143 research outputs found
The Force-Velocity Relation for Growing Biopolymers
The process of force generation by the growth of biopolymers is simulated via
a Langevin-dynamics approach. The interaction forces are taken to have simple
forms that favor the growth of straight fibers from solution. The
force-velocity relation is obtained from the simulations for two versions of
the monomer-monomer force field. It is found that the growth rate drops off
more rapidly with applied force than expected from the simplest theories based
on thermal motion of the obstacle. The discrepancies amount to a factor of
three or more when the applied force exceeds 2.5kT/a, where a is the step size
for the polymer growth. These results are explained on the basis of restricted
diffusion of monomers near the fiber tip. It is also found that the mobility of
the obstacle has little effect on the growth rate, over a broad range.Comment: Latex source, 9 postscript figures, uses psfig.st
Contractility Dominates Adhesive Ligand Density in Regulating Cellular De-adhesion and Retraction Kinetics
Cells that are enzymatically detached from a solid substrate rapidly round up as the tensile prestress in the cytoskeleton is suddenly unopposed by cellāECM adhesions. We recently showed that this retraction follows sigmoidal kinetics with time constants that correlate closely with cortical stiffness values. This raises the promising prospect that these de-adhesion measurements may be used for high-throughput screening of cell mechanical properties; however, an important limitation to doing so is the possibility that the retraction kinetics may also be influenced and potentially rate-limited by the time needed to sever matrix adhesions. In this study, we address this open question by separating contributions of contractility and adhesion to cellular de-adhesion and retraction kinetics. We first develop serum-free conditions under which U373 MG glioma cells can be cultured on substrates of fixed fibronectin density without direct matrix contributions from the medium. We show that while spreading area increases with ECM protein density, cortical stiffness and the time constants of retraction do not. Conversely, addition of lysophosphatidic acid (LPA) to stimulate cell contractility strongly speeds retraction, independent of the initial matrix protein density and LPAās contributions to spreading area. All of these trends hold in serum-rich medium commonly used in tissue culture, with the time constants of retraction much more closely tracking cortical stiffness than adhesive ligand density or cell spreading. These results support the use of cellular de-adhesion measurements to track cellular mechanical properties
Stiffness Gradients Mimicking In Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate
Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ā¼8.7Ā±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ā¼0.6Ā±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0Ā±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior
CellāMatrix De-Adhesion Dynamics Reflect Contractile Mechanics
Measurement of the mechanical properties of single cells is of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this study, we show that tracking cell shape dynamics during trypsin-induced de-adhesion can serve as a simple but extremely useful tool for probing the contractility of adherent cells. When treated with trypsin, both SW13ā/ā epithelial cells and U373 MG glioma cells exhibit a brief lag period followed by a concerted retraction to a rounded shape. The timeāresponse of the normalized cell area can be fit to a sigmoidal curve with two characteristic time constants that rise and fall when cells are treated with blebbistatin and nocodazole, respectively. These differences can be attributed to actomyosin-based cytoskeletal remodeling, as evidenced by the prominent buildup of stress fibers in nocodazole-treated SW13ā/ā cells, which are also two-fold stiffer than untreated cells. Similar results observed in U373 MG cells highlights the direct association between cell stiffness and the de-adhesion response. Faster de-adhesion is obtained with higher trypsin concentration, with nocodazole treatment further expediting the process and blebbistatin treatment blunting the response. A simple finite element model confirms that faster contraction is achieved with increased stiffness
Age-Related Changes in the Mechanical Properties of Human Fibroblasts and Its Prospective Reversal After Anti-Wrinkle Tripeptide Treatment
Amyloid-b peptide on sialyl-LewisX-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface
Increased deposition of amyloid-b peptide (Ab) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimerās disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized
by sialyl-Lewisx (sLex) were employed to investigate Ab-altered mechanics of membrane tethers formed by bonding
between sLex and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Ab to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Ab to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Ab lowered the overall force of membrane tether formation (Fmtf), and produced a bimodal population of Fmtf, suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Ab and latrunculin A decreased membrane stiffness, suggesting a
lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf. In addition, these cerebral endothelial alterations induced by Ab were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Ab to enhance p-selectin expression at the CEC surface and induce cytoskeleton
reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.This work was supported by Alzheimer Association Grant NIRG-06-24448; NIH Grant 1P01 AG18357, R21NS052385, 5R21AG032579 and in part by
1P01HL095486 and AHA 0835676N; āāBolashakāā scholarship and Ministry of Education and Science of the Republic of Kazakhstan 1029/GF2. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Biophysical mechanisms of single-cell interactions with microtopographical cues
Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures (āmicropegsā) can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling
Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells
Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ā¼1 Āµm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis
Cell types can be distinguished by measuring their viscoelastic recovery times using a micro-fluidic device
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