scholarly journals Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication.

1996 ◽  
Vol 132 (5) ◽  
pp. 887-901 ◽  
Author(s):  
A D Donaldson ◽  
J V Kilmartin
Keyword(s):  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Spindle Pole ◽  
Normal Function ◽  
Temperature Sensitive ◽  
Pole Body ◽  
Polymeric Layer ◽  
Essential Function ◽  
Mutant Cells

The 42-kD component of the S. cerevisiae spindle pole body (SPB) localizes to the electron-dense central plaque of the SPB. We have cloned the corresponding gene SPC42 (spindle pole component) and show that it is essential. Seven temperature-sensitive (ts) mutants in SPC42 were prepared by error-prone PCR. We found that a change to a proline residue in a potential coiled-coil region of Spc42p was responsible for the ts phenotype in at least three alleles, suggesting that formation of the coiled-coil is essential in normal function. The mutant cells showed a phenotype of predominantly single or bilobed SPBs often with an accumulation of unstructured electron-dense material associated with the bridge structure adjacent to the SPB. This phenotype suggests a defect in SPB duplication. This was confirmed by examining synchronized mutant cells that lose viability when SPB duplication is attempted. Spc42p is a phosphoprotein which shows some cell cycle-regulated phosphorylation. Overexpression of Spc42p causes the formation of a disc- or dome-shaped polymer composed of phosphorylated Spc42p, which is attached to the central plaque and associated with the outer nuclear membrane. Taken together, these data suggest that Spc42p forms a polymeric layer at the periphery of the SPB central plaque which has an essential function during SPB duplication and may facilitate attachment of the SPB to the nuclear membrane.

scholarly journals Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

2003 ◽  
Vol 162 (7) ◽  
pp. 1211-1221 ◽  
Author(s):  
John V. Kilmartin
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Binding Motif ◽  
Temperature Sensitive ◽  
Pole Body ◽  
Human Proteins ◽  
Z Domain ◽  
Essential Function

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.

scholarly journals Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents.

1996 ◽  
Vol 133 (1) ◽  
pp. 111-124 ◽  
Author(s):  
H A Sundberg ◽  
L Goetsch ◽  
B Byers ◽  
T N Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Immunofluorescent Staining ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nonpermissive Temperature ◽  
Carboxy Terminus ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Mutant Cells

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.

scholarly journals Nuf2, a spindle pole body-associated protein required for nuclear division in yeast.

1994 ◽  
Vol 125 (4) ◽  
pp. 853-866 ◽  
Author(s):  
M A Osborne ◽  
G Schlenstedt ◽  
T Jinks ◽  
P A Silver
Keyword(s):  
Nuclear Division ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Temperature Shift ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Culture Cells ◽  
Pole Body ◽  
Associated Proteins

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.

scholarly journals The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.

1995 ◽  
Vol 129 (4) ◽  
pp. 1033-1047 ◽  
Author(s):  
I Hagan ◽  
M Yanagida
Keyword(s):  
Fission Yeast ◽  
Nuclear Membrane ◽  
Essential Gene ◽  
Nuclear Periphery ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Spindle Formation ◽  
Isolation And Characterization ◽  
Pole Body

Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.

scholarly journals A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Masaki Okazaki ◽  
Kazunori Kume ◽  
Ngang Heok Tang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cross Linker ◽  
Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

scholarly journals Requirement of the spindle pole body for targeting and/or tethering proteins to the inner nuclear membrane

Nucleus ◽  
2014 ◽  
Vol 5 (4) ◽  
pp. 352-366 ◽  
Author(s):  
Greetchen Diaz-Muñoz ◽  
Terri A Harchar ◽  
Tsung-Po Lai ◽  
Kuo-Fang Shen ◽  
Anita K Hopper
Keyword(s):  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Inner Nuclear Membrane ◽  
Pole Body

scholarly journals Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication

2011 ◽  
Vol 30 (16) ◽  
pp. 3337-3352 ◽  
Author(s):  
Thomas Kupke ◽  
Leontina Di Cecco ◽  
Hans-Michael Müller ◽  
Annett Neuner ◽  
Frank Adolf ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Inner Nuclear Membrane ◽  
Pole Body

Ultrastructure of mitosis and the spindle pole body cycle in the smut fungus, Tilletia foetida

1985 ◽  
Vol 63 (1) ◽  
pp. 86-96 ◽  
Author(s):  
James A. Hoffmann ◽  
Blair J. Goates
Keyword(s):  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dense Layer ◽  
Double Structure ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
Dense Chromatin ◽  
Tilletia Foetida

The interphase nucleus in secondary sporidia of Tilletia foetida consists of mostly diffuse chromatin, one or two nucleoli, and an area of heterochromatin located opposite an electron-dense, extranuclear spindle pole body (SPB). The interphase SPB is an oval- to bar-shaped, double-structured disc that has a crystallinelike substructure. During nuclear migration into nascent sporidia, SPBs and nucleoli are randomly oriented. At the onset of division, chromatin begins to condense and the SPB becomes located on a nuclear protuberance. Cytoplasmic microtubules terminate at the SPBs and multivesicular bodies surround the SPBs from the early stages of SPB division to early postdivision. SPB discs become spheroid and each develops a medial, dense layer. Then, a basal, dense layer develops and elongates as the SPBs separate and become positioned on opposite sides of the nuclear protuberance. The nuclear membrane opens opposite the SPB during SPB division. The nucleolus is extruded into a nuclear bleb and degenerates. SPBs migrate to opposing sides of the nucleus and become diffuse as a microtubular spindle develops between them. Some spindle microtubules terminate at dense chromatin patches that are contiguous with the major mass of chromatin surrounding the spindle. During late division stages, spindle microtubules often appear to be closely juxtaposed. Except for polar openings adjacent to the SPBs, the nuclear membrane is entire until late division when it degenerates in the midregion of the nucleus. During early postdivision, the SPB condenses into a small, dense sphere as the chromatin and heterochromatin opposite the SPB become diffuse. The SPB then elongates into a dense bar and SPB material increases, except at the midportion, reforming the double structure of interphase.

scholarly journals Analysis of a Spindle Pole Body Mutant Reveals a Defect in Biorientation and Illuminates Spindle Forces

2005 ◽  
Vol 16 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Tennessee J. Yoder ◽  
Mark A. McElwain ◽  
Susan E. Francis ◽  
Joy Bagley ◽  
Eric G.D. Muller ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Mutant Cells

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-μm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.

Somatic nuclear division in the sporidia of Ustilago violacea. IV. Microtubules and the spindle-pole body

1976 ◽  
Vol 22 (4) ◽  
pp. 507-522 ◽  
Author(s):  
N. H. Poon ◽  
A. W. Day
Keyword(s):  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Control Center ◽  
Ustilago Violacea ◽  
Membrane Breakdown ◽  
Pole Body ◽  
Free Region ◽  
Nuclear Rotation ◽  
Anther Smut

In unbudded cells of the anther smut fungus Ustilago violacea there is a dome-shaped spindle-pole body (SPB) consisting of a core 0.1 μm in diameter surrounded by a ribosome-free region 0.3–0.4 μm in diameter lying in a pocket of the nuclear membrane. After budding the nucleus moves towards the bud and begins to rotate rapidly. At about this stage the SPB divides into two parallel bars each about 0.1–0.15 μm in diameter and 0.3 μm long, separated by a distance of about 0.3 μm. Microtubules associated with the nuclear membrane but not with the SPB are present at the time of nuclear rotation. These microtubules disappear when rotation stops. Microtubules attached to the SPB are found during migration of the chromatinic portion of the nucleus into the bud cell. These microtubules disappear when migration stops and the nuclear membrane begins to break down. The twin SPB bars appear to move into the nucleus through a break in the membrane and begin to move apart forming a spindle about 1 μm long. Chromosomal microtubules (one per kinetochore) were found in several serial sections, and in addition there appeared to be several continuous microtubules present. The separation of the two chromatinic masses appeared to result from elongation of the continuous microtubules to about 3 μm long. Cytoplasmic microtubules and spindle microtubules were both found attached to the SPB as it elongated and one nucleus returned to the mother cell.The paper concludes with a discussion of the SPB as a multifunctional control center affecting nuclear migration, spindle formation, membrane breakdown and synthesis, karyogamy, conjugation, budding, chromosomal movement, replication, and disjunction.

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