Molecular and mechanical exploration of large-scale cell motility coordination in zebrafish embryonic keratocytes [electronic resource]

Cell migration requires the large-scale coordination of force generation. This coordination can occur on a mechanical level by physical coupling of interconnected cytoskeletal components, and on a biochemical level by feedback interactions among the signaling molecules that direct actin polymerization. The study of large-scale coordination in cell motility has been hampered by the fact that the cell types best suited for experimental exploration by mechanical perturbations are usually not ideal for experimental exploration by molecular perturbations, and vice versa. Fish epidermal keratocytes have proved to be particularly useful for experimentation and modeling of the mechanics of large-scale coordination because of their simple and stereotyped shapes, their large uniform actin-rich protrusive lamellipodia, and their extremely rapid and persistent movement. However, molecular manipulations in these primary cells, typically cultured from adult members of fish species that have not been genetically well-characterized, have been limited. I have developed a new method for keratocyte culture from zebrafish embryos, enabling me to take full advantage of the molecular experimental methods including morpholino-based gene knockdown that are available for zebrafish, which also has a fully sequenced genome. Using this new molecularly tractable experimental system, I have identified a novel role for myosin light chain kinase in regulating overall cell polarization independently of previously known polarity regulators such as the small GTPase Rho and membrane tension. My investigations of the biology of embryonic zebrafish keratocytes have also shed new light on other aspects of large-scale cell movement coordination. For example, I have found that some zebrafish embryonic keratocytes exhibit an interesting traveling wave behavior, where an actin-rich protrusion appears to propagate laterally around the perimeter of the cell. While superficially similar behavior has been previously observed in keratocytes isolated from adults from other fish species when they are cultured on highly adhesive substrates, I have been able to demonstrate that the mechanism of wave propagation differs in the two cases. These observations have the potential to illuminate the biochemical feedback interactions that are most crucial for the rapid actin polymerization found in keratocytes and other fast-moving cell types. Finally, I have used the embryonic zebrafish keratocyte system to study the mechanical origin of the regular wrinkles that can form perpendicular to the leading edge in fish keratocyte lamellipodia, which were first described more than 90 years ago. I have found that characteristics of these wrinkles can be well explained by a physical model assuming that the mechanical coupling within the lamellipodial cytoskeleton involved in actin-based cell motility is characterized by largely elastic behavior, suggesting that forces exerted on one side of the cell are rapidly transmitted over the entire length of the lamellipod. This result stands in contrast to previous expectations based on observations in other cell types suggesting that the actin cytoskeleton behaves as a viscous fluid over the relatively slow time scales associated with whole-cell motility.

Keywords: Chemical and Systems Biology.
Advisor: Theriot Julie primary advisor. Thesis advisor
Ferrell James Ellsworth advisor. Thesis advisor
Meyer Tobias advisor. Thesis advisor
Talbot William