Faithful chromosome segregation in Trypanosoma brucei requires a cohort of divergent spindle-associated proteins with distinct functions.

Faithful chromosome segregation depends on correct spindle microtubule-kinetochore attachment and requires certain spindle-associated proteins (SAPs) involved in regulating spindle dynamics and chromosome segregation. Little is known about the spindle-associated proteome in the early divergent Trypanosoma brucei and its roles in chromosome segregation. Here we report the identification of a cohort of divergent SAPs through localization-based screening and proximity-dependent biotin identification. We identified seven new SAPs and seventeen new nucleolar proteins that associate with the spindle, and demonstrated that the kinetochore protein KKIP4 also associates with the spindle. These SAPs localize to distinct subdomains of the spindle during mitosis, and all but one localize to nucleus during interphase and post-mitotic phases. Functional analyses of three nucleus- and spindle-associated proteins (NuSAPs) revealed distinct functions in chromosome segregation. NuSAP1 is a kinetoplastid-specific protein required for equal chromosome segregation and for maintaining the stability of the kinetochore proteins KKIP1 and KKT1. NuSAP2 is a highly divergent ASE1/PRC1/MAP65 homolog playing an essential role in promoting the G2/M transition. NuSAP3 is a kinetoplastid-specific Kif13-1-binding protein maintaining Kif13-1 protein stability and regulating the G2/M transition. Together, our work suggests that chromosome segregation in T. brucei requires a cohort of kinetoplastid-specific and divergent SAPs with distinct functions.