Project Summary Cytokinesis is the final stage of cell division, and is regulated by an evolutionarily conserved signaling pathway in eukaryotic organisms ranging from yeast to humans. Trypanosoma brucei, a parasitic protozoan and causative agent of human sleeping sickness, undergoes a distinct mode of cytokinesis by dividing along the longitudinal cell axes uni-directionally from the anterior end of the new-flagellum daughter cell towards the nascent posterior end of the old-flagellum daughter cell. T. brucei adopts a cytokinesis signaling pathway that is totally different from its human host; therefore, cytokinesis can be exploited as a drug target for combating this dreadful human pathogen. Despite the identification and the functional characterization of multiple cytokinesis regulators, the functional interplay and the order of action among these regulators remain poorly understood. Moreover, it remains also unclear how cleavage furrow ingression is initiated and regulated and whether these identified cytokinesis regulators cooperate with certain cytoskeletal proteins at the cleavage furrow to promote furrow ingression. The current proposal is built on the recently discovered cytokinesis regulatory pathway, and aims to determine the functional interplay among known cytokinesis regulators and to explore the mechanistic roles of kinesin proteins in promoting cytokinesis initiation and progression. In specific Aim 1, we plan to carry out a systematic analysis of the functional interplay among the cytokinesis regulatory proteins before and during cytokinesis. In Aim 2, we plan to dissect the function of a kinesin-13 family protein in regulating cytokinesis initiation and integrate this kinesin into the existing cytokinesis regulatory pathway. In Aim 3, we plan to investigate the mechanistic role of KLIF-associated cytoskeletal proteins and to explore their functional interplay with KLIF and other cytokinesis regulators in driving cleavage furrow ingression. These studies will facilitate the understanding of the molecular mechanism underlying the unusual mode of cytokinesis in T. brucei, and the outcomes from these investigations may provide new targets for chemotherapeutic intervention.