PROJECT SUMMARY In animal cells, cytokinesis is driven by constriction of an actomyosin contractile ring, which is positioned and controlled by signaling from spindle microtubules. Cytokinesis requires a high degree of spatial and temporal molecular regulation to ensure each daughter cell inherits a single nucleus. Although the essential molecular players required for cytokinesis are known, many cytokinesis proteins localize dynamically to multiple subcellular niches throughout cell division, potentially allowing multiple subcellular functions and making these proteins difficult study using traditional genetic approaches. Optogenetics enable spatiotemporal studies by locally targeting light to control protein function in specific subcellular regions. For this reason, we developed FLIRT (Fast Local Infrared Thermogenetics), which uses an infrared (IR) laser to locally heat and thus locally inactivate genetically-encoded fast-acting temperature sensitive (ts) mutant proteins with high spatiotemporal precision. FLIRT is also reversible: the IR laser can be turned off at any point to stop local heating and allow for protein reactivation. Furthermore, using FLIRT, non-ts-mutants (wildtype) can be used as controls for any laser-induced damage induced by a given FLIRT procedure. In preliminary data using C. elegans embryos, we calibrated the temperature induction achieved using FLIRT, validated the use of FLIRT on the subcellular level in both the 1-cell embryo and the 16-cell embryo, and demonstrated that FLIRT can inhibit other cell biological processes such as cell fate signaling in multicellular embryos and membrane partitioning in the adult gonad. Having laid the foundation for further studies, we now propose experiments to define the spatiotemporal regulation of actomyosin contractility and spindle microtubule-associated signaling complexes during cytokinesis and address longstanding questions in the field, such as the relative contributions of equatorial vs. polar actomyosin contractility during cell division. FLIRT experiments will also be conducted on cells in C. elegans early embryos and in somatic cells with multicellular developing worm tissue. These experiments will define the precise spatiotemporal regulation of key players in cytokinesis, test specific hypotheses regarding their mechanisms of action, and test the universality of rules governing cytokinesis, whether the same principles that apply to the early embryo also apply to somatic cells within a multicellular context.