Kinesin-5 is required for bipolar spindle assembly; yet in the absence of kinesins-5 and -14, cells can form spindles. In fission yeast, three distinct pathways compensate for their loss. Microtubule polymerase, kinesin-6, and microtubule cross-linker execute individual roles in concert at different mitotic stages in place of the two kinesins.

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...

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal “cut” phenotype. By implementing an artificial targeting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Interestingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal “cut” phenotype. By implementing an artificial targetting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Intriguingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.