Novel localization and possible functions of cyclin E in early sea urchin development
Bradley J. Schnackenberg1,2 and
William F. Marzluff1,2,3,*
1 Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
3 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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Fig. 1. Cyclin E associates with the sperm head following fertilization and remains associated with male chromatin throughout pronuclear fusion. L. pictus (A-C) and S. purpuratus (D-F) eggs were fertilized and cultured in artificial seawater at 15°C. Eggs and embryos were immobilized on glass coverslips, fixed at various timepoints throughout the first cell cycle and processed for double-label immunofluorescence microscopy using antibodies to cyclin E (shown in red) and  -tubulin (shown in green). Images were collected using a Zeiss LSM-410 laser-scanning confocal microscope. Cyclin E localization is seen as a punctate stain in the female pronucleus of unfertilized eggs (A). Following fertilization, cyclin E associates with the sperm head (B) and is incorporated into the zygotic nucleus following pronuclear fusion (C). It can be seen that the paternal chromatin remains condensed until pronuclear fusion (D,E), at which time decondensation is completed, and cyclin E disperses throughout the nucleus (F). Bar, 25 µm.
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Fig. 2. Cyclin E localizes to mitotic chromosomes and interphase nuclei throughout early development. Early sea urchin embryos (L. pictus) were fixed at timepoints throughout M-phase of the first cell cycle (A-C), and at the 2- (D-F), 4- (G), and 16-cell stages (H). The embryos were then processed for confocal-immunofluorescence microscopy using antibodies to cyclin E (shown in red) and -tubulin (shown in green). Bars, 25 µm.
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Fig. 3. Cyclin E does not concentrate at centrosomes. L. pictus embryos (A. 40’, B. 50’, C. 60’, and D. 90’ after fertilization) were processed for double-label immunofluorescence microscopy using anti-cyclin E (A1-D1) and 4D2, a monoclonal antibody generated against sea urchin centrosomes (A2-D2). Merged images showing cyclin E (red) and 4D2 (green) are shown in panels A3-D3. Finally, embryos from the same batch were stained with 4D2 (red) and anti--tubulin (green) to demonstrate the activity of the centrosomes at these stages in the first cell cycle (A4-D4). There is no colocalization of anti-cyclin E and 4D2. Bar, 25 µm.
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Fig. 4. Cyclin E localizes to the chromosomes of isolated mitotic spindles. Isolated metaphase (A and C) and anaphase (B and D) spindles were observed using DIC microscopy (A and B) and immunofluorescence microscopy (C and D) using antibodies to cyclin E (shown in red) and -tubulin (shown in green). The bright red spots within the spindle asters in panel D are probably fragments of chromosomes. Bar, 10 µm.
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Fig. 5. Cyclin E copurifies with isolated mitotic spindles. Equal amounts of whole-cell extract (E) and isolated mitotic spindles (S) were loaded on a 12% SDS-polyacrylamide gel. Following electrophoresis, gels were stained with colloidal Coomassie (lanes 2 and 3) or were transferred to nitrocellulose and probed with antibodies to -tubulin (lanes 4 and 5), cyclin E (lanes 6 and 7), cdk2 (lanes 8 and 9), cdk4 (lanes 10 and 11) and SLBP (lanes 12 and 13).
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Fig. 6. Chromosomal cyclin E-cdk2 is active during mitosis. Whole-spindle fractions were tested for kinase activity in the absence (lane 1) and presence of the exogenous substrate pRb-GST (lane 2). Cyclin E was immunoprecipitated from whole-spindle fractions, and the immunoprecipitates were assayed for kinase activity using pRb as the substrate. Minimal kinase activity was associated nonspecifically with protein A-agarose beads (lane 3). However, pRb was strongly phosphorylated by anti-cyclin E immunoprecipitates (lane 4). This activity was blocked by the addition of competing peptide to the antibody incubation (lane 5) or by the addition of 5 µg of the cdk2 inhibitor p27 (lane 6) to the kinase reaction.
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© The Company of Biologists Ltd 2002
