scholarly journals Cytoplasmic Dynein Is Required for the Nuclear Attachment and Migration of Centrosomes during Mitosis in Drosophila

1999 ◽  
Vol 146 (3) ◽  
pp. 597-608 ◽  
Author(s):  
John T. Robinson ◽  
Edward J. Wojcik ◽  
Mark A. Sanders ◽  
Maura McGrail ◽  
Thomas S. Hays
Keyword(s):  
Heavy Chain ◽  
Spatial Organization ◽  
Critical Role ◽  
Genetic System ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
And Migration

Cytoplasmic dynein is a multisubunit minus-end–directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immu-nofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.

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

1995 ◽  
Vol 108 (5) ◽  
pp. 1883-1893 ◽  
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.

scholarly journals Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

2004 ◽  
Vol 3 (6) ◽  
pp. 1574-1588 ◽  
Author(s):  
R. Martin ◽  
A. Walther ◽  
J. Wendland
Keyword(s):  
Candida Albicans ◽  
Heavy Chain ◽  
Time Lapse ◽  
Yeast Cells ◽  
Chain Gene ◽  
Wild Type ◽  
Heavy Chain Gene ◽  
Mutant Strains ◽  
Mutant Phenotypes

ABSTRACT Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals A family of dynein genes in Drosophila melanogaster.

1994 ◽  
Vol 5 (1) ◽  
pp. 45-55 ◽  
Author(s):  
K Rasmusson ◽  
M Serr ◽  
J Gepner ◽  
I Gibbons ◽  
T S Hays
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Amino Acid Level ◽  
Single Copy ◽  
Cytoplasmic Dynein ◽  
Amino Acid Identity ◽  
Chain Gene ◽  
Phosphate Binding ◽  
Atp Binding Site ◽  
Dynein Heavy Chain

We report the identification and initial characterization of seven Drosophila dynein heavy chain genes. Each gene is single copy and maps to a unique genomic location. Sequence analysis of partial clones reveals that each encodes a highly conserved portion of the putative dynein hydrolytic ATP-binding site in dyneins that includes a consensus phosphate-binding (P-loop) motif. One of the clones is derived from a Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, that shows extensive amino acid identity to cytoplasmic dynein isoforms from other organisms. Two other Drosophila dynein clones are 85 and 90% identical at the amino acid level to the corresponding region of the beta heavy chain of sea urchin axonemal dynein. Probes for all seven of the dynein-related sequences hybridize to transcripts that are of the appropriate size, approximately 14 kilobases, to encode the characteristic high molecular weight dynein heavy chain polypeptides. The Dhc64C transcript is readily detected in RNA from ovaries, embryos, and testes. Transcripts from five of the six remaining genes are also detected in much lesser amounts in tissues other than testes. All but one of the dynein transcripts are expressed at comparable levels in testes suggesting their participation in flagellar axoneme assembly and motility.

Cytoplasmic Dynein Function Is Essential in Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 865-878 ◽  
Author(s):  
Janice Gepner ◽  
Min-gang Li ◽  
Susan Ludmann ◽  
Cynthia Kortas ◽  
Kristin Boylan ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Intracellular Transport ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Complementation Group ◽  
Transport Processes ◽  
Female Sterility ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Dynein Heavy Chain

Abstract The microtubule motor cytoplasmic dynein has been implicated in a variety of intracellular transport processes. We previously identified and characterized the Drosophila gene Dhc64C, which encodes a cytoplasmic dynein heavy chain. To investigate the function of the cytoplasmic dynein motor, we initiated a mutational analysis of the Dhc64C dynein gene. A small deletion that removes the chromosomal region containing the heavy chain gene was used to isolate EMS-induced lethal mutations that define at least eight essential genes in the region. Germline transformation with a Dhc64C transgene rescued 16 mutant alleles in the single complementation group that identifies the dynein heavy chain gene. All 16 alleles were hemizygous lethal, which demonstrates that the cytoplasmic dynein heavy chain gene Dhc64C is essential for Drosophila development. Furthermore, our failure to recover somatic clones of cells homozygous for a Dhc64C mutation indicates that cytoplasmic dynein function is required for cell viability in several Drosophila tissues. The intragenic complementation of dynein alleles reveals multiple mutant phenotypes including male and/or female sterility, bristle defects, and defects in eye development.

scholarly journals Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein.

1992 ◽  
Vol 119 (6) ◽  
pp. 1597-1604 ◽  
Author(s):  
M P Koonce ◽  
P M Grissom ◽  
J R McIntosh
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Cellular Slime Mold ◽  
Reading Frame ◽  
Dynein Heavy Chain ◽  
Binding Domains

We report here the cloning and sequencing of a cytoplasmic dynein heavy chain gene from the cellular slime mold Dictyostelium discoideum. Using a combination of approaches, we have isolated 14,318 bp of DNA sequence which contains an open-reading frame of 4,725 amino acids. The deduced molecular weight of the polypeptide predicted by this reading frame is 538,482 D. Overall, the polypeptide sequence is 51% similar and 28% identical to the recently published sequences of the beta-dynein heavy chain from sea urchin flagella (Gibbons, I. R., B. H. Gibbons, G. Mocz, and D. J. Asai. 1991. Nature (Lond.). 352: 640-643; Ogawa, K. 1991. Nature (Lond.). 352:643-645). It contains four GXXXXGKT/S motifs that form part of a consensus sequence for ATP-binding domains; these motifs are clustered near the middle of the polypeptide. The distribution of the regions sharing sequence similarity between the Dictyostelium and sea urchin heavy chain polypeptides suggests that the amino termini of dyneins may contain domains that specify axonemal or cytoplasmic functions.

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

1999 ◽  
Vol 147 (1) ◽  
pp. 135-150 ◽  
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.

scholarly journals Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.

1995 ◽  
Vol 131 (2) ◽  
pp. 411-425 ◽  
Author(s):  
M McGrail ◽  
J Gepner ◽  
A Silvanovich ◽  
S Ludmann ◽  
M Serr ◽  
...  
Keyword(s):  
Temporal Distribution ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Gene Products ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Dynein Motor ◽  
Associated Proteins

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.

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