Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

The generation of two daughter cells with the same genetic information requires error-free chromosome segregation during mitosis. Chromosome transmission fidelity is dependent on spindle structure/function, which requires Asp1 in the fission yeast Asp1 belongs to the diphosphoinositol pentakisphosphate kinase (PPIP5K)/Vip1 family which generates high-energy inositol pyrophosphate (IPP) molecules. Here, we show that Asp1 is a bifunctional enzyme: Asp1 kinase generates specific IPPs which are the substrates of the Asp1 pyrophosphatase. Intracellular levels of these IPPs directly correlate with microtubule stability: pyrophosphatase loss-of-function mutants raised Asp1-made IPP levels 2-fold, thus increasing microtubule stability, while overexpression of the pyrophosphatase decreased microtubule stability. Absence of Asp1-generated IPPs resulted in an aberrant, increased spindle association of the kinesin-5 family member Cut7, which led to spindle collapse. Thus, chromosome transmission is controlled via intracellular IPP levels. Intriguingly, identification of the mitochondrion-associated Met10 protein as the first pyrophosphatase inhibitor revealed that IPPs also regulate mitochondrial distribution.

Cells are regularly exposed to stress conditions that may lead to protein misfolding. To cope with this challenge, molecular chaperones selectively target structurally perturbed proteins for degradation via the ubiquitin-proteasome pathway. In mammals the co-chaperone BAG-1 plays an important role in this system. BAG-1 has two orthologues, Bag101 and Bag102, in the fission yeast Schizosaccharomyces pombe. We show that both Bag101 and Bag102 interact with 26S proteasomes and Hsp70. By epistasis mapping we identify a mutant in the conserved kinetochore component Spc7 (Spc105/Blinkin) as a target for a quality control system that also involves, Hsp70, Bag102, the 26S proteasome, Ubc4 and the ubiquitin-ligases Ubr11 and San1. Accordingly, chromosome missegregation of spc7 mutant strains is alleviated by mutation of components in this pathway. In addition, we isolated a dominant negative version of the deubiquitylating enzyme, Ubp3, as a suppressor of the spc7-23 phenotype, suggesting that the proteasome-associated Ubp3 is required for this degradation system. Finally, our data suggest that the identified pathway is also involved in quality control of other kinetochore components and therefore likely to be a common degradation mechanism to ensure nuclear protein homeostasis and genome integrity.

Spc7, a member of the conserved Spc105/KNL-1 family of kinetochore proteins, was identified as an interaction partner of the EB1 homologue Mal3. Spc7 associates with the central centromere region of the chromosome but does not affect transcriptional silencing. Here, we show that Spc7 is required for the integrity of the spindle as well as for targeting of MIND but not of Ndc80 complex components to the kinetochore. Spindle defects in spc7 mutants were severe ranging from the inability to form a bipolar spindle in early mitosis to broken spindles in midanaphase B. spc7 mutant phenotypes were partially rescued by extra alpha-tubulin or extra Mal2. Thus, Spc7 interacts genetically with the Mal2-containing Sim4 complex.

Chromosome transmission fidelity during mitosis is of critical importance for the fitness of an organism, as mistakes will lead to aneuploidy, which has a causative role in numerous severe diseases. Proper segregation of chromosomes depends on interdependent processes at the microtubule-kinetochore interface and the spindle assembly checkpoint. Here we report the discovery of a new element essential for chromosome transmission fidelity that implicates inositol pyrophosphates (IPPs) as playing a key role in this process. The protein is Asp1, the Schizosaccharomyces pombe member of the highly conserved Vip1 family. Vip1 enzymes are bifunctional: they consist of an IPP-generating kinase domain and a pyrophosphatase domain that uses such IPPs as substrates. We show that Asp1 kinase function is required for bipolar spindle formation. The absence of Asp1-generated IPPs resulted in errors in sister chromatid biorientation, a prolonged checkpoint-controlled delay of anaphase onset, and chromosome missegregation. Remarkably, expression of Asp1 variants that generated higher-than-wild-type levels of IPPs led to a fasterthan-wild-type entry into anaphase A without an increase in chromosome missegregation. In fact, the chromosome transmission fidelity of a nonessential chromosome was enhanced with increased cellular IPPs. Thus, we identified an element that optimized the wild-type chromosome transmission process. In eukaryotes, the faithful transmission of genetic information from one generation to the next is tightly controlled. Genome instability generally has a strong negative impact on the fitness of an organism. Decreased chromosome segregation fidelity during cell division will give rise to cell progeny which have lost or gained chromosomes. Aneuploidy is a distinguishing feature of many types of cancer and neurological diseases and has been implicated as an important factor in the aging process (1, 2). It is a poorly understood paradox that genome plasticity can also be advantageous for the propagation of cells or an organism. Aneuploid tumor cells have a proliferative advantage, and large-scale chromosome changes are a common feature of human-pathogenic fungi such as Candida. In the latter case, these alterations facilitate an adaption to the environment resulting in increased survival (3).Chromosome segregation is a precise process: in model yeasts such as the fission yeast Schizosaccharomyces pombe where in vivo chromosome loss rates can be determined easily, it is approximately 0.01% (reviewed in reference 4). Chromosome transmission fidelity depends on the formation and function of the bipolar spindle, the multicomponent kinetochore complex assembled on centromeric chromatin, and the correct connections between these two structures. Biorientation of sister chromatids requires the binding of plus ends of spindle microtubules (MTs) to attachment sites on the kinetochore in such a way that sisters are bound to MTs polymerized from opposing spindle poles. Incorrect attachments are common and are corrected by the Aur...