Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes [1][2][3][4][5][6][7][8][9] . Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore-microtubule attachment using quantitative fluorescence microscopy of GFPtagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone 1 , the copy number ranges from one or two for inner kinetochore proteins such as Mif2p 2 , to 16 for the 9 at the kinetochoremicrotubule interface. These counts allow us to visualize the overall arrangement of a kinetochoremicrotubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments.Accurate segregation of sister chromosomes during mitosis depends on the assembly of structural proteins at the kinetochore that link spindle microtubule plus-ends to centromeric DNA (CEN DNA). The structural arrangement of these proteins within the kinetochore underlies its function in force generation. It may also influence how the spindle assembly checkpoint senses kinetochore-microtubule attachment, and how errors in attachment are corrected to prevent chromosome mis-segregation. Although serial-section transmission electron microscopy has revealed the overall three-dimensional architecture of vertebrate kinetochores, the structure of individual kinetochore-microtubule attachment remains poorly characterized. Consequently, a mechanistic model of kinetochore function that integrates the details of its structure cannot currently be constructed. To understand the molecular architecture of a kinetochore-microtubule attachment site, we focused on counting the copy number for the core structural kinetochore proteins and protein complexes that are necessary for stable kinetochore-microtubule attachment. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Localization of antibodies to Ndc80 in vertebrate cells suggests that the Ndc80p-Nuf2p end of the NDC80 complex localizes proximal to the microtubule attachment site, whereas the other end localizes proximal to the inner centromere7 , 15. In budding yeast, the NDC80 complex and the microtubule associated protein complex, DAM-DASH, are both necessary for microtubule attachment10 , 11. The DAM-DASH complex is a heterodecamer and contains the protein Ask1p. Purified DAM-DASH complexes assemble into rings aro...

Abstract. We have used time-lapse digital-and videoenhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-13-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex; with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

Dynein motor isoforms have been implicated as potential kinetochore-associated motors that power chromosome-to-pole movements during mitosis. The recent identification and sequence determination of genes encoding dynein isoforms has now permitted the in vivo analysis of dynein function in mitosis. In this report we describe the identification and mutational analysis of the gene, DHCI, encoding a dynein heavy chain isoform in Saccharomyces cerevisiae. Sequence analysis of a 9-kb genomic fragment of the DHCI gene predicts a polypeptide highly homologous to dynein sequences characterized from sea urchin, Dictyostelium, Drosophila, and rat.Mutations in the yeast dynein gene disrupt the normal movement of the spindle into budding daughter cells but have no apparent effect on spindle assembly, spindle elongation, or chromosome segregation. Our results suggest that, in yeast, a dynein microtubule motor protein has a nonessential role in spindle assembly and chromosome movement but is involved in establishing the proper spindle orientation during cell division.The major structural and mechanistic features of chromosome segregation on the mitotic spindle appear to be largely conserved between the yeast Saccharomyces cerevisiae and higher eukaryotes. Microtubules nucleated by duplicated spindle pole organelles interact with one another and with the sister kinetochores of duplicated chromosomes to establish a bipolar spindle and ensure the equal division of genetic material to opposite spindle poles. The final goal of generating two cells from one involves the mechanism by which the separated chromosomal complements are placed into two separate cells. In this regard, the similarity between yeast and higher eukaryotes is less clear. In yeast, the process of chromosome segregation takes place entirely within the nucleus. The placement of the segregated chromosomal complements, one set of sister chromatids in the mother cell and the other set into the daughter cell, requires the directed movement of the intranuclear spindle into the growing bud of the daughter cell.

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.

Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.