RESUMO
In Figs. 2b and 3d of this Letter, the labels 'Dynamin 1' and 'Overlay' were inadvertently swapped. This has been corrected online.
RESUMO
Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles1. Here we report a 3.75 Å resolution cryo-electron microscopy structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays. Compared to the lipid-free tetramer form2, membrane-associated dynamin binds to the lipid bilayer with its pleckstrin homology domain (PHD) and self-assembles across the helical rungs via its guanine nucleotide-binding (GTPase) domain3. Notably, interaction with the membrane and helical assembly are accommodated by a severely bent bundle signalling element (BSE), which connects the GTPase domain to the rest of the protein. The BSE conformation is asymmetric across the inter-rung GTPase interface, and is unique compared to all known nucleotide-bound states of dynamin. The structure suggests that the BSE bends as a result of forces generated from the GTPase dimer interaction that are transferred across the stalk to the PHD and lipid membrane. Mutations that disrupted the BSE kink impaired endocytosis. We also report a 10.1 Å resolution cryo-electron microscopy map of a super-constricted dynamin polymer showing localized conformational changes at the BSE and GTPase domains, induced by GTP hydrolysis, that drive membrane constriction. Together, our results provide a structural basis for the mechanism of action of dynamin on the lipid membrane.
Assuntos
Biopolímeros/química , Biopolímeros/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Dinamina I/metabolismo , Dinamina I/ultraestrutura , Biopolímeros/genética , Membrana Celular/química , Dinamina I/química , Dinamina I/genética , Endocitose/genética , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestrutura , Mutação , Domínios Proteicos , Multimerização ProteicaRESUMO
We combine super-resolution localization fluorescence microscopy with transmission electron microscopy of metal replicas to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. We validate robust correlation on the scale of 20 nm by imaging endogenous clathrin (in two and three dimensions) and apply the method to find the previously unknown three-dimensional position of the endocytic protein epsin on clathrin-coated structures at the plasma membrane.
Assuntos
Ouro/química , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nanotubos/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Clatrina/ultraestrutura , Humanos , Proteínas de Membrana/metabolismoRESUMO
Natural antimicrobial peptides (AMPs) provide prototypes for the design of unconventional antimicrobial agents. Existing bulk assays measure AMP activity but do not provide details of the growth-halting mechanism. We use fluorescence microscopy to directly observe the attack of the human antimicrobial peptide LL-37 on single Escherichia coli cells in real time. Our findings strongly suggest that disruption of the cytoplasmic membrane is not the growth-halting mechanism. At 8 µM, LL-37 binding saturates the outer membrane (OM) within 1 min. Translocation across the OM and access to the periplasmic space (5-25 min later) correlates in time with the halting of growth. Septating cells are attacked more readily than nonseptating cells. The halting of growth may occur because of LL-37 interference with cell wall biogenesis. Only well after growth halts does the peptide permeabilize the cytoplasmic membrane to GFP and the small dye Sytox Green. The assay enables dissection of antimicrobial design criteria into two parts: translocation across the OM and the subsequent halting of growth.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Humanos , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/metabolismo , CatelicidinasRESUMO
The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.
Assuntos
Citoesqueleto de Actina , Actinas , Miosina Tipo II , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Camundongos , Fibroblastos , Humanos , Células HEK293 , Miosina Tipo II/metabolismoRESUMO
Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission.
Assuntos
Membrana Celular , Microscopia Crioeletrônica , Dinaminas , Guanosina Trifosfato , Microscopia Crioeletrônica/métodos , Humanos , Membrana Celular/metabolismo , Células HeLa , Dinaminas/metabolismo , Dinaminas/química , Dinaminas/genética , Guanosina Trifosfato/metabolismo , Hidrólise , Guanosina Difosfato/metabolismo , Modelos Moleculares , Endocitose/fisiologiaRESUMO
Conformational changes in endocytic proteins are regulators of clathrin-mediated endocytosis. Three clathrin heavy chains associated with clathrin light chains (CLC) assemble into triskelia that link into a geometric lattice that curves to drive endocytosis. Structural changes in CLC have been shown to regulate triskelia assembly in solution, yet the nature of these changes, and their effects on lattice growth, curvature, and endocytosis in cells are unknown. Here, we develop a new correlative fluorescence resonance energy transfer (FRET) and platinum replica electron microscopy method, named FRET-CLEM. With FRET-CLEM, we measure conformational changes in clathrin at thousands of individual morphologically distinct clathrin-coated structures. We discover that the N-terminus of CLC repositions away from the plasma membrane and triskelia vertex as coats curve. Preventing this conformational switch with chemical tools increases lattice sizes and inhibits endocytosis. Thus, a specific conformational switch in the light chain regulates lattice curvature and endocytosis in mammalian cells.
Assuntos
Cadeias Leves de Clatrina , Endocitose , Animais , Cadeias Leves de Clatrina/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Cadeias Pesadas de Clatrina/metabolismo , Mamíferos/metabolismoRESUMO
The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.
RESUMO
The crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood. Here we use quantitative fluorescence and electron microscopy to reveal a mechanism where flat clathrin lattices partition and activate growth factor signals via a coordinated response that involves crosstalk between epidermal growth factor receptor (EGFR) and the adhesion receptor ß5-integrin. We show that ligand-activated EGFR, Grb2, Src, and ß5-integrin are captured by clathrin coated-structures at the plasma membrane. Clathrin structures dramatically grow in response to EGF into large flat plaques and provide a signaling platform that link EGFR and ß5-integrin through Src-mediated phosphorylation. Disrupting this EGFR/Src/ß5-integrin axis prevents both clathrin plaque growth and dampens receptor signaling. Our study reveals a reciprocal regulation between clathrin lattices and two different receptor systems to coordinate and enhance signaling. These findings have broad implications for the regulation of growth factor signaling, adhesion, and endocytosis.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/química , Proteína Adaptadora GRB2/metabolismo , Cadeias beta de Integrinas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Endocitose , Receptores ErbB/metabolismo , Humanos , Microscopia Eletrônica , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismoRESUMO
Caveolae are small coated plasma membrane invaginations with diverse functions. Caveolae undergo curvature changes. Yet, it is unclear which proteins regulate this process. To address this gap, we develop a correlative stimulated emission depletion (STED) fluorescence and platinum replica electron microscopy imaging (CLEM) method to image proteins at single caveolae. Caveolins and cavins are found at all caveolae, independent of curvature. EHD2 is detected at both low and highly curved caveolae. Pacsin2 associates with low curved caveolae and EHBP1 with mostly highly curved caveolae. Dynamin is absent from caveolae. Cells lacking dynamin show no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by intermittent associations with pacsin2 and EHBP1. These coats can flatten and curve to enable lipid traffic, signaling, and changes to the surface area of the cell.
Assuntos
Cavéolas , Caveolinas , Cavéolas/metabolismo , Membrana Celular/metabolismo , Caveolinas/metabolismo , Endocitose , Dinaminas/metabolismo , Proteínas/metabolismoRESUMO
The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14-1.02 Osm, the mean diffusion coefficient
Assuntos
Difusão , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Periplasma/metabolismo , Meios de Cultura/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Difusão/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Recuperação de Fluorescência Após Fotodegradação , Microscopia de Fluorescência , Pressão Osmótica/efeitos dos fármacos , Periplasma/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacosRESUMO
Protein coats, important for vesicular trafficking in eukaryotic cells, help shape membranes and package cargo. But their dynamic construction cannot be fully understood until the distinct steps of their assembly in their native intracellular context at molecular resolution can be visualized. For this, correlative light and electron microscopy (CLEM) is an essential tool. Here, we discuss how emerging CLEM techniques have been used to study the assembly of protein coats inside cells. We review how current and developing CLEM technologies are poised to answer fundamental questions of protein coat architecture at the nanoscale.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Proteínas de Membrana , Microscopia EletrônicaRESUMO
Rab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we apply a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the 3D axial location of these exocytic proteins and two SNARE proteins (Syntaxin1A and SNAP25) using electron tomography. Rab proteins are distributed across the entire surface and t-SNARE proteins at the base of docked vesicles. We propose that the circumferential distribution of Rabs and Rab-effectors could aid in the efficient transport, capture, docking, and rapid fusion of calcium-triggered exocytic vesicles in excitable cells.
Assuntos
Imagem Molecular/métodos , Células Neuroendócrinas/citologia , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Carbocianinas/química , Células Cultivadas , Exocitose , Ouro , Células HeLa , Humanos , Imageamento Tridimensional , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nanopartículas Metálicas/química , Microscopia/métodos , Células Neuroendócrinas/metabolismo , Células PC12 , Ratos , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína Vermelha FluorescenteRESUMO
Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Esteróis/farmacologia , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/fisiologia , Colesterol/metabolismo , Clatrina/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Receptores da Transferrina/metabolismo , Esteróis/metabolismoRESUMO
Clathrin-mediated endocytosis is the primary pathway for receptor and cargo internalization in eukaryotic cells. It is characterized by a polyhedral clathrin lattice that coats budding membranes. The mechanism and control of lattice assembly, curvature, and vesicle formation at the plasma membrane has been a matter of long-standing debate. Here, we use platinum replica and cryoelectron microscopy and tomography to present a structural framework of the pathway. We determine the shape and size parameters common to clathrin-mediated endocytosis. We show that clathrin sites maintain a constant surface area during curvature across multiple cell lines. Flat clathrin is present in all cells and spontaneously curves into coated pits without additional energy sources or recruited factors. Finally, we attribute curvature generation to loosely connected and pentagon-containing flat lattices that can rapidly curve when a flattening force is released. Together, these data present a universal mechanistic model of clathrin-mediated endocytosis.
Assuntos
Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Clatrina/metabolismo , Adesividade , Animais , Linhagem Celular , Colesterol/metabolismo , Microscopia Crioeletrônica , Humanos , Masculino , Camundongos , Modelos Biológicos , RatosRESUMO
B lymphocytes play a critical role in adaptive immunity. On antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative superresolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab')2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when cluster size increases beyond the size of a single clathrin-coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.
Assuntos
Endocitose/fisiologia , Receptores de Antígenos de Linfócitos B/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Clatrina/metabolismo , Citoesqueleto/metabolismo , Endocitose/imunologia , Humanos , Proteínas de Membrana/metabolismo , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de SinaisRESUMO
Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.
Assuntos
Citoplasma/fisiologia , Escherichia coli K12/fisiologia , Proteínas de Escherichia coli/fisiologia , Animais , Sobrevivência Celular , Meios de Cultura , Escherichia coli K12/citologia , Escherichia coli K12/crescimento & desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Organismos Geneticamente Modificados/fisiologia , Estresse FisiológicoRESUMO
Clathrin-mediated endocytosis (CME) is the primary mechanism eukaryotic cells use to internalize material. New imaging tools are revealing the nanoscale structural dynamics of single clathrin-coated sites. Recently, it has become clear that the structure and dynamics of clathrin - flat clathrin lattices and the transition to highly curved clathrin-coated vesicles - are adaptable and can follow many paths. Thus, understanding this dynamic plasticity will lead to insights into how one molecular machine can participate in multiple pathways and adapt to changing and diverse cellular environments. Here, we review recent studies that have directly addressed this structural plasticity. We discuss the structure of lattices, how clathrin lattices form, and which proteins or biophysical factors might regulate the transition between flat and curved lattices.
Assuntos
Clatrina/química , Clatrina/metabolismo , Animais , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Humanos , Conformação ProteicaRESUMO
Altered regulation of exocytosis is an important mechanism controlling many diseases, including cancer. Defects in exocytosis have been implicated in many cancer cell types and are generally attributed to mutations in cellular transport, trafficking, and assembly of machinery necessary for exocytosis of secretory vesicle cargo. In these cancers, up-regulation of trafficking and secretion of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme, is responsible for degrading the extracellular matrix, a necessary step in tumor progression. Using TIRF microscopy, we identified proteins associated with secretory vesicles containing MMP-9 and imaged the local dynamics of these proteins at fusion sites during regulated exocytosis of MMP-9 from MCF-7 breast cancer cells. We found that many regulators of exocytosis, including several Rab GTPases, Rab effector proteins, and SNARE/SNARE modulator proteins, are stably assembled on docked secretory vesicles before exocytosis. At the moment of fusion, many of these components are quickly lost from the vesicle, while several endocytic proteins and lipids are simultaneously recruited to exocytic sites at precisely that moment. Our findings provide insight into the dynamic behavior of key core exocytic proteins, accessory proteins, lipids, and some endocytic proteins at single sites of secretory vesicle fusion in breast cancer cells.