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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.

Gönczy P, Pichler S, Kirkham M, Hyman AA - J. Cell Biol. (1999)

Bottom Line: Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus.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.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, D-69117 Germany. gonczy@embl-heidelberg.de

ABSTRACT
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 p150(Glued) 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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Mentions: We tested whether anti–DHC-1 antibodies specifically recognize dynein heavy chain protein in C. elegans embryos in two ways. First, we compared the immunofluorescence staining intensity observed with anti–DHC-1 antibodies in wild type to that seen in embryos in which dhc-1 gene expression was silenced in a sequence-specific manner using RNAi (hereafter referred to as dhc-1 (RNAi) embryos; see Materials and Methods). As shown in Fig. 1B and Fig. c, 88% of the anti–DHC-1 signal was lost on average in dhc-1 (RNAi) embryos. Residual staining might be due to incomplete silencing of the dhc-1 gene by RNAi. Second, we determined that anti–DHC-1 immunostaining was entirely absent from embryos incubated with anti–DHC-1 antibodies in the presence of 0.1 mg/ml DHC-1 peptide (data not shown). Taken together, these results demonstrate that most, if not all, of the signal detected with anti–DHC-1 antibodies in wild-type embryos is specific for the cytoplasmic dynein heavy chain.


Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo.

Gönczy P, Pichler S, Kirkham M, Hyman AA - J. Cell Biol. (1999)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164971&req=5

Mentions: We tested whether anti–DHC-1 antibodies specifically recognize dynein heavy chain protein in C. elegans embryos in two ways. First, we compared the immunofluorescence staining intensity observed with anti–DHC-1 antibodies in wild type to that seen in embryos in which dhc-1 gene expression was silenced in a sequence-specific manner using RNAi (hereafter referred to as dhc-1 (RNAi) embryos; see Materials and Methods). As shown in Fig. 1B and Fig. c, 88% of the anti–DHC-1 signal was lost on average in dhc-1 (RNAi) embryos. Residual staining might be due to incomplete silencing of the dhc-1 gene by RNAi. Second, we determined that anti–DHC-1 immunostaining was entirely absent from embryos incubated with anti–DHC-1 antibodies in the presence of 0.1 mg/ml DHC-1 peptide (data not shown). Taken together, these results demonstrate that most, if not all, of the signal detected with anti–DHC-1 antibodies in wild-type embryos is specific for the cytoplasmic dynein heavy chain.

Bottom Line: Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus.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.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, D-69117 Germany. gonczy@embl-heidelberg.de

ABSTRACT
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 p150(Glued) 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.

Show MeSH
Related in: MedlinePlus