scholarly journals Budding Yeast Chromosome Structure and Dynamics during Mitosis

2001 ◽  
Vol 152 (6) ◽  
pp. 1255-1266 ◽  
Author(s):  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Fluorescent Protein ◽  
Budding Yeast ◽  
Spindle Pole ◽  
Elastic Recoil ◽  
Rapid Acquisition ◽  
Anaphase Onset ◽  
Spindle Pole Bodies ◽  
Spindle Elongation ◽  
Green Fluorescent ◽  
Anaphase B

Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

scholarly journals Aspergillus nidulans Dis1/XMAP215 Protein AlpA Localizes to Spindle Pole Bodies and Microtubule Plus Ends and Contributes to Growth Directionality

2007 ◽  
Vol 6 (3) ◽  
pp. 555-562 ◽  
Author(s):  
Cathrin Enke ◽  
Nadine Zekert ◽  
Daniel Veith ◽  
Carolin Schaaf ◽  
Sven Konzack ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Spindle Pole Bodies ◽  
Family Protein ◽  
Associated Proteins ◽  
Hyphal Morphology ◽  
Conventional Kinesin ◽  
Green Fluorescent

ABSTRACTThe dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. InSchizosaccharomyces pombeand in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA fromAspergillus nidulansand show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA.alpAdeletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics.

scholarly journals Marker Fusion Tagging, a New Method for Production of Chromosomally Encoded Fusion Proteins

2010 ◽  
Vol 9 (5) ◽  
pp. 827-830 ◽  
Author(s):  
Julian Lai ◽  
Seng Kah Ng ◽  
Fang Fang Liu ◽  
Rajesh Narhari Patkar ◽  
Yanfen Lu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Spindle Pole ◽  
Hygromycin B ◽  
Content Type ◽  
Spindle Pole Bodies ◽  
New Gene ◽  
Green Fluorescent

ABSTRACT A new gene-tagging method (marker fusion tagging [MFT]) is demonstrated for Neurospora crassa and Magnaporthe oryzae. Translational fusions between the hygromycin B resistance gene and various markers are inserted into genes of interest by homologous recombination to produce chromosomally encoded fusion proteins. This method can produce tags at any position and create deletion alleles that maintain N- and C-terminal sequences. We show the utility of MFT by producing enhanced green fluorescent protein (EGFP) tags in proteins localized to nuclei, spindle pole bodies, septal pore plugs, Woronin bodies, developing septa, and the endoplasmic reticulum.

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

2000 ◽  
Vol 148 (3) ◽  
pp. 441-452 ◽  
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.

scholarly journals Saccharomyces cerevisiae Duo1p and Dam1p, Novel Proteins Involved in Mitotic Spindle Function

1998 ◽  
Vol 143 (4) ◽  
pp. 1029-1040 ◽  
Author(s):  
Christian Hofmann ◽  
Iain M. Cheeseman ◽  
Bruce L. Goode ◽  
Kent L. McDonald ◽  
Georjana Barnes ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Spindle Pole Bodies ◽  
Intranuclear Microtubules ◽  
Spindle Microtubules ◽  
Green Fluorescent

In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

scholarly journals The Nuclear Migration Protein NUDF/LIS1 Forms a Complex with NUDC and BNFA at Spindle Pole Bodies

2008 ◽  
Vol 7 (6) ◽  
pp. 1041-1052 ◽  
Author(s):  
Kerstin Helmstaedt ◽  
Karen Laubinger ◽  
Katja Voßkuhl ◽  
Özgür Bayram ◽  
Silke Busch ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Affinity Purification ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Spindle Pole Bodies ◽  
Green Fluorescent ◽  
Two Hybrid

ABSTRACTNuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC inAspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (forbinding toNUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.

scholarly journals Regulation of mitosis by the NIMA kinase involves TINA and its newly discovered partner, An-WDR8, at spindle pole bodies

2013 ◽  
Vol 24 (24) ◽  
pp. 3842-3856 ◽  
Author(s):  
Kuo-Fang Shen ◽  
Stephen A. Osmani
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole ◽  
Cyclin B ◽  
Nuclear Pore ◽  
Mitotic Progression ◽  
Interacting Protein ◽  
Spindle Pole Bodies ◽  
Anchoring System ◽  
Wd40 Domain ◽  
Green Fluorescent

The NIMA kinase is required for mitotic nuclear pore complex disassembly and potentially controls other mitotic-specific events. To investigate this possibility, we imaged NIMA–green fluorescent protein (GFP) using four-dimensional spinning disk confocal microscopy. At mitosis NIMA-GFP locates to spindle pole bodies (SPBs), which contain Cdk1/cyclin B, followed by Aurora, TINA, and the BimC kinesin. NIMA promotes NPC disassembly in a spatially regulated manner starting near SPBs. NIMA is also required for TINA, a NIMA-interacting protein, to locate to SPBs during initiation of mitosis, and TINA is then necessary for locating NIMA back to SPBs during mitotic progression. To help expand the NIMA-TINA pathway, we affinity purified TINA and found it to uniquely copurify with An-WDR8, a WD40-domain protein conserved from humans to plants. Like TINA, An-WDR8 accumulates within nuclei during G2 but disperses from nuclei before locating to mitotic SPBs. Without An-WDR8, TINA levels are greatly reduced, whereas TINA is necessary for mitotic targeting of An-WDR8. Finally, we show that TINA is required to anchor mitotic microtubules to SPBs and, in combination with An-WDR8, for successful mitosis. The findings provide new insights into SPB targeting and indicate that the mitotic microtubule-anchoring system at SPBs involves WDR8 in complex with TINA.

scholarly journals Time-Lapse Microscopy Reveals Unique Roles for Kinesins during Anaphase in Budding Yeast

1998 ◽  
Vol 143 (3) ◽  
pp. 687-694 ◽  
Author(s):  
Aaron F. Straight ◽  
John W. Sedat ◽  
Andrew W. Murray
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Dynamic Structure ◽  
Time Lapse ◽  
Slow Phase ◽  
Rapid Phase ◽  
Green Fluorescent ◽  
Anaphase B

The mitotic spindle is a complex and dynamic structure. Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis. We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein. Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Δ mutants lack the rapid phase of anaphase B, kip1Δ mutants show defects in the slow phase of anaphase B, and kip3Δ mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell. The kip3Δ and kip1Δ mutants affect the duration of anaphase, but cin8Δ does not.

scholarly journals A Novel Role of the Budding Yeast Separin Esp1 in Anaphase Spindle Elongation

2001 ◽  
Vol 152 (1) ◽  
pp. 27-40 ◽  
Author(s):  
Sanne Jensen ◽  
Marisa Segal ◽  
Duncan J. Clarke ◽  
Steven I. Reed
Keyword(s):  
Mutational Analysis ◽  
Spindle Pole ◽  
Nuclear Localization Sequence ◽  
Anaphase Promoting Complex ◽  
Nuclear Targeting ◽  
Spindle Pole Bodies ◽  
Localization Sequence ◽  
Spindle Midzone ◽  
Spindle Elongation ◽  
Anaphase B

In Saccharomyces cerevisiae, the metaphase–anaphase transition is initiated by the anaphase-promoting complex–dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase–anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

scholarly journals Disappearance of the budding yeast Bub2–Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit

2006 ◽  
Vol 172 (3) ◽  
pp. 335-346 ◽  
Author(s):  
Roberta Fraschini ◽  
Claudio D'Ambrosio ◽  
Marianna Venturetti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Gtpase Activating Protein ◽  
Septin Ring ◽  
Anaphase Onset ◽  
Spindle Pole Bodies ◽  
Bud Neck ◽  
Spindle Position

Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases.

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