Membrane composition-dependent patterning of Rho and F-actin in an artificial cell cortex

ABSTRACT Cortical excitability, a phenomenon in which the cell cortex is dynamically patterned with waves of F-actin assembly, has been described in a variety of animal model systems, including embryos of mammals, flies, frogs and echinoderms, as well as a variety of cultured cells. While the cortical F-actin network is closely linked with the plasma membrane, it is not known if membrane composition or fluidity regulates dynamic cytokinetic patterning. Phospholipids partition within the plasma membrane during cytokinesis, and phosphoinositides play a key regulatory role in other excitable systems, suggesting a role for membrane-dependent regulation of cytokinetic patterning. Here we use an artificial reconstituted cell cortex comprised of Xenopus egg extract and supported lipid bilayers (SLBs) to show that membrane composition regulates self-organized cortical patterning. We find that manipulating levels of candidate lipids, including phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, sphingomyelin and cholesterol, results in both quantitative and qualitative changes in the dynamics of traveling waves and standing oscillatory patterns of active Rho and F-actin, as well as the kinetics of Rho activation and F-actin assembly on supported lipid bilayers. Our findings demonstrate that membrane composition directly regulates the assembly of cortical F-actin, as well as emergent active Rho and F-actin patterning. SIGNIFICANCE STATEMENT The cell cortex self-organizes dynamic patterns of active Rho and F-actin during cytokinesis, but it remains unknown whether and how the membrane composition impacts these dynamics. This study uses in vitro reconstitution of the cell cortex to directly manipulate membrane composition and finds that introducing different lipids induces changes in cortical dynamics of active Rho and F-actin. These findings reveal that membrane compositions regulates self-organized cortical dynamics, independently of changes to membrane fluidity. This work furthers our understanding of the mechanistic relationship between membrane composition, GTPase signaling, and cortical F-actin assembly. Competing Interest Statement The authors have declared no competing interest. Abbreviations - F-actin - Filamentous actin - SLB - supported lipid bilayer - TIRF - Total internal reflection fluorescence - LSS - low speed supernatant - HSS - high speed supernatant - GFP - green fluorescent protein - rGBD - Rhotekin GTPase-binding domain - UtrCH - Utrophin calponin homology - FRAP - fluorescence recovery after photobleaching - PIP2 - Phosphatidylinositol 4,5-bisphosphate - PC - phosphatidylcholine - PS - phosphatidylserine - PI - phosphatidylinositol - PE - phosphatidylethanolamine - SM - sphingomyelin