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Home Projects 1st call (2008) 3. Simultaneous imaging of substrate deformation and cytoskeletal motion in migrating cells

3. Simultaneous imaging of substrate deformation and cytoskeletal motion in migrating cells

Dr. A. Verkhovsky (EPFL), Dr. I.F. Sbalzarini (ETHZ) - PhD student: Mark Ambühl

Project finished in June 2013.

Cell motility is a physical phenomenon that is based on the forces generated by the assembly and contractility of the cytoskeletal network. These forces are exerted on specific extracellular matrix adhesion sites, and result in cell translocation with respect to the substrate. Recent advances in quantitative characterization of motile cells include the development of methods for measuring the forces exerted by the cells on the substrate and methods to map the intracellular motion of the cytoskeletal components. It is, however, not exactly known where and how the motile forces are generated within the cell, and the relationship between cytoskeletal activity, traction forces exerted by the cell on the substrate, and the efficiency of cell motion remains elusive. Particularly intriguing is the phenomenon of break of symmetry during initiation of cell motility (cell polarization) when the cell switches from an isotropic stationary state to a polarized motile state. Different non-mutually exclusive hypotheses are discussed in the literature.
In one scenario, the polarized assembly of an actin network at the leading edge of the cell generates directional traction forces through substrate adhesions and initiates cell motion (polymerization-centered models) (1). In a different model, strengthening of the substrate adhesions at the leading edge of the cell increases traction forces and shifts the balance between protrusion and retrograde flow of the newly assembled actin network towards productive cell advance (clutch hypothesis) (2). In yet another scenario, strong local contraction of the cytoskeleton results in sliding of the cell adhesion complexes with respect to the substrate and in generation of traction force (adhesion raking) (3). To distinguish between these model schemes, and in order to eventually develop a comprehensive physical model of cell polarization and directional motion, it is necessary to correlate cell behavior, cytoskeletal activity, and substrate traction forces with high accuracy and compare the different models directly on the primary image data using a new class of image analysis methods.
We thus propose, for the first time, to simultaneously map the substrate traction forces and motion of the cytoskeletal elements in polarizing and migrating cells. We will use a model system of fish epidermal keratocytes, which are characterized by fast, persistent migration and stable shape, making them a favorable system for quantitative studies and computational modeling. Keratocytes and their lamellar fragments also exhibit a remarkable selfpolarization behavior, i.e. the transition from the isotropic stationary state to sustained directional locomotion (4).
The specific aims of the project are:
1. Perform simultaneous time-lapse digital imaging of the cytoskeletal motion within the cell and the deformation of the elastic substrate, which the cell is crawling over.
2. Develop a new class of model-based image-processing methods for high accuracy mapping of the cytoskeletal motion and substrate traction forces.
3. Develop computational approaches to infer cytoskeletal forces from the mapping data and to evaluate different models of cell polarization and directional locomotion.

1. Pollard, T. D., Blanchoin, L. and Mullins, R. D. 2000. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29: 545-576
2. Jay, D.G. 2000. The clutch hypothesis revisited: ascribing the roles of actin-associated proteins in filopodial protrusion in the nerve growth cone. J. Neurobiol. 44: 114-25.
3. Jurado, C., Haserick, J.R. and Lee J. 2005. Slipping or gripping? Fluorescent speckle microscopy in fish keratocytes reveals two different mechanisms for generating a retrograde flow of actin. Mol. Biol. Cell. 16: 507-18.
4. Verkhovsky, A.B., Svitkina, T.M. and Borisy, G.G. 1999. Self-polarization and directional motility of cytoplasm. Curr. Biol. 14: 11-20

Contact: Mark Ambühl