Intracellular Mechanics of Migrating Fibroblasts

Abstract

Cell migration is a highly coordinated process that occurs through the translation of biochemical signals into specific biomechanical events. The biochemical and structural properties of the proteins involved in cell motility, as well as their subcellular localization, have been studied extensively. However, how these proteins work in concert to generate the mechanical properties required to produce global motility is not well understood. Using intracellular microrheology and a fibroblast scratch-wound assay, we show that cytoskeleton reorganization produced by motility results in mechanical stiffening of both the leading lamella and the perinuclear region of motile cells. This effect is significantly more pronounced in the leading edge, suggesting that the mechanical properties of migrating fibroblasts are spatially coordinated. Disruption of the microtubule network by nocodazole treatment results in the arrest of cell migration and a loss of subcellular mechanical polarization; however, the overall mechanical properties of the cell remain mostly unchanged. Furthermore, we find that activation of Rac and Cdc42 in quiescent fibroblasts elicits mechanical behavior similar to that of migrating cells. We conclude that a polarized mechanics of the cytoskelton is essential for directed cell migration and is coordinated through microtubules. INTRODUCTIONCell migration is a cellular process that plays a critical role in health and disease, including embryogenesis, wound healing, immune response, and tissue development (Lauffenburger and Horwitz, 1996;Mitchison and Cramer, 1996;Pollard and Borisy, 2003). It is a highly regulated and coordinated process, which occurs through a myriad of signaling and structural proteins that orchestrate spatiotemporal reorganization of the actin cytoskeleton. Abnormal and/or unregulated cell motile behavior is known to contribute to several potentially fatal illnesses, including vascular disease and cancer (Ridley et al., 2003).Cell migration can be modeled as a repetitive, multistep process that begins with the establishment of spatial polarity and extension of membrane protrusions in the direction of movement. Newly formed protrusions are subsequently stabilized through firm adhesions to the underlying substrate and initiate contractile mechanisms that generate net forward motion and retraction of the trailing edge (Lauffenburger and Horwitz, 1996;Mitchison and Cramer, 1996;Pollard and Borisy, 2003). Every step of this cycle critically depends on the timely assembly, disassembly, and reorganization of actin filament structures that are mediated by members of the Rho family of small GTPases (Nobes and Hall, 1999). Overexpression of dominant negative variants of Rac and Cdc42 in fibroblasts reveals that Rac is necessary for membrane protrusion and the formation of wide lamella, whereas Cdc42 is required for the maintenance of cell polarity (Nobes and Hall, 1999).Research over the past three decades has identified a myriad of structural and signaling proteins that regulate cell motility. ...

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