Project description:Chemotaxing Dictyostelium discoideum cells adapt their morphology and migration speed in response to intrinsic and extrinsic cues. Using Fourier traction force microscopy, we measured the spatiotemporal evolution of shape and traction stresses and constructed traction tension kymographs to analyze cell motility as a function of the dynamics of the cell's mechanically active traction adhesions. We show that wild-type cells migrate in a step-wise fashion, mainly forming stationary traction adhesions along their anterior-posterior axes and exerting strong contractile axial forces. We demonstrate that lateral forces are also important for motility, especially for migration on highly adhesive substrates. Analysis of two mutant strains lacking distinct actin cross-linkers (mhcA(-) and abp120(-) cells) on normal and highly adhesive substrates supports a key role for lateral contractions in amoeboid cell motility, whereas the differences in their traction adhesion dynamics suggest that these two strains use distinct mechanisms to achieve migration. Finally, we provide evidence that the above patterns of migration may be conserved in mammalian amoeboid cells.
Project description:Cell migration in confined environments is fundamental for diverse biological processes from cancer invasion to leukocyte trafficking. The cell body is propelled by the contractile force of actomyosin networks transmitted from the cell membrane to the external substrates. However, physical determinants of actomyosin-based migration capacity in confined environments are not fully understood. Here, we develop an in vitro migratory cell model, where cytoplasmic actomyosin networks are encapsulated into droplets surrounded by a lipid monolayer membrane. We find that the droplet can move when the actomyosin networks are bound to the membrane, in which the physical interaction between the contracting actomyosin networks and the membrane generates a propulsive force. The droplet moves faster when it has a larger contact area with the substrates, while narrower confinement reduces the migration speed. By combining experimental observations and active gel theory, we propose a mechanism where the balance between sliding friction force, which is a reaction force of the contractile force, and viscous drag determines the migration speed, providing a physical basis of actomyosin-based motility in confined environments.
Project description:Vascular smooth muscle cell (VSMC) migration play a key role in the development of intimal hyperplasia and atherosclerosis. Galectin-1 (Gal-1) is a redox-sensitive ?-galactoside-binding lectin expressed in VSMCs with intracellular and extracellular localizations. Here we show that VSMCs deficient in Gal-1 (Gal-1-KO) exhibited greater motility than wild type (WT) cells. Likewise, Gal-1-KO-VSMC migration was inhibited by a redox-insensitive but activity-preserved Gal-1 (CSGal-1) in a glycan-dependent manner. Gal-1-KO-VSMCs adhered slower than WT cells on fibronectin. Cell spreading and focal adhesion (FA) formation examined by phalloidin and vinculin staining were less in Gal-1-KO-VSMCs. Concomitantly, FA kinase (FAK) phosphorylation was induced to a lower extent in Gal-1-KO cells. Analysis of FA dynamics by nocodazole washout assay demonstrated that FA disassembly, correlated with FAK de-phosphorylation, was faster in Gal-1-KO-VSMCs. Surface plasmon resonance assay demonstrated that CSGal-1 interacted with ?5?1integrin and fibronectin in a glycan-dependent manner. Chemical crosslinking experiment and atomic force microscopy further revealed the involvement of extracellular Gal-1 in strengthening VSMC-fibronectin interaction. In vivo experiment showed that carotid ligation-induced neointimal hyperplasia was more severe in Gal-1-KO mice than WT counterparts. Collectively, these data disclose that Gal-1 restricts VSMC migration by modulating cell-matrix interaction and focal adhesion turnover, which limits neointimal formation post vascular injury.
Project description:The mammary gland develops from the surface ectoderm during embryogenesis and proceeds through morphological phases defined as placode, hillock, bud, and bulb stages followed by branching morphogenesis. During this early morphogenesis, the mammary bud undergoes an invagination process where the thickened bud initially protrudes above the surface epithelium and then transforms to a bulb and sinks into the underlying mesenchyme. The signaling pathways regulating the early morphogenetic steps have been identified to some extent, but the underlying cellular mechanisms remain ill defined. Here, we use 3D and 4D confocal microscopy to show that the early growth of the mammary rudiment is accomplished by migration-driven cell influx, with minor contributions of cell hypertrophy and proliferation. We delineate a hitherto undescribed invagination mechanism driven by thin, elongated keratinocytes-ring cells-that form a contractile rim around the mammary bud and likely exert force via the actomyosin network. Furthermore, we show that conditional deletion of nonmuscle myosin IIA (NMIIA) impairs invagination, resulting in abnormal mammary bud shape.
Project description:We have developed our original tissue engineering technology "cell sheet engineering" utilizing temperature-responsive culture dishes. The cells are confluently grown on a temperature-responsive culture dish and can be harvested as a cell sheet by lowering temperature without enzymatic digestion. Cell sheets are high-cell-density tissues similar to actual living tissues, maintaining their structure and function. Based on this "cell sheet engineering", we are trying to create functional cardiac tissues from human induced pluripotent stem cells, for regenerative therapy and in vitro drug testing. Toward this purpose, it is necessary to evaluate the contractility of engineered cardiac cell sheets. Therefore, in the present study, we developed a contractile force measurement system and evaluated the contractility of human iPSC-derived cardiac cell sheet-tissues. By attaching the cardiac cell sheets on fibrin gel sheets, we created dynamically beating cardiac cell sheet-tissues. They were mounted to the force measurement system and the contractile force was measured stably and clearly. The absolute values of contractile force were around 1 mN, and the mean force value per cross-sectional area was 3.3 mN/mm2. These values are equivalent to or larger than many previously reported values, indicating the functionality of our engineered cardiac cell sheets. We also confirmed that both the contractile force and beating rate were significantly increased by the administration of adrenaline, which are the physiologically relevant responses for cardiac tissues. In conclusion, the force measurement system developed in the present study is valuable for the evaluation of engineered cardiac cell sheet-tissues, and for in vitro drug testing as well.
Project description:Fluid fills intracellular, extracellular, and capillary spaces within muscle. During normal physiological activity, intramuscular fluid pressures develop as muscle exerts a portion of its developed force internally. These pressures, typically ranging between 10 and 250 mmHg, are rarely considered in mechanical models of muscle but have the potential to affect performance by influencing force and work produced during contraction. Here, we test a model of muscle structure in which intramuscular pressure directly influences contractile force. Using a pneumatic cuff, we pressurize muscle midcontraction at 260 mmHg and report the effect on isometric force. Pressurization reduced isometric force at short muscle lengths (e.g., -11.87% of P0 at 0.9 L0), increased force at long lengths (e.g., +3.08% of P0 at 1.25 L0), but had no effect at intermediate muscle lengths ∼1.1-1.15 L0 This variable response to pressurization was qualitatively mimicked by simple physical models of muscle morphology that displayed negative, positive, or neutral responses to pressurization depending on the orientation of reinforcing fibers representing extracellular matrix collagen. These findings show that pressurization can have immediate, significant effects on muscle contractile force and suggest that forces transmitted to the extracellular matrix via pressurized fluid may be important, but largely unacknowledged, determinants of muscle performance in vivo.
Project description:Many cells crawl by extending an actin-rich pseudopod. We have devised a simulation that describes how the polymerization kinetics of a branched actin filament network, coupled with excluded volume effects, powers the motility of crawling cells such as amoebae and fish keratocytes. Our stochastic simulation is based on the key fundamental properties of actin polymerization, namely growth, shrinkage, capping, branching, and nucleation, and also includes contributions from the creation and breaking of adhesive contacts with the substrate together with excluded volume effects related to filament packing. When reasonable values for appropriate constants were employed, this simulation generated a force-velocity relationship that resembled closely that observed experimentally. Our simulations indicated that excluded volume effects associated with actin filament branching lead to a decreased packing efficiency and resultant swelling of the cytoskeleton gel that contributes substantially to lamellipod protrusion.
Project description:Resident cardiac macrophages are critical mediators of cardiac function. Despite their known importance to cardiac electrophysiology and tissue maintenance, there are currently no stem-cell derived models of human engineered cardiac tissues (hECT) that include resident macrophages. In this study, we made an iPSC-derived hECT model with a resident population of macrophages (iM0) to better recapitulate the native myocardium, and characterized their impact on tissue function. Macrophage retention within the hECTs was confirmed via immunofluorescence after 28 days of cultivation. Inclusion of iM0 significantly impacted hECT function, increasing contractile force production. A potential mechanism underlying these changes was revealed by interrogation of calcium signaling, which demonstrated modulation of β-adrenergic signaling in +iM0 hECTs. Collectively, these findings demonstrate that macrophages significantly enhance cardiac function in iPSC-derived hECT models, emphasizing the need to further explore their contributions not only in healthy hECT models, but also in the contexts of disease and injury.
Project description:The stress fiber network within contractile fibroblasts structurally reinforces and provides tension, or "tone", to tissues such as those found in healing wounds. Stress fibers have previously been observed to polymerize in response to mechanical forces. We observed that, when stretched sufficiently, contractile fibroblasts diminished the mechanical tractions they exert on their environment through depolymerization of actin filaments then restored tissue tension and rebuilt actin stress fibers through staged Ca(++)-dependent processes. These staged Ca(++)-modulated contractions consisted of a rapid phase that ended less than a minute after stretching, a plateau of inactivity, and a final gradual phase that required several minutes to complete. Active contractile forces during recovery scaled with the degree of rebuilding of the actin cytoskeleton. This complementary action demonstrates a programmed regulatory mechanism that protects cells from excessive stretch through choreographed active mechanical and biochemical healing responses.
Project description:Blood clot contraction plays an important role in wound healing and hemostasis. Although clot contraction is known to be driven by platelets, how single platelet forces relate to the forces generated by macroscopic clots remains largely unknown. Using our microfabricated high-throughput platelet contraction cytometer, we find that single platelets have an average force of 34 nN ( n=10 healthy individuals). However, multiple bulk clot experiments predict a mean single platelet force lower than 0.5 nN. To resolve this discrepancy, we use a mesoscale computational model to probe the mechanism by which individual platelets induce forces in macroscopic clots. Our experimentally informed model shows that the number of platelets in the clot cross-section defines the net clot force. We provide a relationship between single platelet force and the clot force that is useful for better understanding of blood disorders associated with bleeding and thrombosis, and facilitates the development of platelet-based and platelet-mimetic biomaterials.