In Vitro Reconstitution of Centralspindlin-Mediated Transport

Project summary/abstract This proposal aims to address the knowledge gap in our understanding of the molecular mechanisms through which centralspindlin regulates cytokinesis. Cytokinesis is the last act of cell division, in which cleavage furrow ingression produces physically distinct daughter cells. Errors in cytokinesis can lead to cancer. During cytokinesis, centralspindlin, a motor protein complex, is crucial for the localization of key furrowing components and other proteins to the site of cleavage furrow formation. Although previous in vivo studies have highlighted centralspindlin’s essential role, the exact mechanisms remain unclear. We propose to clarify these through the in vitro reconstitution of centralspindlin-mediated transport. The overall objective of this project is to define the molecular mechanisms through which centralspindlin transports required cargos along microtubules to direct furrow formation. Our lab has established conditions to stably express full-length centralspindlin and developed methods for the in vitro reconstitution of centralspindlin-mediated transport. Our long-term goal is to reconstitute cytokinesis using purified centralspindlin and other regulatory components. Our central hypotheses is that centralspindlin-mediated transport along microtubules is a regulated mechanism to localize the components that direct furrow formation and cytoplasmic abscission. We will test our hypothesis by identifying the features of centralspindlin required for robust microtubule plus-end localization, determining the target cargos transported by centralspindlin, and exploring the factors that regulate centralspindlin-mediated transport. This study is innovative because it employs novel methods to address previously unresolved questions about centralspindlin. The project is significant as it will provide a precise understanding of cytokinesis and offer new potential targets for cancer treatments. Additionally, this project will help establish a research community of underrepresented minority students through my laboratory at the University of Hawaii at Hilo, the most ethnically diverse campus among national universities.