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1.
It has been previously established that living cells, including mesenchymal stem cells, stiffen in response to elevation of substrate stiffness. This stiffening is largely attributed to the elevation of the tractions at the cell base that is associated with increases in cell spreading on more-rigid substrates. We show here, surprisingly, that mouse embryonic stem cells (ESCs) do not stiffen when substrate stiffness increases. As shown recently, these cells do not increase spreading on more-rigid substrates either. However, these ESCs do increase their basal tractions as substrate stiffness increases. We conclude that these ESCs exhibit mechanical behaviors distinct from those of mesenchymal stem cells and of terminally differentiated cells, and decouple its apical cell stiffness from its basal tractional stresses during the substrate rigidity response.  相似文献   

2.
Prolonged exposure to fluid shear stress alters leukocyte functions associated with the immune response. We examined the initial response of freshly isolated human leukocytes to fluid shear stress under high magnification. Adherent leukocytes exhibit a rapid biomechanical response to physiological levels of fluid shear stress. After passive displacement in the direction of a constant fluid shear stress, adherent leukocytes actively recoil back in the opposite direction of the fluid flow. Recoil is observed within seconds of the applied fluid shear stress. Simultaneously, fluid shear stress induces a stiffening of the cell. The immediate cell displacement in response to a step increase in fluid shear stress is greatly attenuated in subsequent steps compared to the initial fluid shear stress step. Recoil is not mediated by actin polymerization-dependent mechanisms, as cytochalasin D had no effect on this early response. However, stiffening was determined in part by an intact actin cytoskeleton. Inhibiting myosin force generation with ML-7 abolished the recoil and stiffening responses, implicating force generation by myosin as an important contributor to the early leukocyte response to fluid shear stress. This initial shear stress response may be particularly important in facilitating leukocyte attachment under sustained fluid shear stress by the flowing blood in the microcirculation.  相似文献   

3.
《The Journal of cell biology》1987,105(6):2933-2943
RBL cells provide a useful model of the IgE and antigen-dependent stimulus-secretion coupling of mast cells and basophils. We have measured cellular deformability to investigate the participation of cytoskeletal mechanical changes. Cross-linking cell-surface IgE- receptor complexes with multivalent ligands not only triggered secretion but also caused the cells to stiffen, i.e., to become more resistant to deformation. This mechanical response required receptor cross-linking, had a time course similar to that of secretion, and was reversed by DNP-L-lysine, a competitive inhibitor of antigen binding. Hence the same stimulus seems to elicit both stiffening and secretion. Cytochalasin D, which inhibits actin filament assembly, prevented or reversed stiffening, thereby implicating the cytoskeleton in the mechanical response. Increasing intracellular calcium ion concentration with the ionophore A23187 stiffened cells and stimulated secretion. Activation of protein kinase C with a phorbol ester also stiffened cells and enhanced both the stiffening and secretion caused by the ionophore. Yet cytochalasin D enhances secretion whereas activation of protein kinase c alone is insufficient for secretion. Therefore stiffening is neither necessary nor sufficient for secretion. These results characterize a cytoskeletal mechanical response triggered by the same receptor-dependent stimulus that elicits secretion and by second messengers that are thought to mediate between the receptor signal and secretion. The function of the mechanical response, however, remains to be determined.  相似文献   

4.
Stiffness changes in cultured airway smooth muscle cells   总被引:7,自引:0,他引:7  
Airwaysmooth muscle (ASM) cells in culture stiffen when exposed tocontractile agonists. Such cell stiffening may reflect activation ofthe contractile apparatus as well as polymerization of cytoskeletalbiopolymers. Here we have assessed the relative contribution of thesemechanisms in cultured ASM cells stimulated with serotonin(5-hydroxytryptamine; 5-HT) in the presence or absence of drugs thatinhibit either myosin-based contraction or polymerization offilamentous (F) actin. Magnetic twisting cytometry was used to measurecell stiffness, and associated changes in structural organization ofactin cytoskeleton were evaluated by confocal microscopy. We found that5-HT increased cell stiffness in a dose-dependent fashion and alsoelicited rapid formation of F-actin as marked by increased intensity ofFITC-phalloidin staining in these cells. A calmodulin antagonist (W-7),a myosin light chain kinase inhibitor (ML-7) and a myosin ATPaseinhibitor (BDM) each ablated the stiffening response but not theF-actin polymerization induced by 5-HT. Agents that inhibited theformation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, andY-27632) attenuated both baseline stiffness and the extent of cellstiffening in response to 5-HT. Together, these data suggest thatagonist-evoked stiffening of cultured ASM cells requires actinpolymerization as well as myosin activation and that neitheractin polymerization nor myosin activation by itself is sufficient toaccount for the cell stiffening response.

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5.
During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.  相似文献   

6.
During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.  相似文献   

7.
Cells remodel extracellular matrix during tissue development and wound healing. Similar processes occur when cells compress and stiffen collagen gels. An important task for cell biologists, biophysicists, and tissue engineers is to guide these remodeling processes to produce tissue constructs that mimic the structure and mechanical properties of natural tissues. This requires an understanding of the mechanisms by which this remodeling occurs. Quantitative measurements of the contractile force developed by cells and the extent of compression and stiffening of the matrix describe the results of the remodeling processes. Not only do forces exerted by cells influence the structure of the matrix but also external forces exerted on the matrix can modulate the structure and orientation of the cells. The mechanisms of these processes remain largely unknown, but recent studies of the regulation of myosin-dependent contractile force and of cell protrusion driven by actin polymerization provide clues about the regulation of cellular functions during remodeling.  相似文献   

8.
The chemokine receptor, CCR-5, a G protein-coupled receptor (GPCR) which mediates chemotactic responses of certain leukocytes, has been shown to serve as the primary co-receptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1). Here we describe functional coupling of CCR-5 to inhibition of forskolin-stimulated cAMP formation via a pertussis toxin-sensitive G(i) protein mechanism in transfected HEK 293 cells. In response to chemokines, CCR-5 was desensitized, phosphorylated and sequestered like a prototypic GPCR only following overexpression of G protein-coupled receptor kinases (GRKs) and beta-arrestins in HEK 293 cells. The lack of CCR-5 desensitization in HEK 293 cells in the absence of GRK overexpression suggests that differences in cellular complements of GRK and/or beta-arrestin proteins could represent an important mechanism determining cellular responsiveness. When tested, the activity of CCR-5 as an HIV-1 co-receptor was dependent neither upon its ability to signal nor its ability to be desensitized and internalized following agonist stimulation. Thus, while chemokine-promoted cellular signaling, phosphorylation and internalization of CCR-5 may play an important role in regulation of chemotactic responses in leukocytes, these functions are dissociable from its HIV-1 co-receptor function.  相似文献   

9.
Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity. Our analyses of the global responses to chronic stressor exposure, at multiple biological levels, revealed a viable neural phenotype in-part reminiscent of aged or damaged neural tissue. Our paradigm indicates how cellular physiology can subtly change in different contexts and potentially aid the appreciation of stress response adaptations.  相似文献   

10.
Protein phosphorylation: hormones, drugs, and bioregulation   总被引:7,自引:0,他引:7  
S Shenolikar 《FASEB journal》1988,2(12):2753-2764
Reversible protein phosphorylation is widely recognized as an important mechanism for the regulation of cell function by a variety of physiological stimuli. Exposure of cells to hormones, neurotransmitters, and growth factors initiates a cascade of events facilitated by intracellular second messengers and mediated in many cases by protein kinases and/or phosphatases. The subsequent covalent modification of target proteins and the associated changes in their function account for the physiological response. Considerable evidence points to cross-talk between multiple membrane-associated signaling processes leading to coordinated regulation of cellular processes. The role of protein phosphorylation at multiple points in the pathways that integrate these signals is becoming increasingly apparent. Pharmacological modulation of cellular protein phosphorylation has yielded useful information on the molecular events involved. This review surveys some of the recent progress in hormonal regulation of cell function, focusing on examples that may provide new insight into the role of protein phosphorylation in the coordinated control of cellular processes by physiological stimuli.  相似文献   

11.
Living cells exhibit, as most biological tissues, a stiffening (strain-hardening) response which reflects the nonlinearity of the stress-strain relationship. Tensegrity structures have been proposed as a comprehensive model of such a cell's mechanical response. Based on a theoretical model of a 30-element tensegrity structure, we propose a quantitative analysis of its nonlinear mechanical behavior under static conditions and large deformations. This study provides theoretical foundation to the passage from large-scale tensegrity models to microscale living cells, as well as the comparison between results obtained in biological specimens of different sizes. We found two non-dimensional parameters (L*-normalized element length and T*-normalized elastic tension) which govern the mechanical response of the structure for three types of loading tested (extension, compression and shear). The linear strain-hardening is uniquely observed for extension but differed for the two other types of loading tested. The stiffening response of the theoretical model was compared and discussed with the living cells stiffening response observed by different methods (shear flow experiments, micromanipulation and magnetocytometry).  相似文献   

12.
Mitochondria are highly dynamic organelles of crucial importance to the proper functioning of neuronal, cardiac and other cell types dependent upon aerobic efficiency. Mitochondrial dysfunction has been implicated in numerous human conditions, to include cancer, metabolic diseases, neurodegeneration, diabetes, and aging. In recent years, mitochondrial turnover by macroautophagy (mitophagy) has captured the limelight, due in part to discoveries that genes linked to Parkinson disease regulate this quality control process. A rapidly growing literature is clarifying effector mechanisms that underlie the process of mitophagy; however, factors that regulate positive or negative cellular outcomes have been less studied. Here, we review the literature on two major pathways that together may determine cellular adaptation vs. cell death in response to mitochondrial dysfunction. Mitochondrial biogenesis and mitophagy represent two opposing, but coordinated processes that determine mitochondrial content, structure, and function. Recent data indicate that the capacity to undergo mitochondrial biogenesis, which is dysregulated in disease states, may play a key role in determining cell survival following mitophagy-inducing injuries. The current literature on major pathways that regulate mitophagy and mitochondrial biogenesis is summarized, and mechanisms by which the interplay of these two processes may determine cell fate are discussed. We conclude that in primary neurons and other mitochondrially dependent cells, disruptions in any phase of the mitochondrial recycling process can contribute to cellular dysfunction and disease. Given the emerging importance of crosstalk among regulators of mitochondrial function, autophagy, and biogenesis, signaling pathways that coordinate these processes may contribute to therapeutic strategies that target or regulate mitochondrial turnover and regeneration.  相似文献   

13.
Strain amplitude and strain rate dependent nonlinear behavior and load-induced mechanical property alterations of full-thickness bovine articular cartilage attached to bone were investigated in unconfined compression. A sequence of test compressions of finite deformation (ranging from 0.9% to 34.5% nominal strain) was performed at strain rates ranging from approximately 0.053%/s to 5.8%/s. Peak and equilibrium loads were analyzed to determine strain amplitude and strain rate dependence of linear versus nonlinear responses. The test protocol was designed to reveal changes in mechanical properties due to these finite deformations by interspersing small-amplitude witness ramps of approximately 1.1% deformation and approximately 0.44%/s strain rate between the test ramps ("witness" meaning to assess any mechanical property changes). We found that peak loads displayed high nonlinearity, stiffening with both increasing compression amplitude and more so with increasing strain rate. The response to witness ramps suggested that mechanical weakening occurred when compression amplitude reached 1.9-2.9% strain and beyond, and that weakening was much more significant at higher strain rate. These findings delineate regimes of linear versus nonlinear behavior of cartilage, and indicate the types of loads which can cause mechanical property alterations. Biological implications of this study are that strain amplitude and strain rate dependent stiffening may be essential to bear physiological loads and to protect cells and matrix from mechanical damage. Structural changes reflected by mechanical weakening at small compression could also initiate remodeling or disease processes.  相似文献   

14.
Rheological characterization of cells using passive particle tracking techniques can yield substantial information regarding local cellular material properties. However, limited work has been done to establish the changes in material properties of mechanically-responsive cells that experience external stimuli. In this study, cardiac fibroblasts plated on either fibronectin or collagen were treated with cytochalasin, mechanically stretched, or both, and their trajectories and complex moduli were extracted. Results demonstrate that both solid and fluid components were altered by such treatments in a receptor-dependent manner, and that, interestingly, cells treated with cytochalasin were still capable of stiffening in response to mechanical stimuli despite gross stress fiber disruption. These results suggest that the material properties of cells are dependent on a variety of environmental cues and can provide insight into physiological and disease processes.  相似文献   

15.
16.
《Biophysical journal》2022,121(8):1381-1394
Phagocytic cells form the first line of defense in an organism, engulfing microbial pathogens. Phagocytosis involves cell mechanical changes that are not yet well understood. Understanding these mechanical modifications promises to shed light on the immune processes that trigger pathological complications. Previous studies showed that phagocytes undergo a sequence of spreading events around their target followed by an increase in cell tension. Seemingly in contradiction, other studies observed an increase in cell tension concomitant with membrane expansion. Even though phagocytes are viscoelastic, few studies have quantified viscous changes during phagocytosis. It is also unclear whether cell lines behave mechanically similarly to primary neutrophils. We addressed the question of simultaneous versus sequential spreading and mechanical changes during phagocytosis by using immunoglobulin-G-coated 8- and 20-μm-diameter beads as targets. We used a micropipette-based single-cell rheometer to monitor viscoelastic properties during phagocytosis by both neutrophil-like PLB cells and primary human neutrophils. We show that the faster expansion of PLB cells on larger beads is a geometrical effect reflecting a constant advancing speed of the phagocytic cup. Cells become stiffer on 20- than on 8-μm beads, and the relative timing of spreading and stiffening of PLB cells depends on target size: on larger beads, stiffening starts before maximal spreading area is reached but ends after reaching maximal area. On smaller beads, the stiffness begins to increase after cells have engulfed the bead. Similar to PLB cells, primary cells become stiffer on larger beads but start spreading and stiffen faster, and the stiffening begins before the end of spreading on both bead sizes. Our results show that mechanical changes in phagocytes are not a direct consequence of cell spreading and that models of phagocytosis should be amended to account for causes of cell stiffening other than membrane expansion.  相似文献   

17.
Autophagy is an evolutionarily conserved catabolic process through which different components of the cells are sequestered into double-membrane cytosolic vesicles called autophagosomes, and fated to degradation through fusion with lysosomes. Autophagy plays a major function in many physiological processes including response to different stress factors, energy homeostasis, elimination of cellular organelles and tissue remodeling during development. Consequently, autophagy is strictly controlled and post-translational modifications such as phosphorylation and ubiquitination have long been associated with autophagy regulation. In contrast, the importance of acetylation in autophagy control has only emerged in the last few years. In this review, we summarize how previously identified histone acetylases and deacetylases modify key autophagic effector proteins, and discuss how this has an impact on physiological and pathological cellular processes.  相似文献   

18.
Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.  相似文献   

19.
Genetic analysis of early endocrine pancreas formation in zebrafish   总被引:3,自引:0,他引:3  
Endocrine pancreas of zebrafish consist of at least four different cell types that function similarly to mammalian pancreatic islet. No mutants specifically affecting formation of the endocrine pancreas have been identified during the previous large-scale mutagenesis screens in zebrafish due to invisibility of a pancreatic islet. We combined in situ hybridization method to visualize pancreatic islet with an ethyl-nitroso-urea mutagenesis screen to identify novel genes involved in pancreatic islet formation in zebrafish. We screened 900 genomes and identified 11 mutations belonging to nine different complementation groups. These mutants fall into three major phenotypic classes displaying severely reduced insulin expression, reduced insulin expression with abnormal islet morphology, or abnormal islet morphology with relatively normal number of insulin expressing cells. Seven of these mutants do not have any other visible phenotypes associated. These mutations affect different processes in pancreatic islet development. Additional analysis on glucagon and somatostatin cell specification revealed that somatostatin cells are specified at a separate domain from insulin cells whereas glucagon cells are specified adjacent to insulin cells. Furthermore, glucagon cells and somatostatin cells are always associated with insulin cells in mutants that have scattered insulin expression. These data indicate that there are separate mechanisms regulating endocrine cell migration, proliferation, and differentiation. Further study on these mutants will reveal important information on novel genes involved in pancreatic islet cell specification and morphogenesis.  相似文献   

20.
D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.  相似文献   

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