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 The offloading model for dynein function

2005 ◽  
Vol 168 (2) ◽  
pp. 201-207 ◽  
Author(s):  
Wei-Lih Lee ◽  
Michelle A. Kaiser ◽  
John A. Cooper
Keyword(s):  
Heavy Chain ◽  
Mitotic Spindle ◽  
Cell Cortex ◽  
Cytoplasmic Microtubule ◽  
Functional Studies ◽  
Dynein Intermediate Chain ◽  
Bud Neck ◽  
Cytoplasmic Microtubules ◽  
Insight Into ◽  
Intermediate Chain

During mitosis in budding yeast, dynein moves the mitotic spindle into the mother-bud neck. We have proposed an offloading model to explain how dynein works. Dynein is targeted to the dynamic plus end of a cytoplasmic microtubule, offloads to the cortex, becomes anchored and activated, and then pulls on the microtubule. Here, we perform functional studies of dynein intermediate chain (IC) and light intermediate chain (LIC). IC/Pac11 and LIC/Dyn3 are both essential for dynein function, similar to the heavy chain (HC/Dyn1). IC and LIC are targeted to the distal plus ends of dynamic cytoplasmic microtubules, as is HC, and their targeting depends on HC. Targeting of HC to the plus end depends on IC, but not LIC. IC also localizes as stationary dots at the cell cortex, the presumed result of offloading in our model, as does HC, but not LIC. Localization of HC to cortical dots depends on both IC and LIC. Thus, the IC and LIC accessory chains have different but essential roles in dynein function, providing new insight into the offloading model.

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 Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism

2010 ◽  
Vol 21 (21) ◽  
pp. 3680-3692 ◽  
Author(s):  
Sandrine Grava ◽  
Peter Philippsen
Keyword(s):  
Budding Yeast ◽  
Ashbya Gossypii ◽  
Wild Type ◽  
Major Function ◽  
Bud Neck ◽  
Pulling Forces ◽  
Cytoplasmic Microtubules ◽  
Hyphal Tip

Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.

scholarly journals The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast

2003 ◽  
Vol 160 (3) ◽  
pp. 355-364 ◽  
Author(s):  
Wei-Lih Lee ◽  
Jessica R. Oberle ◽  
John A. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Mitotic Spindle ◽  
Genetic Interactions ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Bud Neck ◽  
Cytoplasmic Microtubules ◽  
In Cells

During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother–bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.

scholarly journals Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

2016 ◽  
Vol 113 (34) ◽  
pp. E4995-E5004 ◽  
Author(s):  
Wen Lu ◽  
Michael Winding ◽  
Margot Lakonishok ◽  
Jill Wildonger ◽  
Vladimir I. Gelfand
Keyword(s):  
Cytoplasmic Streaming ◽  
Cell Types ◽  
Nurse Cells ◽  
Wild Type ◽  
Actin Cortex ◽  
Microtubule Motor ◽  
Multiple Cell ◽  
Cytoplasmic Microtubules ◽  
Two Populations

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

scholarly journals SEC3 Mutations Are Synthetically Lethal With Profilin Mutations and Cause Defects in Diploid-Specific Bud-Site Selection

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 495-510 ◽  
Author(s):  
B K Haarer ◽  
A Corbett ◽  
Y Kweon ◽  
A S Petzold ◽  
P Silver ◽  
...  
Keyword(s):  
Site Selection ◽  
Secretory Pathway ◽  
Wild Type ◽  
Synthetic Lethal ◽  
Abnormal Growth ◽  
Colony Color ◽  
Synthetically Lethal ◽  
Homozygous Diploid ◽  
Bud Site ◽  
Wild Type Yeast

Abstract Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids.

scholarly journals A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

1997 ◽  
Vol 136 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Shirley Yang ◽  
Kathryn R. Ayscough ◽  
David G. Drubin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Site Selection ◽  
Mitotic Checkpoint ◽  
Cell Cortex ◽  
Spatial Cues ◽  
Genes Encoding ◽  
Mother And Daughter ◽  
Mother Cells ◽  
Bud Site

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.

scholarly journals Mcp5, a meiotic cell cortex protein, is required for nuclear movement mediated by dynein and microtubules in fission yeast

2006 ◽  
Vol 173 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Takamune T. Saito ◽  
Daisuke Okuzaki ◽  
Hiroshi Nojima
Keyword(s):  
Fission Yeast ◽  
Meiotic Prophase ◽  
Coiled Coil ◽  
Cell Cortex ◽  
Ph Domain ◽  
Nuclear Movement ◽  
Recombination Rates ◽  
Homologous Chromosome Pairing ◽  
Cortical Localization ◽  
Pulling Force

During meiotic prophase I of the fission yeast Schizosaccharomyces pombe, oscillatory nuclear movement occurs. This promotes homologous chromosome pairing and recombination and involves cortical dynein, which plays a pivotal role by generating a pulling force with the help of an unknown dynein anchor. We show that Mcp5, the homologue of the budding yeast dynein anchor Num1, may be this putative dynein anchor. mcp5+ is predominantly expressed during meiotic prophase, and GFP-Mcp5 localizes at the cell cortex. Moreover, the mcp5Δ strain lacks the oscillatory nuclear movement. Accordingly, homologous pairing and recombination rates of the mcp5Δ strain are significantly reduced. Furthermore, the cortical localization of dynein heavy chain 1 appears to be reduced in mcp5Δ cells. Finally, the full function of Mcp5 requires its coiled-coil and pleckstrin homology (PH) domains. Our results suggest that Mcp5 localizes at the cell cortex through its PH domain and functions as a dynein anchor, thereby facilitating nuclear oscillation.

scholarly journals Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

1995 ◽  
Vol 130 (3) ◽  
pp. 687-700 ◽  
Author(s):  
E Yeh ◽  
R V Skibbens ◽  
J W Cheng ◽  
E D Salmon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Translocation ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Wild Type ◽  
Immunofluorescent Localization ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Dynamics ◽  
Spindle Elongation ◽  
Anaphase B

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

scholarly journals Characterization of Suspension Cultures of Arabidopsis thaliana (L.) Heynh Cells with Altered Expression of the Gene of Alternative Mitochondrial Oxidase Aox1a and Analysis of their Frost Resistance

2021 ◽  
Vol 35 ◽  
pp. 3-18
Author(s):  
A. V. Stepanov ◽  
◽  
S. A. Kashin ◽  
N. S. Zabanova ◽  
O. A. Fedotova ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Suspension Culture ◽  
Mitochondrial Respiration ◽  
Frost Resistance ◽  
Alternative Oxidase ◽  
Uncoupling Protein ◽  
Living Cells ◽  
Subzero Temperature ◽  
Wild Type ◽  
Culture Cells

The enzyme alternative cyanide-resistant oxidase (AOX) localized in mitochondria is involved in the processes of plant adaptation to various unfavorable biotic and abiotic factors. Transfer of electrons from ubiquinone to oxygen by alternative oxidase has a nonprotonmotive character and, by bypassing two sites of H+ pumping in complexes III and IV, lowers the energy efficiency of respiration and energy of electron flow through AOX is released as heat. In this work, we characterized heterotrophic suspension cultures of Arabidopsis thaliana (L.) Heynh cells obtained from seeds of plants with altered (reduced (AS-12 line) and increased (XX-2 line)) expression of the alternative oxidase gene AOX1a and studied their viability under subzero temperature (-10 °С for 3, 6, 9 hours). Cell viability and reactive oxygen species (ROS) production were assessed using fluorescence microscopy with fluorescein diacetate (FDA) and propidium iodide (PI) for cell viability measurement and H2DCF-DA for ROS measurement. The proportion of living cells was calculated as the proportion of FDApositive and PI-negative cells. Differences between the studied lines were determined in the content of mitochondrial proteins of the respiratory chain (AOX, COXII, NDB) and uncoupling protein (UCP), as well as in the intensity of formation of ROS and frost resistance. The obtained results confirmed the higher content of the AOX protein and its high contribution to mitochondrial respiration in line XX-2. Suspension culture cells of the AS-12 line showed a decrease in the AOX protein content and its contribution to mitochondrial respiration, compared to the wild type (Col-0) and line XX-2. Simultaneously with a decrease in the AOX protein content in the AS-12 cell culture, an increase in the content of the uncoupling protein UCP and subunit II of cytochrome oxidase (COXII) was observed. ROS generation was reduced in cell cultures of both XX-2 and AS-12. The obtained results indicate that the cells of the wildtype (Col-0) suspension culture were subjected to the most significant effect of subzero temperature. Long-term exposure (for 9 h) under -10 °С revealed significant differences in the viability of wild-type culture cells and lines with altered AOX1a gene expression. Cells of line XX-2 with an increased content of AOX turned out to be more resistant to subzero temperature compared to wild-type and AS-12 cells. However, while the proportion of living cells in the culture of the AS-12 line 48 h after exposure remained at the same level as immediately after it, in the suspension culture of the wild type cell death developed over time. The obtained results indicate the importance of alternative oxidase in the development of frost resistance in plant cell.

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