scholarly journals Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

1997 ◽  
Vol 139 (4) ◽  
pp. 985-994 ◽  
Author(s):  
Sidney L. Shaw ◽  
Elaine Yeh ◽  
Paul Maddox ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Dynamic Properties ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Microtubule Dynamics ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Nuclear Movement ◽  
Bud Neck

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at ≅0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

scholarly journals Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex

1997 ◽  
Vol 138 (3) ◽  
pp. 629-641 ◽  
Author(s):  
Janet L. Carminati ◽  
Tim Stearns
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Dynamic Properties ◽  
Yeast Cells ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Animal Cells ◽  
Green Fluorescent ◽  
Mutant Cells

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)–tubulin fusion protein to observe microtubules in living yeast cells. GFP–tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

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.

scholarly journals Microtubule Interactions with the Cell Cortex Causing Nuclear Movements in Saccharomyces cerevisiae

2000 ◽  
Vol 149 (4) ◽  
pp. 863-874 ◽  
Author(s):  
Neil R. Adames ◽  
John A. Cooper
Keyword(s):  
Living Cells ◽  
Cell Cortex ◽  
Cortical Region ◽  
Wild Type ◽  
Microtubule Depolymerization ◽  
Nuclear Movement ◽  
Bud Neck ◽  
Free Microtubules ◽  
Cytoplasmic Microtubules ◽  
Bud Site

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.

scholarly journals Microtubules in Candida albicans Hyphae Drive Nuclear Dynamics and Connect Cell Cycle Progression to Morphogenesis

2005 ◽  
Vol 4 (10) ◽  
pp. 1697-1711 ◽  
Author(s):  
Kenneth R. Finley ◽  
Judith Berman
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Basal Cell ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Microtubule Dynamics ◽  
Growth Defect ◽  
Cell Cortex ◽  
Long Distance ◽  
Time Lapse Microscopy

ABSTRACT Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 μm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.

scholarly journals A Cytoplasmic Dynein Heavy Chain Is Required for Oscillatory Nuclear Movement of Meiotic Prophase and Efficient Meiotic Recombination in Fission Yeast

1999 ◽  
Vol 145 (6) ◽  
pp. 1233-1250 ◽  
Author(s):  
Ayumu Yamamoto ◽  
Robert R. West ◽  
J. Richard McIntosh ◽  
Yasushi Hiraoka
Keyword(s):  
Fission Yeast ◽  
Heavy Chain ◽  
Meiotic Recombination ◽  
Meiotic Prophase ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Nuclear Movement ◽  
Homologous Chromosomes ◽  
Dynein Heavy Chain

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.

scholarly journals Cdk1-dependent destabilization of long astral microtubules is required for spindle orientation

2020 ◽  
Author(s):  
Divya Singh ◽  
Nadine Schmidt ◽  
Franziska Müller ◽  
Tanja Bange ◽  
Alexander W. Bird
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Microtubule Dynamics ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Tissue Organization ◽  
Microtubule Stability ◽  
Astral Microtubule ◽  
Early Mitosis

AbstractThe precise execution of mitotic spindle orientation in response to cell shape cues is important for tissue organization and development. The presence of astral microtubules extending from the centrosome towards the cell cortex is essential for this process, but little is understood about the contribution of astral microtubule dynamics to spindle positioning, or how astral microtubule dynamics are regulated spatiotemporally. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells transition from interphase to mitosis, but how Cdk1 activity specifically modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules to ensure spindle reorientation in response to cell shape. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus-ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus-ends in mitosis. This decreases the catastrophe frequency of astral microtubules, and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules must thus not only be present, but also dynamic to allow the spindle to reorient in response to cell shape, a state achieved by selective destabilization of long astral microtubules via Cdk1.

scholarly journals Dynein light chain 1 and a spindle-associated adaptor promote dynein asymmetry and spindle orientation

2012 ◽  
Vol 198 (6) ◽  
pp. 1039-1054 ◽  
Author(s):  
Anja K. Dunsch ◽  
Dean Hammond ◽  
Jennifer Lloyd ◽  
Lothar Schermelleh ◽  
Ulrike Gruneberg ◽  
...  
Keyword(s):  
Light Chain ◽  
Mitotic Spindle ◽  
Regulatory System ◽  
Cytoplasmic Dynein ◽  
Spindle Orientation ◽  
Growth Surface ◽  
Cell Cortex ◽  
Dynein Light Chain ◽  
Dynein Motor ◽  
Pulling Forces

The cytoplasmic dynein motor generates pulling forces to center and orient the mitotic spindle within the cell. During this positioning process, dynein oscillates from one pole of the cell cortex to the other but only accumulates at the pole farthest from the spindle. Here, we show that dynein light chain 1 (DYNLL1) is required for this asymmetric cortical localization of dynein and has a specific function defining spindle orientation. DYNLL1 interacted with a spindle-microtubule–associated adaptor formed by CHICA and HMMR via TQT motifs in CHICA. In cells depleted of CHICA or HMMR, the mitotic spindle failed to orient correctly in relation to the growth surface. Furthermore, CHICA TQT motif mutants localized to the mitotic spindle but failed to recruit DYNLL1 to spindle microtubules and did not correct the spindle orientation or dynein localization defects. These findings support a model where DYNLL1 and CHICA-HMMR form part of the regulatory system feeding back spindle position to dynein at the cell cortex.

scholarly journals Cortical Num1p Interacts with the Dynein Intermediate Chain Pac11p and Cytoplasmic Microtubules in Budding Yeast

2001 ◽  
Vol 152 (2) ◽  
pp. 251-262 ◽  
Author(s):  
Marian Farkasovsky ◽  
Hans Küntzel
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Migration ◽  
Cytoplasmic Microtubule ◽  
Late Anaphase ◽  
Dynein Intermediate Chain ◽  
Bud Neck ◽  
Early Anaphase ◽  
Cytoplasmic Microtubules ◽  
Green Fluorescent ◽  
Intermediate Chain

Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the α-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p–Kar9p complex at the bud tip of early anaphase cells.

scholarly journals Roles of NUDE and NUDF Proteins of Aspergillus nidulans: Insights from Intracellular Localization and Overexpression Effects

2003 ◽  
Vol 14 (3) ◽  
pp. 871-888 ◽  
Author(s):  
Vladimir P. Efimov
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Nidulans ◽  
Heavy Chain ◽  
Fluorescent Protein ◽  
Specific Interaction ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Dynein Heavy Chain ◽  
Terminal Domain ◽  
Green Fluorescent

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes (“comets”) that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudEgene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditionalnudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF andnudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between thenudF and apsA genes suggests a role for NUDF at the cell cortex.

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