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Biochemical Reconstitution of an Actin-Driven Positive Feedback Loop

  • Author(s): Wong, Derek Tak-Lai
  • Advisor(s): Taunton, Jack
  • Fletcher, Daniel A
  • et al.
Abstract

Actin polymerization provides the physical force for membrane protrusion and determines the directionality of membrane movement in a variety of cellular processes, such as cell migration, establishment of cell polarity, podosome and invadopodia assembly, and endosome motility. To ensure proper directionality is achieved, actin assembly occurs at distinct regions along the membrane surface. Actin nucleation promoting factors, which include members of the WASP/WAVE family, must first be recruited to the membrane from the cytoplasm and activated at the surface in a localized fashion; the specific location of their activation precisely dictates where along the membrane the actin network is produced.

Theoretical studies suggest that positive feedback mechanisms play an essential role in the spatial regulation of signaling events at the membrane. Furthermore, experimental observations demonstrate that the actin cytoskeleton is involved in feedback loops that locally cluster and amplify signaling components that regulate actin assembly. However, despite our extensive knowledge in the biochemistry of actin polymerization, it remains unclear how molecular interactions between F-actin, actin-binding proteins, membrane-bound proteins, and lipids produce the micron-scale spatial organization of clustered signaling complexes observed in cells.

To dissect the molecular mechanisms of actin-dependent feedback loops, I have reconstituted N-WASP-driven actin assembly at a model membrane using purified components. I find that during actin polymerization along the surface of lipid-coated microspheres, the local surface density of membrane-bound N-WASP is increased in an actin-dependent manner. While initial localization and activation of N-WASP requires first binding to Cdc42 at the membrane, this interaction is not required to maintain N-WASP at the interface between the actin network and the membrane. N-WASP is released from Cdc42 and remains tethered at the membrane by the actin network, thus allowing Cdc42 to undergo subsequent cycles of N-WASP activation. Catalytic activation by Cdc42 and local amplification by the product of N-WASP's nucleation promoting factor activity, i.e. F-actin, together provide the mechanism for spontaneous clustering of N-WASP and localized actin assembly along the membrane, even in the absence of any asymmetric cues at the surface. This work provides mechanistic insights into how dendritic actin networks are organized into spatially distinct regions, and in turn, how they may exert feedback control on signaling transduction events at the membrane.

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