RESUMO
The direction in which a cell divides is set by the orientation of its mitotic spindle and is important for determining cell fate, controlling tissue shape, and maintaining tissue architecture. Divisions parallel to the epithelial plane sustain tissue expansion. By contrast, divisions perpendicular to the plane promote tissue stratification and lead to the loss of epithelial cells from the tissue-an event that has been suggested to promote metastasis. Much is known about the molecular machinery involved in orienting the spindle, but less is known about the contribution of mechanical factors, such as tissue tension, in ensuring spindle orientation in the plane of the epithelium. This is important as epithelia are continuously subjected to mechanical stresses. To explore this further, we subjected suspended epithelial monolayers devoid of extracellular matrix to varying levels of tissue tension to study the orientation of cell divisions relative to the tissue plane. This analysis revealed that lowering tissue tension by compressing epithelial monolayers or by inhibiting myosin contractility increased the frequency of out-of-plane divisions. Reciprocally, increasing tissue tension by elevating cell contractility or by tissue stretching restored accurate in-plane cell divisions. Moreover, a characterization of the geometry of cells within these epithelia suggested that spindles can sense tissue tension through its impact on tension at subcellular surfaces, independently of their shape. Overall, these data suggest that accurate spindle orientation in the plane of the epithelium relies on a threshold level of tension at intercellular junctions.
Assuntos
Células Epiteliais , Junções Intercelulares , Epitélio , Divisão Celular , Matriz ExtracelularRESUMO
Throughout embryonic development and adult life, epithelia are subjected to compressive deformations. While these have been shown to trigger mechanosensitive responses such as cell extrusion and differentiation, which span tens of minutes, little is known about how epithelia adapt to compression over shorter timescales. Here, using suspended epithelia, we uncover the immediate response of epithelial tissues to the application of in-plane compressive strains (5-80%). We show that fast compression induces tissue buckling followed by actomyosin-dependent tissue flattening that erases the buckle within tens of seconds, in both mono- and multi-layered epithelia. Strikingly, we identify a well-defined limit to this response, so that stable folds form in the tissue when compressive strains exceed a 'buckling threshold' of ~35%. A combination of experiment and modelling shows that this behaviour is orchestrated by adaptation of the actomyosin cytoskeleton as it re-establishes tissue tension following compression. Thus, tissue pre-tension allows epithelia to both buffer against deformation and sets their ability to form and retain folds during morphogenesis.
Assuntos
Actomiosina/química , Epitélio/fisiologia , Animais , Caderinas/fisiologia , Força Compressiva , Citoesqueleto , Cães , Elasticidade , Células Epiteliais/citologia , Epitélio/embriologia , Proteínas de Fluorescência Verde , Células Madin Darby de Rim Canino , Microscopia Confocal , Modelos Biológicos , Morfogênese , Estresse Mecânico , ViscosidadeRESUMO
Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a long-lived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstream of RhoA (Ras homolog gene family, member A), stabilized moesin and directional memory while depolymerization of microtubules (MTs) disoriented moesin deposition and also reduced directional memory. Our study reveals that long-lived polarized cytoskeletal structures, specifically moesin, actomyosin, and MTs, provide a directional memory in neutrophil-like cells even as they respond on short time scales to external chemical cues.
Assuntos
Polaridade Celular , Quimiotaxia , Citoesqueleto/metabolismo , Memória Imunológica , Células HL-60 , HumanosRESUMO
Cell division plays an important role in animal tissue morphogenesis, which depends, critically, on the orientation of divisions. In isolated adherent cells, the orientation of mitotic spindles is sensitive to interphase cell shape and the direction of extrinsic mechanical forces. In epithelia, the relative importance of these two factors is challenging to assess. To do this, we used suspended monolayers devoid of ECM, where divisions become oriented following a stretch, allowing the regulation and function of epithelial division orientation in stress relaxation to be characterized. Using this system, we found that divisions align better with the long, interphase cell axis than with the monolayer stress axis. Nevertheless, because the application of stretch induces a global realignment of interphase long axes along the direction of extension, this is sufficient to bias the orientation of divisions in the direction of stretch. Each division redistributes the mother cell mass along the axis of division. Thus, the global bias in division orientation enables cells to act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state. Further, this behavior could be quantitatively reproduced using a model designed to assess the impact of autonomous changes in mitotic cell mechanics within a stretched monolayer. In summary, the propensity of cells to divide along their long axis preserves epithelial homeostasis by facilitating both stress relaxation and isotropic growth without the need for cells to read or transduce mechanical signals.
Assuntos
Divisão Celular , Células Epiteliais/citologia , Epitélio/metabolismo , Animais , Caderinas/metabolismo , Forma Celular , Cães , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Células Madin Darby de Rim Canino , Mitose , Morfogênese , Software , Estresse MecânicoRESUMO
L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.
Assuntos
Polaridade Celular , Selectina L/metabolismo , Monócitos/citologia , Sequência de Aminoácidos , Adesão Celular , Movimento Celular , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação , Leucócitos/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Dados de Sequência Molecular , Monócitos/metabolismo , Fosforilação , Serina/químicaRESUMO
Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium. In doing so, intercellular junctions endow tissues with the strength needed to withstand the mechanical stresses encountered in normal physiology and to coordinate tension during morphogenesis. Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established. Here, we use deep atomic force microscopy (AFM) indentation to measure the apparent stiffness of epithelial monolayers reforming from dissociated cells and examine which cellular processes give rise to tissue-scale mechanics. We show that the formation of intercellular junctions coincided with an increase in the apparent stiffness of reforming monolayers that reflected the generation of a tissue-level tension. Tension rapidly increased, reaching a maximum after 150â min, before settling to a lower level over the next 3â h as monolayers established homeostasis. The emergence of tissue tension correlated with the formation of adherens junctions but not desmosomes. As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.
Assuntos
Junções Aderentes/metabolismo , Citoesqueleto/metabolismo , Epitélio/metabolismo , Actinas/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular , Colágeno Tipo I/metabolismo , Desmossomos/metabolismo , Cães , Técnicas de Imagem por Elasticidade , Géis/metabolismo , Humanos , Queratina-18/genética , Queratina-18/metabolismo , Microscopia de Força Atômica , Morfogênese , Tensão SuperficialRESUMO
Cell monolayers line most of the surfaces and cavities in the human body. During development and normal physiology, monolayers sustain, detect and generate mechanical stresses, yet little is known about their mechanical properties. We describe a cell culture and mechanical testing protocol for generating freely suspended cell monolayers and examining their mechanical and biological response to uniaxial stretch. Cells are cultured on temporary collagen scaffolds polymerized between two parallel glass capillaries. Once cells form a monolayer covering the collagen and the capillaries, the scaffold is removed with collagenase, leaving the monolayer suspended between the test rods. The suspended monolayers are subjected to stretching by prying the capillaries apart with a micromanipulator. The applied force can be measured for the characterization of monolayer mechanics. Monolayers can be imaged with standard optical microscopy to examine changes in cell morphology and subcellular organization concomitant with stretch. The entire preparation and testing protocol requires 3-4 d.
Assuntos
Fenômenos Biomecânicos , Técnicas de Cultura de Células , Modelos Biológicos , Animais , Linhagem Celular , Colágeno/química , Cães , Alicerces TeciduaisRESUMO
The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at short timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology at short timescales.
Assuntos
Citoplasma/fisiologia , Modelos Biológicos , Fenômenos Biomecânicos , Forma Celular , Tamanho Celular , Citoesqueleto/fisiologia , Elasticidade , Porosidade , Reologia , Estresse MecânicoRESUMO
Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.
Assuntos
Células Epiteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Pseudópodes/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Colágeno/química , Combinação de Medicamentos , Células Epiteliais/patologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Fibronectinas/química , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Laminina/química , Cadeias Leves de Miosina/genética , Miosina não Muscular Tipo IIA/genética , Fosforilação , Proteoglicanas/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/patologia , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The constitutively active mutant of the Wiskott-Aldrich Syndrome protein (CA-WASp) is the cause of X-linked neutropenia and is linked with genomic instability and myelodysplasia. CA-WASp generates abnormally high levels of cytoplasmic F-actin through dysregulated activation of the Arp2/3 complex leading to defects in cell division. As WASp has no reported role in cell division, we hypothesized that alteration of cell mechanics because of increased F-actin may indirectly disrupt dynamic events during mitosis. Inhibition of the Arp2/3 complex revealed that excess cytoplasmic F-actin caused increased cellular viscosity, slowed all phases of mitosis, and perturbed mitotic mechanics. Comparison of chromosome velocity to the cytoplasmic viscosity revealed that cells compensated for increased viscosity by up-regulating force applied to chromosomes and increased the density of microtubules at kinetochores. Mitotic abnormalities were because of overload of the aurora signaling pathway as subcritical inhibition of Aurora in CA-WASp cells caused increased cytokinesis failure, while overexpression reduced defects. These findings demonstrate that changes in cell mechanics can cause significant mitotic abnormalities leading to genomic instability, and highlight the importance of mechanical sensors such as Aurora B in maintaining the fidelity of hematopoietic cell division.
Assuntos
Actinas/metabolismo , Citocinese/fisiologia , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Mitose/fisiologia , Neutropenia/congênito , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Linhagem Celular Tumoral , Instabilidade Cromossômica/genética , Reparo do DNA/fisiologia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Mutação , Neutropenia/genética , Neutropenia/metabolismo , Transdução Genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics.
Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células , Citoesqueleto/metabolismo , Junções Intercelulares/fisiologia , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/fisiologia , Técnicas de Cultura de Células/instrumentação , Colágeno/metabolismo , Cães , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Células Madin Darby de Rim Canino , Microscopia Confocal , Estresse MecânicoRESUMO
Water is the dominant ingredient of cells and its dynamics are crucial to life. We and others have suggested a physical picture of the cell as a soft, fluid-infiltrated sponge, surrounded by a water-permeable barrier. To understand water movements in an animal cell, we imposed an external, inhomogeneous osmotic stress on cultured cancer cells. This forced water through the membrane on one side, and out on the other. Inside the cell, it created a gradient in hydration, that we visualized by tracking cellular responses using natural organelles and artificially introduced quantum dots. The dynamics of these markers at short times were the same for normal and metabolically poisoned cells, indicating that the cellular responses are primarily physical rather than chemical. Our finding of an internal gradient in hydration is inconsistent with a continuum model for cytoplasm, but consistent with the sponge model, and implies that the effective pore size of the sponge is small enough to retard water flow significantly on time scales ( approximately 10-100 seconds) relevant to cell physiology. We interpret these data in terms of a theoretical framework that combines mechanics and hydraulics in a multiphase poroelastic description of the cytoplasm and explains the experimentally observed dynamics quantitatively in terms of a few coarse-grained parameters that are based on microscopically measurable structural, hydraulic and mechanical properties. Our fluid-filled sponge model could provide a unified framework to understand a number of disparate observations in cell morphology and motility.
Assuntos
Células/metabolismo , Água/metabolismo , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Difusão/efeitos dos fármacos , Elasticidade/efeitos dos fármacos , Células HeLa , Humanos , Queratinas/metabolismo , Metáfase/efeitos dos fármacos , Nocodazol/farmacologia , Pontos Quânticos , Reologia/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Vimentina/metabolismoRESUMO
Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315-324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.
Assuntos
4-Butirolactona/análogos & derivados , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cofilina 1/metabolismo , Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas/metabolismo , 4-Butirolactona/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína de Capeamento de Actina CapZ/metabolismo , Proteínas de Transporte , Linhagem Celular , Chlorocebus aethiops , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Guanilato Quinases , Haplorrinos , Células HeLa , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Coelhos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tiazolidinas/farmacologiaRESUMO
Blebs are spherical cellular protrusions that occur in many physiological situations. Two distinct phases make up the life of a bleb, each of which have their own biology and physics: expansion, which lasts approximately 30 s, and retraction, which lasts approximately 2 min. We investigate these phases using optical microscopy and simple theoretical concepts, seeking information on blebbing itself, and on cytomechanics in general. We show that bleb nucleation depends on pressure, membrane-cortex adhesion energy, and membrane tension, and test this experimentally. Bleb growth occurs through a combination of bulk flow of lipids and delamination from the cell cortex via the formation and propagation of tears. In extreme cases, this can give rise to a traveling wave around the cell periphery, known as "circus movement." When growth stalls, an actin cortex reforms under the bleb membrane, and retraction starts, driven by myosin-II. Using flicker spectroscopy, we find that retracting blebs are fivefold more rigid than expanding blebs, an increase entirely explained by the properties of the newly formed cortical actin mesh. Finally, using artificially nucleated blebs as pressure sensors, we show that cells rounded up in mitosis possess a substantial intracellular pressure.
Assuntos
Vesícula/metabolismo , Vesícula/patologia , Membrana Celular/metabolismo , Fenômenos Fisiológicos Celulares , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Animais , Vesícula/etiologia , Células Cultivadas , Proteínas Contráteis/metabolismo , Citoesqueleto/metabolismo , Humanos , Líquido Intracelular/metabolismo , Proteínas dos Microfilamentos/deficiência , Microscopia , Miosina Tipo II/metabolismoRESUMO
Contractile actin cortex is involved in cell morphogenesis, movement, and cytokinesis, but its organization and assembly are poorly understood. During blebbing, the membrane detaches from the cortex and inflates. As expansion ceases, contractile cortex re-assembles under the membrane and drives bleb retraction. This cycle enabled us to measure the temporal sequence of protein recruitment to the membrane during cortex reassembly and to explore dependency relationships. Expanding blebs were devoid of actin, but proteins of the erythrocytic submembranous cytoskeleton were present. When expansion ceased, ezrin was recruited to the membrane first, followed by actin, actin-bundling proteins, and, finally, contractile proteins. Complete assembly of the contractile cortex, which was organized into a cagelike mesh of filaments, took approximately 30 s. Cytochalasin D blocked recruitment of actin and alpha-actinin, but had no effect on membrane association of ankyrin B and ezrin. Ezrin played no role in actin nucleation, but was essential for tethering the membrane to the cortex. The Rho pathway was important for cortex assembly in blebs.
Assuntos
Actinas/metabolismo , Proteínas Contráteis/metabolismo , Citoesqueleto/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/genética , Proteínas Contráteis/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Membrana Eritrocítica/ultraestrutura , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Cinética , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Microinjeções , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Mutação , Miosina Tipo II/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína Vermelha FluorescenteRESUMO
Current models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry. In plants and fungi, protrusion is driven primarily by hydrostatic pressure. For hydrostatic pressure to drive localized protrusion in animal cells, it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum or viscous liquid require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortex-relaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of approximately 10 microm and 10 s. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogeneous transients in the pressure field, and can be described in the framework of poroelasticity.
Assuntos
Extensões da Superfície Celular/fisiologia , Citoplasma/fisiologia , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Extensões da Superfície Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Elasticidade , Pressão Hidrostática , Microscopia de Vídeo , Modelos Biológicos , Movimento/efeitos dos fármacos , Movimento/fisiologia , Perfusão , Estaurosporina/farmacologia , Tiazóis/farmacologia , Tiazolidinas , Fatores de TempoRESUMO
Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.
Assuntos
Movimento Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Células 3T3 BALB , Movimento Celular/fisiologia , Relação Dose-Resposta a Droga , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microscopia Confocal/métodos , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Associadas a rhoRESUMO
Bone adapts to its environment by a process in which osteoblasts and osteocytes sense applied mechanical strain. One possible pathway for the detection of strain involves mechanosensitive channels and we sought to determine their sensitivity to membrane strain and tension. We used a combination of experimental and computational modeling techniques to gain new insights into cell mechanics and the regulation of mechanosensitive channels. Using patch-clamp electrophysiology combined with video microscopy, we recorded simultaneously the evolution of membrane extensions into the micropipette, applied pressure, and membrane currents. Nonselective mechanosensitive cation channels with a conductance of 15 pS were observed. Bleb aspiration into the micropipette was simulated using finite element models incorporating the cytoplasm, the actin cortex, the plasma membrane, cellular stiffening in response to strain, and adhesion between the membrane and the micropipette. Using this model, we examine the relative importance of the different cellular components in resisting suction into the pipette and estimate the membrane strains and tensions needed to open mechanosensitive channels. Radial membrane strains of 800% and tensions of 5 10(-4) N.m(-1) were needed to open 50% of mechanosensitive channels. We discuss the relevance of these results in the understanding of cellular reactions to mechanical strain and bone physiology.
Assuntos
Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Canais Iônicos/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/fisiologia , Animais , Animais Recém-Nascidos , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Simulação por Computador , Elasticidade , Ativação do Canal Iônico/fisiologia , Fluidez de Membrana/fisiologia , Micromanipulação/métodos , Pressão , Ratos , Estresse Mecânico , Resistência à TraçãoRESUMO
Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease.
Assuntos
Microscopia de Força Atômica/métodos , Osteoblastos/citologia , Actinas/metabolismo , Animais , Fenômenos Biofísicos , Biofísica , Células Cultivadas , Citoesqueleto/metabolismo , Magnetismo , Microscopia Confocal , Modelos Teóricos , Distribuição de Poisson , Ratos , Transdução de Sinais , Software , Estresse MecânicoRESUMO
The skeleton adapts to its mechanical usage, although at the cellular level, the distribution and magnitude of strains generated and their detection are ill-understood. The magnitude and nature of the strains to which cells respond were investigated using an atomic force microscope (AFM) as a microindentor. A confocal microscope linked to the setup enabled analysis of cellular responses. Two different cell response pathways were identified: one, consequent upon contact, depended on activation of stretch-activated ion channels; the second, following stress relaxation, required an intact microtubular cytoskeleton. The cellular responses could be modulated by selectively disrupting cytoskeletal components thought to be involved in the transduction of mechanical stimuli. The F-actin cytoskeleton was not required for responses to mechanical strain, whereas the microtubular and vimentin networks were. Treatments that reduced membrane tension, or its transmission, selectively reduced contact reactions. Immunostaining of the cell cytoskeleton was used to interpret the results of the cytoskeletal disruption studies. We provide an estimate of the cellular strain magnitude needed to elicit intracellular calcium responses and propose a model that links single cell responses to whole bone adaptation. This technique may help to understand adaptation to mechanical usage in other organs.