NuSAP1 promotes spindle assembly in Trypanosoma brucei by bundling spindle microtubules

The parasitic protozoan Trypanosoma brucei assembles an intranuclear spindle during mitosis to segregate its chromosomes through mechanisms that are distinct from its mammalian host. This parasite employs unusual nucleus- and spindle-associated proteins (NuSAPs) to regulate mitosis, but their mechanistic roles remain poorly understood. Here, we characterized NuSAP1 and analyzed the functional interplay of NuSAP1 with its interacting and proximal proteins. NuSAP1 localizes to the entire spindle with spindle pole enrichment, interacts with NuSAP4 and the spindle-associated protein SPB1 through distinct structural motifs, and regulates the stability of NuSAP4 and the localization of SPB1. Knockdown of NuSAP1 disrupts the localization of MAP103 and TbMlp2 to spindle poles, suggesting its requirement for spindle pole protein complex formation and spindle pole complex-nuclear basket association. Finally, NuSAP1 bundles microtubules in vitro, and depletion of NuSAP1 disrupts spindle assembly and kinetochore biorientation. Together, these results uncover the mechanistic role of NuSAP1 in spindle assembly by bundling spindle microtubules and promoting spindle pole complex formation, underscoring an unusual regulatory mechanism for mitosis in this unicellular eukaryote.