Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent—or independent—on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.

Importin β Negatively Regulates Nuclear Membrane Fusion and Nuclear Pore Complex Assembly

Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin β negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin β is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin β down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin β to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin β 45–462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin β-regulation of NPC assembly. Excess full-length importin β and β 45–462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin β NPC block, which maps downstream of GTPγS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 μM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin β. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.

Regulation of anchoring of the RIIα regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIα: implications for chromosome dynamics at mitosis

CDK1 phosphorylates the A-kinase regulatory subunit RIIα on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localization of RIIα. Using an RIIα-deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RIIα or an RIIα(T54E) mutant, we show that RIIα associates with chromatin-bound A-kinase anchoring protein AKAP95 at mitosis and that this interaction involves phosphorylation of RIIα on T54. During interphase, both RIIα and RIIα(T54E) exhibit a centrosome-Golgi localization, whereas AKAP95 is intranuclear. At mitosis and in a mitotic extract, most RIIα, but not RIIα(T54E), co-fractionates with chromatin, onto which it associates with AKAP95. This correlates with T54 phosphorylation of RIIα. Disrupting AKAP95-RIIα anchoring or depleting RIIα from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstitution assay that mimics mitotic nuclear reformation, RIIα is threonine dephosphorylated and dissociates from AKAP95 prior to assembly of nuclear membranes. Lastly, the Reh cell line exhibits premature chromatin decondensation in vitro, which can be rescued by addition of wild-type RIIα or an RIIα(T54D) mutant, but not RIIα(T54E, A, L or V) mutants. Our results suggest that CDK1-mediated T54 phosphorylation of RIIα constitutes a molecular switch controlling anchoring of RIIα to chromatin-bound AKAP95, where the PKA-AKAP95 complex participates in remodeling chromatin during mitosis.