Functional analyses of the CIF1-CIF2 complex in Trypanosoma brucei identify the structural motifs required for complex formation and cytokinesis

Abstract

Cytokinesis in trypanosome occurs unidirectionally along the longitudinal axis from the cell anterior towards the cell posterior and requires a trypanosome-specific CIF1-CIF2 protein complex. However, little is known about the contribution of the structural motifs in CIF1 and CIF2 to complex assembly and cytokinesis. Here, we demonstrated that the two zinc-finger motifs but not the coiled-coil motif in CIF1 are required for interaction with the EF-hand motifs in CIF2. We further showed that localization of CIF1 depends on the coiled-coil motif and the first zincfinger motif and that localization of CIF2 depends on the EF-hand motifs. Deletion of the coiled-coil motif and mutation of either zinc-finger motifs in CIF1 disrupted cytokinesis. Further, mutation of either zinc-finger motif in CIF1 mis-localized CIF2 to the cytosol and destabilized CIF2, whereas deletion of the coiled-coil motif in CIF1 spread CIF2 over to the new flagellum attachment zone and stabilized CIF2. Together, these results uncovered the requirement of the coiled-coil motif and zinc-finger motifs for CIF1 function in cytokinesis and for CIF2 localization and stability, providing structural insights into the functional interplay between the two cytokinesis regulators.Trypanosoma brucei, an early branching parasitic protozoan, causes lethal infections in humans and animals in sub-Saharan Africa. The parasite has a complex life cycle by alternating between insect vectors and mammalian hosts, and proliferates through binary cell fission in the midgut of insects and the bloodstream of mammals. Cell division occurs uni-directionally along the longitudinal axis from the cell anterior to the cell posterior, as visualized by time-lapse video microscopy of dividing cells (1), and the division plane appears to be determined by the length of the new flagellum and a proteinaceous filament termed flagellum attachment zone (FAZ) (2,3). The anterior tip of the newly assembled FAZ filament constitutes the site from which cytokinesis cleavage furrow starts to ingress towards the posterior cell end along the division fold, which is formed through membrane invagination by unknown mechanisms (4,5). Due to the apparent lack of involvement of actin in cytokinesis (6), trypanosome likely does not assemble an actomyosin contractile ring, the evolutionarily conserved machinery for cleavage furrow ingression found in amoebas, fungi, and animals (7,8).The molecular mechanism for the unidirectional, longitudinal binary cell fission in trypanosome remains poorly understood. Based on the observed cell cycle defects through RNAimediated gene ablation, many proteins have been reported to be involved in cytokinesis, which localize to various subcellular structures and possess diverse biochemical functions (5,9-23). However, many of these proteins localize to the cytosol, raising the question of whether they play a . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was ...