Dynamic Phase Transitions in Cell Spreading

We monitored isotropic spreading of mouse embryonic fibroblasts on fibronectin-coated substrates. Cell adhesion area versus time was measured via total internal reflection fluorescence microscopy. Spreading proceeds in well-defined phases. We found a power-law area growth with distinct exponents a_i in three sequential phases, which we denote basal (a_1=0.4+-0.2), continous (a_2=1.6+-0.9) and contractile (a_3=0.3+-0.2) spreading. High resolution differential interference contrast microscopy was used to characterize local membrane dynamics at the spreading front. Fourier power spectra of membrane velocity reveal the sudden development of periodic membrane retractions at the transition from continous to contractile spreading. We propose that the classification of cell spreading into phases with distinct functional characteristics and protein activity patterns serves as a paradigm for a general program of a phase classification of cellular phenotype. Biological variability is drastically reduced when only the corresponding phases are used for comparison across species/different cell lines.Comment: 4 pages, 5 figure

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

High Refractive Index Silicone Gels for Simultaneous Total Internal Reflection Fluorescence and Traction Force Microscopy of Adherent Cells

Substrate rigidity profoundly impacts cellular behaviors such as migration, gene expression, and cell fate. Total Internal Reflection Fluorescence (TIRF) microscopy enables selective visualization of the dynamics of substrate adhesions, vesicle trafficking, and biochemical signaling at the cell-substrate interface. Here we apply high-refractive-index silicone gels to perform TIRF microscopy on substrates with a wide range of physiological elastic moduli and simultaneously measure traction forces exerted by cells on the substrate

Ena/VASP proteins have an anti-capping independent function in filopodia formation

Author Posting. © American Society for Cell Biology, 2007. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 18 (2007): 2579-2591, doi:10.1091/mbc.E06-11-0990.Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin–binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.This work was supported in part by National Institutes of Health Grants GM7542201 to D.A.A., GM58801 to F.B.G., and GM62431 to G.G.B. and by Cell Migration Consortium Grants GM64346 to D.A.A and G.G.B

A Polarised Population of Dynamic Microtubules Mediates Homeostatic Length Control in Animal Cells

An analysis of cells grown on micro-patterned lines, and of cells during zebrafish development, identifies a population of microtubules that align along the long axis of cells to mediate homeostatic length control

Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied via local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ~2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing

Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse.

The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation

Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse.

The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation