Kinesin-1 and Cytoplasmic Dynein Act Sequentially to Move the Meiotic Spindle to the Oocyte Cortex in Caenorhabditis elegans

During female meiosis in animals, the meiotic spindle is attached to the egg cortex by one pole during anaphase to allow selective disposal of half the chromosomes in a polar body. In Caenorhabditis elegans, this anaphase spindle position is achieved sequentially through kinesin-1–dependent early translocation followed by anaphase-promoting complex (APC)-dependent spindle rotation. Partial depletion of cytoplasmic dynein heavy chain by RNA interference blocked spindle rotation without affecting early translocation. Dynein depletion also blocked the APC-dependent late translocation that occurs in kinesin-1–depleted embryos. Time-lapse imaging of green fluorescent protein-tagged dynein heavy chain as well as immunofluorescence with dynein-specific antibodies revealed that dynein starts to accumulate at spindle poles just before the initiation of rotation or late translocation. Accumulation of dynein at poles was kinesin-1 independent and APC dependent, just like dynein driven spindle movements. This represents a case of kinesin-1/dynein coordination in which these two motors of opposite polarity act sequentially and independently on a cargo to move it in the same direction.

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Functional Analysis of Cytoplasmic Dynein Heavy Chain in Caenorhabditis elegans with Fast-acting Temperature-sensitive Mutations

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0523 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1200-1212 ◽

Cited By ~ 68

Author(s):

Diane J. Schmidt ◽

Debra J. Rose ◽

William M. Saxton ◽

Susan Strome

Keyword(s):

Caenorhabditis Elegans ◽

Heavy Chain ◽

Metaphase Plate ◽

Cytoplasmic Dynein ◽

Model Systems ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Timely Initiation ◽

Dynein Heavy Chain ◽

Dominant Temperature Sensitive

Cytoplasmic dynein, a minus-end–directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of a Caenorhabditis elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of Saccharomyces cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems.

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Cytoplasmic Dynein Heavy Chain 1b Is Required for Flagellar Assembly in Chlamydomonas

Molecular Biology of the Cell ◽

10.1091/mbc.10.3.693 ◽

1999 ◽

Vol 10 (3) ◽

pp. 693-712 ◽

Cited By ~ 244

Author(s):

Mary E. Porter ◽

Raqual Bower ◽

Julie A. Knott ◽

Pamela Byrd ◽

William Dentler

Keyword(s):

Caenorhabditis Elegans ◽

Expression Pattern ◽

Heavy Chain ◽

Insertional Mutagenesis ◽

Sea Urchins ◽

Amorphous Material ◽

Cytoplasmic Dynein ◽

Genetic Approach ◽

Dynein Heavy Chain ◽

Flagellar Assembly

A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. TheChlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1bgene have been deleted. The two mutants assemble short flagellar stumps (<1–2 μm) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

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

The Journal of Cell Biology ◽

10.1083/jcb.145.6.1233 ◽

1999 ◽

Vol 145 (6) ◽

pp. 1233-1250 ◽

Cited By ~ 175

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e02-06-0359 ◽

2003 ◽

Vol 14 (3) ◽

pp. 871-888 ◽

Cited By ~ 42

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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Cytoplasmic Dynein Is Required for Distinct Aspects of Mtoc Positioning, Including Centrosome Separation, in the One Cell Stage Caenorhabditis elegans Embryo

The Journal of Cell Biology ◽

10.1083/jcb.147.1.135 ◽

1999 ◽

Vol 147 (1) ◽

pp. 135-150 ◽

Cited By ~ 306

Author(s):

Pierre Gönczy ◽

Silke Pichler ◽

Matthew Kirkham ◽

Anthony A. Hyman

Keyword(s):

Caenorhabditis Elegans ◽

Heavy Chain ◽

Cytoplasmic Dynein ◽

Chain Gene ◽

Differential Interference Contrast Microscopy ◽

Microtubule Organizing Center ◽

Cell Stage ◽

Dynein Heavy Chain ◽

Centrosome Separation ◽

The One

We have investigated the role of cytoplasmic dynein in microtubule organizing center (MTOC) positioning using RNA-mediated interference (RNAi) in Caenorhabditis elegans to deplete the product of the dynein heavy chain gene dhc-1. Analysis with time-lapse differential interference contrast microscopy and indirect immunofluorescence revealed that pronuclear migration and centrosome separation failed in one cell stage dhc-1 (RNAi) embryos. These phenotypes were also observed when the dynactin components p50/dynamitin or p150Glued were depleted with RNAi. Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus. When dynein heavy chain function was diminished only partially with RNAi, centrosome separation took place, but orientation of the mitotic spindle was defective. Therefore, cytoplasmic dynein is required for multiple aspects of MTOC positioning in the one cell stage C. elegans embryo. In conjunction with our observation of cytoplasmic dynein distribution at the periphery of nuclei, these results lead us to propose a mechanism in which cytoplasmic dynein anchored on the nucleus drives centrosome separation.

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Nudel functions in membrane traffic mainly through association with Lis1 and cytoplasmic dynein

The Journal of Cell Biology ◽

10.1083/jcb.200308058 ◽

2004 ◽

Vol 164 (4) ◽

pp. 557-566 ◽

Cited By ~ 83

Author(s):

Yun Liang ◽

Wei Yu ◽

Yan Li ◽

Zhenye Yang ◽

Xiumin Yan ◽

...

Keyword(s):

Heavy Chain ◽

Neuronal Migration ◽

Time Lapse ◽

Cytoplasmic Dynein ◽

Wild Type ◽

Dynein Heavy Chain ◽

Time Lapse Microscopy ◽

Dynein Motor ◽

Defective Mutant ◽

Directed Motility

Nudel and Lis1 appear to regulate cytoplasmic dynein in neuronal migration and mitosis through direct interactions. However, whether or not they regulate other functions of dynein remains elusive. Herein, overexpression of a Nudel mutant defective in association with either Lis1 or dynein heavy chain is shown to cause dispersions of membranous organelles whose trafficking depends on dynein. In contrast, the wild-type Nudel and the double mutant that binds to neither protein are much less effective. Time-lapse microscopy for lysosomes reveals significant reduction in both frequencies and velocities of their minus end–directed motions in cells expressing the dynein-binding defective mutant, whereas neither the durations of movement nor the plus end–directed motility is considerably altered. Moreover, silencing Nudel expression by RNA interference results in Golgi apparatus fragmentation and cell death. Together, it is concluded that Nudel is critical for dynein motor activity in membrane transport and possibly other cellular activities through interactions with both Lis1 and dynein heavy chain.

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Identification and molecular evolution of new dynein-like protein sequences in rat brain

Journal of Cell Science ◽

10.1242/jcs.108.5.1883 ◽

1995 ◽

Vol 108 (5) ◽

pp. 1883-1893 ◽

Cited By ~ 7

Author(s):

Y. Tanaka ◽

Z. Zhang ◽

N. Hirokawa

Keyword(s):

Rat Brain ◽

Heavy Chain ◽

Phylogenetic Trees ◽

Cytoplasmic Dynein ◽

Amino Acid Sequences ◽

Adult Brain ◽

Chain Gene ◽

Evolutionary Analysis ◽

Degenerate Primers ◽

Dynein Heavy Chain

RT-PCR cloning was performed to find unknown members of the dynein superfamily expressed in rat brain. Six kinds of degenerate primers designed for the dynein catalytic domain consensuses were used for extensive PCR amplifications. We have sequenced 550 plasmid clones which turned out to include 13 kinds of new dynein-like sequences (DLP1-8, 9A/B, 10–12) and cytoplasmic dynein heavy chain. In these clones, alternative splicing was detected for a 105 nt-domain containing the CFDEFNRI consensus just downstream of the most N-terminal P-loop (DLP9A and 9B). By using these obtained sequences, initial hybridization studies were performed. Genomic Southern blotting showed each sequence corresponds to a single copy of the gene, while northern blotting of adult brain presented more than one band for some subtypes. We further accomplished molecular evolutionary analysis to recognize their phylogenetic origins for the axonemal and non-axonemal (cytoplasmic) functions. Different methods (UPGMA, NJ and MP) presented well coincident phylogenetic trees from 44 partial amino acid sequences of dynein heavy chain from various eukaryotes. The trunk for all the cytoplasmic dynein heavy chain homologues diverged directly from the root of the phylogenetic tree, suggesting that the first dynein gene duplication defined two distinct functions as respective subfamilies. Of particular interest, we found a duplication event of the cytoplasmic dynein heavy chain gene giving rise to another subtype, DLP4, located between the divergence of yeast and that of Dictyostelium. Such evolutionary topology builds up an inceptive hypothesis that there are at least two non-axonemal dynein heavy chains in mammals.

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