| Actin-based cell motility is important to a wide variety of basic biological processes. Although the dynamic behavior of pure actin has been heavily studied in vitro, relatively little is known about the dynamics of actin filaments inside living moving cells. This work describes direct observations of actin filament movement and turnover during two different types of actin-based motility using the technique of fluorescence photoactivation.; Many animal cells move by extending lamellipodia, thin veil-like structures rich in actin filaments. Previous work had shown that in stationary fibroblast lamellipodia, both the meshwork of actin filaments and particles on the cell's dorsal surface move slowly backwards towards the cell body. By marking actin filaments in the lamellipodia of motile cells using a photoactivatably fluorescent derivative of actin, I have shown that the rate of rearward actin flux varies in different cell types (from 0 to 0.7 microns per minute), but is independent of the rate of forward lamellipodial protrusion. Cell movement is therefore directly and tightly coupled to new actin filament formation at the leading edge. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid, and filaments are continuously polymerizing and depolymerizing everywhere throughout the structure. Particles on the cell's dorsal surface move several times more rapidly than the actin filaments within the lamellipodium. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.; Listeria monocytogenes, a Gram-positive bacterium, is a facultative intracellular pathogen capable of rapid actin-based movement through the host cell cytoplasm. Using photoactivation, I have shown that filaments in the actin-rich "comet tails" formed by moving bacteria behave dynamically much like filaments in lamellipodia. In addition, I have devised a cell-free extract system capable of faithfully reconstituting L. monocytogenes motility, and have used this system to demonstrate that profilin, a host actin monomer-binding protein, is necessary for bacterial actin-based motility. |
