Investigating the mechanism of self-organized cortical patterning in an artificial cortex - The cell cortex underlies essential cellular functions, including cell shape changes that facilitate cell division. Comprised of a meshwork of filamentous actin (F-actin) and the plasma membrane, the cortex is remodeled during cytokinesis, physically dividing the cell in two. Recent work has shown that prior to large-scale remodeling, the cortex is also dynamically patterned with coherent subcellular waves of the small GTPase RhoA and F-actin, a phenomenon termed “cortical excitability”. In developing embryos, these waves appear over the entire surface of the cell and then feed into the cytokinetic furrow as cell division progresses. Investigating the mechanisms that support and regulate cortical patterning has been greatly improved by the development of an “artificial cortex”, made from supported lipid bilayers (SLBs) and Xenopus egg extract, which successfully reconstitutes active Rho and F-actin dynamics in a cell-free system. Like in vivo cortical excitability, patterning in the artificial cortex depends on Rho activity and F-actin polymerization. This novel, synthetic approach to investigating cortical patterning is an ideal system for systematically examining the role of individual factors (such as upstream GTPase regulators, membrane composition and fluidity, cell cycle state) in regulating cortical dynamics. Using the artificial cortex as a model for cortical patterning, this proposal seeks to understand how cortical pattern formation is regulated and how patterning remodels the cell cortex to perform essential functions like cytokinesis. This system will enable us to bridge the biochemical and biophysical principles that drive pattern formation in vitro to the molecular mechanisms that drive patterning in cells. The primary goals of this project will be to investigate the factors that promote cortical wave formation (Aim 1), cytoskeletal remodeling at the cortex (Aim 2), and the role of cortical patterning in supporting successful cell division (Aim 3). This project will build on the findings from the K99 phase of the award, including the observation that SLB composition modulates the behavior and dynamics of excitable waves in the artificial cortex. As modifying membrane composition is difficult in vivo, this result highlights the importance of a synthetic system for dissecting the contributions of individual components, such as the membrane, to self-organized patterning. The work will expand our knowledge of the molecular regulation of the cortex underlying the emergence of cortical excitability, and the role of dynamic patterning in cell division.
