Publication
Crawling cell motility is characteristic of most animal cells and is involved in many important biological processes such as embryogenesis, immune response, and wound healing. It involves several steps: protrusion (dynamic surface extension) at the front of the cell, attachment to the substratum, and forward translocation of the cell body accompanied by the detachment and retraction of the rear portion of the cell. Crawling motion is believed to be based on actin, a major component of the cytoskeleton, and myosin II, a motor protein that is believed to produce contraction force by moving along actin filaments. Actin and myosin II are the two major structural and force-generating components of the cell's motile machinery. Protrusion is thought to be driven by the assembly of a dense actin network that is attached to the substrate through integrin-containing adhesions. Previous studies estimated the forces generated by actin polymerization and the adhesion forces, but the net force developed by the protruding leading edge has not been yet determined. The forward translocation of the cell body is believed to depend on the interaction of myosin with the actin network, but the exact mechanisms behind this interaction are still unclear. Moreover, it is still poorly understood how the molecular reactions involved in each step of the crawling motion are coordinated to result in the integrated cellular response. In this thesis work, we have quantitatively characterized the assembly and motion of actin and myosin II in the stationary and migratory states of the cell, and during the transition between these two steady-states. We have also measured the force developed by the protruding leading edge. The experimental system used is the fish epidermal keratocytes, one of the most spectacular models of cell locomotion. These cells are characterized by fast and persistent migration, and a stable and simple cytoskeletal organization. Enhanced phase contrast, interference reflection, classical fluorescence and fluorescent speckles microscopy were used as imaging techniques along with computer-based tracking approaches. We have first studied actin movement and assembly in the lamellipodium of keratocyte (thin, sheet-like extension at the leading edge of the cell). Actin dynamics in the lamellipodia of many cell types are characterized by retrograde flow of the actin network away from the leading edge. This flow is believed to result from membrane resistance to actin assembly and contractile forces in the network, and was speculated to play a role in controlling the cell shape and motion. Retrograde flow was previously observed in every crawling cell with the exception of keratocytes. We have for the first time detected actin retrograde flow throughout the keratocyte lamellipodium at velocities of 1–3 µm/min and analyzed its organization and relation to the cell motion during both unobstructed persistent migration and events of cell collision. We have found that fre