Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline.

Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.

Actin puts the squeeze on Drosophila glue secretion.

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.

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.

Endocytic accessory factors and regulation of clathrin-mediated endocytosis.

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.

Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered ß2-adrenergic receptor (ß2AR) or Mu-opioid receptor (MOR) endocytosis.

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) ß2-adrenergic receptor (ß2AR) 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 ß2AR 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 ß2AR or MOR. Agonist-triggered ß2AR 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 ß2AR 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.

An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy.

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.

Fishing for clathrin-coated pit nucleators.

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.

A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase) dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR) proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.

A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2ß1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.

Dynamics of endocytic vesicle creation.

Clathrin-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.

Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells.

During 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.

Seeing is believing: imaging actin dynamics at single sites of endocytosis.

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.

Neural Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts.

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.

Annexin 2 binding to phosphatidylinositol 4,5-bisphosphate on endocytic vesicles is regulated by the stress response pathway.

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.

Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.

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.