Kerry Bloom

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

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.

Molecular architecture of a kinetochore-microtubule attachment site

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. 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 GFP-tagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone, the copy number ranges from one or two for inner kinetochore proteins such as Mif2p, to 16 for the DAM–DASH complex at the kinetochore–microtubule interface. These counts allow us to visualize the overall arrangement of a kinetochore–microtubule 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.

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to α-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.

Genetic manipulation of centromere function.

A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL1 promoter from S. cerevisiae. By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucose-carbon source) from the GAL1 promoter, centromere function can be switched off or on, respectively. With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids. Moreover, it was possible to selectively induce the loss of a single chromosome from a mitotically dividing population of cells. We observed that the induction of chromosome III aneuploidy resulted in a dramatic change in cell morphology. The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element.

In vivo protein architecture of the eukaryotic kinetochore with nanometer scale accuracy.

The kinetochore is a macromolecular protein machine [1] that links centromeric chromatin to the plus ends of one or more microtubules (MTs) and segregates chromosomes during cell division. Its core structure consists of eight multicomponent protein complexes, most of which are conserved in all eukaryotes. We use an in vivo two-color fluorescence microscopy technique to determine, for the first time, the location of these proteins along the budding yeast kinetochore axis at nanometer resolution. Together with kinetochore protein counts [2, 3], these localizations predict the 3D protein architecture of a metaphase kinetochore-microtubule attachment and provide new functional insights. We also find that the kinetochore becomes much shorter in anaphase as metaphase tension is lost. Shortening is due mainly to a decrease in the length of the Ndc80 complex, which may result either from intramolecular bending of the Ndc80 complex at the kink within the stalk region of the Ndc80-Nuf2 dimer [4, 5] or from a change in its orientation relative to the microtubule axis. Conformational changes within the Ndc80 and Mtw1 complexes may serve as mechanical cues for tension-dependent regulation of MT attachment and the spindle-assembly checkpoint. The geometry of the core structure of the budding yeast kinetochore reported here is remarkably similar to that found in mammalian kinetochores, indicating that kinetochore structure is conserved in eukaryotes with either point or regional centromeres.

Stable Kinetochore-Microtubule Attachment Constrains Centromere Positioning in Metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.

Pericentric chromatin is organized into an intramolecular loop in mitosis.

BACKGROUND Cohesin proteins link sister chromatids and provide the basis for tension between bioriented sister chomatids in mitosis. Cohesin is concentrated at the centromere region of the chromosome despite the fact that sister centromeres can be separated by 800 nm in vivo. The function of cohesin at sites of separated DNA is unknown. RESULTS We provide evidence that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA adopts an intramolecular loop, whereas sister-chromatid arms are paired intermolecularly. Visualization of cohesin subunits by fluorescence microscopy revealed a cylindrical structure that encircles the central spindle and spans the distance between sister kinetochores. Kinetochore assembly at the apex of the loop initiates intrastrand loop formation that extends approximately 25 kb (12.5 kb on either side of the centromere). Two centromere loops (one from each sister chromatid) are stretched between the ends of sister-kinetochore microtubules along the spindle axis. At the base of the loop there is a transition to intermolecular sister-chromatid pairing. CONCLUSIONS 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.

Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome

Quantitative measurement of the number of Cse4, CBF3, and Ndc80 proteins at kinetochores reveals a 2.5–3-fold increased copy number relative to prior estimates.

Dynamic Microtubules Lead the Way for Spindle Positioning

Coordination between the asymmetric partitioning of cell-fate determinants and equal partitioning of genetic material is crucial to the generation of diverse cell types in a developing organism, and to the maintenance of genomic integrity. The emerging model is of a highly organized and dynamic cellular landscape, the form of which is defined by polarized signals within the cell. Cytoskeletal elements are necessary to generate this landscape and to provide motive forces for proper spindle positioning. These forces are generated by interactions between microtubules and the cell cortex.

Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres

Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site.