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

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.141.5.1217 ◽

1998 ◽

Vol 141 (5) ◽

pp. 1217-1228 ◽

Cited By ~ 110

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.

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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

Molecular Biology of the Cell ◽

10.1091/mbc.01-11-0543 ◽

2002 ◽

Vol 13 (3) ◽

pp. 930-946 ◽

Cited By ~ 65

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.

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Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

Molecular Biology of the Cell ◽

10.1091/mbc.12.7.2031 ◽

2001 ◽

Vol 12 (7) ◽

pp. 2031-2046 ◽

Cited By ~ 124

Author(s):

Nina Tolonen ◽

Laura Doglio ◽

Sibylle Schleich ◽

Jacomine Krijnse Locker

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Dna Synthesis ◽

Vaccinia Virus ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Nuclear Envelope Assembly ◽

Green Fluorescent Protein Tagging ◽

Green Fluorescent

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ∼45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

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Time-Lapse Video Microscopy Analysis Reveals Astral Microtubule Detachment in the Yeast Spindle Pole Mutantcnm67

Molecular Biology of the Cell ◽

10.1091/mbc.11.4.1197 ◽

2000 ◽

Vol 11 (4) ◽

pp. 1197-1211 ◽

Cited By ~ 38

Author(s):

Dominic Hoepfner ◽

Arndt Brachat ◽

Peter Philippsen

Keyword(s):

Fluorescent Protein ◽

Spindle Pole Body ◽

Attachment Site ◽

Time Lapse ◽

Spindle Pole ◽

Cell Cycles ◽

Astral Microtubule ◽

Normal Behavior ◽

Green Fluorescent ◽

Diploid Cells

Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.

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Coordinated Spindle Assembly and Orientation Requires Clb5p-Dependent Kinase in Budding Yeast

The Journal of Cell Biology ◽

10.1083/jcb.148.3.441 ◽

2000 ◽

Vol 148 (3) ◽

pp. 441-452 ◽

Cited By ~ 43

Author(s):

Marisa Segal ◽

Duncan J. Clarke ◽

Paul Maddox ◽

E.D. Salmon ◽

Kerry Bloom ◽

...

Keyword(s):

Fluorescent Protein ◽

Budding Yeast ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Time Lapse ◽

Spindle Pole ◽

Cell Cortex ◽

Dynamic Interactions ◽

Yeast Saccharomyces Cerevisiae ◽

Green Fluorescent

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Δ cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Δ cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

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A Mechanism for Nuclear Positioning in Fission Yeast Based on Microtubule Pushing

The Journal of Cell Biology ◽

10.1083/jcb.153.2.397 ◽

2001 ◽

Vol 153 (2) ◽

pp. 397-412 ◽

Cited By ~ 326

Author(s):

P.T. Tran ◽

L. Marsh ◽

V. Doye ◽

S. Inoué ◽

F. Chang

Keyword(s):

Spatial Organization ◽

Fluorescent Protein ◽

Spindle Pole Body ◽

Spindle Pole ◽

Protein Markers ◽

Division Plane ◽

Attachment Sites ◽

Green Fluorescent ◽

Transient Forces ◽

Cell Division Plane

The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.

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Saccharomyces cerevisiae MPS2Encodes a Membrane Protein Localized at the Spindle Pole Body and the Nuclear Envelope

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2393 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2393-2406 ◽

Cited By ~ 38

Author(s):

Marı́a de la Cruz Muñoz-Centeno ◽

Susan McBratney ◽

Antonio Monterrosa ◽

Breck Byers ◽

Carl Mann ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Protein ◽

Nuclear Envelope ◽

Fluorescent Protein ◽

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Yeast Saccharomyces Cerevisiae ◽

Pole Body ◽

Monopolar Spindle

The MPS2 (monopolar spindle two) gene is one of several genes required for the proper execution of spindle pole body (SPB) duplication in the budding yeast Saccharomyces cerevisiae ( Winey et al., 1991 ). We report here that the MPS2 gene encodes an essential 44-kDa protein with two putative coiled-coil regions and a hydrophobic sequence. Although MPS2 is required for normal mitotic growth, some null strains can survive; these survivors exhibit slow growth and abnormal ploidy. The MPS2 protein was tagged with nine copies of the myc epitope, and biochemical fractionation experiments show that it is an integral membrane protein. Visualization of a green fluorescent protein (GFP) Mps2p fusion protein in living cells and indirect immunofluorescence microscopy of 9xmyc-Mps2p revealed a perinuclear localization with one or two brighter foci of staining corresponding to the SPB. Additionally, immunoelectron microscopy shows that GFP-Mps2p localizes to the SPB. Our analysis suggests that Mps2p is required as a component of the SPB for insertion of the nascent SPB into the nuclear envelope.

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CPNA-1, a copine domain protein, is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e12-06-0478 ◽

2013 ◽

Vol 24 (5) ◽

pp. 601-616 ◽

Cited By ~ 17

Author(s):

Adam Warner ◽

Ge Xiong ◽

Hiroshi Qadota ◽

Teresa Rogalski ◽

A. Wayne Vogl ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Dense Body ◽

Yellow Fluorescent Protein ◽

Thick Filament ◽

The Body ◽

Body Protein ◽

C Elegans ◽

Domain Protein

We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1–null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans.

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Saccharomyces cerevisiaeCells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Molecular Biology of the Cell ◽

10.1091/mbc.9.5.977 ◽

1998 ◽

Vol 9 (5) ◽

pp. 977-991 ◽

Cited By ~ 53

Author(s):

Arndt Brachat ◽

John V. Kilmartin ◽

Achim Wach ◽

Peter Philippsen

Keyword(s):

Fluorescent Protein ◽

Spindle Pole Body ◽

Time Lapse ◽

Nuclear Migration ◽

Spindle Pole ◽

Microtubule Organizing Center ◽

Multinucleated Cells ◽

Pole Body ◽

Cytoplasmic Microtubules ◽

Low Efficiency

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion ofCNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. AlthoughCNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy ofcnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

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Microtubule Organization Requires Cell Cycle-dependent Nucleation at Dispersed Cytoplasmic Sites: Polar and Perinuclear Microtubule Organizing Centers in the Plant Pathogen Ustilago maydis

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0513 ◽

2003 ◽

Vol 14 (2) ◽

pp. 642-657 ◽

Cited By ~ 80

Author(s):

Anne Straube ◽

Marianne Brill ◽

Berl R. Oakley ◽

Tetsuya Horio ◽

Gero Steinberg

Keyword(s):

Cell Cycle ◽

Fluorescent Protein ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Organization ◽

Microtubule Organizing Centers ◽

Nucleation Sites ◽

Cycle Dependent ◽

Green Fluorescent

Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungusUstilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding γ-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the γ-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited γ-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.

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