Limits...
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.

Show MeSH

Related in: MedlinePlus

Centrosomes and male pronucleus are not tightly associated in some dhc-1 (RNAi) embryos. (A, C, and E) Time-lapse DIC microscopy of wild-type (A) and dhc-1 (RNAi) embryos (C and E). A is from the wild-type embryo shown in Fig. 5. (B, D, and F) Wild-type (B) and dhc-1 (RNAi) embryos (D and F) stained with anti–ZYG-9 antibodies and counterstained with Hoechst 33258 to reveal DNA. B and D are from embryos shown in Fig. 6. Merged images, ZYG-9, red; DNA, blue. A, C, and E are at the same magnification, as are B, D, and F. (A and B) In wild type, centrosomes are initially associated with the male pronucleus, and with both male and female pronuclei after pronuclear meeting. (A) The center of the asters is visible by DIC microscopy as areas excluding yolk granules (arrowheads); arrows point to associated pronuclei. (B) Anti–ZYG-9 labeling demonstrates that the two centrosomes (arrowheads) are associated with the male pronucleus (arrow). (C and D) In 85% of dhc-1 (RNAi) embryos, unseparated centrosomes are in the immediate vicinity of the male pronucleus. (C) An area lacking yolk granules and corresponding to the center of the asters (arrowheads) is visible just posterior of the male pronucleus (arrow). (D) Anti–ZYG-9 labeling demonstrates that unseparated centrosomes (arrowheads) are located in the immediate vicinity and posterior of the male pronucleus (arrow). (E and F) In 15% of dhc-1 (RNAi) embryos, unseparated centrosomes are located 3–11-μm away from the male pronucleus. (E) The area lacking yolk granules and corresponding to the center of the asters (arrowheads) is located 6.5 μm away from the male pronucleus (arrow) in this particular embryo. (F) Anti–ZYG-9 labeling reveals that unseparated centrosomes (arrowheads) are located 7.94-μm away from the male pronucleus (arrow) in this particular embryo. In 1 of 7 embryos where association was compromised, the two centrosomes were separated from one another, and only one of them was not associated with the male pronucleus. Although the embryo shown in F is too early in the cell cycle to be unambiguously scoreable for the occurrence of centrosome duplication, there were two centrosomes in 91/91 one cell stage dhc-1 (RNAi) embryos examined during prophase or after NEB. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2164971&req=5

Figure 8: Centrosomes and male pronucleus are not tightly associated in some dhc-1 (RNAi) embryos. (A, C, and E) Time-lapse DIC microscopy of wild-type (A) and dhc-1 (RNAi) embryos (C and E). A is from the wild-type embryo shown in Fig. 5. (B, D, and F) Wild-type (B) and dhc-1 (RNAi) embryos (D and F) stained with anti–ZYG-9 antibodies and counterstained with Hoechst 33258 to reveal DNA. B and D are from embryos shown in Fig. 6. Merged images, ZYG-9, red; DNA, blue. A, C, and E are at the same magnification, as are B, D, and F. (A and B) In wild type, centrosomes are initially associated with the male pronucleus, and with both male and female pronuclei after pronuclear meeting. (A) The center of the asters is visible by DIC microscopy as areas excluding yolk granules (arrowheads); arrows point to associated pronuclei. (B) Anti–ZYG-9 labeling demonstrates that the two centrosomes (arrowheads) are associated with the male pronucleus (arrow). (C and D) In 85% of dhc-1 (RNAi) embryos, unseparated centrosomes are in the immediate vicinity of the male pronucleus. (C) An area lacking yolk granules and corresponding to the center of the asters (arrowheads) is visible just posterior of the male pronucleus (arrow). (D) Anti–ZYG-9 labeling demonstrates that unseparated centrosomes (arrowheads) are located in the immediate vicinity and posterior of the male pronucleus (arrow). (E and F) In 15% of dhc-1 (RNAi) embryos, unseparated centrosomes are located 3–11-μm away from the male pronucleus. (E) The area lacking yolk granules and corresponding to the center of the asters (arrowheads) is located 6.5 μm away from the male pronucleus (arrow) in this particular embryo. (F) Anti–ZYG-9 labeling reveals that unseparated centrosomes (arrowheads) are located 7.94-μm away from the male pronucleus (arrow) in this particular embryo. In 1 of 7 embryos where association was compromised, the two centrosomes were separated from one another, and only one of them was not associated with the male pronucleus. Although the embryo shown in F is too early in the cell cycle to be unambiguously scoreable for the occurrence of centrosome duplication, there were two centrosomes in 91/91 one cell stage dhc-1 (RNAi) embryos examined during prophase or after NEB. Bars, 10 μm.

Mentions: The same batch of yk161f11 dsRNA was used to quantify all the phenotypic manifestations reported in the text. However, other batches of yk161f11 dsRNA gave identical phenotypes, as did dhc-1 dsRNAs generated from four other sources: (1) a 1.6-kb cDNA insert in λZAPII from Yuji Kohara, yk166g8; (2) T21E12.4-5, a PCR fragment corresponding to exon 2 and part of exon 3 of dhc-1, positions 358–1803 in cosmid T21E12; (3) T21E12.4-M, a PCR fragment corresponding to the end of exon 7 and most of exon 8, positions 6636–7844 in T21E12; and (4) T21E12.4-3, a PCR fragment corresponding to exons 13–15, positions 13420–14854 in T21E12 dsRNAs 2–4 were generated by Alan Coulson at the Sanger Center. dsRNA (2) was used for injections that yielded dhc-1 (RNAi) embryos shown in Fig. 6 and Fig. 8.


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)

Centrosomes and male pronucleus are not tightly associated in some dhc-1 (RNAi) embryos. (A, C, and E) Time-lapse DIC microscopy of wild-type (A) and dhc-1 (RNAi) embryos (C and E). A is from the wild-type embryo shown in Fig. 5. (B, D, and F) Wild-type (B) and dhc-1 (RNAi) embryos (D and F) stained with anti–ZYG-9 antibodies and counterstained with Hoechst 33258 to reveal DNA. B and D are from embryos shown in Fig. 6. Merged images, ZYG-9, red; DNA, blue. A, C, and E are at the same magnification, as are B, D, and F. (A and B) In wild type, centrosomes are initially associated with the male pronucleus, and with both male and female pronuclei after pronuclear meeting. (A) The center of the asters is visible by DIC microscopy as areas excluding yolk granules (arrowheads); arrows point to associated pronuclei. (B) Anti–ZYG-9 labeling demonstrates that the two centrosomes (arrowheads) are associated with the male pronucleus (arrow). (C and D) In 85% of dhc-1 (RNAi) embryos, unseparated centrosomes are in the immediate vicinity of the male pronucleus. (C) An area lacking yolk granules and corresponding to the center of the asters (arrowheads) is visible just posterior of the male pronucleus (arrow). (D) Anti–ZYG-9 labeling demonstrates that unseparated centrosomes (arrowheads) are located in the immediate vicinity and posterior of the male pronucleus (arrow). (E and F) In 15% of dhc-1 (RNAi) embryos, unseparated centrosomes are located 3–11-μm away from the male pronucleus. (E) The area lacking yolk granules and corresponding to the center of the asters (arrowheads) is located 6.5 μm away from the male pronucleus (arrow) in this particular embryo. (F) Anti–ZYG-9 labeling reveals that unseparated centrosomes (arrowheads) are located 7.94-μm away from the male pronucleus (arrow) in this particular embryo. In 1 of 7 embryos where association was compromised, the two centrosomes were separated from one another, and only one of them was not associated with the male pronucleus. Although the embryo shown in F is too early in the cell cycle to be unambiguously scoreable for the occurrence of centrosome duplication, there were two centrosomes in 91/91 one cell stage dhc-1 (RNAi) embryos examined during prophase or after NEB. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Centrosomes and male pronucleus are not tightly associated in some dhc-1 (RNAi) embryos. (A, C, and E) Time-lapse DIC microscopy of wild-type (A) and dhc-1 (RNAi) embryos (C and E). A is from the wild-type embryo shown in Fig. 5. (B, D, and F) Wild-type (B) and dhc-1 (RNAi) embryos (D and F) stained with anti–ZYG-9 antibodies and counterstained with Hoechst 33258 to reveal DNA. B and D are from embryos shown in Fig. 6. Merged images, ZYG-9, red; DNA, blue. A, C, and E are at the same magnification, as are B, D, and F. (A and B) In wild type, centrosomes are initially associated with the male pronucleus, and with both male and female pronuclei after pronuclear meeting. (A) The center of the asters is visible by DIC microscopy as areas excluding yolk granules (arrowheads); arrows point to associated pronuclei. (B) Anti–ZYG-9 labeling demonstrates that the two centrosomes (arrowheads) are associated with the male pronucleus (arrow). (C and D) In 85% of dhc-1 (RNAi) embryos, unseparated centrosomes are in the immediate vicinity of the male pronucleus. (C) An area lacking yolk granules and corresponding to the center of the asters (arrowheads) is visible just posterior of the male pronucleus (arrow). (D) Anti–ZYG-9 labeling demonstrates that unseparated centrosomes (arrowheads) are located in the immediate vicinity and posterior of the male pronucleus (arrow). (E and F) In 15% of dhc-1 (RNAi) embryos, unseparated centrosomes are located 3–11-μm away from the male pronucleus. (E) The area lacking yolk granules and corresponding to the center of the asters (arrowheads) is located 6.5 μm away from the male pronucleus (arrow) in this particular embryo. (F) Anti–ZYG-9 labeling reveals that unseparated centrosomes (arrowheads) are located 7.94-μm away from the male pronucleus (arrow) in this particular embryo. In 1 of 7 embryos where association was compromised, the two centrosomes were separated from one another, and only one of them was not associated with the male pronucleus. Although the embryo shown in F is too early in the cell cycle to be unambiguously scoreable for the occurrence of centrosome duplication, there were two centrosomes in 91/91 one cell stage dhc-1 (RNAi) embryos examined during prophase or after NEB. Bars, 10 μm.
Mentions: The same batch of yk161f11 dsRNA was used to quantify all the phenotypic manifestations reported in the text. However, other batches of yk161f11 dsRNA gave identical phenotypes, as did dhc-1 dsRNAs generated from four other sources: (1) a 1.6-kb cDNA insert in λZAPII from Yuji Kohara, yk166g8; (2) T21E12.4-5, a PCR fragment corresponding to exon 2 and part of exon 3 of dhc-1, positions 358–1803 in cosmid T21E12; (3) T21E12.4-M, a PCR fragment corresponding to the end of exon 7 and most of exon 8, positions 6636–7844 in T21E12; and (4) T21E12.4-3, a PCR fragment corresponding to exons 13–15, positions 13420–14854 in T21E12 dsRNAs 2–4 were generated by Alan Coulson at the Sanger Center. dsRNA (2) was used for injections that yielded dhc-1 (RNAi) embryos shown in Fig. 6 and Fig. 8.

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