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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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Distribution of cytoplasmic dynein in early wild-type embryos. Embryos stained with anti–DHC-1 and antitubulin (TUB; high magnification images only) antibodies and counterstained with Hoechst 33258 to reveal DNA. A, B, O, and P are at the same magnification, as are C–N. Merged images, DHC-1, red; TUB, green; and DNA, blue. (A and B) One cell stage embryo during pronuclear migration. DHC-1 is distributed throughout the cytoplasm in a punctate manner, and is enriched at the periphery of the male (A and B, arrowhead) and female (A and B, arrow) pronuclei. DHC-1 is also slightly enriched at the cortex of one cell stage embryos, but this is not rendered in this particular focal plane. Small arrowhead in B points to out-of-focus polar body DNA. (C–F) Prometaphase, one cell stage embryo. DHC-1 is enriched on both sides (C and F, arrows) of congressing chromosomes (D and F, arrowhead). (G–J) Metaphase, one cell stage embryo. DHC-1 is enriched on the spindle on both sides of the metaphase plate. (K–N) Early anaphase, P1 blastomere of two cell stage embryo. DHC-1 is enriched on the spindle between the chromosomes (L and N, arrowheads) and the spindle poles, as well as centrally (K and N, arrow) between the two sets of chromosomes. (O and P) Two cell stage embryo, P1 blastomere (right) is in late anaphase, AB blastomere (left) in late telophase. In P1, DHC-1 is enriched on the spindle (O, arrow) between the chromosomes and the spindle poles and in the central spindle. In AB, DHC-1 is enriched at the periphery of reforming nuclei (O, black arrowhead) as well as in an area above the spindle poles. In addition, DHC-1 is localized throughout the cortex between the AB and P1 blastomeres (O, arrowheads). Small arrowhead in P points to polar body DNA. Bars, 10 μm.
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Figure 2: Distribution of cytoplasmic dynein in early wild-type embryos. Embryos stained with anti–DHC-1 and antitubulin (TUB; high magnification images only) antibodies and counterstained with Hoechst 33258 to reveal DNA. A, B, O, and P are at the same magnification, as are C–N. Merged images, DHC-1, red; TUB, green; and DNA, blue. (A and B) One cell stage embryo during pronuclear migration. DHC-1 is distributed throughout the cytoplasm in a punctate manner, and is enriched at the periphery of the male (A and B, arrowhead) and female (A and B, arrow) pronuclei. DHC-1 is also slightly enriched at the cortex of one cell stage embryos, but this is not rendered in this particular focal plane. Small arrowhead in B points to out-of-focus polar body DNA. (C–F) Prometaphase, one cell stage embryo. DHC-1 is enriched on both sides (C and F, arrows) of congressing chromosomes (D and F, arrowhead). (G–J) Metaphase, one cell stage embryo. DHC-1 is enriched on the spindle on both sides of the metaphase plate. (K–N) Early anaphase, P1 blastomere of two cell stage embryo. DHC-1 is enriched on the spindle between the chromosomes (L and N, arrowheads) and the spindle poles, as well as centrally (K and N, arrow) between the two sets of chromosomes. (O and P) Two cell stage embryo, P1 blastomere (right) is in late anaphase, AB blastomere (left) in late telophase. In P1, DHC-1 is enriched on the spindle (O, arrow) between the chromosomes and the spindle poles and in the central spindle. In AB, DHC-1 is enriched at the periphery of reforming nuclei (O, black arrowhead) as well as in an area above the spindle poles. In addition, DHC-1 is localized throughout the cortex between the AB and P1 blastomeres (O, arrowheads). Small arrowhead in P points to polar body DNA. Bars, 10 μm.

Mentions: We used anti–DHC-1 antibodies to determine the subcellular distribution of cytoplasmic dynein in early wild-type embryos by immunofluorescence microscopy (Fig. 2). We found that cytoplasmic dynein was present in a punctate manner throughout the cytoplasm at all stages of the cell cycle. In addition, a stronger signal was detected at the periphery of pronuclei in one cell stage embryos (Fig. 2 A, arrow and arrowhead) and of nuclei in later stage embryos (Fig. 2 O, black arrowhead). Moreover, cytoplasmic dynein was present at the cell cortex; this was especially apparent at boundaries between cells, for instance, between the AB and P1 blastomeres of the two cell stage embryo (Fig. 2 O, white arrowheads). The distribution of cytoplasmic dynein changed as cells progressed through mitosis. During prometaphase, cytoplasmic dynein accumulated along both sides of prometaphase chromosomes (Fig. 2C and Fig. D, arrows and arrowhead, respectively). Since chromosomes in C. elegans are holocentric (Albertson and Thomson 1993), this possibly corresponds to kinetochore staining. During metaphase, cytoplasmic dynein became enriched on the spindle (Fig. 2, G–J). During early anaphase (Fig. 2, K–N), strong spindle signal was still detected, both between segregating chromosomes and spindle poles, as well as centrally (Fig. 2 K, arrow), between the two sets of chromosomes (Fig. 2 L, arrowheads). A similar staining pattern persisted throughout anaphase (Fig. 2 O, arrow). At telophase, cytoplasmic dynein was enriched in two areas of the cytoplasm adjacent to the spindle poles (Fig. 2 O, cell to the left). In addition, a strong signal was detected at the periphery of reforming nuclei (Fig. 2 O, black arrowhead).


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)

Distribution of cytoplasmic dynein in early wild-type embryos. Embryos stained with anti–DHC-1 and antitubulin (TUB; high magnification images only) antibodies and counterstained with Hoechst 33258 to reveal DNA. A, B, O, and P are at the same magnification, as are C–N. Merged images, DHC-1, red; TUB, green; and DNA, blue. (A and B) One cell stage embryo during pronuclear migration. DHC-1 is distributed throughout the cytoplasm in a punctate manner, and is enriched at the periphery of the male (A and B, arrowhead) and female (A and B, arrow) pronuclei. DHC-1 is also slightly enriched at the cortex of one cell stage embryos, but this is not rendered in this particular focal plane. Small arrowhead in B points to out-of-focus polar body DNA. (C–F) Prometaphase, one cell stage embryo. DHC-1 is enriched on both sides (C and F, arrows) of congressing chromosomes (D and F, arrowhead). (G–J) Metaphase, one cell stage embryo. DHC-1 is enriched on the spindle on both sides of the metaphase plate. (K–N) Early anaphase, P1 blastomere of two cell stage embryo. DHC-1 is enriched on the spindle between the chromosomes (L and N, arrowheads) and the spindle poles, as well as centrally (K and N, arrow) between the two sets of chromosomes. (O and P) Two cell stage embryo, P1 blastomere (right) is in late anaphase, AB blastomere (left) in late telophase. In P1, DHC-1 is enriched on the spindle (O, arrow) between the chromosomes and the spindle poles and in the central spindle. In AB, DHC-1 is enriched at the periphery of reforming nuclei (O, black arrowhead) as well as in an area above the spindle poles. In addition, DHC-1 is localized throughout the cortex between the AB and P1 blastomeres (O, arrowheads). Small arrowhead in P points to polar body DNA. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Distribution of cytoplasmic dynein in early wild-type embryos. Embryos stained with anti–DHC-1 and antitubulin (TUB; high magnification images only) antibodies and counterstained with Hoechst 33258 to reveal DNA. A, B, O, and P are at the same magnification, as are C–N. Merged images, DHC-1, red; TUB, green; and DNA, blue. (A and B) One cell stage embryo during pronuclear migration. DHC-1 is distributed throughout the cytoplasm in a punctate manner, and is enriched at the periphery of the male (A and B, arrowhead) and female (A and B, arrow) pronuclei. DHC-1 is also slightly enriched at the cortex of one cell stage embryos, but this is not rendered in this particular focal plane. Small arrowhead in B points to out-of-focus polar body DNA. (C–F) Prometaphase, one cell stage embryo. DHC-1 is enriched on both sides (C and F, arrows) of congressing chromosomes (D and F, arrowhead). (G–J) Metaphase, one cell stage embryo. DHC-1 is enriched on the spindle on both sides of the metaphase plate. (K–N) Early anaphase, P1 blastomere of two cell stage embryo. DHC-1 is enriched on the spindle between the chromosomes (L and N, arrowheads) and the spindle poles, as well as centrally (K and N, arrow) between the two sets of chromosomes. (O and P) Two cell stage embryo, P1 blastomere (right) is in late anaphase, AB blastomere (left) in late telophase. In P1, DHC-1 is enriched on the spindle (O, arrow) between the chromosomes and the spindle poles and in the central spindle. In AB, DHC-1 is enriched at the periphery of reforming nuclei (O, black arrowhead) as well as in an area above the spindle poles. In addition, DHC-1 is localized throughout the cortex between the AB and P1 blastomeres (O, arrowheads). Small arrowhead in P points to polar body DNA. Bars, 10 μm.
Mentions: We used anti–DHC-1 antibodies to determine the subcellular distribution of cytoplasmic dynein in early wild-type embryos by immunofluorescence microscopy (Fig. 2). We found that cytoplasmic dynein was present in a punctate manner throughout the cytoplasm at all stages of the cell cycle. In addition, a stronger signal was detected at the periphery of pronuclei in one cell stage embryos (Fig. 2 A, arrow and arrowhead) and of nuclei in later stage embryos (Fig. 2 O, black arrowhead). Moreover, cytoplasmic dynein was present at the cell cortex; this was especially apparent at boundaries between cells, for instance, between the AB and P1 blastomeres of the two cell stage embryo (Fig. 2 O, white arrowheads). The distribution of cytoplasmic dynein changed as cells progressed through mitosis. During prometaphase, cytoplasmic dynein accumulated along both sides of prometaphase chromosomes (Fig. 2C and Fig. D, arrows and arrowhead, respectively). Since chromosomes in C. elegans are holocentric (Albertson and Thomson 1993), this possibly corresponds to kinetochore staining. During metaphase, cytoplasmic dynein became enriched on the spindle (Fig. 2, G–J). During early anaphase (Fig. 2, K–N), strong spindle signal was still detected, both between segregating chromosomes and spindle poles, as well as centrally (Fig. 2 K, arrow), between the two sets of chromosomes (Fig. 2 L, arrowheads). A similar staining pattern persisted throughout anaphase (Fig. 2 O, arrow). At telophase, cytoplasmic dynein was enriched in two areas of the cytoplasm adjacent to the spindle poles (Fig. 2 O, cell to the left). In addition, a strong signal was detected at the periphery of reforming nuclei (Fig. 2 O, black arrowhead).

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