scholarly journals Localization of Core Spindle Pole Body (SPB) Components during SPB Duplication in Saccharomyces cerevisiae

1999 ◽  
Vol 145 (4) ◽  
pp. 809-823 ◽  
Author(s):  
Ian R. Adams ◽  
John V. Kilmartin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Large Plaque ◽  
Temperature Sensitive Mutants ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Preceding Cell

We have examined the process of spindle pole body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear envelope. We analyzed the role of the main SPB components in the formation of these structures by identifying them from an SPB core fraction by mass spectrometry. Temperature-sensitive mutants for two of the components, Spc29p and Nud1p, were prepared to partly define their function. The composition of two of the intermediates in SPB duplication, the satellite and the duplication plaque, was examined by immunoelectron microscopy. Both contain cytoplasmic SPB components showing that duplication has already been partly achieved by the end of the preceding cell cycle when the satellite is formed. We show that by overexpression of SPB components the structure of the satellite can be changed and SPB duplication inhibited by disrupting the attachment of the plaque-like intermediate to the half-bridge. We present a model for SPB duplication where binding of SPB components to either end of the bridge structure ensures two separate SPBs.

scholarly journals Outer plaque assembly and spore encapsulation are defective during sporulation of adenylate cyclase-deficient mutants of Saccharomyces cerevisiae.

1985 ◽  
Vol 100 (6) ◽  
pp. 1854-1862 ◽  
Author(s):  
I Uno ◽  
K Matsumoto ◽  
A Hirata ◽  
T Ishikawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylate Cyclase ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Sensitive Period ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
Diploid Cells

Sporulation in diploid cells homozygous for the cyr1-2 mutation of the yeast Saccharomyces cerevisiae was examined. This mutation causes a defect in adenylate cyclase and temperature-sensitive arrest in the G1 phase of the mitotic cell cycle. The cyr1-2/cyr1-2 diploid cells were able to initiate meiotic divisions, but produced predominantly two-spored asci at the restrictive temperature. Temperature-sensitive period for production of two-spored asci was approximately 12 h after the transfer of cells to the sporulation medium. The levels of cAMP increased during this period in the wild type and cyr1-2/cyr1-2 diploid cells incubated at the permissive temperature, but remained at an extremely low level in the cyr1-2/cyr1-2 diploid cells incubated at the restrictive temperature. Dyad analysis of the cyr1-2 strain indicated that meiotic products were randomly included into ascospores. Fluorescent microscopy of the cyr1-2/cyr1-2 diploid cells incubated at the restrictive temperature revealed that individual haploid nuclei were enclosed in each of the two spores after meiosis. About half of the cyr1-2/cyr1-2 diploid cells entered normal meiosis 1 producing two normal spindle pole bodies with inner and outer plaques, and the other half entered abnormal meiosis 1 producing one normal spindle pole body and one defective spindle pole body without out plaque. At meiosis II, some cells contained a pair of normal spindle pole bodies and other cells contained pairs of normal and abnormal spindle pole bodies.

scholarly journals Structural role of Sfi1p–centrin filaments in budding yeast spindle pole body duplication

2006 ◽  
Vol 173 (6) ◽  
pp. 867-877 ◽  
Author(s):  
Sam Li ◽  
Alan M. Sandercock ◽  
Paul Conduit ◽  
Carol V. Robinson ◽  
Roger L. Williams ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Crystal Structures ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
N Terminus ◽  
Α Helix

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–centrin complexes containing several repeats show Sfi1p as an α helix with centrins wrapped around each repeat and similar centrin–centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.

scholarly journals YOR129c, a new element interacting with Cnm67p, a component of the spindle pole body of Saccharomyces cerevisiae.

2003 ◽  
Vol 50 (3) ◽  
pp. 883-890 ◽  
Author(s):  
Monika Wysocka ◽  
Agnieszka Białkowska ◽  
Arkadiusz Miciałkiewicz ◽  
Anna Kurlandzka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Double Deletion ◽  
Pole Body ◽  
Cation Homeostasis ◽  
Accessory Factor ◽  
C Terminus ◽  
Cytoplasmic Face

The Saccharomyces cerevisiae spindle pole body (SPB) consists of numerous proteins forming the outer, inner and central plaques. The protein Cnm67 is an important component of the outer plaque. The C-terminus of this protein contains a determinant important for its SPB localization. We identified a protein encoded by YOR129c which interacts with this C-terminus in the two-hybrid system. YOR129c and CNM67 exhibit weak genetic interaction. The double deletion strain yor129cdelta cnm67delta exhibits moderately increased resistance to 0.1M LiCl and hygromycin B compared with the cnm67delta single mutant. We propose that the YOR129c protein is an accessory factor associated with the cytoplasmic face of SPB and plays a role in cation homeostasis and/or multidrug resistance.

scholarly journals Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body

2017 ◽  
Author(s):  
Ngang Heok Tang ◽  
Chii Shyang Fong ◽  
Hirohisa Masuda ◽  
Isabelle Jourdain ◽  
Masashi Yukawa ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Genetic Manipulation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Cellular Processes ◽  
Temperature Sensitive Mutants ◽  
Pole Body ◽  
Genes Encoding ◽  
Mitosis And Meiosis

The spindle pole body (SPB) in fungi is the equivalent of the animal centrosome. A number of previous studies have identified many, if not all, components of the SPB. The SPB is the structural platform for microtubule nucleation and plays important roles, both in mitosis and meiosis. The SPB is absolutely essential for cell survival and its abnormalities give rise to aberrant cell division and morphogenesis. Therefore, it is crucial to understand how the SPB organises itself and how the functions of individual SPB components are regulated. We report here a procedure to generate temperature sensitive mutants in the fission yeast, Schizosaccharomyces pombe. The approach has proved useful to characterise functions of individual SPB components. This original genetic manipulation is however not restricted to analysis of SPB functions, and can be suited to investigate other cellular processes in S. pombe.

scholarly journals Generation and characterisation of temperature sensitive mutants of genes encoding the fission yeast spindle pole body

2017 ◽  
Author(s):  
Ngang Heok Tang ◽  
Chii Shyang Fong ◽  
Hirohisa Masuda ◽  
Isabelle Jourdain ◽  
Masashi Yukawa ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Genetic Manipulation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Cellular Processes ◽  
Temperature Sensitive Mutants ◽  
Pole Body ◽  
Genes Encoding ◽  
Mitosis And Meiosis

The spindle pole body (SPB) in fungi is the equivalent of the animal centrosome. A number of previous studies have identified many, if not all, components of the SPB. The SPB is the structural platform for microtubule nucleation and plays important roles, both in mitosis and meiosis. The SPB is absolutely essential for cell survival and its abnormalities give rise to aberrant cell division and morphogenesis. Therefore, it is crucial to understand how the SPB organises itself and how the functions of individual SPB components are regulated. We report here a procedure to generate temperature sensitive mutants in the fission yeast, Schizosaccharomyces pombe. The approach has proved useful to characterise functions of individual SPB components. This original genetic manipulation is however not restricted to analysis of SPB functions, and can be suited to investigate other cellular processes in S. pombe.

Ady3p Links Spindle Pole Body Function to Spore Wall Synthesis in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1439-1450
Author(s):  
Mark E Nickas ◽  
Aaron M Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Spore Wall ◽  
Pole Body ◽  
Prospore Membrane ◽  
Sporulation Efficiency ◽  
Spore Walls

Abstract Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Δ/ady3Δ asci that do form contain fewer than four spores. The sporulation defect in ady3Δ/ady3Δ cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Δ/ady3Δ cells. In mpc70Δ/mpc70Δ cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.

scholarly journals Mutant Membrane Protein of the Budding Yeast Spindle Pole Body Is Targeted to the Endoplasmic Reticulum Degradation Pathway

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 567-578 ◽  
Author(s):  
Susan McBratney ◽  
Mark Winey
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Degradation Pathway ◽  
Spindle Pole ◽  
Wild Type ◽  
Er Quality Control ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugation

Abstract Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle. We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB. Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1. Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome. Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6. The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels. Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway.

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.

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