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1.
PLoS One ; 15(12): e0231606, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382707

RESUMO

Keratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.


Assuntos
Citoesqueleto de Actina/metabolismo , Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Queratinas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Linhagem Celular , Citocalasina D/farmacologia , Elasticidade/efeitos dos fármacos , Expressão Gênica , Dureza/efeitos dos fármacos , Humanos , Filamentos Intermediários/ultraestrutura , Queratinócitos/efeitos dos fármacos , Queratinócitos/ultraestrutura , Queratinas/genética , Microtúbulos/ultraestrutura , Pinças Ópticas , Especificidade de Órgãos
2.
Nat Commun ; 11(1): 6234, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277490

RESUMO

Pattern-triggered immunity and effector-triggered immunity are two primary forms of innate immunity in land plants. The molecular components and connecting nodes of pattern-triggered immunity and effector-triggered immunity are not fully understood. Here, we report that the Arabidopsis calcium-dependent protein kinase CPK3 is a key regulator of both pattern-triggered immunity and effector-triggered immunity. In vitro and in vivo phosphorylation assays, coupled with genetic and cell biology-based analyses, show that actin-depolymerization factor 4 (ADF4) is a physiological substrate of CPK3, and that phosphorylation of ADF4 by CPK3 governs actin cytoskeletal organization associated with pattern-triggered immunity. CPK3 regulates stomatal closure induced by flg22 and is required for resistance to Pst DC3000. Our data further demonstrates that CPK3 is required for resistance to Pst DC3000 carrying the effector AvrPphB. These results suggest that CPK3 is a missing link between cytoskeleton organization, pattern-triggered immunity and effector-triggered immunity.


Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fatores de Despolimerização de Actina/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Microscopia Confocal , Mutação , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Plantas Geneticamente Modificadas , Pseudomonas syringae/fisiologia , Homologia de Sequência de Aminoácidos
3.
Sci Rep ; 10(1): 20003, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203986

RESUMO

Cell migration is a fundamental process that is crucial for many biological functions in the body such as immune responses and tissue regeneration. Dysregulation of this process is associated with cancer metastasis. In this study, polydimethylsiloxane platforms with various topographical features were engineered to explore the influence of guiding patterns on MC3T3-E1 osteoblast cell migration. Focusing on the guiding effects of grating patterns, variations such as etch depth, pattern discontinuity, and bending angles were investigated. In all experiments, MC3T3-E1 cells on patterned surfaces demonstrated a higher migration speed and alignment when compared to flat surfaces. The study revealed that an increase in etch depth from 150 nm to 4.5 µm enhanced cell alignment and elongation along the grating patterns. In the presence of discontinuous elements, cell migration speed was accelerated when compared to gratings of the same etch depth. These results indicated that cell directionality preference was influenced by a high level of pattern discontinuity. On patterns with bends, cells were more inclined to reverse on 45° bends, with 69% of cells reversing at least once, compared to 54% on 135° bends. These results are attributed to cell morphology and motility mechanisms that are associated with surface topography, where actin filament structures such as filopodia and lamellipodia are essential in sensing the surrounding environment and controlling cell displacement. Knowledge of geometric guidance cues could provide a better understanding on how cell migration is influenced by extracellular matrix topography in vivo.


Assuntos
Movimento Celular/fisiologia , Osteoblastos/citologia , Células 3T3 , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Sinais (Psicologia) , Matriz Extracelular/metabolismo , Camundongos , Osteoblastos/metabolismo , Propriedades de Superfície , Engenharia Tecidual/métodos
4.
PLoS One ; 15(11): e0242167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33201890

RESUMO

Esophageal atresia (EA) and tracheoesophageal fistula (TEF) are relatively frequently occurring foregut malformations. EA/TEF is thought to have a strong genetic component. Not much is known regarding the biological processes disturbed or which cell type is affected in patients. This hampers the detection of the responsible culprits (genetic or environmental) for the origin of these congenital anatomical malformations. Therefore, we examined gene expression patterns in the TEF and compared them to the patterns in esophageal, tracheal and lung control samples. We studied tissue organization and key proteins using immunohistochemistry. There were clear differences between TEF and control samples. Based on the number of differentially expressed genes as well as histological characteristics, TEFs were most similar to normal esophagus. The BMP-signaling pathway, actin cytoskeleton and extracellular matrix pathways are downregulated in TEF. Genes involved in smooth muscle contraction are overexpressed in TEF compared to esophagus as well as trachea. These enriched pathways indicate myofibroblast activated fibrosis. TEF represents a specific tissue type with large contributions of intestinal smooth muscle cells and neurons. All major cell types present in esophagus are present-albeit often structurally disorganized-in TEF, indicating that its etiology should not be sought in cell fate specification.


Assuntos
Fístula Traqueoesofágica/metabolismo , Transcriptoma , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Adulto , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Esôfago/metabolismo , Esôfago/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibrose , Humanos , Pulmão/metabolismo , Masculino , Transdução de Sinais , Traqueia/metabolismo , Fístula Traqueoesofágica/genética , Fístula Traqueoesofágica/patologia
5.
Nat Commun ; 11(1): 5897, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33214556

RESUMO

Since the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.


Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Difosfato de Adenosina/metabolismo , Fosfatos/metabolismo , Pirenos/química , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Iodoacetamida/análogos & derivados , Iodoacetamida/química , Cinética , Proteínas dos Microfilamentos/metabolismo , Polimerização , Ligação Proteica
6.
Nat Commun ; 11(1): 5973, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239610

RESUMO

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.


Assuntos
Citoesqueleto de Actina/metabolismo , Multimerização Proteica , Utrofina/metabolismo , Citoesqueleto de Actina/química , Fatores de Despolimerização de Actina/metabolismo , Actinas , Animais , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia Intravital , Cinética , Microscopia de Fluorescência , Mutação , Neuropeptídeos/metabolismo , Mutação Puntual , Ligação Proteica/genética , Domínios Proteicos/genética , Coelhos , Imagem Individual de Molécula , Utrofina/química , Utrofina/genética
7.
Proc Natl Acad Sci U S A ; 117(45): 28307-28315, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33122440

RESUMO

Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in ß-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic ß-cells. ß-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic ß-cells.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Glucose/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Silenciamento de Genes , Intolerância à Glucose , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas SNARE/metabolismo , Transcriptoma , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
8.
PLoS Biol ; 18(9): e3000848, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898131

RESUMO

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Miocárdio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Sarcômeros/metabolismo
9.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
10.
Sci Rep ; 10(1): 13596, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788615

RESUMO

Familial amyloid polyneuropathy (FAP) is a neurodegenerative disorder whose major hallmark is the deposition of mutated transthyretin (TTR) in the form of amyloid fibrils in the peripheral nervous system (PNS). The exposure of PNS axons to extracellular TTR deposits leads to an axonopathy that culminates in neuronal death. However, the molecular mechanisms underlying TTR-induced neurodegeneration are still unclear, despite the extensive studies in vertebrate models. In this work we used a Drosophila FAP model, based on the expression of the amyloidogenic TTR (V30M) in the fly retina, to uncover genetic interactions with cytoskeleton regulators. We show that TTR interacts with actin regulators and induces cytoskeleton alterations, leading to axonal defects. Moreover, our study pinpoints an interaction between TTRV30M and members of Rho GTPase signaling pathways, the major actin regulators. Based on these findings we propose that actin cytoskeleton alterations may mediate the axonopathy observed in FAP patients, and highlight a molecular pathway, mediated by Rho GTPases, underlying TTR-induced neurodegeneration. We expect this work to prompt novel studies and approaches towards FAP therapy.


Assuntos
Citoesqueleto de Actina/metabolismo , Neuropatias Amiloides Familiares/metabolismo , Pré-Albumina/metabolismo , Transdução de Sinais , Neuropatias Amiloides Familiares/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Drosophila , Mutação , Pré-Albumina/genética , Retina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
11.
Sci Rep ; 10(1): 13352, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770097

RESUMO

Glioblastoma (GBM) is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy. GBM tumors show nuclear factor-κB (NF-κB) activity that has been associated with tumor formation, growth, and increased resistance to therapy. We investigated the effect of NF-κB inhibitor BAY 11-7082 with Temozolomide (TMZ) on the signaling pathways in GBM pathogenesis. GBM cells and patient-derived GBM cells cultured in 3D microwells were co-treated with BAY 11-7082 and TMZ or BAY 11-7082 and TMZ alone, and combined experiments of cell proliferation, apoptosis, wound healing assay, as well as reverse-phase protein arrays, western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells. The results revealed that the co-treatment significantly altered cell proliferation by decreasing GBM viability, suppressed NF-κB pathway and enhanced apoptosis. Moreover, it was found that the co-treatment of BAY 11-7082 and TMZ significantly contributed to a decrease in the migration pattern of patient-derived GBM cells by modulating actin cytoskeleton pathway. These findings suggest that in addition to TMZ treatment, NF-κB can be used as a potential target to increase the treatment's outcomes. The drug combination strategy, which is significantly improved by NF-κB inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatment.


Assuntos
Citoesqueleto de Actina/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Temozolomida/uso terapêutico , Fator de Transcrição RelA/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , NF-kappa B/antagonistas & inibidores , Nitrilos/farmacologia , Sulfonas/farmacologia
12.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
13.
Nat Commun ; 11(1): 4187, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826901

RESUMO

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/fisiologia , Alinhamento de Sequência , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética
14.
Nat Commun ; 11(1): 4259, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848156

RESUMO

The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available to manipulate membrane protein location at the single-molecule level. Here, we use fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to control their movement. FMNPs allow single-particle tracking (SPT) at 10 nm and 5 ms spatiotemporal resolution, and using a magnetic needle, we pull membrane components laterally with femtonewton-range forces. In this way, we drag membrane proteins over the surface of living cells. Doing so, we detect barriers which we could localize to the submembrane actin cytoskeleton by super-resolution microscopy. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.


Assuntos
Nanopartículas de Magnetita , Proteínas de Membrana/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas de Fluorescência Verde/metabolismo , Campos Magnéticos , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência , Nanotecnologia , Imagem Individual de Molécula/métodos
16.
Sci Rep ; 10(1): 11960, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686703

RESUMO

The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal activity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into longitudinal fibers. We show that this activity-dependent remodeling involves [Formula: see text] and NMDA receptor-dependent mechanisms. This highly dynamic restructuring of dendritic F-actin based submembrane lattice into longitudinal fibers may serve to support activity-dependent membrane remodeling, protein trafficking and neuronal plasticity.


Assuntos
Actinas/metabolismo , Axônios/metabolismo , Membrana Celular/metabolismo , Dendritos/metabolismo , Hipocampo/citologia , Citoesqueleto de Actina/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Aprendizado Profundo , Modelos Neurológicos , Nanoestruturas/química , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo
17.
J Vis Exp ; (160)2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32628167

RESUMO

In this article, we describe the steps required to isolate a single permeabilized ("skinned") cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Fenômenos Fisiológicos Cardiovasculares , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Animais , Camundongos , Miócitos Cardíacos/citologia , Miofibrilas
18.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32699089

RESUMO

Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms multiple, dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is a major site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Finally, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways and cell-specific pathways for membrane reorganization.IMPORTANCE Herpesvirus particles are complex and contain many different proteins that must come together in an organized and coordinated fashion. Many viruses solve this coordination problem by creating a specialized assembly factory in the host cell, and the formation of such factories provides a promising target for interfering with virus production. Herpes simplex virus 1 (HSV-1) infects several types of cells, including neurons, but has not previously been shown to form such an organized factory in the nonneuronal cells in which its assembly has been best studied. Here, we show that HSV-1 forms an organized assembly factory in neuronal cells, and we identify some of the viral and host cell factors that are important for its formation.


Assuntos
Membrana Celular/fisiologia , Herpesvirus Humano 1/fisiologia , Neurônios/virologia , Proteínas Virais/metabolismo , Montagem de Vírus/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoplasma/virologia , Complexo de Golgi/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Células Vero , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(31): 18511-18520, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690698

RESUMO

It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.


Assuntos
Actinas/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Divisão Celular , Chlamydomonas/química , Citocinese , Microtúbulos/metabolismo , Miosinas/química , Miosinas/metabolismo , Ligação Proteica
20.
Proc Natl Acad Sci U S A ; 117(29): 17399-17408, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641513

RESUMO

The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be tested in cellulo Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells of Arabidopsis thaliana We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.


Assuntos
Fenômenos Fisiológicos Celulares , Forma Celular/fisiologia , Citoesqueleto/fisiologia , Células Vegetais/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas , Arabidopsis/metabolismo , Citocalasina D/farmacologia , Microtúbulos/metabolismo , Células Vegetais/efeitos dos fármacos , Células Vegetais/ultraestrutura , Protoplastos
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