NuSAP1 promotes bipolar spindle assembly in Trypanosoma brucei by bundling spindle microtubules

The parasitic protozoan Trypanosoma brucei assembles a bipolar mitotic spindle and undergoes a closed mitosis to segregate its megabase chromosomes and mini-chromosomes through mechanisms that are distinct from its mammalian host. This parasite employs a subset of trypanosome-specific nucleus- and spindle-associated proteins (NuSAPs) to regulate mitosis, but the mechanistic roles of these proteins remain poorly understood. Here, we performed biochemical and molecular characterization of NuSAP1 and analyzed the functional interplay of NuSAP1 with its interacting and proximal proteins. NuSAP1 localizes to the mitotic spindle with spindle pole enrichment, and interacts with the spindle-associated and spindle pole-enriched proteins NuSAP4 and SPB1 through distinct structural motifs. NuSAP1 and NuSAP4 are interdependent for protein stability, and NuSAP1 is required for SPB1 localization. Further, NuSAP1 bundles microtubules in vitro, and depletion of NuSAP1 disrupts bipolar spindle assembly. Finally, knockdown of NuSAP1 disrupts the localization of its proximal proteins MAP103 and TbMlp2 to spindle poles. Together, these results uncover the mechanistic role of NuSAP1 in bipolar spindle assembly by bundling spindle microtubules and promoting spindle pole complex formation, underscoring unusual regulatory mechanisms for mitosis in this early divergent unicellular eukaryote.