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 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 Cytoplasmic dynein is required for normal nuclear segregation in yeast

1993 ◽  
Vol 90 (23) ◽  
pp. 11172-11176 ◽  
Author(s):  
D Eshel ◽  
L A Urrestarazu ◽  
S Vissers ◽  
J C Jauniaux ◽  
J C van Vliet-Reedijk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Heavy Chain ◽  
Mitotic Spindle ◽  
Cytoplasmic Dynein ◽  
Predicted Amino Acid Sequence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Abnormal Distribution ◽  
Bud Neck ◽  
Cytoplasmic Microtubules

We have identified the gene DYN1, which encodes the heavy chain of cytoplasmic dynein in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence (M(r) 471,305) reveals the presence of four P-loop motifs, as in all dyneins known so far, and has 28% overall identity to the dynein heavy chain of Dictyostelium [Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. Cell Biol. 119, 1597-1604] with 40% identity in the putative motor domain. Disruption of DYN1 causes misalignment of the spindle relative to the bud neck during cell division and results in abnormal distribution of the dividing nuclei between the mother cell and the bud. Cytoplasmic dynein, by generating force along cytoplasmic microtubules, may play an important role in the proper alignment of the mitotic spindle in yeast.

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 Somatic CRISPR–Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia

2015 ◽  
Vol 208 (6) ◽  
pp. 683-692 ◽  
Author(s):  
Wenjing Li ◽  
Peishan Yi ◽  
Guangshuo Ou
Keyword(s):  
Caenorhabditis Elegans ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Regulatory Mechanisms ◽  
Light Chains ◽  
Induced Mutations ◽  
Functional Studies ◽  
Cilium Formation ◽  
Dynein Intermediate Chain ◽  
Intermediate Chain

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2–driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT–dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

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 Dynein Intermediate Chain Mediated Dynein–Dynactin Interaction Is Required for Interphase Microtubule Organization and Centrosome Replication and Separation inDictyostelium

1999 ◽  
Vol 147 (6) ◽  
pp. 1261-1274 ◽  
Author(s):  
Shuo Ma ◽  
Leda Triviños-Lagos ◽  
Ralph Gräf ◽  
Rex L. Chisholm
Keyword(s):  
Heavy Chain ◽  
Physiological Role ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Microtubule Organization ◽  
Dynein Intermediate Chain ◽  
Organizing Center ◽  
Intermediate Chain

Cytoplasmic dynein intermediate chain (IC) mediates dynein–dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806–28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507–1516). To investigate the physiological role of IC and dynein–dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICΔC associated with dynactin but not with dynein heavy chain, whereas ICΔN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

scholarly journals The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis.

1986 ◽  
Vol 102 (5) ◽  
pp. 1688-1698 ◽  
Author(s):  
L Wordeman ◽  
K L McDonald ◽  
W Z Cande
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Centric Diatom ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Cytoplasmic Microtubules

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.

scholarly journals Complexities of Microtubule Population Dynamics within the Mitotic Spindle

2019 ◽  
Author(s):  
Aaron R. Tipton ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Cell Cortex ◽  
Spindle Microtubule ◽  
Sister Chromatids ◽  
Two Component ◽  
Chromosome Attachment ◽  
Complex Landscape ◽  
Spindle Microtubules ◽  
Insight Into ◽  
Slow Turnover

AbstractThe mitotic spindle functions to move chromosomes to alignment at metaphase, then segregate sister chromatids during anaphase. Analysis of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, historically taken to reflect kinetochore versus non-kinetochore microtubules respectively. This two component demarcation seems likely oversimplified. Microtubule turnover may vary among different spindle microtubules, regulated by spatial distribution and interactions with other microtubules and with organelles such as kinetochores, chromosome arms, and the cell cortex. How turnover among various spindle microtubules is differentially regulated and its significance remains unclear. We tested the concept of kinetochore versus non-kinetochore microtubules by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibited slow and fast turnover populations, though proportions and timings of turnover were altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. Dissection of the dynamics of microtubule populations will provide a greater understanding of mitotic spindle kinetics and insight into roles in facilitating chromosome attachment, movement, and segregation during mitosis.

scholarly journals The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

1999 ◽  
Vol 144 (5) ◽  
pp. 963-975 ◽  
Author(s):  
Rita K. Miller ◽  
Dina Matheos ◽  
Mark D. Rose
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Nuclear Positioning ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoplasmic Microtubule ◽  
Microtubule Orientation ◽  
Cortical Localization ◽  
Cytoplasmic Microtubules ◽  
Green Fluorescent ◽  
Mitotic Cells

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)– Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Δ, pea2Δ, bud6Δ, and bni1Δ, required for normal polarization and actin cytoskeleton functions and, of these, bni1Δ affected Kar9p localization most severely. Like kar9Δ, bni1Δ mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Δ, the bni1Δ mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Δ bni1Δ double mutants suggested that Kar9p retained some function in bni1Δ mitotic cells. Unlike the polarization mutants, kar9Δ shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Δ. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.

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