scholarly journals FH3, A Domain Found in Formins, Targets the Fission Yeast Formin Fus1 to the Projection Tip During Conjugation

1998 ◽  
Vol 141 (5) ◽  
pp. 1217-1228 ◽  
Author(s):  
Janni Petersen ◽  
Olaf Nielsen ◽  
Richard Egel ◽  
Iain M. Hagan
Keyword(s):  
Fluorescent Protein ◽  
Protein Localization ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminal ◽  
Gfp Fusion Protein ◽  
Pole Body ◽  
Body Component ◽  
Green Fluorescent ◽  
Amino Terminal Domain

Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain–GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.

scholarly journals The spacer protein Spc110p targets calmodulin to the central plaque of the yeast spindle pole body

1996 ◽  
Vol 109 (9) ◽  
pp. 2229-2237 ◽  
Author(s):  
A. Spang ◽  
K. Grein ◽  
E. Schiebel
Keyword(s):  
Binding Site ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminus ◽  
Carboxy Terminus ◽  
Calmodulin Binding ◽  
Amino Terminal ◽  
Pole Body ◽  
Body Component ◽  
Nanomolar Range

Yeast calmodulin (CaM) was found to be localized to the microtubule organizing centre, the spindle pole body. The spindle pole body is a multi-layered structure consisting of outer, central and inner plaques. In this paper, we report that a fraction of CaM is in association with the central plaque of the spindle pole body. This localization is dependent on the calmodulin-binding site of another spindle pole body component, Spc110p, which serves as a spacer connecting the inner plaque with the central plaque. Since the CaM-binding site of Spc110p is located near the carboxy terminus, Spc110p-dependent localization of calmodulin defines the orientation of Spc110p with the carboxy terminus towards the central plaque and the amino terminus towards the inner plaque. This orientation of Spc110p was confirmed using antibodies specific for the amino-terminal end of Spc110p, which predominantly labelled the inner plaque. In addition, synthetic peptides corresponding to the calmodulin-binding site of Spc110p bound to calmodulin with a Kd in the nanomolar range and nearly independent of Ca2+.

scholarly journals Slk19p Is a Centromere Protein That Functions to Stabilize Mitotic Spindles

1999 ◽  
Vol 146 (2) ◽  
pp. 415-425 ◽  
Author(s):  
Xuemei Zeng ◽  
Jason A. Kahana ◽  
Pamela A. Silver ◽  
Mary K. Morphew ◽  
J. Richard McIntosh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Synthetic Lethal ◽  
Pole Body ◽  
Spindle Midzone ◽  
Green Fluorescent ◽  
Spindle Structure

We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability. When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed. A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body. Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules.

scholarly journals Bim1p/Yeb1p Mediates the Kar9p-dependent Cortical Attachment of Cytoplasmic Microtubules

2000 ◽  
Vol 11 (9) ◽  
pp. 2949-2959 ◽  
Author(s):  
Rita K. Miller ◽  
Soo-Chen Cheng ◽  
Mark D. Rose
Keyword(s):  
Fluorescent Protein ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Attachment Site ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Microtubule Binding ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Two Hybrid

In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, andKAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion ofBIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein–Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.

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.

UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3171-3181 ◽  
Author(s):  
C.J. Malone ◽  
W.D. Fixsen ◽  
H.R. Horvitz ◽  
M. Han
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Spindle Pole Body ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Dna Lesions ◽  
Body Protein ◽  
C Elegans ◽  
Green Fluorescent

Nuclear migrations are essential for metazoan development. Two nuclear migrations that occur during C. elegans development require the function of the unc-84 gene. unc-84 mutants are also defective in the anchoring of nuclei within the hypodermal syncytium and in the migrations of the two distal tip cells of the gonad. Complementation analyses of 17 unc-84 alleles defined two genetically separable functions. Both functions are required for nuclear and distal tip cell migrations, but only one is required for nuclear anchorage. The DNA lesions associated with these 17 mutations indicate that the two genetically defined functions correspond to two distinct regions of the UNC-84 protein. The UNC-84 protein has a predicted transmembrane domain and a C-terminal region with similarity to the S. pombe spindle pole body protein Sad1 and to two predicted mammalian proteins. Analysis of a green fluorescent protein reporter indicated that UNC-84 is widely expressed and localized to the nuclear envelope. We propose that UNC-84 functions to facilitate a nuclear-centrosomal interaction required for nuclear migration and anchorage.

scholarly journals Dynamics of Nuclear Pore Distribution in Nucleoporin Mutant Yeast Cells

1997 ◽  
Vol 136 (4) ◽  
pp. 747-759 ◽  
Author(s):  
Naïma Belgareh ◽  
Valérie Doye
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Pore Distribution ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Dividing Cells ◽  
Green Fluorescent ◽  
Amino Terminal Domain

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133− dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p- induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133− mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

scholarly journals The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast

2002 ◽  
Vol 13 (3) ◽  
pp. 930-946 ◽  
Author(s):  
Futaba Miki ◽  
Koei Okazaki ◽  
Mizuki Shimanuki ◽  
Ayumu Yamamoto ◽  
Yasushi Hiraoka ◽  
...  
Keyword(s):  
Light Chain ◽  
Meiotic Prophase ◽  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Dynein Light Chain ◽  
Nuclear Movement ◽  
Green Fluorescent

A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family. Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle. During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1. In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction. Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant. Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant. Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase. Strains mutated in both the dlc1 and dhc1loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1. S. pombe contains a homolog of the 8-kDa dynein light chain, Dlc2. This class of dynein light chain, however, is not essential in either the vegetative or sexual phases.

Role of gamma-tubulin in mitosis-specific microtubule nucleation from the Schizosaccharomyces pombe spindle pole body

1996 ◽  
Vol 109 (1) ◽  
pp. 165-177 ◽  
Author(s):  
H. Masuda ◽  
T. Shibata
Keyword(s):  
Protein Kinase ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Amino Terminal ◽  
Pole Body ◽  
Gamma Tubulin ◽  
Mitotic Spindle Pole

The ability of the Schizosacchromyces pombe spindle pole body to nucleate microtubules is activated at the onset of mitosis for forming a mitotic spindle, but it is inactivated during interphase. We have previously developed an in vitro assay for studying the molecular mechanism of spindle pole body activation using permeabilized interphase S. pombe cells and Xenopus mitotic extracts. We have shown that the interphase spindle pole body is activated indirectly by p34cdc2 protein kinase in Xenopus mitotic extracts. In this study we examined the role of gamma-tubulin, a component of both interphase and mitotic spindle pole body, in formation of the microtubule nucleating complex at the mitotic spindle pole body. A polyclonal antibody specific to S. pombe gamma-tubulin inhibited both activation of the interphase spindle pole body and microtubule nucleation from the mitotic spindle pole body. Addition of bacterially expressed S. pombe gamma-tubulin or its amino-terminal fragments to Xenopus mitotic extracts inhibited spindle pole body activation. Affinity chromatography of partially fractionated Xenopus mitotic extracts with the amino-terminal fragment of S. pombe gamma-tubulin showed that fractions bound to the fragment supported the activation. The fractions did not contain Xenopus gamma-tubulin, showing that activation of the spindle pole body is not due to recruitment of Xenopus gamma-tubulin to the spindle pole body. The spindle pole body activation occurred in extracts depleted of p34cdc2 protein kinase or MAP kinase. The activity of the fractions bound to the fragment was inhibited by a protein kinase inhibitor, staurosporine. These results suggest that S. pombe gamma-tubulin is a component of the microtubule nucleating complex, and that the function of proteins that interact with gamma-tubulin is required for activation of the spindle pole body. We present possible models for the activation that convert the immature microtubule nucleating complex at interphase into the mature microtubule nucleating complex at mitosis.

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