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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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Spindle orientation defect in dhc-1 (ssRNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–C) and dhc-1 (ssRNAi) (D–F) embryos. In each image, time elapsed since the beginning of the sequence is displayed in minutes and seconds and arrowheads point to center of asters and spindle poles. All images are at the same magnification. (A and D) In both wild-type and dhc-1 (ssRNAi) embryos, pronuclei meet at ∼70% egg length. (B and E) In wild type, the centrosome pair and associated pronuclei move towards the center while undergoing a 90° rotation. This does not happen in the dhc-1 (ssRNAi) embryo. As a result, while the spindle sets up in the cell center and along the longitudinal axis in wild-type, it does so in the posterior half and perpendicular to the longitudinal axis in the dhc-1 (ssRNAi) embryo. (C and F) Both wild-type and dhc-1 (ssRNAi) embryos divide asymmetrically into a larger anterior blastomere and a smaller posterior one. In the dhc-1 (ssRNAi) embryo, this occurs after rescue of the spindle orientation onto the longitudinal axis during anaphase, possibly because of the physical constraints of the eggshell. Note that the cleavage furrow ingresses sooner on one side of the dhc-1 (ssRNAi) embryo (bottom side), because the spindle was closer to that side during rescue of spindle orientation. In some dhc-1 (ssRNAi) embryos, >1 nucleus reformed in each daughter blastomere, indicative of defects in chromosome segregation. Bar, 10 μm.
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Figure 9: Spindle orientation defect in dhc-1 (ssRNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–C) and dhc-1 (ssRNAi) (D–F) embryos. In each image, time elapsed since the beginning of the sequence is displayed in minutes and seconds and arrowheads point to center of asters and spindle poles. All images are at the same magnification. (A and D) In both wild-type and dhc-1 (ssRNAi) embryos, pronuclei meet at ∼70% egg length. (B and E) In wild type, the centrosome pair and associated pronuclei move towards the center while undergoing a 90° rotation. This does not happen in the dhc-1 (ssRNAi) embryo. As a result, while the spindle sets up in the cell center and along the longitudinal axis in wild-type, it does so in the posterior half and perpendicular to the longitudinal axis in the dhc-1 (ssRNAi) embryo. (C and F) Both wild-type and dhc-1 (ssRNAi) embryos divide asymmetrically into a larger anterior blastomere and a smaller posterior one. In the dhc-1 (ssRNAi) embryo, this occurs after rescue of the spindle orientation onto the longitudinal axis during anaphase, possibly because of the physical constraints of the eggshell. Note that the cleavage furrow ingresses sooner on one side of the dhc-1 (ssRNAi) embryo (bottom side), because the spindle was closer to that side during rescue of spindle orientation. In some dhc-1 (ssRNAi) embryos, >1 nucleus reformed in each daughter blastomere, indicative of defects in chromosome segregation. Bar, 10 μm.

Mentions: In wild type, the centrosome pair is positioned at 70% egg length and transverse to the longitudinal axis after pronuclear meeting (Fig. 9 A, arrowheads). The centrosome pair and associated pronuclei subsequently move to the embryo center while undergoing a 90° rotation (Fig. 9 B, arrowheads). As a result, after breakdown of the pronuclear envelopes, the spindle is positioned in the cell center and oriented along the longitudinal axis (Fig. 9 C, arrowheads).


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)

Spindle orientation defect in dhc-1 (ssRNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–C) and dhc-1 (ssRNAi) (D–F) embryos. In each image, time elapsed since the beginning of the sequence is displayed in minutes and seconds and arrowheads point to center of asters and spindle poles. All images are at the same magnification. (A and D) In both wild-type and dhc-1 (ssRNAi) embryos, pronuclei meet at ∼70% egg length. (B and E) In wild type, the centrosome pair and associated pronuclei move towards the center while undergoing a 90° rotation. This does not happen in the dhc-1 (ssRNAi) embryo. As a result, while the spindle sets up in the cell center and along the longitudinal axis in wild-type, it does so in the posterior half and perpendicular to the longitudinal axis in the dhc-1 (ssRNAi) embryo. (C and F) Both wild-type and dhc-1 (ssRNAi) embryos divide asymmetrically into a larger anterior blastomere and a smaller posterior one. In the dhc-1 (ssRNAi) embryo, this occurs after rescue of the spindle orientation onto the longitudinal axis during anaphase, possibly because of the physical constraints of the eggshell. Note that the cleavage furrow ingresses sooner on one side of the dhc-1 (ssRNAi) embryo (bottom side), because the spindle was closer to that side during rescue of spindle orientation. In some dhc-1 (ssRNAi) embryos, >1 nucleus reformed in each daughter blastomere, indicative of defects in chromosome segregation. Bar, 10 μm.
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Figure 9: Spindle orientation defect in dhc-1 (ssRNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–C) and dhc-1 (ssRNAi) (D–F) embryos. In each image, time elapsed since the beginning of the sequence is displayed in minutes and seconds and arrowheads point to center of asters and spindle poles. All images are at the same magnification. (A and D) In both wild-type and dhc-1 (ssRNAi) embryos, pronuclei meet at ∼70% egg length. (B and E) In wild type, the centrosome pair and associated pronuclei move towards the center while undergoing a 90° rotation. This does not happen in the dhc-1 (ssRNAi) embryo. As a result, while the spindle sets up in the cell center and along the longitudinal axis in wild-type, it does so in the posterior half and perpendicular to the longitudinal axis in the dhc-1 (ssRNAi) embryo. (C and F) Both wild-type and dhc-1 (ssRNAi) embryos divide asymmetrically into a larger anterior blastomere and a smaller posterior one. In the dhc-1 (ssRNAi) embryo, this occurs after rescue of the spindle orientation onto the longitudinal axis during anaphase, possibly because of the physical constraints of the eggshell. Note that the cleavage furrow ingresses sooner on one side of the dhc-1 (ssRNAi) embryo (bottom side), because the spindle was closer to that side during rescue of spindle orientation. In some dhc-1 (ssRNAi) embryos, >1 nucleus reformed in each daughter blastomere, indicative of defects in chromosome segregation. Bar, 10 μm.
Mentions: In wild type, the centrosome pair is positioned at 70% egg length and transverse to the longitudinal axis after pronuclear meeting (Fig. 9 A, arrowheads). The centrosome pair and associated pronuclei subsequently move to the embryo center while undergoing a 90° rotation (Fig. 9 B, arrowheads). As a result, after breakdown of the pronuclear envelopes, the spindle is positioned in the cell center and oriented along the longitudinal axis (Fig. 9 C, arrowheads).

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