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1.
Phys Rev Lett ; 125(6): 068101, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845697

RESUMO

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.


Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , Viscosidade
2.
Nat Cell Biol ; 22(8): 1011-1023, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32719553

RESUMO

Detection and conversion of mechanical forces into biochemical signals controls cell functions during physiological and pathological processes. Mechanosensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell-stretching device compatible with super-resolution microscopy and single-protein tracking, we explored the nanoscale deformations and reorganizations of individual proteins inside mechanosensitive structures. We achieved super-resolution microscopy after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous single-protein tracking and stretching showed that while integrins followed the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodelling and talin deformations. Capturing acute reorganizations of single molecules during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements, enabling protein deformation and recruitment in mechanosensitive structures.


Assuntos
Actinas/fisiologia , Forma Celular , Animais , Fenômenos Biomecânicos , Células Cultivadas , Técnicas Citológicas , Humanos , Integrinas/metabolismo , Camundongos , Microscopia/métodos , Nanoestruturas , Dobramento de Proteína , Transporte Proteico , Talina/metabolismo , Vinculina/metabolismo
3.
PLoS Comput Biol ; 16(6): e1007693, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520928

RESUMO

Understanding cellular remodeling in response to mechanical stimuli is a critical step in elucidating mechanical activation of biochemical signaling pathways. Experimental evidence indicates that external stress-induced subcellular adaptation is accomplished through dynamic cytoskeletal reorganization. To study the interactions between subcellular structures involved in transducing mechanical signals, we combined experimental data and computational simulations to evaluate real-time mechanical adaptation of the actin cytoskeletal network. Actin cytoskeleton was imaged at the same time as an external tensile force was applied to live vascular smooth muscle cells using a fibronectin-functionalized atomic force microscope probe. Moreover, we performed computational simulations of active cytoskeletal networks under an external tensile force. The experimental data and simulation results suggest that mechanical structural adaptation occurs before chemical adaptation during filament bundle formation: actin filaments first align in the direction of the external force by initializing anisotropic filament orientations, then the chemical evolution of the network follows the anisotropic structures to further develop the bundle-like geometry. Our findings present an alternative two-step explanation for the formation of actin bundles due to mechanical stimulation and provide new insights into the mechanism of mechanotransduction.


Assuntos
Citoesqueleto de Actina/fisiologia , Resistência à Tração , Actinas/fisiologia , Animais , Anisotropia , Fenômenos Biomecânicos , Células Cultivadas , Simulação por Computador , Fibronectinas/fisiologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Mecanotransdução Celular , Microscopia de Força Atômica , Miócitos de Músculo Liso/metabolismo , Miosinas/fisiologia , Ratos , Estresse Mecânico
4.
Nat Cell Biol ; 22(3): 297-309, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066907

RESUMO

Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. Here we report a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor γ-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.


Assuntos
Membrana Basal/metabolismo , Núcleo Celular , Centro Organizador dos Microtúbulos/fisiologia , Actinas/fisiologia , Animais , Membrana Celular , Núcleo Celular/ultraestrutura , Centrossomo , Drosophila/metabolismo , Drosophila/ultraestrutura , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Dineínas/fisiologia , Endossomos/metabolismo , Corpo Adiposo/metabolismo , Corpo Adiposo/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/fisiologia , Proteínas Musculares/metabolismo , Tubulina (Proteína)/fisiologia
5.
Microscopy (Oxf) ; 69(1): 44-52, 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-31990031

RESUMO

Cortactin regulates actin polymerization and stabilizes branched actin network. In neurons, cortactin is enriched in dendritic spines that contain abundant actin polymers. To explore the function of cortactin in dendritic spines, we examined spine morphology and dynamics in cultured neurons taken from cortactin knockout (KO) mice. Histological analysis revealed that the density and morphology of dendritic spines were not significantly different between wild-type (WT) and cortactin KO neurons. Time-lapse imaging of hippocampal slice cultures showed that the extent of spine volume change was similar between WT and cortactin KO neurons. Despite little effect of cortactin deletion on spine morphology and dynamics, actin turnover in dendritic spines was accelerated in cortactin KO neurons. Furthermore, we detected a suppressive effect of cortactin KO on spine head size under the condition of excessive spine enlargement induced by overexpression of a prominent postsynaptic density protein Shank2. These results suggest that cortactin may have a role in maintaining actin organization by stabilizing actin filaments near the postsynaptic density.


Assuntos
Actinas/fisiologia , Cortactina/genética , Espinhas Dendríticas/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Animais , Células Cultivadas , Espinhas Dendríticas/ultraestrutura , Hipocampo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Imagem com Lapso de Tempo
6.
PLoS Comput Biol ; 16(1): e1007250, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929522

RESUMO

Actin protrusion dynamics plays an important role in the regulation of three-dimensional (3D) cell migration. Cells form protrusions that adhere to the surrounding extracellular matrix (ECM), mechanically probe the ECM and contract in order to displace the cell body. This results in cell migration that can be directed by the mechanical anisotropy of the ECM. However, the subcellular processes that regulate protrusion dynamics in 3D cell migration are difficult to investigate experimentally and therefore not well understood. Here, we present a computational model of cell migration through a degradable viscoelastic ECM. This model is a 2D representation of 3D cell migration. The cell is modeled as an active deformable object that captures the viscoelastic behavior of the actin cortex and the subcellular processes underlying 3D cell migration. The ECM is regarded as a viscoelastic material, with or without anisotropy due to fibrillar strain stiffening, and modeled by means of the meshless Lagrangian smoothed particle hydrodynamics (SPH) method. ECM degradation is captured by local fluidization of the material and permits cell migration through the ECM. We demonstrate that changes in ECM stiffness and cell strength affect cell migration and are accompanied by changes in number, lifetime and length of protrusions. Interestingly, directly changing the total protrusion number or the average lifetime or length of protrusions does not affect cell migration. A stochastic variability in protrusion lifetime proves to be enough to explain differences in cell migration velocity. Force-dependent adhesion disassembly does not result in faster migration, but can make migration more efficient. We also demonstrate that when a number of simultaneous protrusions is enforced, the optimal number of simultaneous protrusions is one or two, depending on ECM anisotropy. Together, the model provides non-trivial new insights in the role of protrusions in 3D cell migration and can be a valuable contribution to increase the understanding of 3D cell migration mechanics.


Assuntos
Actinas , Movimento Celular/fisiologia , Matriz Extracelular , Modelos Biológicos , Actinas/química , Actinas/metabolismo , Actinas/fisiologia , Biologia Computacional , Simulação por Computador , Elasticidade/fisiologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Viscosidade
7.
J Neurosci ; 40(1): 131-142, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31767677

RESUMO

Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission.SIGNIFICANCE STATEMENT The presence and functional role of MTs in the presynaptic terminal are controversial. Here, we demonstrate that MTs are present near SVs in calyceal presynaptic terminals and that MT depolymerization specifically prolongs the slow-recovery component of EPSCs from short-term depression. In contrast, F-actin depolymerization specifically prolongs fast-recovery component. Depolymerization of MT or F-actin has no direct effect on SV exocytosis/endocytosis or basal transmission, but significantly impairs the fidelity of high-frequency transmission, suggesting that presynaptic cytoskeletal filaments play essential roles in SV replenishment for the maintenance of high-frequency neurotransmission.


Assuntos
Citoesqueleto de Actina/fisiologia , Exocitose/fisiologia , Microtúbulos/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Actinas/fisiologia , Animais , Vias Auditivas/fisiologia , Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos , Tiazolidinas/farmacologia , Corpo Trapezoide/fisiologia , Vimblastina/farmacologia
8.
J Neurosci ; 40(3): 542-556, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31754011

RESUMO

Synapse formation, maturation, and turnover require a finely regulated transport system that delivers selected cargos to specific synapses. However, the supporting mechanisms of this process are not fully understood. The present study unravels a new molecular system for vesicle-based axonal transport of proteins in male and female flies (Drosophila melanogaster). Here, we identify the gene CG14579 as the transcription unit corresponding to the regulatory mutations known as central complex broad (ccb). These mutations were previously isolated for their morphological phenotype in R-neurons of the ellipsoid body, a component of the central complex. Mutant axons from R-neurons fail to cross the midline, which is indicative of an aberrant composition of the growth cone. However, the molecular mechanism remained to be deciphered. In this manuscript, we show that CCB is involved in axonal trafficking of FasII and synaptobrevin, but not syntaxin. These results suggest that axonal transport of certain proteins is required for the correct pathfinding of R-neurons. We further investigated the molecular network supporting the CCB system and found that CCB colocalizes and coimmunoprecipitates with Rab11. Epistasis studies indicated that Rab11 is positioned downstream of CCB within this axonal transport system. Interestingly, ccb also interacts with actin and the actin nucleator spire The data revealed that this interaction plays a key role in the development of axonal connections within the ellipsoid body. We propose that the CCB/Rab11/SPIRE system regulates axonal trafficking of synaptic proteins required for proper connectivity and synaptic function.SIGNIFICANCE STATEMENT Proper function of the nervous system requires the establishment of mature, functional synapses. Differential protein composition in the synapse enables optimal performance of cognitive tasks. Therefore, it is critical to have a finely regulated transport system to deliver selected synaptic proteins to synapses. Remarkably, impairments in cytoskeleton-based protein-transport systems often underlie cognitive deficits, such as those associated with aging and neurodegenerative diseases. This study reveals that CCB is part of a novel transport system that delivers certain synaptic proteins via the actin cytoskeleton within the Rab11-related domain of slow recycling endosomes.


Assuntos
Actinas/fisiologia , Transporte Axonal/genética , Transporte Axonal/fisiologia , Proteínas de Drosophila/fisiologia , Proteínas de Membrana/fisiologia , Sinapses/fisiologia , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Retículo Endoplasmático/metabolismo , Feminino , Cones de Crescimento/fisiologia , Masculino , Proteínas de Membrana/genética , Mutação/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/fisiologia
9.
J Vis Commun Med ; 43(1): 35-46, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31642358

RESUMO

Cell biology and imaging technology have vastly improved over the past decades, enabling scientists to dissect the inner workings of a cell. In addition to technical limits on spatial and temporal resolution, which obscure the picture at the molecular level, the sheer density and complexity of information impede clear understanding. 3D molecular visualisation has therefore blossomed as a way to translate molecular data in a more tangible form. Whilst the molecular machinery involved in cell locomotion has been extensively studied, existing narratives describing how cells generate the forces that drive movement remain unclear. Polymerisation of a protein called actin is clearly essential. The general belief in the cell migration field is that actin polymerisation's main role is to push the leading edge of the cell forwards, while the rest of the cell follows passively. The cell migration & chemotaxis group at the CRUK Beatson Institute propose an alternative hypothesis, in which actin filaments constitute cables. Motor proteins pull on these cables, causing them to behave like the treads of a tank and drive cell movement. This article describes the development of a 3D animation that uses analogical reasoning to contrast the 'tank' hypothesis for cell locomotion with the current dogma.


Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Ilustração Médica , Modelos Biológicos , Biologia Celular , Humanos
10.
Am J Respir Cell Mol Biol ; 62(3): 331-341, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31513749

RESUMO

Hydrated magnesium silicate (or "talc" particles) is a sclerosis agent commonly used in the management of malignant pleural effusions, a common symptom of metastatic diseases, including lung cancers. However, the direct effects of talc particles to lung carcinoma cells, which can be found in the malignant pleural effusion fluids from patients with lung cancer, are not fully understood. Here, we report a study of the signaling pathways that can modulate the cell death and IL-6 secretion induced by talc particles in human lung carcinoma cells. We found that talc-sensitive cells have higher mRNA and protein expression of PI3K catalytic subunits α and ß. Further experiments confirmed that modulation (inhibition or activation) of the PI3K pathway reduces or enhances cellular sensitivity to talc particles, respectively, independent of the inflammasome. By knocking down specific PI3K isoforms, we also confirmed that both PI3Kα and -ß mediate the observed talc effects. Our results suggest a novel role of the PI3K pathway in talc-induced cell death and IL-6 secretion in lung carcinoma cells. These cellular events are known to drive fibrosis, and thus further studies of the PI3K pathway may provide a better understanding of the mechanisms of talc sclerosis in the malignant pleural space.


Assuntos
Adenocarcinoma/enzimologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Classe II de Fosfatidilinositol 3-Quinases/fisiologia , Neoplasias Pulmonares/enzimologia , Proteínas de Neoplasias/fisiologia , Soluções Esclerosantes/farmacologia , Talco/farmacologia , Fatores de Transcrição/fisiologia , Actinas/fisiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Morte Celular , Linhagem Celular Tumoral , Classe II de Fosfatidilinositol 3-Quinases/biossíntese , Classe II de Fosfatidilinositol 3-Quinases/genética , Resistência a Medicamentos , Indução Enzimática , Humanos , Interleucina-6/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Derrame Pleural Maligno/química , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
11.
J Formos Med Assoc ; 119(1 Pt 2): 300-309, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31202500

RESUMO

BACKGROUND/PURPOSE: Pulmonary veno-occlusive disease (PVOD) is a rare but fatal cause of pulmonary hypertension reported to be linked to mutations of eukaryotic initiation factor 2 alpha kinase 4 (EIF2AK4), also known as general control nonderepressible 2 kinase (GCN2). PVOD is difficult to diagnose and often initially misdiagnosed as other types of idiopathic pulmonary arterial hypertension (IPAH). To rapidly and correctly identify PVOD patients and explore the possible pathogenesis, we thoroughly investigated histopathological features and GCN2 protein levels in non-PAH, PVOD and PAH patients. METHODS: Lung specimens were examined for histopathological changes, including those of pulmonary arteries and veins, by Masson's trichrome, modified Verhoeff's and α-SMA staining in the PVOD, IPAH, and non-PAH groups. GCN2 and α-SMA expression in lung tissue was examined by immunohistochemistry and western blotting. RESULTS: PVOD and IPAH patients showed significant intimal and medial thickening of muscular pulmonary arteries compared with non-PAH patients. PVOD patients had more prominent intimal and medial thickening of muscular pulmonary veins than the other two groups. Interestingly, specialized muscle bundles surrounding the tunica adventitia of the pulmonary artery and vein were observed in PVOD patients. A significant decrease in GCN2 expression in the PVOD group was confirmed by immunohistochemistry and western blotting. CONCLUSION: Our study is the first to show remarkable histological structures, including the wreath-like arrangement of a hyperplastic muscle bundle in the adventitia of pulmonary arteries, in PVOD patients as a diagnostic clue and to disclose the biological difference between PAH and PVOD in a Taiwanese population.


Assuntos
Pulmão/patologia , Músculo Liso/patologia , Hipertensão Arterial Pulmonar/patologia , Pneumopatia Veno-Oclusiva/patologia , Actinas/genética , Actinas/fisiologia , Adulto , Idoso , Proliferação de Células , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/patologia , Veias Pulmonares/patologia , Pneumopatia Veno-Oclusiva/genética , Remodelação Vascular
12.
J Orthop Res ; 38(1): 105-116, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228280

RESUMO

Traditional tendon-to-bone repair where the tendon is reattached to bone via suture anchors often results in disorganized scar production rather than the formation of a zonal insertion. In contrast, ligament reconstructions where tendon grafts are passed through bone tunnels can yield zonal tendon-to-bone attachments between the graft and adjacent bone. Therefore, ligament reconstructions can be used to study mechanisms that regulate zonal tendon-to-bone repair in the adult. Anterior cruciate ligament (ACL) reconstructions are one of the most common reconstruction procedures and while we know that cells from outside the graft produce the attachments, we have not yet established specific cell populations that give rise to this tissue. To address this knowledge gap, we performed ACL reconstructions in lineage tracing mice where α-smooth muscle actin (αSMACreERT2) was used to label αSMA-expressing progenitors within the bone marrow that produced zonal attachments. Expression of αSMA was increased during early stages of the repair process such that the contribution of SMA-labeled cells to the tunnel integration was highest when tamoxifen was delivered in the first week post-surgery. The zonal attachments shared features with normal entheses, including tidemarks oriented perpendicularly to collagen fibers, Col1a1-expressing cells, alkaline phosphatase activity, and proteoglycan-rich staining. Finally, the integration strength increased with time, requiring 112% greater force to remove the graft from the tunnel at 28 days compared with 14 days post-surgery. Future studies will target these progenitor cells to define the pathways that regulate zonal tendon-to-bone repair in the adult. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:105-116, 2020.


Assuntos
Actinas/análise , Reconstrução do Ligamento Cruzado Anterior/métodos , Células da Medula Óssea/metabolismo , Osso e Ossos/cirurgia , Células-Tronco/metabolismo , Tendões/cirurgia , Actinas/fisiologia , Animais , Células da Medula Óssea/química , Camundongos , Cicatrização
13.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801239

RESUMO

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Contração Isométrica/fisiologia , Músculo Estriado/fisiologia , Miosinas/fisiologia , Sarcômeros/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Animais , Simulação por Computador , Conectina/fisiologia , Conectina/ultraestrutura , Modelos Biológicos , Método de Monte Carlo , Músculo Estriado/diagnóstico por imagem , Miosinas/ultraestrutura , Rana catesbeiana/fisiologia , Sarcômeros/ultraestrutura , Espalhamento a Baixo Ângulo , Técnicas de Cultura de Tecidos , Difração de Raios X
14.
Proc Natl Acad Sci U S A ; 116(48): 23894-23900, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719206

RESUMO

In this work, we explore fundamental energy requirements during mammalian cell movement. Starting with the conservation of mass and momentum for the cell cytosol and the actin-network phase, we develop useful identities that compute dissipated energies during extensions of the cell boundary. We analyze 2 complementary mechanisms of cell movement: actin-driven and water-driven. The former mechanism occurs on 2-dimensional cell-culture substrate without appreciable external hydraulic resistance, while the latter mechanism is prominent in confined channels where external hydraulic resistance is high. By considering various forms of energy input and dissipation, we find that the water-driven cell-migration mechanism is inefficient and requires more energy. However, in environments with sufficiently high hydraulic resistance, the efficiency of actin-polymerization-driven cell migration decreases considerably, and the water-based mechanism becomes more efficient. Hence, the most efficient way for cells to move depends on the physical environment. This work can be extended to higher dimensions and has implication for understanding energetics of morphogenesis in early embryonic development and cancer-cell metastasis and provides a physical basis for understanding changing metabolic requirements for cell movement in different conditions.


Assuntos
Movimento Celular , Actinas/metabolismo , Actinas/fisiologia , Permeabilidade da Membrana Celular , Forma Celular , Metabolismo Energético , Modelos Biológicos , Polimerização , Água/metabolismo
15.
Cytoskeleton (Hoboken) ; 76(11-12): 586-599, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31600850

RESUMO

Polymerization and depolymerization of cytoskeletal filaments against cellular structures can generate forces that are key to many cellular processes, such as cell motility and chromosomes movements during cell division. Motions generated by these forces induce global cytoplasmic flows and couple the dynamics of the polymerizing filaments and other bodies immersed in the fluid through their long-range hydrodynamic interactions (HIs). Previous theoretical and computational studies have largely ignored HIs. We use three dimensional discrete simulations to study the relationship between polymerization forces and their resulting flows and HIs. As a model system, we choose a filament that is polymerizing against an obstacle, and is embedded in a cylindrical array of parallel filaments of the same length. We consider three distinct mechanical scenarios for the filaments within the array: (a) all of the filaments are polymerizing with the same velocity; (b) they are all fixed in space, and (c) they are freely suspended. We show that each of these conditions produce their unique cytoplasmic flows and each result in differentiable polymerization forces and velocities. We also study the effect of buckling of filaments on polymerization forces and velocities and discuss the effect of HIs on the onset of buckling transition. Finally, we show that HIs result in the bundling of the buckled filaments within the array.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Hidrodinâmica , Modelos Biológicos , Polímeros/química , Citoesqueleto de Actina/química , Animais , Humanos , Polimerização
16.
Nat Commun ; 10(1): 4651, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604948

RESUMO

Mammalian oocytes assemble a bipolar acentriolar microtubule spindle to segregate chromosomes during asymmetric division. There is increasing evidence that actin in the spindle interior not only participates in spindle migration and positioning but also protects oocytes from chromosome segregation errors leading to aneuploidy. Here we show that actin is an integral component of the meiotic machinery that closely interacts with microtubules during all major events of human oocyte maturation from the time point of spindle assembly till polar body extrusion and metaphase arrest. With the aid of drugs selectively affecting cytoskeleton dynamics and transiently disturbing the integrity of the two cytoskeleton systems, we identify interdependent structural rearrangements indicative of a close communication between actin and microtubules as fundamental feature of human oocytes. Our data support a model of actin-microtubule interplay that is essential for bipolar spindle assembly and correct partitioning of the nuclear genome in human oocyte meiosis.


Assuntos
Actinas/fisiologia , Segregação de Cromossomos/fisiologia , Oócitos/citologia , Fuso Acromático/metabolismo , Feminino , Humanos , Meiose , Microtúbulos/fisiologia , Oócitos/ultraestrutura , Corpos Polares/citologia , Corpos Polares/metabolismo , Corpos Polares/ultraestrutura , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismo
17.
EMBO Rep ; 20(12): e48896, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31584242

RESUMO

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.


Assuntos
Actinas/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/fisiologia , Toxoplasma/parasitologia , Toxoplasma/patogenicidade , Actinas/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/parasitologia , Núcleo Celular/fisiologia , Células Cultivadas , Técnicas de Inativação de Genes , Humanos , Merozoítos/genética , Merozoítos/patogenicidade , Merozoítos/fisiologia , Modelos Biológicos , Mutação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Transdução de Sinais , Toxoplasma/genética , Virulência/fisiologia
18.
Philos Trans R Soc Lond B Biol Sci ; 374(1786): 20190081, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31587648

RESUMO

The mechanical performance of cardiomyocytes (CMs) is an important indicator of their maturation state and of primary importance for the development of therapies based on cardiac stem cells. As the mechanical analysis of adherent cells at high-throughput remains challenging, we explore the applicability of real-time deformability cytometry (RT-DC) to probe cardiomyocytes in suspension. RT-DC is a microfluidic technology allowing for real-time mechanical analysis of thousands of cells with a throughput exceeding 1000 cells per second. For CMs derived from human-induced pluripotent stem cells, we determined a Young's modulus of 1.25 ± 0.08 kPa which is in close range to previous reports. Upon challenging the cytoskeleton with cytochalasin D (CytoD) to induce filamentous actin depolymerization, we distinguish three different regimes in cellular elasticity. Transitions are observed below 10 nM and above 103 nM and are characterized by a decrease in Young's modulus. These regimes can be linked to cytoskeletal and sarcomeric actin contributions by CM contractility measurements at varying CytoD concentrations, where we observe a significant reduction in pulse duration only above 103 nM while no change is found for compound exposure at lower concentrations. Comparing our results to mechanical cell measurements using atomic force microscopy, we demonstrate for the first time to our knowledge, the feasibility of using a microfluidic technique to measure mechanical properties of large samples of adherent cells while linking our results to the composition of the cytoskeletal network. This article is part of a discussion meeting issue 'Single cell ecology'.


Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Miócitos Cardíacos/fisiologia , Fenômenos Biomecânicos , Microfluídica
19.
Dev Biol ; 456(2): 201-211, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31479647

RESUMO

In many spiralians, asymmetry in the first two cleavages is achieved through the formation of a polar lobe (PL), which transiently constricts to sequester vegetal cytoplasm into the CD and D blastomeres. While microtubules and actin filaments are required for polar lobe formation, little else is known regarding the structural and functional similarities with the contractile ring, or how the PL constriction is able to form perpendicular to the cleavage plane. Examination of scallop embryos revealed that while activated myosin II could be detected in both the cleavage furrow and early PL constriction, astral or central spindle microtubules were not observed associated with the PL neck until the constriction was nearly complete. Further, inhibition of Aurora B had no effect on polar lobe initiation, but blocked both contractile ring ingression and PL constriction beyond phase II. The cortex destined for PL sequestration was marked by enrichment of the Arp2/3 complex, which was first detected during meiosis and remained enriched at the vegetal pole through the first two cleavages. Inhibition of Arp2/3 affected PL formation and partitioning of cytoplasm into the two daughter cells, suggesting that Arp2/3 plays a functional role in defining the zone of cortex to be sequestered into the polar lobe. Together, these data offer for the first time a mechanism by which a cytoskeletal specialization defines the polar lobe in this atypical form of asymmetric cell division.


Assuntos
Divisão Celular/fisiologia , Crassostrea/embriologia , Pectinidae/embriologia , Actinas/metabolismo , Actinas/fisiologia , Animais , Blastômeros , Polaridade Celular/fisiologia , Crassostrea/metabolismo , Citocinese , Citoesqueleto/metabolismo , Microtúbulos/fisiologia , Morfogênese , Miosina Tipo II/metabolismo , Miosina Tipo II/fisiologia , Pectinidae/metabolismo , Transdução de Sinais
20.
Am J Physiol Cell Physiol ; 317(6): C1304-C1312, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31553646

RESUMO

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO-) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO- donor (SIN-1) or directly with ONOO-, which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO- treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate (gapp) in combination with a conservation of the force redevelopment rate (kTr) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.


Assuntos
Contração Isométrica/efeitos dos fármacos , Molsidomina/análogos & derivados , Miofibrilas/efeitos dos fármacos , Miosinas/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Oxidantes/farmacologia , Ácido Peroxinitroso/farmacologia , Actinas/antagonistas & inibidores , Actinas/química , Actinas/fisiologia , Animais , Contração Isométrica/fisiologia , Molsidomina/química , Molsidomina/farmacologia , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Miosinas/química , Miosinas/fisiologia , Doadores de Óxido Nítrico/química , Estresse Oxidativo , Músculos Psoas/efeitos dos fármacos , Músculos Psoas/fisiologia , Músculos Psoas/ultraestrutura , Coelhos , Técnicas de Cultura de Tecidos
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