N-WASP at the Membrane-Actin Interface

Actin polymerization occurs on membrane surfaces in cells. Signal transduction cascades activate nucleation promoting factors (NPFs) that are recruited to and activated at the membrane to produce polarized actin networks. These cytoskeletal structures generate force against membranes such as at the leading edge of chemotaxing cells, at the basal membrane of invasive podosomes in cancerous cells, and at the surface of rocketing intracellular vesicles. The molecular mechanisms that regulate the establishment and maintenance of polarized actin networks on the membrane are not well understood. Specifically, it is unknown how the actin network remains attached to the membrane as it pushes against it. Formation of polarized actin networks has been reconstituted in vitro on the surface of polystyrene beads. Because these systems used NPFs that are fixed to the bead surface, the physiological relevance of the membrane and of signal transduction activation could not be assessed on the formation and/or maintenance of polarized actin networks.We used the vesicle motility system to study the role of the NPF, N-WASP, at the membrane-actin interface of rocketing vesicles. We found that the actin monomer binding domain, the WH2 domain, was necessary and sufficient to localize to the membrane-actin interface. To dissect the mechanism of this localization, we created a novel biomimetic motility system using pure components. This system included a lipid bilayer substrate and a set of soluble signaling proteins including autoinhibited N-WASP and inactive Cdc42/RhoGDI complex. Using this system, we made two critical observations: first, we demonstrated that WH2 mutants that were defective in localization to the membrane-actin interface formed comet tails that prematurely detached from the membrane. N-WASP maintained membrane-actin attachment by WH2-mediated capture of actin filament barbed ends. Further, we observed in cells that these mutations resulted in defects in organization of large podosome structures. The second observation has initiated a new study. We demonstrated that the concurrent activation of Cdc42 and N-WASP with actin polymerization yielded a new form of symmetry breaking on membranes. This result, which mimics a more physiological experimental scenario, highlights the necessity to study signal-dependent actin assembly on membranes in vitro