Atomic Force Microscopy of height fluctuations of fibroblast cells

We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope (AFM) under physiological conditions. Correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1--2 nm amplitude on a cell with low motility showing emphasized structure of stress fibres. Myosin driven contractions of stress fibres are thought to induce this pulsation.Comment: 6 pages, 5 figures, 1 tabl

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

The role of the cytoskeleton in volume regulation and beading transitions in PC12 neurites

We present investigations on volume regulation and beading shape transitions in PC12 neurites conducted using a flow-chamber technique. By disrupting the cell cytoskeleton with specific drugs we investigate the role of its individual components in the volume regulation response. We find that microtubule disruption increases both swelling rate and maximum volume attained, but does not affect the ability of the neurite to recover its initial volume. In addition, investigation of axonal beading --also known as pearling instability-- provides additional clues on the mechanical state of the neurite. We conclude that the initial swelling phase is mechanically slowed down by microtubules, while the volume recovery is driven by passive diffusion of osmolites. Our experiments provide a framework to investigate the role of cytoskeletal mechanics in volume homeostasis

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 ∼8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ∼0.6±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 1.0±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

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 cell–ECM 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 LPA’s 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

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 Alzheimer’s 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; ‘‘Bolashak’’ 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

Cell–Matrix 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 SW13−/− epithelial cells and U373 MG glioma cells exhibit a brief lag period followed by a concerted retraction to a rounded shape. The time–response 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 SW13−/− 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

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 ∼1 µm2 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