Chromosome arm length and nuclear constraints determine the dynamic relationship of yeast subtelomeres

Physical interactions between distinct chromosomal genomic loci are important for genomic functions including recombination and gene expression, but the mechanisms by which these interactions occur remain obscure. Using telomeric association as a model system, we analyzed here the in vivo organization of chromosome ends of haploid yeast cells during interphase. We separately labeled most of the 32 subtelomeres and analyzed their positions both in nuclear space and relative to three representative reference subtelomeres by high-throughput 3D microscopy and image processing. We show that subtelomeres are positioned nonrandomly at the nuclear periphery, depending on the genomic size of their chromosome arm, centromere attachment to the microtubule organizing center (spindle pole body, SPB), and the volume of the nucleolus. The distance of subtelomeres to the SPB increases consistently with chromosome arm length up to ≈300 kb; for larger arms the influence of chromosome arm length is weaker, but the effect of the nucleolar volume is stronger. Distances between pairs of subtelomeres also exhibit arm-length dependence and suggest, together with dynamic tracking experiments, that potential associations between subtelomeres are unexpectedly infrequent and transient. Our results suggest that interactions between subtelomeres are nonspecific and instead governed by physical constraints, including chromosome structure, attachment to the SPB, and nuclear crowding.

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Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae ofAshbya gossypii

Molecular Biology of the Cell ◽

10.1091/mbc.e09-01-0063 ◽

2010 ◽

Vol 21 (1) ◽

pp. 18-28 ◽

Cited By ~ 29

Author(s):

Claudia Lang ◽

Sandrine Grava ◽

Tineke van den Hoorn ◽

Rhonda Trimble ◽

Peter Philippsen ◽

...

Keyword(s):

Spindle Pole Body ◽

Hyphal Growth ◽

Spindle Pole ◽

Microtubule Organizing Center ◽

Microtubule Nucleation ◽

Cytoplasmic Microtubule ◽

Organizing Center ◽

Cytoplasmic Side ◽

Independent Mode

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

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Telomeric chromosome ends are highly mobile and behave like free double-strand DNA breaks

10.1101/720821 ◽

2019 ◽

Author(s):

Mathias Toulouze ◽

Assaf Amitai ◽

Ofir Shukron ◽

David Holcman ◽

Karine Dubrana

Keyword(s):

Nuclear Periphery ◽

Spindle Pole Body ◽

Strand Break ◽

Spindle Pole ◽

Dna Breaks ◽

Computational Approaches ◽

Pole Body ◽

Double Strand Dna Breaks ◽

Chromosome Arm ◽

Approaches To Study

AbstractChromosome organization and dynamics are critical for DNA transactions, including gene expression, replication, and DNA repair. In yeast, the chromosomes are anchored through their centromeres to the spindle pole body, and their telomeres are grouped into clusters at the nuclear periphery, constraining chromosome mobility. Here, we have used experimental and computational approaches to study the effects of chromosome-nuclear envelope (NE) attachments on the dynamics of S. cerevisiae chromosomes. We found that although centromere proximal loci were, as predicted, more dynamically constrained than distal loci, telomeres were highly mobile, even when positioned at the nuclear periphery. Polymer modeling indicated that polymer ends are intrinsically more mobile than internal sites. We tested this model by measuring the mobility of a double strand break (DSB) end within a chromosome arm. Upon separation of the DSB ends, their mobility significantly increased. Altogether, our results reveal that telomeres behave as highly mobile polymer ends, despite interactions with the nuclear membrane.

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In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis

Journal of Cell Science ◽

10.1242/jcs.114.14.2627 ◽

2001 ◽

Vol 114 (14) ◽

pp. 2627-2640 ◽

Cited By ~ 1

Author(s):

Anabelle Decottignies ◽

Patrick Zarzov ◽

Paul Nurse

Keyword(s):

Fission Yeast ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Nuclear Periphery ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cyclin Dependent Kinase ◽

Spindle Pole Bodies ◽

Mitosis And Meiosis

We investigated the in vivo localisation of fission yeast cyclin-dependent kinase cdc2p during mitosis and meiosis. Fusion to yellow fluorescent protein (YFP) revealed that cdc2-YFP is present in the cytoplasm at all stages of the cell cycle. Nuclear cdc2-YFP fluorescence oscillates with that of cdc13-YFP cyclin. At G1/S, at least one of cdc13p, cig1p or cig2p B-type cyclins is required for the accumulation of cdc2-YFP into the nucleus. Cdc2-YFP and cdc13-YFP are highly enriched on the spindle pole body of cells in late G2 or arrested at S phase. Both accumulate on the spindle pole bodies and the spindle in prophase and metaphase independently of the microtubule-associated protein dis1p. In anaphase, the cdc2p/cdc13p complex leaves the spindle prior to sister chromatid separation, and cdc13-YFP is enriched at the nuclear periphery before fluorescence disappears. If cdc13p cannot be recognized by the anaphase-promoting complex, cdc2-YFP and cdc13-YFP remain associated with the spindle. In mating cells, cdc2-YFP enters the nucleus as soon as the cells undergo fusion. During karyogamy and meiotic prophase, cdc2-YFP is highly enriched on the centromeres. In meiosis I, association of cdc2-YFP with the spindle and the spindle pole bodies shows differences to mitotic cells, suggesting different mechanisms of spindle formation. This study suggests that changes in cdc2p localisation are important for both mitosis and meiosis regulation.

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Nuclear Export Receptor Xpo1/Crm1 Is Physically and Functionally Linked to the Spindle Pole Body in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.02043-07 ◽

2008 ◽

Vol 28 (17) ◽

pp. 5348-5358 ◽

Cited By ~ 9

Author(s):

Anja Neuber ◽

Jacqueline Franke ◽

Angelika Wittstruck ◽

Gabriel Schlenstedt ◽

Thomas Sommer ◽

...

Keyword(s):

Nuclear Export ◽

Budding Yeast ◽

Nuclear Export Signal ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Organizing Center ◽

Functional Link ◽

Pole Body ◽

Nuclear Export Receptor

ABSTRACT The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the γ-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.

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A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2201 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2201-2216 ◽

Cited By ~ 64

Author(s):

Thu Nguyen ◽

Dani B.N. Vinh ◽

Douglas K. Crawford ◽

Trisha N. Davis

Keyword(s):

Spindle Pole Body ◽

Spindle Pole ◽

Amino Terminus ◽

Temperature Sensitive ◽

Microtubule Organizing Center ◽

Synthetic Lethal ◽

Lethal Phenotype ◽

Amino Terminal ◽

N Terminus ◽

Two Hybrid

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations inSPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, thespc97 alleles are synthetic lethal withspc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

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Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/137.2.407 ◽

1994 ◽

Vol 137 (2) ◽

pp. 407-422 ◽

Cited By ~ 8

Author(s):

E A Vallen ◽

W Ho ◽

M Winey ◽

M D Rose

Keyword(s):

Copy Number ◽

Gene Dosage ◽

Spindle Pole Body ◽

Direct Interaction ◽

Spindle Pole ◽

High Copy Number ◽

Temperature Sensitive ◽

Microtubule Organizing Center ◽

Recessive Mutations ◽

Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

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Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.114.3.515 ◽

1991 ◽

Vol 114 (3) ◽

pp. 515-532 ◽

Cited By ~ 78

Author(s):

M Snyder ◽

S Gehrung ◽

B D Page

Keyword(s):

Cell Cycle ◽

Cell Polarity ◽

Spindle Pole Body ◽

Spindle Pole ◽

Yeast Cells ◽

Cell Cycle Distribution ◽

Restrictive Temperature ◽

Microtubule Bundle ◽

Pole Body ◽

Capture Site

The establishment of cell polarity was examined in the budding yeast, S. cerevisiae. The distribution of a polarized protein, the SPA2 protein, was followed throughout the yeast cell cycle using synchronized cells and cdc mutants. The SPA2 protein localizes to a patch at the presumptive bud site of G1 cells. Later it concentrates at the bud tip in budded cells. At cytokinesis, the SPA2 protein is at the neck between the mother and daughter cells. Analysis of unbudded haploid cells has suggested a series of events that occurs during G1. The SPA2 patch is established very early in G1, while the spindle pole body residues on the distal side of the nucleus. Later, microtubules emanating from the spindle pole body intersect the SPA2 crescent, and the nucleus probably rotates towards the SPA2 patch. By middle G1, most cells contain the SPB on the side of the nucleus proximal to the SPA2 patch, and a long extranuclear microtubule bundle intersects this patch. We suggest that a microtubule capture site exists in the SPA2 staining region that stabilizes the long microtubule bundle; this capture site may be responsible for rotation of the nucleus. Cells containing a polarized distribution of the SPA2 protein also possess a polarized distribution of actin spots in the same region, although the actin staining is much more diffuse. Moreover, cdc4 mutants, which form multiple buds at the restrictive temperature, exhibit simultaneous staining of the SPA2 protein and actin spots in a subset of the bud tips. spa2 mutants contain a polarized distribution of actin spots, and act1-1 and act1-2 mutants often contain a polarized distribution of the SPA2 protein suggesting that the SPA2 protein is not required for localization of the actin spots and the actin spots are not required for localization of the SPA2 protein. cdc24 mutants, which fail to form buds at the restrictive temperature, fail to exhibit polarized localization of the SPA2 protein and actin spots, indicating that the CDC24 protein is directly or indirectly responsible for controlling the polarity of these proteins. Based on the cell cycle distribution of the SPA2 protein, a "cytokinesis tag" model is proposed to explain the mechanism of the non-random positioning of bud sites in haploid yeast cells.

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Redistribution of centrosomal proteins by centromeres and Polo kinase controls nuclear envelope breakdown

10.1101/2020.12.01.406553 ◽

2020 ◽

Author(s):

Andrew J. Bestul ◽

Zulin Yu ◽

Jay R. Unruh ◽

Sue L. Jaspersen

Keyword(s):

Nuclear Envelope ◽

Spindle Pole Body ◽

Spindle Pole ◽

Ring Structure ◽

Yeast Cells ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Spindle Formation ◽

Nuclear Envelope Breakdown ◽

Polo Kinase

AbstractProper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB – yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy (SIM), we show that the conserved SUNprotein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for SPB ring protein redistribution and for complete NEBD to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate NEBD and spindle formation through building of an SPB ring structure.SummaryNuclear envelope breakdown is necessary for fission yeast cells to go through mitosis. Bestul et al. show that the SUN protein, Sad1, is vital in carrying out this breakdown and is regulated by the centromere and Polo kinase.

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Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes in fission yeast

Journal of Cell Science ◽

10.1242/jcs.114.13.2427 ◽

2001 ◽

Vol 114 (13) ◽

pp. 2427-2435 ◽

Cited By ~ 2

Author(s):

Bunshiro Goto ◽

Koei Okazaki ◽

Osami Niwa

Keyword(s):

Fission Yeast ◽

De Novo ◽

Nuclear Periphery ◽

Spindle Pole Body ◽

Spindle Pole ◽

Limited Area ◽

Cytoplasmic Microtubule ◽

Random Arrangement ◽

Triplex Structure ◽

Microtubular System

Chromosomes are not packed randomly in the nucleus. The Rabl orientation is an example of the non-random arrangement of chromosomes, centromeres are grouped in a limited area near the nuclear periphery and telomeres are located apart from centromeres. This orientation is established during mitosis and maintained through subsequent interphase in a range of species. We report that a Rabl-like configuration can be formed de novo without a preceding mitosis during the transition from the sexual phase to the vegetative phase of the life cycle in fission yeast. In this process, each of the dispersed centromeres is often associated with a novel Sad1-containing body that is contacting a cytoplasmic microtubule laterally (Sad1 is a component of the spindle pole body (SPB)). The Sad1-containing body was colocalized with other known SPB components, Kms1 and Spo15 but not with Cut12, indicating that it represents a novel SPB-related complex. The existence of the triplex structure (centromere-microtubule-Sad1 body) suggests that the clustering of centromeres is controlled by a cytoplasmic microtubular system. Accordingly, when microtubules are destabilized, clustering is markedly reduced.

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Reorientation of Mispositioned Spindles in Short Astral Microtubule Mutantspc72Δ Is Dependent on Spindle Pole Body Outer Plaque and Kar3 Motor Protein

Molecular Biology of the Cell ◽

10.1091/mbc.01-07-0338 ◽

2002 ◽

Vol 13 (4) ◽

pp. 1366-1380 ◽

Cited By ~ 16

Author(s):

Dominic Hoepfner ◽

Florian Schaerer ◽

Arndt Brachat ◽

Achim Wach ◽

Peter Philippsen

Keyword(s):

Spindle Pole Body ◽

Time Lapse ◽

Nuclear Migration ◽

Spindle Pole ◽

Astral Microtubule ◽

Spindle Pole Bodies ◽

Microtubule Motor ◽

Microtubule Formation ◽

Mother Cells

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic γ-tubulin complex, can only generate very short (<1 μm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However,SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.

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