Initial Characterization of two novel spindle proteins, CHICA and HURP

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.