RESUMO
Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.
Assuntos
Actinas , Septinas , MicrotúbulosRESUMO
Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Sítios de Ligação , Bovinos , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas , Simulação de Dinâmica MolecularRESUMO
Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D in vitro and are delayed in reaching the draining lymph node (DLN) in vivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.
Assuntos
Proteínas de Capeamento de Actina/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas do Citoesqueleto/imunologia , Células Dendríticas/imunologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apresentação de Antígeno , Movimento Celular/imunologia , Proliferação de Células , Células Cultivadas , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Dermatite de Contato/imunologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologiaRESUMO
Dendritic spines are postsynaptic structures in neurons often having a mushroom-like shape. Physiological significance and cytoskeletal mechanisms that maintain this shape are poorly understood. The spectrin-based membrane skeleton maintains the biconcave shape of erythrocytes, but whether spectrins also determine the shape of nonerythroid cells is less clear. We show that ßIII spectrin in hippocampal and cortical neurons from rodent embryos of both sexes is distributed throughout the somatodendritic compartment but is particularly enriched in the neck and base of dendritic spines and largely absent from spine heads. Electron microscopy revealed that ßIII spectrin forms a detergent-resistant cytoskeletal network at these sites. Knockdown of ßIII spectrin results in a significant decrease in the density of dendritic spines. Surprisingly, the density of presynaptic terminals is not affected by ßIII spectrin knockdown. However, instead of making normal spiny synapses, the presynaptic structures in ßIII spectrin-depleted neurons make shaft synapses that exhibit increased amplitudes of miniature EPSCs indicative of excessive postsynaptic excitation. Thus, ßIII spectrin is necessary for formation of the constricted shape of the spine neck, which in turn controls communication between the synapse and the parent dendrite to prevent excessive excitation. Notably, mutations of SPTNB2 encoding ßIII spectrin are associated with neurodegenerative syndromes, spinocerebellar ataxia Type 5, and spectrin-associated autosomal recessive cerebellar ataxia Type 1, but molecular mechanisms linking ßIII spectrin functions to neuronal pathologies remain unresolved. Our data suggest that spinocerebellar ataxia Type 5 and spectrin-associated autosomal recessive cerebellar ataxia Type 1 pathology likely arises from poorly controlled synaptic activity that leads to excitotoxicity and neurodegeneration.SIGNIFICANCE STATEMENT Dendritic spines are small protrusions from neuronal dendrites that make synapses with axons of other neurons in the brain. Dendritic spines usually have a mushroom-like shape, which is essential for brain functions, because aberrant spine morphology is associated with many neuropsychiatric disorders. The bulbous head of a mushroom-shaped spine makes the synapse, whereas the narrow neck transmits the incoming signals to the dendrite and supposedly controls the signal propagation. We show that a cytoskeletal protein ßIII spectrin plays a key role for the formation of narrow spine necks. In the absence of ßIII spectrin, dendritic spines collapse onto dendrites. As a result, synaptic strength exceeds acceptable levels and damages neurons, explaining pathology of human syndromes caused by ßIII spectrin mutations.
Assuntos
Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Neurogênese/fisiologia , Neurônios/fisiologia , Espectrina/metabolismo , Transmissão Sináptica/fisiologia , Animais , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Células Cultivadas , Masculino , Neurônios/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Nonmuscle myosin II is an actin-based motor that executes numerous mechanical tasks in cells including spatiotemporal organization of the actin cytoskeleton, adhesion, migration, cytokinesis, tissue remodeling, and membrane trafficking. Nonmuscle myosin II is ubiquitously expressed in mammalian cells as a tissue-specific combination of three paralogs. Recent studies reveal novel specific aspects of their kinetics, intracellular regulation and functions. On the other hand, the three paralogs also can copolymerize and cooperate in cells. Here we review the recent advances from the prospective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cell.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/química , Matriz Extracelular/química , Cadeias Pesadas de Miosina/química , Miosina Tipo II/química , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIB/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Adesão Celular , Movimento Celular , Citocinese/genética , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Expressão Gênica , Humanos , Mamíferos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Multimerização ProteicaRESUMO
Axon initial segments (AISs) and nodes of Ranvier are sites of clustering of voltage-gated sodium channels (VGSCs) in nervous systems of jawed vertebrates that facilitate fast long-distance electrical signaling. We demonstrate that proximal axonal polarity as well as assembly of the AIS and normal morphogenesis of nodes of Ranvier all require a heretofore uncharacterized alternatively spliced giant exon of ankyrin-G (AnkG). This exon has sequence similarity to I-connectin/Titin and was acquired after the first round of whole-genome duplication by the ancestral ANK2/ANK3 gene in early vertebrates before development of myelin. The giant exon resulted in a new nervous system-specific 480-kDa polypeptide combining previously known features of ANK repeats and ß-spectrin-binding activity with a fibrous domain nearly 150 nm in length. We elucidate previously undescribed functions for giant AnkG, including recruitment of ß4 spectrin to the AIS that likely is regulated by phosphorylation, and demonstrate that 480-kDa AnkG is a major component of the AIS membrane "undercoat' imaged by platinum replica electron microscopy. Surprisingly, giant AnkG-knockout neurons completely lacking known AIS components still retain distal axonal polarity and generate action potentials (APs), although with abnormal frequency. Giant AnkG-deficient mice live to weaning and provide a rationale for survival of humans with severe cognitive dysfunction bearing a truncating mutation in the giant exon. The giant exon of AnkG is required for assembly of the AIS and nodes of Ranvier and was a transformative innovation in evolution of the vertebrate nervous system that now is a potential target in neurodevelopmental disorders.
Assuntos
Anquirinas , Axônios/metabolismo , Evolução Molecular , Éxons , Nós Neurofibrosos , Transdução de Sinais , Potenciais de Ação/genética , Animais , Anquirinas/genética , Anquirinas/metabolismo , Camundongos , Camundongos Knockout , Mutação , Estrutura Terciária de Proteína , Nós Neurofibrosos/genética , Nós Neurofibrosos/metabolismo , RatosRESUMO
Several bacterial pathogens hijack the actin assembly machinery and display intracellular motility in the cytosol of infected cells. At the cell cortex, intracellular motility leads to bacterial dissemination through formation of plasma membrane protrusions that resolve into vacuoles in adjacent cells. Here, we uncover a crucial role for actin network disassembly in dissemination of Listeria monocytogenes. We found that defects in the disassembly machinery decreased the rate of actin tail turnover but did not affect the velocity of the bacteria in the cytosol. By contrast, defects in the disassembly machinery had a dramatic impact on bacterial dissemination. Our results suggest a model of L. monocytogenes dissemination in which the disassembly machinery, through local recycling of the actin network in protrusions, fuels continuous actin assembly at the bacterial pole and concurrently exhausts cytoskeleton components from the network distal to the bacterium, which enables membrane apposition and resolution of protrusions into vacuoles.
Assuntos
Citoesqueleto de Actina/genética , Actinas/genética , Listeria monocytogenes/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Cofilina 1/genética , Cofilina 1/metabolismo , Citosol/metabolismo , Citosol/microbiologia , Regulação da Expressão Gênica , Fator de Maturação da Glia/genética , Fator de Maturação da Glia/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Vacúolos/metabolismo , Vacúolos/microbiologiaRESUMO
The axon initial segment (AIS) is a specialized structure in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in neurons is ideal for its two major functions: it serves as the site of action potential firing and helps to maintain neuron polarity. It has become increasingly clear that the AIS cytoskeleton is fundamental to AIS functions. In this review, we discuss current understanding of the AIS cytoskeleton with particular interest in its unique architecture and role in maintenance of neuron polarity. The AIS cytoskeleton is divided into two parts, submembrane and cytoplasmic, based on localization, function, and molecular composition. Recent studies using electron and subdiffraction fluorescence microscopy indicate that submembrane cytoskeletal components (ankyrin G, ßIV-spectrin, and actin filaments) form a sophisticated network in the AIS that is conceptually similar to the polygonal/triangular network of erythrocytes, with some important differences. Components of the AIS cytoplasmic cytoskeleton (microtubules, actin filaments, and neurofilaments) reside deeper within the AIS shaft and display structural features distinct from other neuronal domains. We discuss how the AIS submembrane and cytoplasmic cytoskeletons contribute to different aspects of AIS polarity function and highlight recent advances in understanding their AIS cytoskeletal assembly and stability.
Assuntos
Segmento Inicial do Axônio/fisiologia , Axônios/fisiologia , Polaridade Celular/fisiologia , Citoesqueleto/metabolismo , Neurônios/fisiologia , Animais , Humanos , Microtúbulos/metabolismoRESUMO
Accumulation of filamentous actin (F-actin) at the immunological synapse (IS) is a prerequisite for the cytotoxic function of natural killer (NK) cells. Subsequent to reorganization of the actin network, lytic granules polarize to the IS where their contents are secreted directly toward a target cell, providing critical access to host defense. There has been limited investigation into the relationship between the actin network and degranulation. Thus, we have evaluated the actin network and secretion using microscopy techniques that provide unprecedented resolution and/or functional insight. We show that the actin network extends throughout the IS and that degranulation occurs in areas where there is actin, albeit in sub-micron relatively hypodense regions. Therefore we propose that granules reach the plasma membrane in clearances in the network that are appropriately sized to minimally accommodate a granule and allow it to interact with the filaments. Our data support a model whereby lytic granules and the actin network are intimately associated during the secretion process and broadly suggest a mechanism for the secretion of large organelles in the context of a cortical actin barrier.
Assuntos
Actinas/metabolismo , Degranulação Celular , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/metabolismo , Via Secretória , Algoritmos , Linhagem Celular , Membrana Celular/metabolismo , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Células Matadoras Naturais/fisiologia , Células Matadoras Naturais/ultraestrutura , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Microscopia Confocal , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Centro Organizador dos Microtúbulos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Sensibilidade e Especificidade , Transdução de Sinais , TransfecçãoRESUMO
Clathrin-mediated endocytosis (CME) is essential for maintaining cellular homeostasis. Previous studies have reported more than 50 CME accessory proteins; however, the mechanism driving the invagination of clathrin-coated pits (CCPs) remains elusive. Quantitative live cell imaging reveals that CCDC32, a poorly characterized endocytic accessory protein, regulates CCP stabilization and is required for efficient CCP invagination. CCDC32 interacts with the α-appendage domain (AD) of AP2 via its coiled-coil domain to exert this function. Furthermore, we showed that the clinically observed nonsense mutations in CCDC32, which result in the development of cardio-facio-neuro-developmental syndrome (CFNDS), inhibit CME by abolishing CCDC32-AP2 interactions. Overall, our data demonstrates the function and molecular mechanism of a novel endocytic accessory protein, CCDC32, in CME regulation. Significance Statement: Clathrin-mediated endocytosis (CME) happens via the initiation, stabilization, and invagination of clathrin-coated pits (CCPs). In this study, we used a combination of quantitative live cell imaging, ultrastructure electron microscopy and biochemical experiments to show that CCDC32, a poorly studied and functional ambiguous protein, acts as an important endocytic accessory protein that regulates CCP stabilization and invagination. Specifically, CCDC32 exerts this function via its interactions with AP2, and the coiled-coil domain of CCDC32 and the α-appendage domain (AD) of AP2 are essential in mediating CCDC32-AP2 interactions. Importantly, we demonstrate that clinically observed loss-of-function mutations in CCDC32 lose AP2 interaction capacity and inhibit CME, resulting in the development of cardio-facio-neuro-developmental syndrome (CFNDS).
RESUMO
The formation of specific cellular protrusions, plasma membrane blebs, underlies the amoeboid mode of cell motility, which is characteristic for free-living amoebae and leukocytes, and can also be adopted by stem and tumor cells to bypass unfavorable migration conditions and thus facilitate their long-distance migration. Not all cells are equally prone to bleb formation. We have previously shown that membrane blebbing can be experimentally induced in a subset of HT1080 fibrosarcoma cells, whereas other cells in the same culture under the same conditions retain non-blebbing mesenchymal morphology. Here we show that this heterogeneity is associated with the distribution of vimentin intermediate filaments (VIFs). Using different approaches to alter the VIF organization, we show that blebbing activity is biased toward cell edges lacking abundant VIFs, whereas the VIF-rich regions of the cell periphery exhibit low blebbing activity. This pattern is observed both in interphase fibroblasts, with and without experimentally induced blebbing, and during mitosis-associated blebbing. Moreover, the downregulation of vimentin expression or displacement of VIFs away from the cell periphery promotes blebbing even in cells resistant to bleb-inducing treatments. Thus, we reveal a new important function of VIFs in cell physiology that involves the regulation of non-apoptotic blebbing essential for amoeboid cell migration and mitosis.
Assuntos
Filamentos Intermediários , Vimentina , Movimento Celular , Citoplasma , Membrana CelularRESUMO
Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.
Assuntos
Exossomos , Exossomos/metabolismo , Endossomos/metabolismo , Fosfatidilinositóis/metabolismo , Corpos Multivesiculares/metabolismoRESUMO
Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four ß-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in ß-trefoil domains 1 and 3. The site in ß-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in ß-trefoil-3 is related by pseudo-2-fold symmetry to that in ß-trefoil-1. The two sites are â¼5 nm apart, resulting in a distance between actin filaments in the bundle of â¼8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/genética , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Mutação , Fosforilação/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Pseudópodes/química , Pseudópodes/genética , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Relação Estrutura-AtividadeRESUMO
Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms submicrometer-sized clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca(2+) and Mg(2+) to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca(2+) but not Mg(2+), Zn(2+), or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force, and electron microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal divalent metal ion (Me(2+))-induced cluster formation or diffusion retardation, which follows the trend: Ca(2+) â« Mg(2+) > Zn(2+), and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.
Assuntos
Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Magnésio/metabolismo , Membranas Artificiais , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Competitiva , Difusão , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher-order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton.This article describes application of rotary shadowing (or platinum replica ) EM (PREM) for visualization of the cytoskeleton . The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction (or mechanical "unroofing") of cells to expose their cytoskeleton , chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved and individual proteins can be identified by immunogold labeling. More importantly, PREM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high-resolution structural organization of the cytoskeleton in the same cell.
Assuntos
Microscopia Eletrônica , Citoesqueleto de Actina , Actinas , Citoesqueleto , Detergentes , Microtúbulos , PlatinaRESUMO
Adenomatous Polyposis Coli (APC) protein is mostly known as a tumor suppressor that regulates Wnt signaling, but is also an important cytoskeletal protein. Mutations in the APC gene are linked to colorectal cancer and various neurological disorders and intellectual disabilities. Cytoskeletal functions of APC appear to have significant contributions to both types of these disorders. As a cytoskeletal protein, APC can regulate both actin and microtubule cytoskeletons, which together form the main machinery for cell migration. As APC is a multifunctional protein with numerous interaction partners, the complete picture of how APC regulates cell motility is still unavailable. However, some molecular mechanisms begin to emerge. Here, we review available information about roles of APC in cell migration and propose a model explaining how microtubules, using APC as an intermediate, can initiate leading edge protrusion in response to external signals by stimulating Arp2/3 complex-dependent nucleation of branched actin filament networks via a series of intermediate events.
Assuntos
Proteína da Polipose Adenomatosa do Colo , Movimento Celular , Genes APC , Actinas/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Humanos , Microtúbulos/metabolismoRESUMO
Clathrin-mediated endocytosis (CME) requires energy input from actin polymerization in mechanically challenging conditions. The roles of actin in CME are poorly understood due to inadequate knowledge of actin organization at clathrin-coated structures (CCSs). Using platinum replica electron microscopy of mammalian cells, we show that Arp2/3 complex-dependent branched actin networks, which often emerge from microtubule tips, assemble along the CCS perimeter, lack interaction with the apical clathrin lattice, and have barbed ends oriented toward the CCS. This structure is hardly compatible with the widely held "apical pulling" model describing actin functions in CME. Arp2/3 complex inhibition or epsin knockout produce large flat non-dynamic CCSs, which split into invaginating subdomains upon recovery from Arp2/3 inhibition. Moreover, epsin localization to CCSs depends on Arp2/3 activity. We propose an "edge pushing" model for CME, wherein branched actin polymerization promotes severing and invagination of flat CCSs in an epsin-dependent manner by pushing at the CCS boundary, thus releasing forces opposing the intrinsic curvature of clathrin lattices.
Assuntos
Actinas , Platina , Complexo 2-3 de Proteínas Relacionadas à Actina , Animais , Clatrina , Vesículas Revestidas por Clatrina , Endocitose , Mamíferos , PolimerizaçãoRESUMO
During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However, <5 min after bath-applied NMDA, F-actin depolymerizes within spines and polymerizes into stable filaments within the dendrite shaft and soma. A similar actinification occurs after experimental ischemia in culture, and photothrombotic stroke in mouse. Following transient NMDA incubation, actinification spontaneously reverses. Na+, Cl-, water, and Ca2+ influx, and spine F-actin depolymerization are all necessary, but not individually sufficient, for actinification, but combined they induce activation of the F-actin polymerization factor inverted formin-2 (INF2). Silencing of INF2 renders neurons vulnerable to cell death and INF2 overexpression is protective. Ischemia-induced dendritic actin reorganization is therefore an intrinsic pro-survival response that protects neurons from death induced by cell edema.
Assuntos
Actinas , N-Metilaspartato , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Forminas , Isquemia/metabolismo , Camundongos , N-Metilaspartato/metabolismo , Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Água/metabolismoRESUMO
The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the regulation of endothelial junctions as dependent on the feedback between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Simulations reveal that active cell tension can stabilize cadherin bonds, but excessive RhoA signaling can drive bond dissociation and junction failure. While actin polymerization aids gap closure, high levels of Rac1 can induce junction weakening. Combining the modeling framework with experiments, our model predicts the influence of pharmacological treatments on the junction state and identifies that a critical balance between RhoA and Rac1 expression is required to maintain junction stability. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.
Assuntos
Actinas , Células Endoteliais , Células Endoteliais/metabolismo , Actinas/metabolismo , Retroalimentação , Transdução de Sinais , Citoesqueleto de Actina/metabolismoRESUMO
The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.