribbon could be visualized in a living terminal, both its own dynamics and its relation to calcium and vesicle dynamics could be studied. We designed a short fluorescent peptide with affinity for a known binding domain of RIBEYE, a protein unique to the ribbon. When introduced via a whole-cell patch pipette, the peptide labeled structures at the presynaptic plasma membrane of ribbon-type terminals. The fluorescent spots match in size, location, number, and distribution the known features of synaptic ribbons. Furthermore, fluorescent spots mapped by confocal microscopy directly match the ribbons identified by electron microscopy in the same cell. Clearly the peptide binds to the synaptic ribbon, but even at saturating concentrations it affects neither the morphology of the ribbon nor its tethering of synaptic vesicles. It also does not inhibit exocytosis. Using the peptide label, we observed that the ribbon is immobile over minutes and that calcium influx is concentrated at the ribbon. Finally, we find that each ribbon in a retinal bipolar cell contains ∼4000 molecules of RIBEYE, indicating that it is the major component of the synaptic ribbon.

Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.

The membrane-curvature-inducing protein Fcho was proposed to be part of a ubiquitous nucleation mechanism for clathrin-coated pits. However, studies in developing zebrafish embryos now indicate a role for Fcho as a receptor-specific adaptor in bone morphogenetic protein (BMP) signalling, rather than a global coated-pit nucleator.

The endocytosis of the transferrin receptor (TfR) and β2-adrenergic receptor or Mu-opioid receptor by individual endocytic vesicles was imaged in live cells. Ligand-triggered endocytosis of GPCR has little effect on TfR endocytosis dynamics, and there is little evidence for coated-pit specialization., The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein–coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)–based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.

Annexin 2 is a Ca 2+ binding protein that binds to and aggregates secretory vesicles at physiological Ca 2+ levels [1] and that also associates Ca 2+ independently with early endosomes [2, 3]. These properties suggest roles in both exocytosis and endocytosis, but little is known of the dynamics of Annexin 2 distribution in live cells during these processes. We have used evanescent field microscopy to image Annexin 2-GFP in live, secreting rat basophilic leukemia cells and in cells performing pinocytosis. Although we found no evidence of Annexin 2 involvement in exocytosis, we observed an enrichment of Annexin 2-GFP in actin tails propeling macropinosomes. The association of Annexin 2-GFP with rocketing macropinosomes was specific because Annexin 2-GFP was absent from the actin tails of rocketing Listeria. This finding suggests that the association of Annexin 2 with macropinocytic rockets requires native pinosomal membrane. Annexin 2 is necessary for the formation of macropinocytic rockets since overexpression of a dominant-negative Annexin 2 construct abolished the formation of these structures. The same construct did not prevent the movement of Listeria in infected cells. These results show that recruitment of Annexin 2 to nascent macropinosome membranes 16656is an essential prerequisite for actin polymerization-dependent vesicle locomotion.

A molecular dissection of contractile vacuole (CV) discharge shows that Rab8a is recruited to the CV a few seconds before the exocyst. Together they tether it to the plasma membrane and commit it to fusion. GTP hydrolysis is necessary for vacuole detethering, a process in which LvsA, a protein of the Chédiak–Higashi family, plays a crucial role., Water expulsion by the contractile vacuole (CV) in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion, and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a effector, Rab8a, the exocyst complex, and LvsA, a protein of the Chédiak–Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder, suggesting that a Rab8a guanine nucleotide exchange factor activity is associated with the complex. Absence of Drainin leads to overtethering and blocks fusion, whereas expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in postfusion detethering. Of interest, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype.

Clathrin mediated endocytosis is the principle mechanism by which cells internalize cell surface receptors. It can be thought of as a molecular machine which concentrates receptors into patches at the plasma membrane and which bends the membrane into a vesicle that pinches off. Quite how the many structural, enzymatic and cytoskeletal components of the endocytic machine are spatially and temporally organised is not fully understood. In this talk I will describe our efforts to address this problem by analysing single endocytic events using total internal fluorescence microscopy (TIR-FM).

SummaryClathrin-mediated endocytosis is the main path for receptor internalization in metazoans and is essential for controlling cell integrity and signaling. It is driven by a large array of protein and lipid interactions that have been deciphered mainly by biochemical and genetic means. To place these interactions into context, and ultimately build a fully operative model of endocytosis at the molecular level, it is necessary to know the kinetic details of the role of each protein in this process. In this review, we describe the recent efforts made, by using live cell imaging, to define clear steps in the formation of endocytic vesicles and to observe the recruitment of key proteins during membrane invagination, the scission of a newly formed vesicle, and its movement away from the plasma membrane.

Endocytosis is characterized by movement and precisely controlled changes in membrane geometry during vesicle formation. Recent developments in live-cell imaging have enabled such movements to be monitored in vivo and correlated with the recruitment and dismissal of fluorescently labeled proteins. This experimental strategy has revealed the sequential recruitment of proteins that are involved in actin polymerization, and actin to single sites of endocytosis in both yeast and mammalian cells. Actin polymerization is correlated with the inward movements of endocytic organelles, which suggests that actin polymerization has a conserved role in this process. In this article, I will discuss three models for the role of actin polymerization in endocytosis.

Summary Several findings suggest that actin-mediated motility can play a role in clathrin-mediated endocytosis but it remains unclear whether and when key proteins required for this process are recruited to endocytic sites. Here we investigate this question in live Swiss 3T3 cells using two-colour evanescent field (EF) microscopy. We find that Arp3, a component of the Arp2/3 complex, appears transiently while single clathrin-coated pits internalize. There is also additional recruitment of Neural-Wiskott Aldrich Syndrome Protein (N-WASP), a known activator of the Arp2/3 complex. Both proteins appear at about the same time as actin. We suggest that N-WASP and the Arp2/3 complex trigger actin polymerization during a late step in clathrin-mediated endocytosis, and propel clathrin-coated pits or vesicles from the plasma membrane into the cytoplasm.

As a final step in endocytosis, clathrin-coated pits must separate from the plasma membrane and move into the cytosol as a coated vesicle. Because these events involve minute movements that conventional light microscopy cannot resolve, they have not been observed directly and their dynamics remain unexplored. Here, we used evanescent field (EF) microscopy to observe single clathrin-coated pits or vesicles as they draw inwards from the plasma membrane and finally lose their coats. This inward movement occurred immediately after a brief burst of dynamin recruitment and was accompanied by transient actin assembly. Therefore, dynamin may provide the trigger and actin may provide the force for movement into the cytosol.

Up to 60 different proteins are recruited to the site of clathrin-mediated endocytosis in an ordered sequence. These accessory proteins have roles during all the different stages of clathrin-mediated endocytosis. First, they participate in the initiation of the endocytic event, thereby determining when and where endocytic vesicles are made; later they are involved in the maturation of the clathrin coat, recruitment of specific cargo molecules, bending of the membrane, and finally in scission and uncoating of the nascent vesicle. In addition, many of the accessory components are involved in regulating and coupling the actin cytoskeleton to the endocytic membrane. We will discuss the different accessory components and their various roles. Most of the data comes from studies performed with cultured mammalian cells or yeast cells. The process of endocytosis is well conserved between these different organisms, but there are also many interesting differences that may shed light on the mechanistic principles of endocytosis.

The annexins, are a family of calcium ion (Ca2+)-binding proteins whose physiological functions are poorly understood. Although many diverse functions have been proposed for these proteins, such as in vesicle trafficking, this review focuses on their proposed roles as Ca2+ or other ion channels, or as intracellular ion channel regulators. Such ideas are founded mainly on in vitro and structural analyses, but there is increasing evidence that at least some members of this protein family may indeed play a part in intracellular Ca2+ signaling by acting both as atypical ion channels and as modulators of ion channel activity. This review first introduces the annexin family, then discusses intracellular localization, developmental regulation, and modes of membrane association of annexins, which suggest roles in Ca2+ homeostasis. Finally, it examines the structural and electrophysiological data that argue for key roles for annexins in the control of ion fluxes.

The plasma membrane defines the outer limit of eukaryotic cells and it is here that cells interact with their environment through receptor proteins. Clearly the management of cell surface receptor concentration is critical for normal cellular function, and this is achieved through two key processes, exocytosis and endocytosis. During exocytosis transport vesicles containing both membrane receptors and soluble cargo fuse with the plasma membrane releasing cargo and delivering receptors to the cell surface. During clathrin mediated endocytosis receptors destined for internalisation become concentrated in clathrin coated pits before coat invagination and membrane fission. Since transferrin receptors are recycled and undergo numerous rounds of exocytosis and endocytosis by labeling transferrin receptor with an appropriate fluorophore it is possible to label both exocytic and endocytic organelles in the same cell. We have exploited this phenomenon to visualise the spatial and temporal organisation of constitutive exocytosis and endocytosis at the surface of single cells using dual wavelength evanescent field microscopy.

SummaryDuring clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.