Page 23 - Annual Report 2020

Cultured cells showing position of centrosome (arrows)
Recently we have identified a conserved feedback mechanism that monitors the relative position of lagging chromosomes during anaphase via the differential phosphorylation of the histone variant H3.3 at Ser31. During normal mitosis H3.3 Ser31 is phosphorylated exclusively at pericentromeres, which are rapidly dephosphorylated in anaphase. We induced non-transformed cells to missegre- gate chromosomes by transiently depolymerizing spindle microtubules with cold. These cells undergo the metaphase-anaphase transition in the presence of one or more misaligned chromo- somes that lack BubR1 labeling. These cells transit mitosis with relatively normal timing and lack DNA damage. After re-warming, correlative same cell live and fixed imaging revealed that isolated chromosomes (e.g. lagging in anaphase) have hyper-phosphorylated H3.3 Ser31 (pS31) along their arms that persists into G1 as these chromosomes assemble into a micronucleus. Surprisingly, during telophase Ser31 phosphoryla- tion along individual chromosomes initiates global phosphorylation of H3.3 Ser31 in both reforming nuclei, suggesting both an amplification step of the aneuploid failsafe, and an explanation for
why both daughter cells trigger p53 activation in response to a single chromosome missegregation event. pS31 is mimicked by the hyperlocalization of ATRX to isolated chromosome arms. ATRX – a member of the SWI/SNF family of chromatin bind- ing protein – is known to load histone H3.3 into nucleosomes. Unlike H3.3 S31 phosphorylation during anaphase, the association of ATRX with isolated chromosomes is transient; by nuclear envelope reformation ATRX is absent from the resulting micronucleus.
Finally, we demonstrate that post-anaphase
H3.3 pS31 and ATRX are required to trigger p53 stabilization in the subsequent G1. Microinjection
of monospecific antibodies against either pS31
or ATRX into anaphase cells containing lagging chromosomes blocks p53 accumulation in G1 nuclei. Here we show that p53 cell cycle arrest – triggered by chromosome missegregation – is mediated via a novel signaling mechanism depen- dent upon H3.3 S31 phosphorylation and ATRX recruitment to lagging chromosomes. This work provides insight into how aneuploidy is normally monitored and suppressed. Furthermore, driver mutations in H3.3 (flanking Ser31) and null muta- tions in ATRX are both found in pediatric glioblas- tomas, suggesting that disrupting the aneuploidy failsafe contributes to neoplastic progression.
The role of the centrosome in mitotic spindle assembly: The role of centrosomes/ centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes
and assemble anastral spindles. However, vertebrate somatic cells normally assemble a
connected pair of polarized, astral MT arrays – termed an amphiaster (“a star on both sides”)
– that is formed by the splitting and separation of the microtubule-organizing center (MTOC)
well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC re-assembled, and prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin
are all recruited to the interphase aMTOC, and
the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split/separate before NEB, and these entered mitosis with persistent monastral spindles. The chromatin-mediated RAN-GTP pathway could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but in its absence, the fidelity of bipolar spindle assembly is highly compromised.
THE HORMEL INSTITUTE // UNIVERSITY OF MINNESOTA PG 23