Non-motor microtubule associated proteins (MAPs)
Spindle function and structure does not depend solely on motor proteins and MTs. The spindle and its components are coordinated by proteins that control cell-cycle progression, regulate motor function or promote MT nucleation (Manning and Compton, 2008). Among many regulatory and accessory proteins associated with the spindle and its components, non-motor MAPs are tasked with providing structural integrity to the spindle by crosslinking parallel or anti-parallel MT bundles. In this chapter, I will discuss non-motor proteins in the context of spindle structure and architecture. Cross-linker proteins of the MAP65 family are arguably the most prominent spindle structure maintaining proteins besides kinesin-5. Mammalian member of the MAP65 family, PRC1, is a homodimer with four distinct domains: a dimerization domain, a rod domain, a spectrin domain, and a C-terminal domain (Figure 2.10A) (Subramanian et al., 2010; Kellogg et al., 2016). MAP65 members are crucial elements of the spindle midzone, for they crosslink iMTs, and support the spindle structure (Figure 2.10B) (Mollinari et al., 2002; Kurasawa et al., 2004; Loïodice et al., 2005). Mitotic spindles in cells deficient for MAP65 frequently break upon anaphase onset, producing two unorganized half-spindles.
Non-centrosomal pathways of spindle assembly
As mentioned earlier (Chapter 1.2. and Chapter 2.2.), many female oocytes lack centrosomes, yet manage to nucleate MTs and assemble a spindle. Moreover, mitotic cells in which centrosomes are ablated, or mutants that lack centrosomes, still assemble a mitotic spindle. Regardless of differences, it appears that MT nucleation is mediated by γ-tubulin in all pathways. In the following chapter, I will address the most prominent centrosome-independent spindle assembly pathways.
The small GTPase Ran exists in a gradient around the chromosomes in mitosis and meiosis, and acts as an activator of spindle assembly factors (Figure 3.1). Ran is active in its GTP-bound form and inactive in its guanosine diphosphate (GDP)-bound form. Ran-GTP transition is promoted by chromatin associated RCC1, so the concentration of Ran-GTP is higher in the chromatin vicinity (Carazo-Salas et al., 1999). Studies in Xenopus egg extract have shown that RCC1 coupled to a bead is capable of organizing MTs into a bipolar network around the bead (Halpin et al., 2011). In contrast, Ran-GTP to Ran-GDP transition is promoted by the cytoplasmic RanGAP. Therefore, RCC1 and RanGAP form a gradient of Ran-GTP, with Ran-GTP enriched around the chromatin, and reducing in concentration away from it (Kaláb, Weis and Heald, 2002; Kaláb et al., 2006). Ran-GTP promotes spindle assembly by activating spindle assembly factors, most notably TPX2 (Figure 3.1) (Gruss et al., 2001, 2002). TPX2 then activates Aurora A kinase which phosphorylates the γTuRC adaptor protein NEDD1 and initiates MT nucleation (Pinyol, Scrofani and Vernos, 2013; Scrofani et al., 2015). Perturbations of Ran-GTP in Xenopus extracts result in loss of MT density in spindles, but do not show a great impact on spindle structure in centrosome containing mitotic cells (Kaláb et al., 2006). This hints that Ran-GTP pathway might be more important in acentrosomal spindle assembly than the one relying on centrosomes.
Chromosomal passenger complex (CPC) pathway
A second chromatin associated pathway of spindle assembly, which can operate separately of the Ran-GTP pathway, is called the CPC pathway (Maresca et al., 2009). The CPC consists of (human/fission yeast) INCENP/Pic1, Aurora B/Ark1, Survivin/Bir1/Cut17 and Borealin/Nlb1 (Carmena et al., 2012; van der Waal et al., 2012). The mechanism of CPC-dependent assembly pathway is well explored in Xenopus, and involves Aurora B-dependent inhibition of MT-destabilizing agents, which in turn creates an environment where MT assembly is promoted (Figure 3.1) (Andrews et al., 2004; Lan et al., 2004; Sampath et al., 2004; Gadea and Ruderman, 2006; Kelly et al., 2007).
In Drosophila oocytes, CPC pathway is essential for acentrosomal spindle assembly, as its suppression prevents MI spindle assembly (Colombié et al., 2008; Radford, Jang and McKim, 2012). In C. elegans, CPC was found to contribute to chromosome alignment in meiosis, the release of cohesion, and the proper assembly of the meiotic (Kaitna et al., 2002; Rogers et al., 2002; Wignall and Villeneuve, 2009; Dumont, Oegema and Desai, 2010). As Ran-GTP, inactivation of the CPC-pathway through INCENP knockdown, or chemically by inactivating Aurora B/C, did not abolish MI spindle assembly in mouse oocytes, but it did provoke chromosome misalignment at meiotic metaphase (Shuda et al., 2009; Sharif et al., 2010).
Table of contents :
Table of contents
1. Cell division
2. Spindle components
2.1. Microtubules (MTs)
2.1.1. Kinetochore microtubules (KT-MTs)
2.1.2. Interpolar MTs (iMTs)
2.1.3. Astral MTs (aMTs)
2.2. Centrosome and spindle pole body (SPB)
2.3. Chromosomes and KTs
2.3.1. Chromokinesins & polar ejection force (PEF)
2.4. Motor proteins
2.5. Non-motor microtubule associated proteins (MAPs)
3. Non-centrosomal pathways of spindle assembly
3.1. Ran-GTP pathway.
3.2. Chromosomal passenger complex (CPC) pathway
3.3. Acentriolar MTOCs (aMTOCs)
3.4. Augmin pathway
4. Fission yeast as model system for analysing mitotic and meiotic spindle dynamics
Mitotic and meiotic spindle dynamics comparison in fission yeast
4.1. Phase I – Initial stages of spindle nucleation
4.2. Phase I – Establishment of a bipolar spindle
4.3. PhaseI/phaseII – Chromosome attachment to the spindle and congression
4.4. Phase II – Spindle forces and force-balance maintenance in fission yeast
4.5. Phase III – Final spindle elongation
4.6. Comparison of mitotic and meiotic spindle dynamics in fission yeast
Aim of this work
1.1. Cell division
1.1.1. La mitose
1.1.2. La Méiose
1.2. La levure fissipare S. pombe comme système modèle pour l’analyse de la dynamique du fuseau mitotique et méiotique
1.2.1. Comparaison de la dynamique du fuseau mitotique et méiotique
1.2.2. Le double mutant de délétion kinésine-5 /kinésine-14 (cut7Δpkl1Δ) comme outil permettant la comparaison des fuseaux mitotiques et méiotiques
2.1. La Dynamique du fuseau diffère en mitose et en méiose dans la levure fissipare
2.2. L’intégrité du fuseau est compromise spécifiquement en MI dans les zygotes du double mutant cut7Δpkl1Δ
2.3. Le ratio Cut7-à-Pkl1 est plus élevé dans le fuseau MI qu’en mitose
2.4. La fonction de la kinésine 14 Klp2 exercée sur le fuseau est distincte en MI et en mitose
2.5. La suppression de la dynamique des MTs restaurela bipolarité du fuseau de MI dans les zygotes cut7Δpkl1Δ