The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Organization of a Dynamic Actin Cytoskeleton.

Cell motility, cell shape, and cytoplasmic organization are all determined, in part, by the ability of the actin cytoskeleton to physically organize cells. Our lab is interested in understanding the spatial organization of the actin cytoskeleton and its function in cell physiology. We use a combination of biochemistry, quantitative light microscopy and electron microscopy to probe the molecular mechanisms through which cells control actin filament assembly, disassembly, and structural organization. Whenever possible, we try to reconstitute the complex actin behaviors detected in cells in order to study their underlying mechanisms in greater detail. Our fledgling lab is currently focused on two fundamental questions that apply to all actin biology.

Mechanism of actin depolymerization.

Actin filaments in cells are highly dynamic, rapidly polymerizing in some regions of the cell while depolymerizing in others. The remarkable ability of actin to rapidly disassemble in an environment that simultaneously supports fast actin assembly allows cells to quickly remodel their cytoskeleton in response to both internal and external cues. The physiological mechanism of actin depolymerization, however, is not known. A deeper understanding of depolymerization would help motivate new hypotheses and experiments towards how actin disassembly helps organize the actin cytoskeleton.

Our research on depolymerization is guided by two questions. 1) Why do actin filaments depolymerize faster in cells than in pure solution? and 2) How do actin filaments manage to depolymerize at all in an environment that would seem to favor fast assembly? We have isolated two proteins (coronin and Aip1) that act synergistically with a third protein (cofilin) to rapidly depolymerize actin filaments, even when the reaction is challenged with a high concentration of actin monomer. Our goals now are to understand the kinetic pathway of depolymerization, its structural basis, the role of nucleotide hydrolysis, and the function of each disassembly factor in vivo.

Morphogenesis of the Listeria actin comet tail.

The primary function of the actin cytoskeleton is to physically organize cells by generating mechanical force. The actin cytoskeleton, therefore, faces the challenge of having to fulfill two seemingly incompatible roles. It needs to be stable enough to provide mechanical support for the cell, yet be dynamic enough to be rapidly reconfigured to meet new physiological demands. Since actin filaments are thin and flexible, they are always crosslinked into higher order assemblies that are capable of resisting compression. While these actin arrays are mechanically stable, they are dynamic, steady-state structures that are maintained by balancing assembly and disassembly. Cells make a number of actin arrays to perform different functions that can be distinguished by both their structural organization and dynamics. While differences between actin arrays have been noted for several years, we still do not understand the mechanisms controlling their morphogenesis. One approach to this problem is to reconstitute complex actin arrays in vitro and dissect the biochemical pathways controlling their assembly, disassembly, and structural organization.

Listeria monocytogenes is a bacterial, food borne pathogen that upon infecting its host cell assembles an actin array, referred to as the actin comet tail, to propel itself through the host’s cytoplasm. Listeria motility shares biochemistry with a number of other actin dependent motile processes such as protrusion of the leading edge of migrating cells. Listeria, however, is biochemically and analytically more tractable than other actin-based cellular movements. It therefore serves as a model system for understanding how polymer dynamics can be used to generate mechanical force. Actin dependent Listeria motility can be reconstituted in vitro opening the system up for biochemical analysis. We identify the factors required to recapitulate the morphogenesis of the comet tail by fractionating cell extracts to isolate unknowns and by directly testing known candidate molecules by immunodepletion. Our goals in this project are to understand the relationships between the mechanism of comet tail assembly, filament organization, and its disassembly. Most importantly, we plan to use this system to understand how cells control a full actin cycle of polymerization, depolymerization, and recycling for another round of assembly. Factors and principles implicated in shaping the Listeria comet tail should help inform us as to how other actin arrays are organized and guide future experiments in other systems.