Chromatin-Bound Xenopus Dppa2 Shapes the Nucleus by Locally Inhibiting Microtubule Assembly

Abstract

SUMMARY Nuclear shape and size vary between species, during development and in many tissue pathologies, but the causes and effects of these differences remain poorly understood. During fertilization, sperm nuclei undergo a dramatic conversion from a heavily compacted form into decondensed, spherical pronuclei, accompanied by rapid nucleation of microtubules from centrosomes. Here we report that the assembly of the spherical nucleus depends on a critical balance of microtubule dynamics, which is regulated by the chromatin-binding protein Developmental pluripotency-associated 2 (Dppa2). While microtubules normally promote sperm pronuclear expansion, in Dppa2-depleted Xenopus egg extracts excess microtubules cause pronuclear assembly defects leading to abnormal morphology and disorganized DNA replication. Dppa2 inhibits microtubule polymerization in vitro, and Dppa2 activity is needed at a precise time and location during nascent pronuclear formation. This demonstrates a strict spatiotemporal requirement for local suppression of microtubules during nuclear formation, fulfilled by chromatin-bound microtubule regulators.

Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts

“…Furthermore, the diameter of the incubated nuclei was much smaller than in the absence of the inhibitors but still slightly larger than that of the pre-assembled nuclei ( Figure 5B). Although this slow nuclear expansion might be caused by remaining MTs after exposure to these inhibitors (Verde et al, 1991) and/or very slow nuclear expansion in absence of MTs (Xue et al, 2013), this result indicates that MTs have a major function in nuclear expansion. Next we aimed to elucidate how MTs interact with membranes.…”

“…With increasing incubation times, the space that was occupied by MTs expanded, and the density of MTs increased ( Figure 4B), which is consistent with previous observations (Ishihara et al, 2014). Because the MTs emanate evenly from the centrosome but cannot fill the space occupied by the nucleus (Xue et al, 2013), the observed shapes were not perfectly spherical. Nonetheless, a continuous increase in MT-occupied space could be observed over time, reaching 100À150 mm after 40 min of pre-assembly of the nucleus ( Figure 4B).…”

“…Obvious candidates are microtubules (MTs) as they have been shown to be involved in nuclear assembly in X. laevis egg extracts (Ewald et al, 2001;Xue et al, 2013). To reveal whether, and how exactly, MTs control nuclear expansion, we performed immunostaining after incubating the nuclei in a test tube ( Figure 4A) and quantified the MTs by image processing (Figures S3A and S3B).…”

The nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation

“…MEL-28 thus drives positive feedback in RanGTP-mediated microtubule nucleation 24,25 . The nearly complete absence of microtubules around chromatin in MEL-28-depleted extracts may be attributable to the chromatin-bound protein Dppa2, which locally inhibits microtubule assembly 26 . Dppa2 on chromatin may effectively depolymerize microtubules nucleated by TPX2 and centrosomes in the absence of MEL-28.…”

Recent advances in understanding nuclear size and shape

“…Identified in Xenopus, Dppa2 and REEP3/4 are required to remove microtubules and ER membrane, respectively, from chromatin at the end of mitosis to ensure a nucleus of the proper size and shape forms in the subsequent interphase. 129,130 In C. elegans, partial inactivation of polo-like kinase PLK-1 leads to defects in NE breakdown giving rise to 2 nuclei that fail to merge into one. 131 In yeast, formation of NE flares adjacent to the nucleolus in response to excess membrane production or mitotic arrest is dependent on polo kinase Cdc5.…”