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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

Journal of Cell Biology (1999) 147(1) 135-150
DOI: 10.1083/jcb.147.1.135
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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.

Figures

  • Figure 1. Characterization of anti– DHC-antibodies. (A) Western blot of C. elegans (left lane) or Xenopus (right lane) extracts probed with antibodies raised against a C. elegans DHC-1 peptide (left lane) or a portion of Xenopus DHC (right lane). Both antibodies recognize a similarly migrating very high molecular mass species (arrowhead). In addition, the C. elegans antibodies recognize a species of z180 kD. (B) Wildtype (arrow) and dhc-1 (RNAi) (arrowhead) embryos stained with anti–DHC-1 antibodies (top) and counterstained with Hoechst 33258 to reveal DNA (bottom). Anti–DHC-1 staining in this particular dhc-1 (RNAi) embryo is 16.3% of that in the neighboring wildtype embryo. Embryos were also stained with antitubulin antibodies to unambiguously identify dhc-1 (RNAi)
  • 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, P 1 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 P 1 blastomeres (O, arrowheads). Small arrowhead in P points to polar body DNA. Bars, 10 mm.
  • Figure 3. Characterization of rapid minus end– directed movements of yolk granules in wild type. (A) One cell stage embryo in anaphase; high magnification images show an area just anterior of the anterior aster during a 2-s sequence from a time-lapse DIC microscopy recording. Time is indicated in seconds at the right of the frames, which are 10 mm across. One yolk granule (arrows) moves towards the center of the aster (black arrowheads) at an average velocity of 1.40 mm/sec. White arrowheads point to a neighboring yolk granule that remains immobile during the sequence. (B) Histogram of velocities of minus end–directed movement of yolk granules in wild type. Number of motility events per velocity class is shown. Velocity class 1.1 encompasses values from 1.0 to 1.19, velocity class 1.3 values from 1.2 to 1.39 and so forth. On average, motility events (n 5 37) lasted 2.7 s (SD 0.85), covered 3.91 mm (SD 1.47), and had a peak velocity of 1.44 mm/s (SD 0.23).
  • Figure 4. Cytoplasmic flows are not affected in dhc-1 (RNAi) embryos. (A) The average peak velocity of posteriorly directed flow of yolk granules during the pseudocleavage stage is indistinguishable in wild type (5.55 mm/min; n 5 12 granules in 5 embryos; SD 1.63) and dhc-1 (RNAi) embryos (5.51 mm/min; n 5 15 granules in 5 embryos; SD 1.50). These average velocities are slightly higher than those reported previously in wild type (4.4 mm/min; Hird and White, 1993). (B) Wild-type and dhc-1 (RNAi) embryos stained with anti– PGL-1 antibodies to visualize P granules and counterstained with Hoechst 33258 to reveal DNA. All images are at the same magnification. In both wild-type and dhc-1 (RNAi) one cell stage embryos, PGL-1 is segregated to the posterior. Arrowheads point to anteriorly located polar bodies. The wild-type embryo is in prometaphase (arrow points to chromosomes lining up on the metaphase plate), the dhc-1 (RNAi) embryo later in mitosis (arrow points to chromosomes). Embryos were simultaneously stained with antitubulin antibodies (not shown), which revealed the position of centrosomes and unambiguously identified polarity in dhc-1 (RNAi) embryos (Fig. 6). Bar, 10 mm.
  • 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 908 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 mm.
  • Table I. Summary of Time-lapse DIC Microscopy Analysis
  • Figure 6. Failure of centrosome separation in dhc-1 (RNAi) embryos. Embryos stained with anti–ZYG-9 and antitubulin (TUB) antibodies and counterstained with Hoechst 33258 to reveal DNA. Images in first two columns: early, before nuclear envelope breakdown (NEB); and prophase. Images in last two columns: late, after NEB. Merged images: ZYG-9, red; TUB, green; and DNA, blue. All images are at the same magnification. (A–D) Wild type, prophase. (A) ZYG-9 labels the two centrosomes (arrowheads), which have separated from one another while remaining associated with the male pronucleus; ZYG-9 is also present in the cytoplasm and polar bodies (small arrowheads). (B) Astral microtubules emanate from the centrosomes; the mesh of cortical microtubules is also visible. (C) DNA of both male (arrow) and female (out of focus, arrowhead) pronuclei is condensing; small arrowheads point to polar body material. (E–F) dhc-1 (RNAi), prophase. (E) Daughter centrosomes (arrowheads) fail to separate from one another and are posterior of the male pronucleus. (F) Some astral microtubules are fairly long (arrow). (G) DNA of both the male (arrow) and the three female (arrowheads) pronuclei is condensing. (I–L) Wild type, anaphase. (I) The two spindle poles (arrowheads) have moved away from each other during anaphase B. (J) Numerous and long astral microtubules extend from the spindle poles towards the anterior and posterior cortices; spindle microtubules extend centrally. (K) The two sets of chromosomes segregate towards the spindle poles; small arrowhead points to polar body material. (M–P) dhc-1 (RNAi); after NEB. (M) Centrosomes (arrowheads) are still in close proximity of one another. (N) No bipolar spindle is assembled; astral microtubules seem to grow preferentially towards chromosomes or be stabilized in their vicinity (arrow); such microtubules are directed towards chromosomes coming presumably from the male pronucleus in most dhc-1 (RNAi) embryos. (O) Condensed chromosomes coming most likely from the male pronucleus (arrow) and the female pronucleus (arrowhead) are visible. Small arrowhead points to laterally positioned polar body material. In 1/46 dhc-1 (RNAi) embryo after NEB, the two centrosomes were separated from one another; a bipolar spindle was not apparent in this case either. Bar, 10 mm.
  • Figure 7. Failure of pronuclear migration and MTOC separation in p150Glued (RNAi) and p50/dynamitin (RNAi) embryos. (A and D) Single images from time-lapse DIC microscopy recordings of p150Glued (RNAi) (A) and p50/ dynamitin (RNAi) (D) embryos. (B, C, E, and F) p150Glued (RNAi) (B and C) and p50/dynamitin (RNAi) embryos (E and F) stained with antitubulin antibodies and counterstained with Hoechst 33258 to reveal DNA. A and D are at the same magnification, as are B, C, E, and F. (A and D) In both p150Glued (RNAi) and p50/dynamitin (RNAi) embryos, no bipolar spindle is visible after NEB. However, an area devoid of yolk granules extends towards the anterior of the embryo (arrows point to anterior of this area). The asters appear to be at the very posterior of the embryos (arrowheads). Note that the membranes of the female pronuclei are still intact after that of the male pronuclei broke down. (B, C, E, and F) In both p150Glued (RNAi) and p50/dynamitin (RNAi) embryos, the two MTOCs are in close proximity at the very posterior of the embryo (B and E, arrowheads). p150Glued (RNAi) embryo is just after breakdown of the male pronucleus (C, arrow); arrowhead in C points to condensed chromosomes from the female pronucleus. p50/ dynamitin (RNAi) embryo is towards the end of prophase, before breakdown of the male pronucleus. At these stages in wild type, centrosomes are well separated (Fig. 6). Small arrowheads in C and F point to polar body material. Bars, 10 mm.

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Gönczy, P., Pichler, S., Kirkham, M., & Hyman, A. A. (1999). Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. Journal of Cell Biology, 147(1), 135–150. https://doi.org/10.1083/jcb.147.1.135

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