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1.
Cell ; 179(1): 120-131.e13, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31539492

RESUMO

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.


Assuntos
Adesões Focais/metabolismo , Domínios e Motivos de Interação entre Proteínas , Talina/química , Talina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Sítios de Ligação , Adesão Celular/fisiologia , Microscopia Crioeletrônica , Citoesqueleto/metabolismo , Dimerização , Escherichia coli/metabolismo , Humanos , Integrinas/metabolismo , Modelos Moleculares , Ligação Proteica , Transdução de Sinais/fisiologia , Vinculina/metabolismo
2.
Proc Natl Acad Sci U S A ; 113(38): E5552-61, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27601635

RESUMO

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.


Assuntos
Proteínas de Drosophila/química , Proteínas de Membrana/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Sequência de Aminoácidos/genética , Animais , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Domínios de Homologia de src/genética
3.
Nat Commun ; 15(1): 4986, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862544

RESUMO

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P2-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.


Assuntos
Membrana Celular , Adesões Focais , Talina , Vinculina , Talina/metabolismo , Talina/química , Adesões Focais/metabolismo , Membrana Celular/metabolismo , Vinculina/metabolismo , Vinculina/química , Humanos , Animais , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Integrinas/metabolismo , Integrinas/química , Citoplasma/metabolismo , Ligação Proteica , Separação de Fases
4.
Nat Commun ; 12(1): 2254, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859190

RESUMO

One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.


Assuntos
Actomiosina/metabolismo , Divisão Celular/fisiologia , Modelos Biológicos , Biologia Sintética/métodos , Lipossomas Unilamelares/metabolismo , Actomiosina/genética , Actomiosina/isolamento & purificação , Animais , Linhagem Celular , Drosophila , Humanos , Microscopia Intravital , Microscopia Confocal , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
5.
Elife ; 102021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34324418

RESUMO

Synaptic membrane-remodeling events such as endocytosis require force-generating actin assembly. The endocytic machinery that regulates these actin and membrane dynamics localizes at high concentrations to large areas of the presynaptic membrane, but actin assembly and productive endocytosis are far more restricted in space and time. Here we describe a mechanism whereby autoinhibition clamps the presynaptic endocytic machinery to limit actin assembly to discrete functional events. We found that collective interactions between the Drosophila endocytic proteins Nwk/FCHSD2, Dap160/intersectin, and WASp relieve Nwk autoinhibition and promote robust membrane-coupled actin assembly in vitro. Using automated particle tracking to quantify synaptic actin dynamics in vivo, we discovered that Nwk-Dap160 interactions constrain spurious assembly of WASp-dependent actin structures. These interactions also promote synaptic endocytosis, suggesting that autoinhibition both clamps and primes the synaptic endocytic machinery, thereby constraining actin assembly to drive productive membrane remodeling in response to physiological cues.


Neurons constantly talk to each other by sending chemical signals across the tiny gap, or 'synapse', that separates two cells. While inside the emitting cell, these molecules are safely packaged into small, membrane-bound vessels. Upon the right signal, the vesicles fuse with the external membrane of the neuron and spill their contents outside, for the receiving cell to take up and decode. The emitting cell must then replenish its vesicle supply at the synapse through a recycling mechanism known as endocytosis. To do so, it uses dynamically assembling rod-like 'actin' filaments, which work in concert with many other proteins to pull in patches of membrane as new vesicles. The proteins that control endocytosis and actin assembly abound at neuronal synapses, and, when mutated, are linked to many neurological diseases. Unlike other cell types, neurons appear to 'pre-deploy' these actin-assembly proteins to synaptic membranes, but to keep them inactive under normal conditions. How neurons control the way this machinery is recruited and activated remains unknown. To investigate this question, Del Signore et al. conducted two sets of studies. First, they exposed actin to several different purified proteins in initial 'test tube' experiments. This revealed that, depending on the conditions, a group of endocytosis proteins could prevent or promote actin assembly: assembly occurred only if the proteins were associated with membranes. Next, Del Signore et al. mutated these proteins in fruit fly larvae, and performed live cell microscopy to determine their impact on actin assembly and endocytosis. Consistent with the test tube findings, endocytosis mutants had more actin assembly overall, implying that the proteins were required to prevent random actin assembly. However, the same mutants had reduced levels of endocytosis, suggesting that the proteins were also necessary for productive actin assembly. Together, these experiments suggest that, much like a mousetrap holds itself poised ready to spring, some endocytic proteins play a dual role to restrain actin assembly when and where it is not needed, and to promote it at sites of endocytosis. These results shed new light on how neurons might build and maintain effective, working synapses. Del Signore et al. hope that this knowledge may help to better understand and combat neurological diseases, such as Alzheimer's, which are linked to impaired membrane traffic and cell signalling.


Assuntos
Actinas/genética , Actinas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Endocitose/genética , Sinapses/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endocitose/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/metabolismo
6.
Elife ; 92020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32657269

RESUMO

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Fosfatidilinositóis/metabolismo , Talina/metabolismo , Vinculina/metabolismo , Membranas Artificiais
8.
Commun Integr Biol ; 8(2): e1000703, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26478768

RESUMO

F-BAR domains form crescent-shaped dimers that bind to and deform lipid bilayers, and play a role in many cellular processes requiring membrane remodeling, including endocytosis and cell morphogenesis. Nervous Wreck (NWK) encodes an F-BAR/SH3 protein that regulates synapse growth in Drosophila. Unlike conventional F-BAR proteins that assemble tip-to-tip into filaments and helical arrays around membrane tubules, the Nwk F-BAR domain instead assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. In cells, this membrane deforming activity generates small buds, which can lengthen into extensive protrusions upon actin cytoskeleton polymerization. Here, we show that Nwk-induced cellular protrusions contain dynamic microtubules, distinguishing them from conventional filopodia, and further do not depend on actin filaments or microtubules for their maintenance. Our results indicate new ways in which close cooperation between the membrane remodeling and cytoskeletal machinery underlies large-scale changes in cellular morphology.

9.
Cell Rep ; 13(11): 2597-2609, 2015 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-26686642

RESUMO

F-BAR domain proteins regulate and sense membrane curvature by interacting with negatively charged phospholipids and assembling into higher-order scaffolds. However, regulatory mechanisms controlling these interactions are poorly understood. Here, we show that Drosophila Nervous Wreck (Nwk) is autoregulated by a C-terminal SH3 domain module that interacts directly with its F-BAR domain. Surprisingly, this autoregulation does not mediate a simple "on-off" switch for membrane remodeling. Instead, the isolated Nwk F-BAR domain efficiently assembles into higher-order structures and deforms membranes only within a limited range of negative membrane charge, and autoregulation elevates this range. Thus, autoregulation could either reduce membrane binding or promote higher-order assembly, depending on local cellular membrane composition. Our findings uncover an unexpected mechanism by which lipid composition directs membrane remodeling.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Proteínas de Transporte/química , Dimerização , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Larva/metabolismo , Lipossomos/metabolismo , Microscopia Confocal , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Domínios de Homologia de src
10.
Mol Biol Cell ; 24(15): 2406-18, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23761074

RESUMO

Eukaryotic cells are defined by extensive intracellular compartmentalization, which requires dynamic membrane remodeling. FER/Cip4 homology-Bin/amphiphysin/Rvs (F-BAR) domain family proteins form crescent-shaped dimers, which can bend membranes into buds and tubules of defined geometry and lipid composition. However, these proteins exhibit an unexplained wide diversity of membrane-deforming activities in vitro and functions in vivo. We find that the F-BAR domain of the neuronal protein Nervous Wreck (Nwk) has a novel higher-order structure and membrane-deforming activity that distinguishes it from previously described F-BAR proteins. The Nwk F-BAR domain assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. This activity depends on structural determinants at the tips of the F-BAR dimer and on electrostatic interactions of the membrane with the F-BAR concave surface. In cells, Nwk-induced scallops can be extended by cytoskeletal forces to produce protrusions at the plasma membrane. Our results define a new F-BAR membrane-deforming activity and illustrate a molecular mechanism by which positively curved F-BAR domains can produce a variety of membrane curvatures. These findings expand the repertoire of F-BAR domain mediated membrane deformation and suggest that unique modes of higher-order assembly can define how these proteins sculpt the membrane.


Assuntos
Estruturas da Membrana Celular/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Linhagem Celular , Estruturas da Membrana Celular/ultraestrutura , Simulação por Computador , Proteínas de Drosophila/química , Drosophila melanogaster , Humanos , Lipossomos/química , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
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