Molecular Requirements for Kinetochore-Associated Microtubule Formation in Mammalian Cells

Abstract

In centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly. In addition, microtubule formation at kinetochores has also been observed, most recently under physiological conditions in live cells. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-beta or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture.

Mechanisms of plant spindle formation

“…1), plant kinetochores appear to initiate their own kinetochore fibers early in prometaphase, either by directly nucleating microtubules or promoting microtubule growth in their vicinity. Similar images and interpretations can also be found in the animal literature (Tulu et al 2006;Torosantucci et al 2008).…”

Targeted disruption of Aurora A causes abnormal mitotic spindle assembly, chromosome misalignment and embryonic lethality

“…Additionally, the presence of a relatively elevated number of cells in mitosis in the null embryos indicate that mitosis may be initiated with a delay rather than inhibited in absence of Aurora A as has been reported in case of Xenopus early embryonic cell cycles investigated with egg extracts depleted of endogenous Aurora A (Liu and Ruderman, 2006;Satinover et al, 2006). Aberrant spindle assembly is expected due to impaired formation of k fibers requiring active Aurora A complex with spindle assembly factors, such as TPX2 and HURP, among others (Gruss et al, 2001;Koffa et al, 2006;Sillje et al, 2006;Tulu et al, 2006). It has been hypothesized that microtubules can be organized at multiple, often transient, structures that can catalyze g-tubulin-dependent microtubule nucleation from their minus ends, plus ends or sides (Luders and Stearns, 2007) with the polar spindle microtubules 'collecting' preassembled chromosome-generated minispindles into a single bipolar spindle within the cells (Rieder, 2005).…”

MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

“…A third possibility is that there are new MTs that grow from the chromosomes and become incorporated into the K-fiber bundles (Khodjakov et al, 2003). The MTs that grow from the chromosomes may quickly become coated with HURP or with TPX2, two specialized MT-associated proteins that have been implicated in chromosomemediated spindle assembly (Gruss et al, 2002;Koffa et al, 2006;Sillje et al, 2006;Tulu et al, 2006;Wong and Fang, 2006;Casanova et al, 2008), such that their overall dynamics are more consistent with the stabilized MTs present in a K-fiber. This possibility is consistent with our observation in Figure 5, G and H, where as the concentration of paclitaxel is increased, the percentage of MTs in the slow phase is increased.…”