Cell locomotion is essential for embryonic development, wound healing, and immune system function. Moreover, it is critical factor in the pathogenesis of cancer and of cardiovascular disease. One subprocess of overall cell locomotion, the protrusion of the leading lamellipodia of locomoting cells, is thought to be driven by actin polymerization. The long term goal of this project is to elucidate the molecular mechanisms that control actin polymerization in cells and to determine how polymerization contributes to the forces that drive this aspect of cell locomotion. We have explored this issue by examining the actin-polymerization driven motility of the pathogenic bacterium Listeria monocytogenes, which represent a model for understanding the protrusion of the plasma membrane at the leading edge of motile cells. We purified a protein complex from platelets, the Arp2/3 complex. That nucleates actin assembly at the surface of L. monocytogenes and mediates bacterial motility. The Arp2/3 complex is localized to lamellipodia in locomoting fibroblast cells, suggesting that it also plays a role in nucleating actin polymerization during lamellipodial protrusion. Therefore the complex is likely to represent a central component of the cellular actin polymerization machinery. Moreover, it may also be a key target of signaling pathways that regulate cell motility in response to extracellular cues. The complex has seen polypeptide subunits that are conserved in sequence among diverse eukaryotes, suggesting that its structure and function have been conserved through eukaryotic evolution. However, we do not yet understand how the other proteins in L. monocytogenes motility and lamellipodial protrusion. We will take a biochemical approach to understand the mechanism of Arp2/3 complex nucleates actin assembly, what factors regulate its activity in cells, and how it functions with other proteins in L. monocytogenes motility and lamellipodial protrusion. We will take a biochemical approach to understand the mechanism of Arp2/3 complex function and its interaction with other cytoskeletal and regulatory proteins in the context of L. monocytogenes propulsion and cell locomotion.
The aims are to: (1) Examine the requires for the formation of the Arp2/3 complex, (2) Determine the function of ARP2/3 complex subunits in actin nucleation, (3) Dissect the mechanism by which Arp2/3 complex nucleating activating is stimulated by the L. monocytogenes ActA protein, (4) Identify cellular factors that enhance L. monocytogenes motility and (5) identify cellular factors that activate Arp2/3 complex nucleating activity.
Showing the most recent 10 out of 37 publications
