The CIF1 protein is a master orchestrator of trypanosome cytokinesis that recruits several cytokinesis regulators to the cytokinesis initiation site.

To proliferate, the parasitic protozoan Trypanosoma brucei undergoes binary fission in a unidirectional manner along the cell's longitudinal axis from the cell anterior toward the cell posterior. This unusual mode of cell division is controlled by a regulatory pathway composed of two evolutionarily conserved protein kinases, Polo-like kinase and Aurora B kinase, and three trypanosome-specific proteins, CIF1, CIF2, and CIF3, which act in concert at the cytokinesis initiation site located at the distal tip of the newly assembled flagellum attachment zone (FAZ). However, additional regulators that function in this cytokinesis signaling cascade remain to be identified and characterized. Using proximity biotinylation, co-immunofluorescence microscopy, and co-immunoprecipitation, we identified 52 CIF1-associated proteins and validated six CIF1-interacting proteins, including the putative protein phosphatase KPP1, the katanin p80 subunit KAT80, the cleavage furrow-localized proteins KLIF and FRW1, and the FAZ tip-localized proteins FAZ20 and FPRC. Further analyses of the functional interplay between CIF1 and its associated proteins revealed a requirement of CIF1 for localization of a set of CIF1-associated proteins, an interdependence between KPP1 and CIF1, and an essential role of katanin in the completion of cleavage furrow ingression. Together, these results suggest that CIF1 acts as a master regulator of cytokinesis in T. brucei by recruiting a cohort of cytokinesis regulatory proteins to the cytokinesis initiation site.