Kinetochore-derived Microtubules

: from Molecular Regulation to their Role in Mitosis

Abstract

To maintain genetic integrity in cell division, replicated chromosomes must be segregated accurately into newly formed daughter cells. The faithful segregation of sister chromatids is a crucial event in cell proliferation since it ensures maintenance of a stable set of chromosomes that is critical for the genetic integrity of the daughter cells. Mistakes in sister chromatid segregation have been related to chromosome instability and aneuploidy characteristic of a variety of human diseases, such as cancers and congenital anomalies.

During cell division, spindle-pole microtubules (MTs) must capture kinetochores (KTs) so that the chromosomes can be loaded on the mitotic spindle. However, it remains a mystery how spindle-pole MTs can locate KTs with high efficiency, within realistic capture times (Wollman et al. 2005). The appearance of MTs at KTs is correlated with their capture, which is consistent with KT-derived MTs facilitating the initial encounter of KTs by spindle-pole MTs (Kitamura et al. 2010). However, so far it has been difficult to establish a causal relationship between the appearance of KT-derived MTs and efficient KT capture. It has also been unclear how much contribution KT-derived MTs make to efficient KT capture by spindle-pole MTs.

Here we show that a MT-associated protein Stu1^CLASP is a good molecular tool to study the role of KT-derived MTs. Depletion of Stu1 protein abolished both localisation of the microtubule polymerase Stu2^XMAP215 from KTs, and MT/tubulin nucleation at KTs, without affecting KT assembly (i.e. the ability of KTs to interact with spindle-pole MTs) or generation of spindle-pole MTs. Abolishing these KT-derived MTs in Stu1-depleted cells led to a delay in KT capture with an increase in the average capture time.
To test whether KT-derived MTs were solely responsible for this delay in KT capture, we developed a mathematical model to recapitulate the roles of KT-derived MTs in Stu1-depleted cells. The model suggested that Stu1-depletion indeed delays KT capture due to a lack of KT-derived MTs. Our results also revealed the extent to which KT-derived MTs contribute to a rapid KT capture by spindle-pole MTs.
Furthermore we showed that, after initial KT capture, KT-associated Stu1^CLASP and Stu^2XMAP215 are required to regulate dynamics of their associated spindle-pole MTs. Removal of Stu1^CLASP and Stu^2XMAP215 from KTs lead to defects in KT-dependent switch from MT depolymerisation to polymerisation (rescue). Interfering with KT-dependent MT rescue would compromise the maintenance of the KT–MT interaction.
Our study reveals that KT-derived MTs facilitate efficient KT capture by spindle-pole MTs. Stu1^CLASP promotes MT generation at KTs by recruiting a MT polymerase Stu^2XMAP215. Afterwards, Stu^2XMAP215 recruitment by Stu1^CLASP to KTs is also important for MT rescue and sustained KT–MT interaction. Thus, we reveal crucial regulatory mechanisms of KT–MT interaction in early stages of mitosis.

Date of Award	2015
Original language	English
Awarding Institution	University of Dundee
Sponsors	Wellcome Trust & European Research Council
Supervisor	Tomoyuki Tanaka (Supervisor)