The goal of this project is to develop new techniques to analyze and control biopolymer network microstructures with a holographic laser tweezer array, a powerful new instrument whose potential is just beginning to be explored. This tool will permit complex spatially distributed perturbations and measurements of forces in dynamic biopolymer networks. While the techniques will be developed on actin, they should be suitable to investigate forces and front instabilities in a broad range of network forming biomolecules. The response of the networks to perturbations should reveal the interrelationship between the polymerization front dynamics and the mechanical properties of the network. Understanding this inter-relationship is crucial for understanding how the polymerization dynamics of various cytoskeletal proteins affect cell function, for example, in the morphological changes associated with cell mobility. Understanding and controlling cell mobility has implications for many aspects of human health, from enhancing nerve regeneration after injury to reducing the mobility of metastatic cancer cells.
Specific Aim 1 : Control actin polymerization fronts with submicron resolution using a holographic tweezer array. Spatially extended perturbations of polymerization fronts will be introduced with the tweezer array by thermal heating or by localized uncaging of molecules that affect the polymerization. From the response of the polymerization front to the perturbation the investigators can extract characteristic length scales and timescales, and investigate instabilities.
Specific Aim 2 : Develop technology to measure the spatial distribution of forces using the holographic tweezer array. Pico Newton forces will be measured with trapped beads and trapped extended objects. They will measure the forces generated by a front of polymerizing actin and the correlations between those forces. With this technique they will determine how the polymerization force depends on the shape of the front, growth velocity, and other parameters.
Specific Aim 3 : Deform actin-containing vesicles with multiple laser traps to probe network properties and mechanical instabilities. Holding and deforming the membrane with multiple laser tweezers will permit us to probe and possibly control the development of protrusions and of irregular membrane shapes similar to the lamellipodia or pseudopodia observed in living cells. This work will lay the technical and analytical foundations for a new approach to investigating cell motility.
