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Katrin Martin (1), Noo Li Jeon (2) and Olivier Pertz (1)
(1) Institute of Biochemistry and Genetics, Dept. of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland (2) School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-742, Republic of Korea We have previously shown that different Rho GTPase signaling programs occur at the leading edge of fibroblasts that extend pseudopods in response to an extracellular matrix (ECM) substrate versus PDGF growth factor stimulation [1]. This suggests that different actin and adhesion dynamics occur in these two cell migration modes (hapto- versus chemokinesis). In order to study the dynamics of these different cell migration events, we engineered assays that standardize cell shape and morphodynamics and allow robust induction of cell motility. For that purpose, we used microfabrication techniques to pattern 1D fibronectin lines that mimick the anisotropy inherent to fibrillar structures present in the 3D ECM. In comparison with a classic 2D environment, we found that this 1D environment led to elevated migration speed that became independent of ECM density (2D: 10 um/hour, 1D: 40 um/hour). Migration speed increased even further after PDGF stimulation with the fibroblasts speeding up to 200 um/hour (2D:20 um/hour, 1D 200 um/hour). Total internal reflection live cell imaging with the Lifeact-GFP probe revealed that this was the result of a drastic remodeling of the actin cytoskeleton in which there was: i) a dramatic loss of stress fibers, ii) formation of a discrete zone at the leading edge with highly dynamic, short lived podosome-like F-actin structures and loss of actin retrograde flow observed in the haptokinetic mode, and iii) relocalization of the actin retrograde flow to the lateral sides of the cell behind the "podosome zone", where it now moves inwards. This cell migration mode is characterized by exquisite co-ordination between the front and the back of the cell. Thus, pre-polarized cells on a 1D substrate integrate growth factor stimulation differently than cells in an isotropic 2D substrate. We are now characterizing these two cell migration modes by visualizing the dynamics of a variety of cytoskeletal and adhesion components, and correlating this with spatio-temporal Rho GTPase signaling programs using FRET-based probes. Because the cell morphodynamics on the 1D substrates are exquisitely stereotypic, this will allow to build integrated models of both cell migration modes. [1] Pertz O., Hodgson, L., Klemke, R. L. & Hahn, K. M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069-1072 (2006). |
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