News and Publications

Actin Dynamics in Cell Morphogenesis and Function

Regulation of actin dynamics at the ends of filaments determines the organization and turnover of actin cytoskeletal structures and is critical for cell motility and cell architecture. For example, when cells change shape or crawl, new actin filaments are rapidly assembled at the barbed ends of the filaments and disassembled at the pointed ends during extension of lamellipodia or filopodia. In contrast, in nonmotile cells such as striated muscle and red blood cells, actin filaments are organized into regular architectural arrays that persist for the lifetime of the cell and are important for maintenance of cell shape, mechanical properties, and physiologic function. Our goal is to elucidate and compare the distinct regulatory mechanisms that control the polymerization and dynamics of actin filaments in the rapidly turning over filaments of motile cells with the regulatory mechanisms for the stable, long-lived actin filaments of nonmotile cells. Specifically, we focus on the regulation of the pointed ends of filaments by the tropomodulin family of actin-capping proteins and the roles of the proteins in actin-based morphogenetic processes during cell motility and in development.

The function of tropomodulin is best understood in striated muscle, where the tropomodulin-1 isoform is associated with the free, pointed ends of thin filaments in myofibrils. Previously, we showed that tropomodulin-1 controls the lengths of thin filaments in living cardiac muscle cells by transiently binding to the pointed ends of the filaments and competing for the addition of actin monomers. This finding led to the conclusion that the lengths of actin filaments in muscle cells are maintained via ongoing, dynamic regulation of actin polymerization at the pointed ends of the filaments. A related conclusion was that filaments are not restricted to a unique length and thus most likely are not determined by a molecular-ruler mechanism.

To establish the in vivo function of tropomodulin-1 in muscle, we are analyzing myofibril assembly during heart development in mice that lack the gene for tropomodulin-1. To date, we found that nascent myofibrils can assemble in the absence of tropomodulin-1, but the lengths of thin filaments are unregulated and myofibrils fail to mature. This abnormality leads to aborted cardiac development, failure of the heart to beat, and embryonic lethality by day 10.5 of embryogenesis. This analysis is the first to elucidate a molecular pathway for myofibril assembly in vivo in the developing heart in mice.

A different tropomodulin isoform, tropomodulin-3, is present in human microvascular endothelial cells, where it is enriched in leading-edge ruffles and lamellipodia. Transient overexpression of tropomodulin-3 labeled with green fluorescent protein led to a depolarized cell morphology and decreased cell motility. A 5-fold increase in tropomodulin-3 resulted in an equivalent decrease in free pointed ends and decreases in free barbed ends, F-actin, and the Arp2/3 complex in lamellipodia. Conversely, decreased expression of tropomodulin-3 by RNA interference led to faster mean cell migration and increases in free pointed and barbed ends in lamellipodial actin filaments.

Although counterintuitive, the effects of capping of pointed ends on free barbed ends, Arp2/3, and F-actin are predicted from kinetic modeling, because disassembly of pointed ends is the rate-limiting step in the turnover of actin filament networks in the leading lamellipodia. Thus, these data reveal a novel control point for actin regulation in lamellipodia and indicate that tropomodulin-3 is a negative regulator of cell migration. Currently, we are examining the regulation of tropomodulin-3 activity in endothelial cells and the relationship between the stabilization of pointed ends by tropomodulin-3 and the disassembly of pointed ends by actin-depolymerizing factor/cofilin.

In collaboration with D. Takemoto, University of Kansas, Lawrence, Kansas, we investigated the function of tropomodulin-1 in the eye lens. Our previous work indicated that expression of tropomodulin-1 is upregulated during morphogenetic differentiation of lens fiber cells, when it becomes associated with the plasma membrane. Using a rabbit lens cell culture model of fiber cell differentiation, we found that tropomodulin-1 is a substrate for protein kinase C α. Stimulation of the kinase by phorbol esters or treatment with epidermal growth factor resulted in increased association of the kinase with tropomodulin-1, tropomodulin-1 phosphorylation, and increased association of tropomodulin-1 with the cytoskeleton. These findings are the first evidence of regulation of tropomodulin-1 by phosphorylation in any cell type. Currently, we are determining the molecular consequences of phosphorylation of protein kinase C α for tropomodulin-1 functions in vitro and in vivo.

We also collaborated with H. Zoghbi, Baylor College of Medicine, Houston, Texas, to characterize the phenotype of a mouse strain that lacks the gene for tropomodulin-2, which is expressed exclusively in neurons. Mice that lack the gene are viable and have no gross morphologic or anatomic abnormalities, but they have hyperactivity, reduced sensorimotor gating, and impaired learning and memory. Electrophysiologic analysis revealed enhanced long-term potentiation in these mice. These results suggest a function for tropomodulin-2 in dendritic spines, where actin dynamics most likely are important for synaptic plasticity and for long-term potentiation in the hippocampus. We are now determining the subcellular localization of tropomodulin-2 in neurons and how tropomodulin-1 regulation of actin dynamics is important for neuronal morphogenesis and function.

Publications

Cox, P.R., Fowler, V., Xu, B., Sweatt, J.D., Paylor, R., Zoghbi, H.Y. Mice lacking tropomodulin-2 show enhanced long-term potentiation, hyperactivity, and deficits in learning and memory. Mol. Cell. Neurosci. 23:1, 2003.

Fischer, R.S., Fritz-Six, K., Fowler, V.M. Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility. J. Cell Biol. 161:371, 2003.

Littlefield, R., Fowler, V.M. A minor actin catastrophe. Nat. Cell Biol. 4:E209, 2002.

Wagner, L.M., Fowler, V.M., and Takemoto, D.J. The interaction and phosphorylation of tropomodulin by protein kinase Cα in N/N 1003A lens epithelial cells. Mol. Vis. 8:394, 2002.