15 research outputs found
Motor-Independent Targeting of CLASPs to Kinetochores by CENP-E Promotes Microtubule Turnover and Poleward Flux
Efficient chromosome segregation during mitosis relies on the coordinated activity of molecular motors with proteins that regulate kinetochore attachments to dynamic spindle microtubules [1]. CLASPs are conserved kinetochore- and microtubule-associated proteins encoded by two paralog genes, clasp1 and clasp2, and have been previously implicated in the regulation of kinetochore microtubule dynamics [2-4]. However, it remains unknown how CLASPs work in concert with other proteins to form a functional kinetochore microtubule interface. Here we have identified mitotic inter-actors of human CLASP1 via a proteomic approach. Among these, the microtubule plus-end-directed motor CENP-E [5] was found to form a complex with CLASP1 that colocalizes to multiple structures of the mitotic apparatus in human cells. We found that CENP-E recruits both CLASP1 and CLASP2 to kinetochores independently of its motor activity or the presence of microtubules. Depletion of CLASPs or CENP-E by RNA interference in human cells causes a significant and comparable reduction of kinetochore microtubule poleward flux and turnover rates and rescues spindle bipolarity in Kif2a-depleted cells. We conclude that CENP-E integrates two critical functions that are important for accurate chromosome movement and spindle architecture: one relying directly on its motor activity, and the other involving the targeting of key microtubule regulators to kinetochores
CENP-E targeting of CLASPs to kinetochores
Efficient chromosome segregation during mitosis relies on the coordinated activity of
molecular motors with proteins that regulate kinetochore attachments to dynamic spindle
microtubules [1]. CLASPs are conserved kinetochore- and microtubule-associated
proteins encoded by two paralogue genes, clasp1 and clasp2, and have been previously
implicated in the regulation of kinetochore-microtubule dynamics [2-4]. However, it
remains unknown how CLASPs work in concert with other proteins to form a functional
kinetochore-microtubule interface. Here we have identified mitotic interactors of human
CLASP1 using a proteomic approach. Among these, the microtubule plus-end directed
motor CENP-E [5] was found to form a complex with CLASP1 that co-localizes to
multiple structures of the mitotic apparatus in human cells. We found that CENP-E
recruits both CLASP1 and CLASP2 to kinetochores independent of its motor activity or
the presence of microtubules. Depletion of CLASPs or CENP-E by RNAi in human cells
causes a significant and comparable reduction of kinetochore-microtubule poleward flux
and turnover rates, as well as rescues spindle bipolarity in Kif2a-depleted cells. We
conclude that CENP-E integrates two critical functions that are important for accurate
chromosome movement and spindle architecture: one relying directly on its motor
activity and the other involving the targeting of key microtubule regulators to
kinetochores
CLASP-dependent microtubule nucleation at the TGN
Proper organization of microtubule arrays is essential for intracellular trafficking and cell
motility. It is generally assumed that most if not all microtubules in vertebrate somatic
cells are formed by the centrosome. Here we demonstrate that a large number of
microtubules in untreated human cells originate from the Golgi apparatus in a
centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules
need γ-tubulin for nucleation. Additionally, formation of microtubules at the Golgi
requires CLASPs, microtubule-binding proteins that selectively coat non-centrosomal
microtubule seeds. We show that CLASPs are recruited to trans-Golgi network (TGN) at
the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal
arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially
oriented toward the leading edge in motile cells. We propose that Golgi–emanating
microtubules contribute to the asymmetric microtubule networks in polarized cells and
support diverse processes including post-Golgi transport to the cell front
Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function
CLASPs are widely conserved microtubule plus-end–tracking proteins with essential roles in the local regulation of
microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for
mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1
has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles
remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis
is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary
fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially
rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are
slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle
function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout
cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells.
Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent
aneuploidy in mammals
Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network
SummaryProper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need γ-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front