We study the regulation and dynamics of the actin cytoskeleton; that is, how living cells establish polarity and use actin polymerization to change shape or propel themselves forward.
In 1903 the Wright brothers solved the problem of flight by dividing it into three parts: (1) A structural problem. How do you build a wing that generates lift? (2) A propulsion problem. How do you generate force to drive forward motion? (3) A control problem. How can alterations in wing structure be used to regulate its function? Amoeboid cell motility can be similarly divided: (1) What are the structures of the macromolecular assemblies that drive cell movement and shape change and how does the cell construct them? (2) What is the mechanism by which molecular assemblies generate the force required to deform cell membranes? (3) How do intracellular signaling systems control the assembly and function of these macromolecular machines? We are interested in all three questions.
The basic processes we study are conserved across all eukaryotic phyla and we study them in several organisms including free-living soil amoebas, budding yeast, fruitflies, and mammals.
The forward, or leading edge of a motile cell is a highly specialized structure - containing an extremely dense meshwork of actin filaments built and maintained by a particular subset of actin-associated proteins. Filaments assemble at the membrane and drive it forward (Mogilner and Oster 1996) and behind the advancing membrane filaments are disassembled (Theriot and Mitchison 1991) so that the actin network at the leading edge churns forward like a tank tread. We do not understand exactly how extracellular signals are converted into three dimensional structures that accomplish specific tasks but we suspect that the basic principles, if not the molecular mechanisms, are conserved across evolution.
Here are a few questions we are pondering right now:
1. What is the molecular mechanism of Arp2/3 activation?
The Arp2/3 complex is a remarkably complicated machine. It builds mechanically rigid networks of actin filaments by binding to the sides of pre-formed filaments and nucleating formation of new, crosslinked filaments in a process we call dendritic nucleation. The nucleation activity of the complex is stimulated by both exogenous and endogenous cellular factors that we call nucleation promoting factors (or NPFs if we are feeling particularly jargony). Many nucleation promoting factors have been identified to date but all the really potent ones share some common features. They all contain an actin-binding domain (called a WH2 domain) and an acidic Arp2/3-binding domain. From biochemical experiments we built a kinetic model of the nucleation process and identified a critical, first-order activation step in the mechanism. We also showed that ATP binding and hydrolysis were required for the nucleation reaction. We are following up on this work to determine exactly how the WH2 domain presents actin monomers to the Arp2/3 complex, what structural changes are involved in the activation step, and the role of ATP hydrolysis in the nucleation, crosslinking and uncrosslinking cycle of the Arp2/3 complex.
2. How does actin assembly induce polarity and generate force?
Actin-based force generation is driven by a modular biochemical network whose main players are conserved across all eukaryotic phyla. We can now recapitulate the entire actin assembly/disassembly cycle as well as polymerization-driven motility using purified proteins. We are currently using this purified in vitro system to investigate the mechanisms of actin-based force generation and symmetry breaking. Questions we are addressing include: What is the timing of the disassembly of Arp2/3-generated actin networks? How much elastic energy is stored in force-generating actin networks?
3. Novel mechanisms of actin assembly.
We now know that the Arp2/3 complex is not the only cellular factor capable of de novo nucleation of actin filaments. We are investigating the function and cellular roles of other, novel actin nucleation factors.
4. Structure and function of the prokaryotic cytoskeleton.
Bacteria have actin-like cytoskeletal filaments! Who knew? We are interested in studying the biophysics of these bug-derived actins. We are also busy isolating and characterizing all the components of the bacterial cytoskeleton. It's a brave new world out there! Come join us!