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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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Failure of pronuclear migration in dhc-1 (RNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–D) and dhc-1 (RNAi) embryos (E–H). Time elapsed since the beginning of the sequence is displayed in minutes and seconds in each image. All images are at the same magnification. (A and E) In both wild-type and dhc-1 (RNAi) embryo, the male pronucleus is apposed to the posterior cortex (A and E, rightmost arrow). In wild-type, there is a single female pronucleus located slightly off the anterior cortex (A, leftmost arrow). In contrast, there are five female pronuclei in the dhc-1 (RNAi) embryo (E, arrows towards the left point at three that are visible in this focal plane). Note the pseudocleavage furrow in the middle of both wild-type and dhc-1 (RNAi) embryos. Female pronuclei in some dhc-1 (RNAi) embryos were located towards the middle of the embryo (not shown). (B and F) In wild type, after migration of both male and female pronuclei, the pronuclei have met and move along with the centrosome pair (B, arrowheads) towards the center while undergoing a 90° rotation. In contrast, neither male nor female pronuclei migrate in the dhc-1 (RNAi) embryo. (C and G) In wild type, the spindle sets up in the cell center and along the longitudinal axis (C, arrowheads point to spindle poles). In the dhc-1 (RNAi) embryo, no bipolar structure is visible after nuclear envelope breakdown. However, an area devoid of yolk granules extends towards the anterior of the embryo (arrow in G points to anterior of this area). The asters appear to be at the very posterior of the embryo (G, arrowheads). Note that the membranes of the female pronuclei are still intact after the male pronuclear membrane broke down. (D and H) In wild type, the first cleavage generates two unequally sized daughters, each with a centrally located nucleus (D, arrows). In contrast, no proper cell division occurs in the dhc-1 (RNAi) embryo. While some furrowing activity does take place, this is usually restricted to the anterior and does not result in productive cleavage. Numerous small nuclei reform, presumably around nonsegregated chromosomes, as the cell returns into interphase (H, arrows). Bar, 10 μm.
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Figure 5: Failure of pronuclear migration in dhc-1 (RNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–D) and dhc-1 (RNAi) embryos (E–H). Time elapsed since the beginning of the sequence is displayed in minutes and seconds in each image. All images are at the same magnification. (A and E) In both wild-type and dhc-1 (RNAi) embryo, the male pronucleus is apposed to the posterior cortex (A and E, rightmost arrow). In wild-type, there is a single female pronucleus located slightly off the anterior cortex (A, leftmost arrow). In contrast, there are five female pronuclei in the dhc-1 (RNAi) embryo (E, arrows towards the left point at three that are visible in this focal plane). Note the pseudocleavage furrow in the middle of both wild-type and dhc-1 (RNAi) embryos. Female pronuclei in some dhc-1 (RNAi) embryos were located towards the middle of the embryo (not shown). (B and F) In wild type, after migration of both male and female pronuclei, the pronuclei have met and move along with the centrosome pair (B, arrowheads) towards the center while undergoing a 90° rotation. In contrast, neither male nor female pronuclei migrate in the dhc-1 (RNAi) embryo. (C and G) In wild type, the spindle sets up in the cell center and along the longitudinal axis (C, arrowheads point to spindle poles). In the dhc-1 (RNAi) embryo, no bipolar structure is visible after nuclear envelope breakdown. However, an area devoid of yolk granules extends towards the anterior of the embryo (arrow in G points to anterior of this area). The asters appear to be at the very posterior of the embryo (G, arrowheads). Note that the membranes of the female pronuclei are still intact after the male pronuclear membrane broke down. (D and H) In wild type, the first cleavage generates two unequally sized daughters, each with a centrally located nucleus (D, arrows). In contrast, no proper cell division occurs in the dhc-1 (RNAi) embryo. While some furrowing activity does take place, this is usually restricted to the anterior and does not result in productive cleavage. Numerous small nuclei reform, presumably around nonsegregated chromosomes, as the cell returns into interphase (H, arrows). Bar, 10 μm.

Mentions: Fig. 5A–D, shows the relevant sequence of events in wild type. After fertilization, the two meiotic divisions are completed in the one cell stage embryo. The resulting female pronucleus lies slightly off the anterior cortex (Fig. 5 A, left arrow), whereas the male pronucleus is tightly apposed to the posterior cortex (Fig. 5 A, right arrow). The sperm contributes the single centrosome of the one cell stage embryo (Albertson 1984; Hyman and White 1987). After duplication, the two daughter centrosomes separate, while remaining closely associated with the male pronucleus. The separated centrosomes migrate slightly anteriorly, along with the male pronucleus, whereas the female pronucleus migrates posteriorly towards the centrosomes. As a result, the male and female pronuclei meet at ∼70% egg length (Fig. 5 B; 0% anterior-most, 100% posterior-most).


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)

Failure of pronuclear migration in dhc-1 (RNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–D) and dhc-1 (RNAi) embryos (E–H). Time elapsed since the beginning of the sequence is displayed in minutes and seconds in each image. All images are at the same magnification. (A and E) In both wild-type and dhc-1 (RNAi) embryo, the male pronucleus is apposed to the posterior cortex (A and E, rightmost arrow). In wild-type, there is a single female pronucleus located slightly off the anterior cortex (A, leftmost arrow). In contrast, there are five female pronuclei in the dhc-1 (RNAi) embryo (E, arrows towards the left point at three that are visible in this focal plane). Note the pseudocleavage furrow in the middle of both wild-type and dhc-1 (RNAi) embryos. Female pronuclei in some dhc-1 (RNAi) embryos were located towards the middle of the embryo (not shown). (B and F) In wild type, after migration of both male and female pronuclei, the pronuclei have met and move along with the centrosome pair (B, arrowheads) towards the center while undergoing a 90° rotation. In contrast, neither male nor female pronuclei migrate in the dhc-1 (RNAi) embryo. (C and G) In wild type, the spindle sets up in the cell center and along the longitudinal axis (C, arrowheads point to spindle poles). In the dhc-1 (RNAi) embryo, no bipolar structure is visible after nuclear envelope breakdown. However, an area devoid of yolk granules extends towards the anterior of the embryo (arrow in G points to anterior of this area). The asters appear to be at the very posterior of the embryo (G, arrowheads). Note that the membranes of the female pronuclei are still intact after the male pronuclear membrane broke down. (D and H) In wild type, the first cleavage generates two unequally sized daughters, each with a centrally located nucleus (D, arrows). In contrast, no proper cell division occurs in the dhc-1 (RNAi) embryo. While some furrowing activity does take place, this is usually restricted to the anterior and does not result in productive cleavage. Numerous small nuclei reform, presumably around nonsegregated chromosomes, as the cell returns into interphase (H, arrows). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 5: Failure of pronuclear migration in dhc-1 (RNAi) embryos. Time-lapse DIC microscopy recordings of wild-type (A–D) and dhc-1 (RNAi) embryos (E–H). Time elapsed since the beginning of the sequence is displayed in minutes and seconds in each image. All images are at the same magnification. (A and E) In both wild-type and dhc-1 (RNAi) embryo, the male pronucleus is apposed to the posterior cortex (A and E, rightmost arrow). In wild-type, there is a single female pronucleus located slightly off the anterior cortex (A, leftmost arrow). In contrast, there are five female pronuclei in the dhc-1 (RNAi) embryo (E, arrows towards the left point at three that are visible in this focal plane). Note the pseudocleavage furrow in the middle of both wild-type and dhc-1 (RNAi) embryos. Female pronuclei in some dhc-1 (RNAi) embryos were located towards the middle of the embryo (not shown). (B and F) In wild type, after migration of both male and female pronuclei, the pronuclei have met and move along with the centrosome pair (B, arrowheads) towards the center while undergoing a 90° rotation. In contrast, neither male nor female pronuclei migrate in the dhc-1 (RNAi) embryo. (C and G) In wild type, the spindle sets up in the cell center and along the longitudinal axis (C, arrowheads point to spindle poles). In the dhc-1 (RNAi) embryo, no bipolar structure is visible after nuclear envelope breakdown. However, an area devoid of yolk granules extends towards the anterior of the embryo (arrow in G points to anterior of this area). The asters appear to be at the very posterior of the embryo (G, arrowheads). Note that the membranes of the female pronuclei are still intact after the male pronuclear membrane broke down. (D and H) In wild type, the first cleavage generates two unequally sized daughters, each with a centrally located nucleus (D, arrows). In contrast, no proper cell division occurs in the dhc-1 (RNAi) embryo. While some furrowing activity does take place, this is usually restricted to the anterior and does not result in productive cleavage. Numerous small nuclei reform, presumably around nonsegregated chromosomes, as the cell returns into interphase (H, arrows). Bar, 10 μm.
Mentions: Fig. 5A–D, shows the relevant sequence of events in wild type. After fertilization, the two meiotic divisions are completed in the one cell stage embryo. The resulting female pronucleus lies slightly off the anterior cortex (Fig. 5 A, left arrow), whereas the male pronucleus is tightly apposed to the posterior cortex (Fig. 5 A, right arrow). The sperm contributes the single centrosome of the one cell stage embryo (Albertson 1984; Hyman and White 1987). After duplication, the two daughter centrosomes separate, while remaining closely associated with the male pronucleus. The separated centrosomes migrate slightly anteriorly, along with the male pronucleus, whereas the female pronucleus migrates posteriorly towards the centrosomes. As a result, the male and female pronuclei meet at ∼70% egg length (Fig. 5 B; 0% anterior-most, 100% posterior-most).

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