Two XMAP215/TOG microtubule polymerases, Alp14 and Dis1, play non-exchangeable, distinct roles in microtubule organisation in fission yeast

Abstract

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of 13 microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally 14 coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule 15 polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, 16Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally 17 collaborate remains undetermined. Here we show the functional interplay and specification of 18 Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in 19 the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found 20 that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, 21 fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal "cut" 22 phenotype. By implementing an artificial targetting system, we have shown that Alp14 and Dis1 are 23 not functionally exchangeable and as such are not merely redundant paralogues. Intriguingly, while 24 Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic 25 specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions 26 of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome 27 stability. 28 Keywords: Fission yeast; microtubule polymerase; XMAP215/TOG; mitotic spindle; spindle pole 29 body; kinetochore 30 31 progression, development and differentiation pathways [1]. During mitosis, spindle MTs assemble 34 to form a bipolar structure that emanates from the two spindle poles. The bipolar spindle functions35as the division apparatus for sister chromatids, generating pulling forces to move them towards 36 opposite poles to ensure equal partition of genetic material. Errors in this process can lead to cell 37 death and/or aneuploidy, a major risk factor for miscarriage, birth defects and tumourigenesis [2, 3]. 38MTs are intrinsically dynamic, a characteristic termed dynamic instability [4, 5]. In a living cell, 39 a cohort of proteins, collectively known as microtubule-associated proteins (MAPs), participate in 40 spindle assembly, stability and maintenance as structural and/or regulatory factors. A conserved 41 family of MAPs, here referred to as the XMAP215/TOG family, is deeply rooted in the eukaryotic 42 branch of the tree of life and arguably the most important regulator in MT organisation [6-9]. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 16 [10], homologues have been identified in virtually all eukaryotic species [9]. The seminal 45 advancement of our understanding of the XMAP215/TOG family was the discovery that XMAP215 46 is a MT polymerase [11]; this protein is capable of incorporating α/β-heterodimers at the plus end of 47 pre-existing MTs. Since then, this activity has been proven for other members of the family from a 48 diverse ra...

Loss of kinesin-8 improves the robustness of the self-assembled spindle in Schizosaccharomyces pombe

“…Loss of Klp6 leads to chromosome segregation defects in an SPB-mediated setting (Fig. S7A), as previously described (Pinder et al, 2019;Syrovatkina et al, 2013;Garcia et al, 2002;West et al, 2002). However, in our system, elimination of Klp6 significantly improved chromosome segregation carried out by selfassembled spindles, probably as a consequence of reinforced spindle structure as well as stabilization of kinetochore-microtubule interaction, which might facilitate kinetochore capture (Fig.…”

“…Most klp6Δ SPB-dependent spindles (84%) segregated parental chromosomes into two masses, i.e., normal segregation, although a minority exhibited three to six masses, consistent with chromosome segregation defects intrinsic to the loss of Klp6 (Fig. S7A, n=69) (Pinder et al, 2019;Syrovatkina et al, 2013;Garcia et al, 2002;West et al, 2001). Self-assembled spindles presented severe segregation defects: nearly a third (32%) of the spindles did not segregate chromosomes, leaving the parental nucleus as a single mass, while another third (36%) segregated chromosomes into up to three to six masses, and the remaining spindles (32%) segregated chromosomes into two masses (Fig.…”

“…S5E " type="url"/> ), and we were unable to film meiosis in this triple mutant. To circumvent this problem, we used the thermosensitive allele alp14-26 ( Yukawa et al, 2019 ). Meiosis analysis cannot be performed at temperatures above 32°C, but analysis of alp14-26 meiosis at a semi-permissive temperature of 28°C showed defects in prophase microtubules length (compare Fig.…”

“…7B, 20', yellow arrowhead, and 70', top yellow arrowhead) as well as lagging chromosomes (Fig. 7B, 70', bottom yellow arrowhead) (Pinder et al, 2019;Syrovatkina et al, 2013). Remarkably, elimination of Klp6 rendered MI and MII self-assembled spindles thicker and brighter than those formed in the klp6 + setting (compare Fig.…”

“…Klp5 and Klp6, the representatives of the kinesin-8 family in fission yeast, are motor proteins that form a heterodimer, Klp5/Klp6, which is involved in regulating microtubule dynamics in interphase and mitosis through diverse activities (Unsworth et al, 2008;Gergely et al, 2016;West and McIntosh, 2008) such as microtubule destabilization. Indeed, deletion of either protein leads to depolymerization-resistant interphase microtubules (Garcia et al, 2002) and promotes spindle microtubule polymerization (Pinder et al, 2019). To assess the potentially improved robustness of the spindle, we quantified the effect of Klp6 elimination according to three parameters: i) spindle formation frequency, ii) maximum spindle length as a readout of structural strength, and iii) chromosome segregation efficiency.…”