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Maintenance of nuclear architecture is crucial for gene regulation, cell proliferation and tissue development. However, during every open mitosis and meiosis, chromosomes are exposed to cytoskeletal forces until they are fully reassembled into mature nuclei. Here we discuss our recent study of nuclear assembly in Xenopus egg extracts, where we showed that the DNA binding protein Developmental pluripotency associated 2 (Dppa2) directly inhibits microtubule polymerization during nuclear formation, and that this is essential for normal nuclear shape and replication. We explore mechanisms by which microtubule dynamics could regulate nuclear formation and morphology, and discuss the importance of both spatial and temporal regulation of microtubules in this process. Moreover, expression of Dppa2 is limited to the early embryo and pluripotent tissues, and we highlight the specific demands of mitosis in these often rapidly dividing cells, in which telophase nuclear assembly must be expedited and may facilitate developmental changes in nuclear architecture.
Figure 1. Nuclear assembly during mitosis and fertilization. (A) During mitotic exit, spindle disassembly is coordinated with chromosome decondensation and nuclear envelope formation. During fertilization, completion of female meiosis II follows largely the same events, with disassembly of the meiotic spindle and assembly of the maternal pronucleus; the only major difference is that one meiotic daughter cell is extruded as a polar body (not pictured). (B) Fertilization also requires the dramatic conversion of compact sperm chromatin into decondensed spherical pronuclei. Concomitantly, astral microtubules nucleated from sperm centrosomes capture both sperm and egg pronuclei and transport them toward one another at the center of the egg. ♂ indicates sperm chromosomes and pronucleus, ♀ indicates female pronucleus. (C) Nuclear (and pronuclear) assembly results in assembly of a double nuclear membrane and nuclear pore complexes around decondensed chromatin.
Figure 2. Pronuclear assembly in Xenopus egg extracts. (A) Sperm chromatin was added to metaphase egg extracts, released into interphase and visualized at the indicated time points with 1 μg/ml Hoechst 33342. (B) Fully assembled pronucleus at 60 min. DNA was visualized with Hoechst 33342; nuclear and endoplasmic-reticular membrane with 1 μM CM-DiI lipophilic dye; microtubules with 0.2 μM Alexa-Fluor-488 labeled bovine tubulin. Scale bars, 10 μm.
Figure 3. Dppa2 is required for nuclear shape and organized DNA replication. (A) Nuclei assembled in mock depleted and Dppa2-depleted Xenopus egg extracts were fixed at 60 min after release into interphase. DNA was visualized with Hoechst 33342 and replication by incorporation of 1 μM fluorescein labeled dUTP. Scale bar, 10 μm. (B) Chromatin-bound Dppa2 suppresses local microtubule assembly during nuclear formation. Microtubules play both positive (“+”) and negative (“–”) roles on nuclear formation.
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