Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.     

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.     

Imaging single endocytic events reveals diversity in clathrin, dynamin and vesicle dynamics

The dynamics of clathrin-mediated endocytosis can be assayed using fluorescently tagged proteins and total internal reflection fluorescence microscopy. Many of these proteins, including clathrin and dynamin, are soluble and changes in fluorescence intensity can be attributed either to membrane/vesicle movement or to changes in the numbers of individual molecules. It is important for assays to discriminate between physical membrane events and the dynamics of molecules. Two physical events in endocytosis were investigated: vesicle scission from the plasma membrane and vesicle internalization. Single vesicle analysis allowed the characterization of dynamin and clathrin dynamics relative to scission and internalization. We show that vesicles remain proximal to the plasma membrane for variable amounts of time following scission, and that uncoating of clathrin can occur before or after vesicle internalization. The dynamics of dynamin also vary with respect to scission. Results from assays based on physical events suggest that disappearance of fluorescence from the evanescent field should be re-evaluated as an assay for endocytosis. These results illustrate the heterogeneity of behaviors of endocytic vesicles and the importance of establishing suitable evaluation criteria for biophysical processes.     

External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane

Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane.     

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, synaptojanin2β1, 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.     

A Highlights from MBoC Selection: Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit

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.     

Sequential steps in clathrin-mediated synaptic vesicle endocytosis

Synaptic vesicles are recycled with remarkable speed and precision in nerve terminals. A major recycling pathway involves clathrin-mediated endocytosis at endocytic zones located around sites of release. Different 'accessory' proteins linked to this pathway have been shown to alter the shape and composition of lipid membranes, to modify membrane-coat protein interactions, and to influence actin polymerization. These include the GTPase dynamin, the lysophosphatidic acid acyl transferase endophilin, and the phosphoinositide phosphatase synaptojanin. Protein perturbation studies in living nerve terminals are now beginning to link the actions of these proteins with morphologically defined steps of endocytosis.     

Characterization of rotavirus cell entry

While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-beta-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.     

Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis

Endocytosis is an important way for cells to take up liquids and particles from their environment. It requires membrane bending to be coupled with membrane fission, and the actin cytoskeleton has an active role in membrane remodelling. Here, we review recent research into the function of Bin-Amphiphysin-Rvs (BAR) domain proteins, which can sense membrane curvature and recruit actin to membranes. BAR proteins interact with the endocytic and cytoskeletal machinery, including the GTPase dynamin (which mediates vesicle fission), N-WASP (an Arp2/3 complex regulator) and synaptojanin (a phosphoinositide phosphatase). We describe three classes of BAR domains, BAR, N-BAR and F-BAR, providing examples of each discussing and how they function in linking membranes to the actin cytoskeleton in endocytosis.     

SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis

Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domain-containing proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity.     

Ever-expanding network of dynamin-interacting proteins

Clathrin-mediated endocytosis is a major cellular pathway for internalization of proteins and lipids and for recycling of synaptic vesicles. The GTPase dynamin plays a key role in this process, and the proline-rich domain of dynamin participates in various protein-protein interactions to ensure a proper coordination of endocytic processes. Although dynamin is not directly associated with actin, several dynamin-binding proteins can interact with actin or with proteins that regulate actin assembly, thereby coordinately regulating actin assembly and trafficking events. This article summarizes dynamin interactions with various Src homology 3-containing proteins, many of which are actin-binding proteins. It also discusses the recently identified two new dynamin binding proteins, SH3 protein interacting with Nck, 90 kDa/Wiskott-Aldrich syndrome protein interacting with SH3 protein (SPIN90/WISH) and sorting nexin 9, and outlines their potential role as a link between endocytosis and actin dynamics.     

A Hip1R-cortactin complex negatively regulates actin assembly associated with endocytosis

Actin polymerization plays a critical role in clathrin-mediated endocytosis in many cell types, but how polymerization is regulated is not known. Hip1R may negatively regulate actin assembly during endocytosis because its depletion increases actin assembly at endocytic sites. Here, we show that the C-terminal proline-rich domain of Hip1R binds to the SH3 domain of cortactin, a protein that binds to dynamin, actin filaments and the Arp2/3 complex. We demonstrate that Hip1R deleted for the cortactin-binding site loses its ability to rescue fully the formation of abnormal actin structures at endocytic sites induced by Hip1R siRNA. To determine when this complex might function during endocytosis, we performed live cell imaging. The maximum in vivo recruitment of Hip1R, clathrin and cortactin to endocytic sites was coincident, and all three proteins disappeared together upon formation of a clathrin-coated vesicle. Finally, we showed that Hip1R inhibits actin assembly by forming a complex with cortactin that blocks actin filament barbed end elongation.     

An InCytes from MBC Selection: Membrane Insertion of the Pleckstrin Homology Domain Variable Loop 1 Is Critical for Dynamin-catalyzed Vesicle Scission

The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and for clathrin-mediated endocytosis in vivo.     

Interactions between Piccolo and the actin/dynamin-binding protein Abp1 link vesicle endocytosis to presynaptic active zones

Piccolo is a high molecular weight multi-domain protein shown to be a structural component of the presynaptic CAZ (cytoskeletal matrix assembled at active zones). These features indicate that Piccolo may act to scaffold proteins involved in synaptic vesicle endo- and exocytosis near their site of action. To test this hypothesis, we have utilized a functional cell-based endocytosis assay and identified the N-terminal proline-rich Q domain in Piccolo as a region that interferes with clathrin-mediated endocytosis. Utilizing the Piccolo Q domain as bait in a yeast two-hybrid screen, we have identified the F-actin-binding protein Abp1 (also called SH3P7 or HIP-55) as a potential binding partner for this domain. The physiological relevance of this interaction is supported by in vitro binding studies, colocalization in nerve terminals, in vivo recruitment studies, and immunoprecipitation experiments. Intriguingly, Abp1 binds to both F-actin and the GTPase dynamin and has been implicated in linking the actin cytoskeleton to clathrin-mediated endocytosis. Our results suggest that Piccolo, as a structural protein of the CAZ, may serve to localize Abp1 at active zones where it can actively participate in creating a functional connection between the dynamic actin cytoskeleton and synaptic vesicle recycling.     

Neural Wiskott-Aldrich syndrome protein is recruited to rafts and associates with endophilin A in response to epidermal growth factor

Neural Wiskott-Aldrich syndrome protein (N-WASP) has been implicated in endocytosis; however, little is known about how it interacts functionally with the endocytic machinery. Sucrose gradient fractionation experiments and immunofluorescence studies with anti-N-WASP antibody revealed that N-WASP is recruited together with clathrin and dynamin, which play essential roles in clathrin-mediated endocytosis, to lipid rafts in an epidermal growth factor (EGF)-dependent manner. Endophilin A (EA) binds to dynamin and plays an essential role in the fission step of clathrin-mediated endocytosis. In the present study, we show that the Src homology 3 (SH3) domain of EA associates with the proline-rich domain of N-WASP and dynamin in vitro. Co-immunoprecipitation assays with anti-N-WASP antibody revealed that EGF induces association of N-WASP with EA. In addition, EA enhances N-WASP-induced actin-related protein 2/3 (Arp2/3) complex activation in vitro. Immunofluorescence studies revealed that actin accumulates at sites where N-WASP and EA are co-localized after EGF stimulation. Furthermore, studies of overexpression of the SH3 domain of EA indicate that EA may regulate EGF-induced recruitment of N-WASP to lipid rafts. These results suggest that, upon EGF stimulation, N-WASP interacts with EA through its proline-rich domain to induce the fission step of clathrin-mediated endocytosis.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients

The large GTPase dynamin has an important membrane scission function in receptor‐mediated endocytosis and other cellular processes. Self‐assembly on phosphoinositide‐containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin‐homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C‐terminal α‐helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either ‘sensitize’ dynamin to lipid stimulation or elevate basal GTPase rates by promoting self‐assembly and thus rendering dynamin no longer lipid responsive. We also describe a low‐resolution structure of dimeric dynamin from small‐angle X‐ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self‐assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.     

Essential Function of Dynamin in the Invasive Properties and Actin Architecture of v-Src Induced Podosomes/Invadosomes

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.     

From membranes to organelles: Emerging roles for dynamin-like proteins in diverse cellular processes

Dynamin is a GTPase mechanoenzyme most noted for its role in vesicle scission during endocytosis, and belongs to the dynamin family proteins. The dynamin family consists of classical dynamins and dynamin-like proteins (DLPs). Due to structural and functional similarities DLPs are thought to carry out membrane tubulation and scission in a similar manner to dynamin. Here, we discuss the newly emerging roles for DLPs, which include vacuole fission and fusion, peroxisome maintenance, endocytosis and intracellular trafficking. Specific focus is given to the role of DLPs in the budding yeast Saccharomyces cerevisiae because the diverse function of DLPs has been well characterized in this organism. Recent insights into DLPs may provide a better understanding of mammalian dynamin and its associated diseases.     

The PX-BAR membrane-remodeling unit of sorting nexin 9

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.