Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

Yeh, Elaine; Skibbens, Robert V.; Cheng, Judy W.; Salmon, Edward D.; Bloom, Kerry

doi:10.1083/jcb.130.3.687

Cited by 362 publications

(422 citation statements)

References 24 publications

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“…Flared MT ends were also present in late anaphase cells, where the MTs are only slightly longer than they are wide ( Figure 7C, right). The plus ends of cytoplasmic MTs were not examined, because these MTs tend to be quite long (Yeh et al, 1995) and were not present in the volume of the original thick section. Figure 8 shows representative 3-D models from four haploid cells in various stages of spindle formation.…”

Section: Hvem Tomography Of Yeast Spindlesmentioning

confidence: 99%

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

O’Toole¹,

Winey

McIntosh³

1999

MBoC

266

267

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The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5-10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ϳ150 nm. MTs growing from duplicated but not separated SPBs have a median length of ϳ130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ϳ300 nm and then decreases to ϳ30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells. INTRODUCTIONMicrotubule-organizing centers (MTOCs) nucleate and anchor microtubules (MTs) during cell differentiation and division (for review, see Brinkley, 1985;Rose et al., 1993;Kellogg et al., 1994;Pereira and Schiebel, 1997). Although MTOCs vary widely in structure, their functions are largely conserved. In metazoa, the principal MTOC is the centrosome, which organizes both the interphase MTs and those of the mitotic spindle. In the yeast Saccharomyces cerevisiae, this role is served by the spindle pole body (SPB), a complex and dynamic organelle that undergoes significant structural changes during the yeast cell cycle (for review, see Winey and Byers, 1993;Kilmartin, 1994;Snyder, 1994). Nuclear MTs, which form the meiotic or mitotic spindles, attach to an inner plaque of the SPB, whereas cytoplasmic MTs, important for nuclear movements and position, are attached to an outer plaque (Moens and Rapport, 1971;Byers and Goetsch, 1974, 1975;Rabinow and Marak, 1966). Recent studies of isolated SPBs by cryomicroscopy and electron tomography have shown that SPBs are organized from six major layers, including a central crystalline core that contains the SPC42 gene product (Bullitt et al., 1997).Duplication of the SPB during the cell cycle begins with the formation of a "satellite" on the cytoplasmic face of the half-bridge, a specialized region of the nuclear envelope that lies immediately adjacent to the existing SPB. By processes not yet described in detail, the satellite develops into a...

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Section: Hvem Tomography Of Yeast Spindlesmentioning

confidence: 99%

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

O’Toole¹,

Winey

McIntosh³

1999

MBoC

266

267

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“…DNA was stained with 1 g/ml bisBenzimide (Sigma, St. Louis, MO). For time-lapse microscopy, cells growing exponentially in liquid SC-Ura medium were spotted onto a microscope slide spread with a thin layer of SC-Ura medium solidified with 25% gelatin (Yeh et al, 1995). Individual cells were followed at 15-min intervals using a computer-controlled microscope (Eclipse E800; Nikon, Tokyo, Japan) with motorized focus and a high-resolution CCD camera (model C4742-95; Hamamatsu Photonics, Bridgewater, NJ).…”

Section: Microscopymentioning

confidence: 99%

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

Caviston

Longtine

Pringle

et al. 2003

MBoC

227

281

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The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42 V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42 V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth. INTRODUCTIONThe septin family of proteins was first recognized in yeast and now appears to be ubiquitous in fungi and animals, although not in plants (Longtine et al., 1996;Field and Kellogg, 1999;Trimble, 1999;Nguyen et al., 2000;Momany et al., 2001;Macara et al., 2002). Typical septins have a variable N-terminal region, a conserved core that includes the elements of a GTP-binding site, and a variable C-terminal region that (in all but a few cases) includes a predicted coiled-coil domain. In each species examined to date, there are two or more septins that form heterooligomeric complexes to perform their specific functions. Purified septins from yeast, Drosophila, Xenopus, and mammalian cells form filaments of 7-9 nm diameter and of variable length (Field et al., 1996;Frazier et al., 1998;Hsu et al...

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“…Cells were grown to midlog phase and mounted as described previously (Yeh et al, 1995;Maddox et al, 2000) using SD-complete media (0.67% yeast nitrogen base without amino acids, 2% glucose, 0.5% casamino acids), and 50 g/ml each uracil, tryptophan, and adenine. The equipment and techniques for imaging GFP-fusion proteins have been described in detail (Shaw et al, 1997a).…”

Section: In Vivo Microtubule Dynamics: Microscopic Imagingmentioning

confidence: 99%

β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

Gupta

Bode

Thrower

et al. 2002

MBoC

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Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast ␤-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

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Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

Cited by 362 publications

References 24 publications

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

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