The tethered infinitesimal tori and spheres algorithm: a versatile calculator for axisymmetric problems in equilibrium membrane mechanics. suggests that kinesin-8Cinduced effects on microtubule dynamics, kinetochore attachment stability, and sliding push in the spindle can explain the aberrant chromosome motions and spindle size fluctuations seen. INTRODUCTION Kinesin-8 proteins are engine enzymes that can alter microtubule dynamics (Messin and Millar, 2014 ). Users of the kinesin-8 family include Kip3 in budding candida (DeZwaan (Western (Pereira kinesin-8 (Grissom allele, SPBs labeled with (Yamamoto SPL-707 and Hiraoka, 2003 ). This was our wild-type strain, to which we added deletions of -tubulin allele indicated under a fragile promoter (Yamagishi are different with = 2.3 10?6. We observed hovering in 30% of all kinesin-8 deletion mutant cells; using the Pearson chi-square test for proportions, the wild-type and mutant populations are different with = 4.7 10?4. In the same cell populations, we recorded whether KC reeling in to the SPB experienced occurred at 5-min intervals from 0 SPL-707 to 20 min after temp shift (Number 2K). During initial imaging of cells at 18C, we observed a lost KC in 30C50% of cells. For 5?6+ and 5?6? cells, a larger initial portion of uncaptured KCs was visible compared with crazy type and 5+6?. Earlier work found that KC-MT attachment occurs approximately exponentially in time (Kalinina = 2.6 10?5). We used the two-sample test to compare rate measurements for each pair of strains and found strong, statistically significant variations for crazy type versus 5?6+ (= 4 10?4) and 5+6? versus 5?6+ (= 2.6 10?5) and weaker but significant variations for 5+6? versus 5?6? (= 1.4 10?2) and 5?6+ versus 5?6? (= 3.6 10?2). These results suggest both that kinesin-8 deletion can alter the speeds of reeling motions and that different types of kinesin-8 deletion lead to different speeds SPL-707 of reeling motions. Klp5-null strains occasionally displayed tripolar mitotic spindles Our experimental results showing variations in chromosome SPL-707 motions in 5?6+ versus 5+6? were surprising because earlier work found related mitotic phenotypes for deletion of either Klp5 or 6 (Western cold-sensitive tubulin, low-level MT labeling with under a fragile promoter, SPBs labeled with and and present (crazy type), erased (5?6+), and deleted (5+6?). After chilly treatment and subsequent rewarming within the microscope, these cells showed similar phenotypes to the people observed with our unique tagging strategy. Chilly treatment regularly led to lost chromosomes, which were recaptured to allow mitosis to continue. Spindle size instability occurred in kinesin-8 deletion mutants but not in wild-type cells. We observed aberrant chromosome pushing motions in 5?6+ cells (Number 3A and Supplemental Movies S9 and S10) but not in wild-type or 5+6? cells. This confirmed that our results were not a tagging artifact. Open in a separate window Number 3: Kinetochore pushing motions and tripolar mitotic spindles. Schematics and images of cells comprising SPBs tagged with sid4-mCherry SPB marker and microtubules tagged with mCherry-atb2 under a fragile promoter (reddish, top), kinetochores tagged with mis6-GFP and mis12-GFP (green, middle), and merged images (bottom), all in the 5?6+ background. (A) Chromosome-pushing motions showing TNFRSF10D KC (arrowhead) near the end of a polar MT. Observe Supplemental Movies S9 and S10. (B) Tripolar mitotic spindle showing KC (arrowhead) colocalized with two bright and one dim SPB. Observe Supplemental Movie S11. (C) Chromosome- pushing motions and tripolar spindle formation in the same cell. Initial images.