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1.
Cells can sense and respond to different types of mechanical stimuli that can lead to changes in rate of cell division, cell orientation, cell motility, and gene expression. There is rapidly growing interest in understanding how these processes are regulated by mechano-chemical signaling mechanisms. The movement offish epithelial keratocytes is regulated by the activation of stretch-activated calcium channels, which allow cells to trigger retraction of the rear cell margin, when forward movement is impeded. We have developed a new assay that permits imaging of intracellular calcium concentration simultaneously with the detection of traction forces generated by moving keratocytes. The assay consists of a thin sheet of gelatin embedded with a surface layer of small fluorescent marker beads, on which cells can move. The elastic properties of the gelatin substrata can be reproducibly varied over a wide range and are stable for long periods, while submerged beneath culture medium. Gelatin substrata are thin, transparent, and highly elastic, allowing real-time detection of changes in traction force production that are associated with transient increases in intracellular calcium and that occur in response to mechanical stretching. 相似文献
2.
Growth cone behavior on gradients of substratum bound laminin 总被引:8,自引:0,他引:8
We have tested the ability of a gentle gradient of neurite-promoting activity to orient the extension of embryonic growth cones. Gradients of neurite-promoting activity were made with biologically active, tritium-labeled laminin. The distributions of laminin bound to glass substrata were visualized by autoradiography and quantified with an image processing system. Embryonic chick sympathetic ganglia were explanted onto laminin gradients and cultured. No tendency for neurites to be oriented up-gradient was detected by examining the morphology of explants. Time-lapse studies of individual growth cones detected no up- or down-gradient bias in growth cone motility. These results suggest that growth cone orientation is relatively insensitive to a graded distribution of a naturally occurring neurite-promoting molecule. 相似文献
3.
Cell traction force and measurement methods 总被引:2,自引:0,他引:2
Cell traction forces (CTFs) are crucial to many biological processes such as inflammation, wound healing, angiogenesis, and
metastasis. CTFs are generated by actomyosin interactions and actin polymerization and regulated by intracellular proteins
such as alpha-smooth muscle actin (α-SMA) and soluble factors such as transforming growth factor-β (TGF-β). Once transmitted
to the extracellular matrix (ECM) through stress fibers via focal adhesions, which are assemblies of ECM proteins, transmembrane
receptors, and cytoplasmic structural and signaling proteins (e.g., integrins), CTFs direct many cellular functions, including
cell migration, ECM organization, and mechanical signal generation. Various methods have been developed over the years to
measure CTFs of both populations of cells and of single cells. At present, cell traction force microscopy (CTFM) is among
the most efficient and reliable method for determining CTF field of an entire cell spreading on a two-dimensional (2D) substrate
surface. There are currently three CTFM methods, each of which is unique in both how displacement field is extracted from
images and how CTFs are subsequently estimated. A detailed review and comparison of these methods are presented. Future research
should improve CTFM methods such that they can automatically track dynamic CTFs, thereby providing new insights into cell
motility in response to altered biological conditions. In addition, research effort should be devoted to developing novel
experimental and theoretical methods for determining CTFs in three-dimensional (3D) matrix, which better reflects physiological
conditions than 2D substrate used in current CTFM methods. 相似文献
4.
Transmission of growth cone traction force through apCAM-cytoskeletal linkages is regulated by Src family tyrosine kinase activity.
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Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor-cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM-cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces. 相似文献
5.
A. Jorge-Peñas A. Muñoz-Barrutia E.M. de-Juan-Pardo 《Computer methods in biomechanics and biomedical engineering》2013,16(13):1377-1385
Traction force microscopy (TFM) is commonly used to estimate cells' traction forces from the deformation that they cause on their substrate. The accuracy of TFM highly depends on the computational methods used to measure the deformation of the substrate and estimate the forces, and also on the specifics of the experimental set-up. Computer simulations can be used to evaluate the effect of both the computational methods and the experimental set-up without the need to perform numerous experiments. Here, we present one such TFM simulator that addresses several limitations of the existing ones. As a proof of principle, we recreate a TFM experimental set-up, and apply a classic 2D TFM algorithm to recover the forces. In summary, our simulator provides a valuable tool to study the performance, refine experimentally, and guide the extraction of biological conclusions from TFM experiments. 相似文献
6.
Andrea Zancla Pamela Mozetic Monica Orsini Giancarlo Forte Alberto Rainer 《The Journal of biological chemistry》2022,298(5)
Traction force microscopy (TFM) has emerged as a versatile technique for the measurement of single-cell-generated forces. TFM has gained wide use among mechanobiology laboratories, and several variants of the original methodology have been proposed. However, issues related to the experimental setup and, most importantly, data analysis of cell traction datasets may restrain the adoption of TFM by a wider community. In this review, we summarize the state of the art in TFM-related research, with a focus on the analytical methods underlying data analysis. We aim to provide the reader with a friendly compendium underlying the potential of TFM and emphasizing the methodological framework required for a thorough understanding of experimental data. We also compile a list of data analytics tools freely available to the scientific community for the furtherance of knowledge on this powerful technique. 相似文献
7.
Pompe T Kaufmann M Kasimir M Johne S Glorius S Renner L Bobeth M Pompe W Werner C 《Biophysical journal》2011,(8):1863-1870
The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo. 相似文献
8.
C.N. Holenstein C.R. Lendi Nino Wili 《Computer methods in biomechanics and biomedical engineering》2019,22(8):853-860
Measuring cell-generated forces by Traction Force Microscopy (TFM) has become a standard tool in cell mechanobiology. Although widely used in two dimensional (2D) experiments, only a few methods exist to measure traction in three-dimensional (3D) cell culture, since 3D volumetric high-resolution microscopy and more demanding computational approaches are required. Although it is commonly known that the selected experimental and computational setup highly influence the quality and accuracy of the results, no existing methods can adequately assess the errors involved in this process. We present a fully integrated simulation and evaluation platform that allows one to simulate TFM images and quantify errors of an applied approach for traction stress reconstruction, in order to improve experiments that attempt to measure mechanical interaction in cellular systems. In this context, we show that a careful parameter selection can decrease the reconstructed traction error by up to 40%. 相似文献
9.
Neurons obtain their stereotyped morphologies and connections as a result of growth cone migration. In the past year, studies on growth cone migration and pathfinding have helped to define certain properties of cytoskeletal filaments and cell membranes that may be important in growth cone function. Antisense mRNAs have proved to be particularly useful for examining the roles of specific neurite proteins. 相似文献
10.
Growth cone motility. 总被引:4,自引:0,他引:4
The exact nature of growth cone motility is far from understood but progress has been made in several areas. It now appears that growth cones pull and not push; we will review the biophysical basis of growth cone movement. Current ideas on the regulation of growth cone motility and the relationship between motility and axon pathfinding are also discussed. 相似文献
11.
Using an established corneal stromal cell differentiation model, we manipulated alpha-smooth muscle actin (alpha-SMA) protein expression levels in fibroblasts by treating them with TGF-beta1, bFGF, TGF-beta type I receptor inhibitor (SB-431542), and siRNA against alpha-SMA. The corresponding cell traction forces (CTFs) were determined by cell traction force microscopy. With all these treatments, we found that alpha-SMA is not required for CTF induction, but its expression upregulates CTF. This upregulation involves the modification of stress fibers but does not appear to relate to non-muscle myosin II expression or beta-actin expression. Moreover, there exists a linear relationship between alpha-SMA protein expression level and CTF magnitude. Finally, CTFs were found to vary among a population of myofibroblasts, suggesting that alpha-SMA protein expression levels of individual cells also vary. 相似文献
12.
Tissue engineering science: Consequences of cell traction force 总被引:3,自引:0,他引:3
Blood and tissue cells mechanically interact with soft tissues and tissue-equivalent reconstituted collagen gels in a variety of situations relevant to biomedicine and biotechnology. A key phenomenon in these interactions is the exertion of traction force by cells on local collagen fibers which typically constitute the solid network of these tissues and gels and impart gross mechanical integrity. Two important consequences of cells exerting traction on such collagen networks are first, when the cells co-ordinate their traction, resulting in cell migration, and second, when their traction is sufficient to deform the network. Such cell-collagen network interactions are coupled in a number of ways. Network deformation, for example, can result in net alignment of collagen fibers, eliciting contact guidance, wherein cells move with bidirectional bias along an axis of fiber alignment, potentially leading to a nonuniform cell distribution. This may govern cell accumulation in wounds and be exploited to control cell infiltration of bioartificial tissues and organs. Another consequence of cell traction is the resultant stress and strain in the network which modulate cell protein and DNA synthesis and differentiation. We summarize, here, relevant mathematical theories which we have used to describe the inherent coupling of cell dynamics and tissue mechanics in cell-populated collagen gels via traction. The development of appropriate models based on these theories, in an effort to understand how events in wound healing govern the rate and extent of wound contraction, and to measure cell traction forces in vitro, are described. Relevant observations and speculation from cell biology and medicine that motivate or serve to critique the assumptions made in the theories and models are also summarized.Abbreviations ECM
Extracellular Matrix
- FPCL
Fibroblast-Populated Collagen Lattice
- FPCM
Fibroblast-Populated Collagen Microsphere 相似文献
13.
Studies in vitro have revealed a great deal about growth cone behaviors, especially responses to guidance molecules, both positive and negative, and the signaling systems mediating these responses. Little, however, is known about these events as they take place in vivo. With new imaging methods, growth cone behaviors can be chronicled in the complex settings of intact or semi-intact systems. With the retinal projection through the optic chiasm as a model, we examined the hypothesis previously drawn from static material that growth cone form is position-specific: growth cone form in fact reflects specific behaviors, including rate and tempo of extension, that are more or less prominent in different locales in which growth cones are situated. Other studies show that growth cones interact with cells along the pathway, both specialized nonneuronal cells and other neurons, some expressing known guidance molecules. The present challenge is to bridge dynamic imaging with electron microscopy and molecular localization, in order to link growth cone behaviors with cell and molecular interactions in the natural setting in which growth cones extend. 相似文献
14.
Cell adhesion and migration crucially depend on the transmission of actomyosin-generated forces through sites of focal adhesion to the extracellular matrix. Here we report experimental and computational advances in improving the resolution and reliability of traction force microscopy. First, we introduce the use of two differently colored nanobeads as fiducial markers in polyacrylamide gels and explain how the displacement field can be computationally extracted from the fluorescence data. Second, we present different improvements regarding standard methods for force reconstruction from the displacement field, which are the boundary element method, Fourier-transform traction cytometry, and traction reconstruction with point forces. Using extensive data simulation, we show that the spatial resolution of the boundary element method can be improved considerably by splitting the elastic field into near, intermediate, and far field. Fourier-transform traction cytometry requires considerably less computer time, but can achieve a comparable resolution only when combined with Wiener filtering or appropriate regularization schemes. Both methods tend to underestimate forces, especially at small adhesion sites. Traction reconstruction with point forces does not suffer from this limitation, but is only applicable with stationary and well-developed adhesion sites. Third, we combine these advances and for the first time reconstruct fibroblast traction with a spatial resolution of ∼1 μm. 相似文献
15.
Andreas Bauer Magdalena Prechov Lena Fischer Ingo Thievessen Martin Gregor Ben Fabry 《PLoS computational biology》2021,17(6)
Cellular force generation and force transmission are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. In this work, we also thoroughly analyze the accuracy and performance of the Traction Force Microscopy and Monolayer Stress Microscopy algorithms of pyTFM using synthetic and experimental data from epithelial cell patches. 相似文献
16.
17.
Rho mediates the shear-enhancement of endothelial cell migration and traction force generation 总被引:4,自引:0,他引:4
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Shiu YT Li S Marganski WA Usami S Schwartz MA Wang YL Dembo M Chien S 《Biophysical journal》2004,86(4):2558-2565
The migration of vascular endothelial cells in vivo occurs in a fluid dynamic environment due to blood flow, but the role of hemodynamic forces in cell migration is not yet completely understood. Here we investigated the effect of shear stress, the frictional drag of blood flowing over the cell surface, on the migration speed of individual endothelial cells on fibronectin-coated surfaces, as well as the biochemical and biophysical bases underlying this shear effect. Under static conditions, cell migration speed had a bell-shaped relationship with fibronectin concentration. Shear stress significantly increased the migration speed at all fibronectin concentrations tested and shifted the bell-shaped curve upwards. Shear stress also induced the activation of Rho GTPase and increased the traction force exerted by endothelial cells on the underlying substrate, both at the leading edge and the rear, suggesting that shear stress enhances both the frontal forward-pulling force and tail retraction. The inhibition of a Rho-associated kinase, p160ROCK, decreased the traction force and migration speed under both static and shear conditions and eliminated the shear-enhancement of migration speed. Our results indicate that shear stress enhances the migration speed of endothelial cells by modulating the biophysical force of tractions through the biochemical pathway of Rho-p160ROCK. 相似文献
18.
Tao Liu 《Biomechanics and modeling in mechanobiology》2017,16(4):1255-1268
Finite element (FE) simulations of contractile responses of vascular muscular thin films (vMTFs) and endothelial cells resting on an array of microposts under stimulation of soluble factors were conducted in comparison with experimental measurements reported in the literature. Two types of constitutive models were employed in the simulations, i.e. smooth muscle cell type and non-smooth muscle cell type. The time histories of the effects of soluble factors were obtained via calibration against experimental measurements of contractile responses of tissues or cells. The numerical results for vMTFs with micropatterned tissues suggest that the radius of curvature of vMTFs under stimulation of soluble factors is sensitive to width of the micropatterned tissue, i.e. the radius of curvature increases as the tissue width decreases. However, as the tissue response is essentially isometric, the time history of the maximum principal stress of the micropatterned tissues is not sensitive to tissue width. Good agreement has been achieved for predictions of the vasoconstrictor endothelin-1-induced contraction stress between the FE numerical simulation and the experiment-based approach of Alford (Integr Biol 3:1063–1070, 2011) for the vMTFs with 40, 60, 80 and 100 \(\upmu \hbox {m}\) width patterns. This may suggest the contraction stress is weakly sensitive to the tissue width for these patterns. However, for 20 \(\upmu \hbox {m}\) width tissue patterning, the numerical simulation result for contraction stress is less than the average value of experimental measurements, which may suggest the thinner and more elongated spindle-like cells within the 20 \(\upmu \hbox {m}\) width tissue patterning have higher contractile output. The constitutive model for non-smooth muscle cells was used to simulate the contractile response of the endothelial cells. The substrate was treated as an effective continuum. For agonists such as lysophosphatidic acid and vascular endothelial growth factor, the deformation of the cell diminishes from edge to centre and the central part of the cell is essentially under isometric state. Numerical studies demonstrated the scenarios that cell polarity can be triggered via manipulation of the effective stiffness and Possion’s ratio of the substrate. 相似文献
19.
Effie Bastounis Ruedi Meili Bego?a álvarez-González Joshua Francois Juan C. del álamo Richard A. Firtel Juan C. Lasheras 《The Journal of cell biology》2014,204(6):1045-1061
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. 相似文献
20.
von Philipsborn AC Lang S Loeschinger J Bernard A David C Lehnert D Bonhoeffer F Bastmeyer M 《Development (Cambridge, England)》2006,133(13):2487-2495
Graded distributions of ephrin ligands are involved in the formation of topographic maps. However, it is still poorly understood how growth cones read gradients of membrane-bound guidance molecules. We used microcontact printing to produce discontinuous gradients of substrate-bound ephrinA5. These consist of submicron-sized protein-covered spots, which vary with respect to their sizes and spacings. Growth cones of chick temporal retinal axons are able to integrate these discontinuous ephrin distributions and stop at a distinct zone in the gradient while still undergoing filopodial activity. The position of this stop zone depends on both the steepness of the gradient and on the amount of substrate-bound ephrin per unit surface area. Quantitative analysis of axon outgrowth shows that the stop reaction is controlled by a combination of the local ephrin concentration and the total amount of encountered ephrin, but cannot be attributed to one of these parameters alone. 相似文献