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1.
Figure 1

Figure 1. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Histone H1 kinase activities in control zygotes (squares) and zygotes treated with aphidicolin (diamonds) to prevent DNA synthesis. Both cultures came from a common pool of zygotes. H1 kinase activity in control zygotes (squares) rises at first mitosis and then drops at the metaphase–anaphase transition. Ordinate: H1 kinase activity expressed as “volume,” which is the sum of the pixel values of the H1 band minus background as determined in the PhosphorImager. Abscissa: minutes after fertilization.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
2.
Figure 9

Figure 9. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Indirect immunofluorescence using an anti-BrdU antibody to assay for DNA synthesis. (a) This zygote was treated with translation inhibitors and BrdU at first prophase (55 min after fertilization) and fixed 120 min later when it was arrested in second interphase. There is no incorporation of BrdU into nuclear DNA. Background cytoplasmic fluorescence has become evident due to the long camera exposure used. (b) Control for BrdU permeability in prophase zygotes. BrdU was applied in first prophase (55 min after fertilization) and translation inhibitors were applied at telophase (90 min after fertilization) when second DNA synthesis had begun. The zygotes were fixed 120 min later. Incorporation of BrdU into nuclear DNA, as shown here, indicates that BrdU enters prophase/mitotic zygotes. Fluorescence optics. 10 μm per scale division.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
3.
Figure 2

Figure 2. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Repeated centrosome reproduction with high Cdk1-B activity in a zygote arrested in S phase with aphidicolin. Here we have focused on one aster that has separated from the nucleus and doubles twice starting 221 min after fertilization. (a) Four asters at the nucleus; one aster is detaching (arrow). (b) The detached aster migrates away from the nucleus. (c) The detached aster has begun to double. (d) This aster has split into two asters (arrows). (e) These asters split again (arrows); the fourth aster is out of the plane of focus. Minutes after fertilization are shown in the lower corner of each frame. Polarization optics. 10 μm per scale division.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
4.
Figure 6

Figure 6. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

The development of a zygote treated with protein synthesis inhibitors beginning at first prophase. (a) Prometaphase. (b) Anaphase. (c) Initiation of cytokinesis at telophase. (d) The blastomeres arrest in interphase of the second cell cycle, and the nuclei become enlarged over time. The two asters in each blastomere are weakly birefringent and thus not visible in this micrograph. (e) Another zygote from the same culture, also arrested in second interphase. This zygote has been treated with 2% hexylene glycol to augment astral birefringence. Minutes after fertilization are shown in the lower corner of each panel. Polarization optics. 10 μm per scale division.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
5.
Figure 4

Figure 4. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Anaphase chromosome disjunction in a zygote injected with cyclin B Δ-90 mRNA. (a–c) Polarization micrographs. (d–f) Fluorescence micrographs of the chromosomes of the same cell at the same times. (a and d) A normal prometaphase spindle has formed, with the chromosomes aligned at the metaphase plate. The oil droplet used to cap the injection needle can be seen below the spindle. (b and e) Mid-anaphase. The sister chromatids have disjoined and are moving towards the spindle poles. (c and f) Both spindle poles have split, and a tetrapolar spindle has formed. The disjoined chromatids are dispersed throughout the central region of the tetrapolar spindle. Minutes after fertilization are shown in the lower left corner of the lower frames. 10 μm per scale division.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
6.
Figure 8

Figure 8. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Indirect immunofluorescence using anti-BrdU antibody to assay for DNA synthesis. (a) Nuclear region of a control zygote fixed 50 min after fertilization. (b) Nuclear region of a zygote treated with protein synthesis inhibitors at fertilization and fixed 50 min later. (c) Polarization micrograph of a zygote treated with protein synthesis inhibitors at fertilization and refertilized 180 min after fertilization. Note that the asters associated with the nucleus have reproduced. The late entering sperm pronucleus with its sperm aster can be seen on the left. Minutes after refertilization are indicated in the lower corner of this frame. (d) Anti-BrdU immunofluorescence of a refertilized zygote from the same culture as c. Note that both the zygote nucleus and the late-entering sperm pronucleus have incorporated BrdU. Minutes after refertilization are indicated in the lower corner of this frame. (a, b, and d) Fluorescence optics. (c) Polarization optics. 10 μm per scale division.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
7.
Figure 5

Figure 5. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Histone H1 kinase activity in zygotes treated with protein synthesis inhibitors (E/A) beginning before fertilization (diamonds) and at prophase of first mitosis (squares). Both cultures came from a common pool of zygotes. The arrow (F) indicates time of fertilization. Note that the first time point for each experiment was taken 15 min before fertilization and represents the basal level of H1 kinase activity in unfertilized eggs. Each data point represents the average histone H1 kinase activity for two samples taken at each time. The two curves represent actual PhosphorImager values and were not normalized to each other. Ordinate: H1 kinase activity expressed as “volume,” which is the sum of the pixel values of the H1 band minus background as determined in the PhosphorImager. Abscissa: minutes after fertilization.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
8.
Figure 7

Figure 7. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Serial semithick section ultrastructural analysis of centrosomes in two zygotes treated with protein synthesis inhibitors at first prophase. (a–c) Zygote fixed 164 min after fertilization. (a, upper inset) Zygote before fixation. Three centrosomes were completely reconstructed, and each contained two centrioles (a–c). The lower inset in a shows the second centriole in this centrosome, which was located in a different section. The profile of the nuclear envelope can be seen to the left of the lower inset. (d–g) Zygote fixed 345 min after fertilization. (d, inset) Zygote just before fixation; the refractile spheres are oil drops used to mark the zygote for recovery and fixation. All four centrosomes in this zygote were reconstructed, and each contained two centrioles (d–g). All four centrosomes are close to the nuclear envelopes, which are seen running vertically through the center of each panel. (a and d, insets) Polarization optics. 10 μm per scale division. Bar, 2 μm.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.
9.
Figure 3

Figure 3. From: The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote .

Microinjection of cyclin B Δ-90 mRNA at first prophase arrests zygotes in mitosis. (a) The zygote enters mitosis and forms a normal bipolar spindle. The refractile sphere in the lower left portion of the zygote is a drop of oil used to cap the micropipet. (b) Anaphase onset occurs at the normal time. (c) Both of the spindle poles have split and a tetrapolar spindle forms. (d) This zygote shortly before fixation for serial section ultrastructural analysis. (e) Each spindle pole of this zygote contains only a single centriole. All four centrioles in this zygote are shown in this frame. (a–d) Polarization optics. Minutes after fertilization are shown in the lower left corner of each frame. 10 μm per scale division. Bar: (e) 0.25 μm.

Edward H. Hinchcliffe, et al. J Cell Biol. 1998 March 23;140(6):1417-1426.

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