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

Abstract

To proliferate, the parasitic protozoan 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 by recruiting a cohort of cytokinesis regulatory proteins to the cytokinesis initiation site.

Functional analyses of an axonemal inner‐arm dynein complex in the bloodstream form of Trypanosoma brucei uncover its essential role in cytokinesis initiation

“…Cells were washed once with PBS, incubated with the blocking buffer (3% BSA in PBS) for 1 h at room temperature, and then incubated with the primary antibody for 1 h at room temperature. The following primary antibodies were used: anti‐CIF1 polyclonal antibody (1:1,000 dilution) (Zhou et al , ), anti‐CIF2 polyclonal antibody (1: 1,000 dilution) (Zhang et al , ), anti‐Protein A (anti‐ProtA) polyclonal antibody (1:400 dilution) (Sigma‐Aldrich), and FITC‐conjugated anti‐HA antibody (1:400 dilution) (Sigma‐Aldrich). Cells were washed three times with PBS containing 0.1% Triton X‐100.…”

“…Regulation of the unusual cytokinesis in T. brucei is orchestrated by a novel signaling pathway composed of two evolutionarily conserved proteins kinases, the Polo‐like kinase homolog TbPLK and the Aurora B kinase homolog TbAUK1, and a cohort of trypanosome‐specific regulators, which act at the distal tip of the new FAZ and/or the cleavage furrow to drive cytokinesis initiation and furrow ingression (Li and Wang, 2006; Li et al ., 2009; McAllaster et al , ; Zhou et al , ; ; ; ; Hu et al , ; Hilton et al , ; Kurasawa et al , ). Among these trypanosome‐specific regulators are three cytokinesis initiation factors named CIF1, CIF2, and CIF3 (Zhou et al , ; ; Hu et al , ; Kurasawa et al , ).…”

“…CIF1 and CIF2 are interdependent for maintaining their respective protein stability (Zhou et al , ), whereas CIF1 maintains CIF3 protein stability and CIF3 recruits CIF1 to the new FAZ tip (Kurasawa et al , ). These regulators also include a kinetoplastid‐specific protein phosphatase named KPP1 (Hilton et al , ; Zhou et al , ), a kinesin motor domain‐ and tropomyosin domain‐containing protein named KLIF (Hilton et al , ; Zhou et al , ), a katanin60–katanin80 complex named KAT60a–KAT80 (Zhou et al , ), and two coiled‐coil domain‐containing proteins named FRW1 and FPRC (Zhou et al , ). All of these cytokinesis regulators interact with CIF1, and many of them are recruited by CIF1 to the distal tip of the new FAZ (Zhou et al , ), suggesting a pivotal role of CIF1 in cytokinesis initiation.…”

BOH1 cooperates with Polo-like kinase to regulate flagellum inheritance and cytokinesis initiation in Trypanosoma brucei

“…First, BOH1 interacts with and targets TbPLK to the new FAZ tip (Figs 6 and 7). TbPLK is a key cytokinesis regulator that acts most upstream of the cytokinesis regulatory pathway, involving Aurora B kinase TbAUK1, and the three trypanosome-specific cytokinesis regulators CIF1, CIF2 and CIF3 (Kurasawa et al, 2018;Zhou et al, 2018aZhou et al, , 2016a. Second, BOH1 is required for localization of CIF1 to the new FAZ tip (Fig.…”

“…Cytokinesis in the procyclic (insect) form of T. brucei is initiated from the distal tip of the new FAZ, and requires a signaling cascade mediated by TbPLK, the Aurora B kinase homolog TbAUK1 and a cohort of trypanosome-specific proteins that act in concert at the new FAZ tip from early S phase to telophase of the cell cycle (Kumar and Wang, 2006;Kurasawa et al, 2018;Li et al, 2008;McAllaster et al, 2015;Zhou et al, 2016a,b;Zhou et al, 2018a). Among these cytokinesis regulators, the cytokinesis initiation factor 1 (CIF1) plays a pivotal role in recruiting multiple cytokinesis regulatory proteins to the new FAZ tip (Zhou et al, 2018a). It forms two separate protein complexes with cytokinesis initiation factors 2 and 3 (CIF2 and CIF3, respectively) and the two protein complexes function at the new FAZ tip to promote cytokinesis initiation (Kurasawa et al, 2018).…”