Abstract

During mitotic spindle assembly, the sister chromatids have to be captured by kinetochore (K) -fibers (bundles of kinetochore microtubules; KMTs) to ensure stable attachment of the chromosomes. This is a prerequisite for chromosome congression at the metaphase plate, and the subsequent segregation of separated sister chromatids to the spindle poles. Although, this is a critical step during mitosis, the identity and regulation of the proteins that mediate the formation and stabilization of K-fibers are still largely unknown. This thesis describes a functional characterization of HURP (hepatoma upregulated protein) and CHICA (C20Orf129), two proteins recently identified in a proteomic survey of the human spindle apparatus (Sauer et al., 2005). The spindle association of both proteins was analyzed with polyclonal antibodies, showing that that of HURP and CHICA were mutually exclusive. While HURP decorated the KMT plus ends, CHICA localized to the spindle pole caps and had no major influence on K-fiber stability. The description of the CHICA project will be brief, as this work was primarily continued by Dr. Anna Santamaria. Her investigations demonstrated that CHICA is important for the spindle recruitment of the chromokinesin Kid, which is required for polar ejection forces. Our studies showed that HURP binds to, and bundles microtubules (MTs) in vitro. In vivo, HURP localizes predominantly to K-fibers in the vicinity of chromosomes and is required for K-fiber stabilization. Moreover, we revealed that importin β binds to the N-terminus of HURP and demonstrated that the nucleotide state of the small GTPase Ran controls HURP localization and function. We conclude that the spindle assembly pathway centered on RanGTP contributes to K-fiber stabilization and that HURP is a critical target of this pathway. To better understand the mechanism of HURP recruitment to the K-fibers we subsequently carried out a structure-function analysis of the different HURP domains. This study revealed that the N-terminus, which contains two coiled coil domains binds to, and bundles MTs and is essential for the initial loading of HURP onto the spindle, whereas the C-terminus (including a Guanylate kinase-associated protein domain; GKAP) is involved in the specific KMT plus end targeting. Furthermore, we identified a conserved mitosis-specific Cdk1 phosphorylation site in the GKAP domain of HURP, indicating that in addition to the RanGTP gradient, Cdk1 phosphorylation may also play a role in HURP recruitment to the K-fibers.