Description of Research Expertise

Cell biology of actin and microfilaments.

Actin filaments are recognized as key elements in the dynamic machinery of motile cells and in the scaffolding that maintains the shape of cells. What is still poorly understood is how this scaffold is formed in vivo, what the essential components are out of the countless numbers of actin binding proteins in cells, and how the steps in its assembly and/or dynamics are regulated.

Over the years I have tried to confine my attention to very specialized cells with the idea that they carry out a more limited repertory of functions than a less differentiated cell. Because of the relative simplicity of a specialized cell and thus the precision with which it regulates the cytoskeleton, we can relate more directly the behavior of actin and actin binding proteins in vitro with what occurs in vivo. Thus, over the years I have chosen to study the brush border of intestinal epithelial cells, the acrosome reaction of sperm, the hair cells of the ear, the motile behavior of an intracellular pathogen, Listeria. Currently, making use of the power of molecular genetics, I am studying the formation of bristles in Drosophila pupae as a model system to understand in vivo how actin filaments pack into bundles, how the length and number of actin filaments is determined, and how these bundles interact with the cell surface to help determine cell form.

Within each bristle are 7-10 bundles of actin filaments. These actin filaments are crossbridged at precise intervals by two macromolecules, “fascin", a well characterized crossbridge, and a second, poorly characterized crossbridge, the “forked" transcript. Using null mutants that specifically eliminate one or the other or both crossbridges, singed or forked, we can begin to understand what each one does. By studying how the bristle, and more precisely how the bundles assemble developmentally we are obtaining further insights into the function of these crossbridges and in particular into understanding why two species of crossbridges are used. Here antibodies to the crossbridging proteins (confocal microscopy) and structural analysis (electron microscopy) appear particularly useful.

From these beginnings we should be able to compare what is happening in vitro to how these components behave in vivo as both crossbridges have been cloned and sequenced and are expressed recombinantly. At the same time we are examining and collecting mutants which should help us to identify the components that link the actin bundles to the plasma membrane and specify the number of bundles and the number of filaments within each bundle.

I am also studying oogenesis in Drosophila; in particular the assembly of actin filaments in the circumferential rings of actin filaments (ring canals) that enclose the intracellular bridges between nurse cells and oocytes. Again I am using mutants to determine what actin binding proteins are essential and how they function.