Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.

During mitosis, spindle microtubule force is balanced by the combined activities of the cohesin and condensin SMC complexes and intramolecular pericentric chromatin loops.

The C loop conformation reveals the structural basis for sister-kinetochore clustering in budding yeast and for kinetochore biorientation and thus resolves the paradox of maximal interstrand separation in regions of highest cohesin concentration.

Summary During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.

SummaryThe Ndc80 complex (Ndc80, Nuf2, Spc24, Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus-ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule and MAP binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus-end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule binding interactions with Ndc80 and Dam1 complexes.