Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading

Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments – spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters – a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together, determine the overall motility function. Our data and algorithms are publicly available to encourage further exploration.

[1]  M. Sheetz,et al.  Talin depletion reveals independence of initial cell spreading from integrin activation and traction , 2008, Nature Cell Biology.

[2]  Zachary Pincus,et al.  Emergence of Large-Scale Cell Morphology and Movement from Local Actin Filament Growth Dynamics , 2007, PLoS biology.

[3]  D. Greenhalgh,et al.  Cutaneous Wound Healing , 2007, Journal of burn care & research : official publication of the American Burn Association.

[4]  Jake M. Hofman,et al.  Opposing Effects of PKCθ and WASp on Symmetry Breaking and Relocation of the Immunological Synapse , 2007, Cell.

[5]  N. Gov,et al.  Membrane waves driven by actin and Myosin. , 2007, Physical review letters.

[6]  Manuel Théry,et al.  The Universal Dynamics of Cell Spreading , 2007, Current Biology.

[7]  J. Segall,et al.  Coordinated regulation of pathways for enhanced cell motility and chemotaxis is conserved in rat and mouse mammary tumors. , 2007, Cancer research.

[8]  Yoav Freund,et al.  Lamellipodial Actin Mechanically Links Myosin Activity with Adhesion-Site Formation , 2007, Cell.

[9]  N. Ninov,et al.  Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis , 2007, Development.

[10]  Li-Huei Tsai,et al.  Trekking across the Brain: The Journey of Neuronal Migration , 2007, Cell.

[11]  Oscar Marín,et al.  Neurons in motion: same principles for different shapes? , 2006, Trends in Neurosciences.

[12]  Michael P. Sheetz,et al.  Force Sensing by Mechanical Extension of the Src Family Kinase Substrate p130Cas , 2006, Cell.

[13]  Jake M. Hofman,et al.  Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. , 2006, Biophysical journal.

[14]  C. Wiggins,et al.  The small GTPase R-Ras regulates organization of actin and drives membrane protrusions through the activity of PLCϵ , 2006, Journal of Cell Science.

[15]  1/f ruffle oscillations in plasma membranes of amphibian epithelial cells under normal and inverted gravitational orientations. , 2006, Physical review. E, Statistical, nonlinear, and soft matter physics.

[16]  T. Soldati,et al.  Dissection of amoeboid movement into two mechanically distinct modes , 2006, Journal of Cell Science.

[17]  Chris H Wiggins,et al.  Lateral membrane waves constitute a universal dynamic pattern of motile cells. , 2006, Physical review letters.

[18]  M. Sheetz,et al.  The small GTPase R-Ras regulates organization of actin and drives membrane protrusions through the activity of PLCϵ , 2006, Journal of Cell Science.

[19]  J. Käs,et al.  Neuronal growth: a bistable stochastic process. , 2006, Physical review letters.

[20]  Dylan T Burnette,et al.  Myosin II functions in actin-bundle turnover in neuronal growth cones , 2006, Nature Cell Biology.

[21]  G. Danuser,et al.  Morphodynamic profiling of protrusion phenotypes. , 2006, Biophysical journal.

[22]  S. Rafii,et al.  VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche , 2005, Nature.

[23]  J. Segall,et al.  The great escape: when cancer cells hijack the genes for chemotaxis and motility. , 2005, Annual review of cell and developmental biology.

[24]  P. Friedl,et al.  Tuning immune responses: diversity and adaptation of the immunological synapse , 2005, Nature Reviews Immunology.

[25]  L. Mahadevan,et al.  Non-equilibration of hydrostatic pressure in blebbing cells , 2005, Nature.

[26]  F. Bruckert,et al.  Kinetics of cell spreading. , 2005, Physical review letters.

[27]  M. Sheetz,et al.  Force sensing and generation in cell phases: analyses of complex functions. , 2005, Journal of applied physiology.

[28]  George M Church,et al.  From systems biology to synthetic biology , 2005, Molecular systems biology.

[29]  R. Austin,et al.  Force mapping in epithelial cell migration. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. Baum.  Animal Development: Crowd Control , 2004, Current Biology.

[31]  Gary G. Borisy,et al.  Lamellipodial Versus Filopodial Mode of the Actin Nanomachinery Pivotal Role of the Filament Barbed End , 2004, Cell.

[32]  D. Ruppert.  The Elements of Statistical Learning: Data Mining, Inference, and Prediction , 2004 .

[33]  M. Mann,et al.  RNA and RNA Binding Proteins Participate in Early Stages of Cell Spreading through Spreading Initiation Centers , 2004, Cell.

[34]  Grégory Giannone,et al.  Dynamic phase transitions in cell spreading. , 2004, Physical review letters.

[35]  M. Sheetz,et al.  Nanometer analysis of cell spreading on matrix-coated surfaces reveals two distinct cell states and STEPs. , 2004, Biophysical journal.

[36]  M. Sheetz,et al.  Periodic Lamellipodial Contractions Correlate with Rearward Actin Waves , 2004, Cell.

[37]  Fumio Matsumura,et al.  Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts , 2004, The Journal of cell biology.

[38]  Micah Dembo,et al.  Regulation of mechanical interactions between fibroblasts and the substratum by stretch-activated Ca2+ entry , 2004, Journal of Cell Science.

[39]  Erik Sahai,et al.  Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis , 2003, Nature Cell Biology.

[40]  E. Sahai,et al.  ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumor cell motility. , 2003, Cancer cell.

[41]  J. Castro,et al.  Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency , 2003, Cell Death and Differentiation.

[42]  T. Meyer,et al.  Switch-of-Function Mutants Based on Morphology Classification of Ras Superfamily Small GTPases , 2003, Cell.

[43]  T. Pollard.  The cytoskeleton, cellular motility and the reductionist agenda , 2003, Nature.

[44]  Michael P. Sheetz,et al.  RPTP-α acts as a transducer of mechanical force on αv/β3-integrin–cytoskeleton linkages , 2003, The Journal of cell biology.

[45]  Michael P. Sheetz,et al.  The relationship between force and focal complex development , 2002, The Journal of cell biology.

[46]  Gary G. Borisy,et al.  Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility , 2002, Cell.

[47]  J. Bertoglio,et al.  Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing , 2001, Nature Cell Biology.

[48]  E. Sahai,et al.  Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I , 2001, Nature Cell Biology.

[49]  Michael Loran Dustin,et al.  Environmental control of immunological synapse formation and duration. , 2001, Trends in immunology.

[50]  K. Adamsky,et al.  Glial tumor cell adhesion is mediated by binding of the FNIII domain of receptor protein tyrosine phosphatase β (RPTPβ) to tenascin C , 2001, Oncogene.

[51]  K. Adamsky,et al.  Glial tumor cell adhesion is mediated by binding of the FNIII domain of receptor protein tyrosine phosphatase beta (RPTPbeta) to tenascin C. , 2001, Oncogene.

[52]  K. Rottner,et al.  VASP dynamics during lamellipodia protrusion , 1999, Nature Cell Biology.

[53]  J. Segall,et al.  Chemoattractant‐induced lamellipod extension , 1998, Microscopy research and technique.

[54]  G M Bokoch,et al.  Activation of Rac and Cdc42 by integrins mediates cell spreading. , 1998, Molecular biology of the cell.

[55]  Kenneth M. Yamada,et al.  Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. , 1998, Science.

[56]  Jason C. Mills,et al.  Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation , 1998, The Journal of cell biology.

[57]  M. Kanzaki,et al.  Growth or Differentiation , 1996 .

[58]  H Bebie,et al.  Protrusive activity quantitatively determines the rate and direction of cell locomotion. , 1996, Cell motility and the cytoskeleton.

[59]  R. Winklbauer,et al.  Motile behavior and protrusive activity of migratory mesoderm cells from the Xenopus gastrula. , 1992, Developmental biology.

[60]  Julie A. Theriot,et al.  Actin microfilament dynamics in locomoting cells , 1991, Nature.

[61]  J. Bereiter-Hahn,et al.  Spreading of trypsinized cells: cytoskeletal dynamics and energy requirements. , 1990, Journal of cell science.

[62]  Tomaso Poggio,et al.  Computational vision and regularization theory , 1985, Nature.

[63]  J. Aplin,et al.  Kinetic analysis of cell spreading. I. Theory and modelling of curves. , 1983, Journal of cell science.

[64]  Daniel Axelrod,et al.  Total internal reflection fluorescent microscopy , 1983 .

[65]  N. Thompson,et al.  Total internal inflection fluorescent microscopy. , 1983, Journal of microscopy.

[66]  J. Vasiliev.  Spreading and locomotion of tissue cells: factors controlling the distribution of pseudopodia. , 1982, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[67]  Smolianinov Vv,et al.  Kinetics of fibroblast spreading , 1977 .

[68]  T. Kageyama.  MOTILITY AND LOCOMOTION OF EMBRYONIC CELLS OF THE MEDAKA, ORYZIAS LATIPES, DURING EARLY DEVELOPMENT , 1977 .

[69]  V. Smolianinov,et al.  [Kinetics of fibroblast spreading]. , 1977, Biofizika.

[70]  T. Kageyama,et al.  Circus movement in dissociated embryonic cells of a teleost, Oryzias latipes. , 1975, Journal of cell science.

[71]  J. Boss.  Mitosis in cultures of newt tissues. IV. The cell surface in late anaphase and the movements of ribonucleoprotein. , 1955, Experimental cell research.

[72]  J. Boss.  Mitosis in cultures of newt tissues. III. Cleavage and chromosome movements in anaphase. , 1954, Experimental cell research.