1
An anaphase surveillance mechanism prevents micronuclei formation
from mitotic errors
Bernardo Orr
1,2
, Filipe De Sousa
1,2,†
, Ana Margarida Gomes
1,2
, Luísa T. Ferreira
1,2
, Ana
C. Figueiredo
1,2
and Helder Maiato
1,2,3,*
5
1
Chromosome Instability & Dynamics Group, i3S - Instituto de Investigação e Inovação
em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal
2
Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen
208, 4200-135 Porto, Portugal 10
3
Cell Division Group, Experimental Biology Unit, Department of Biomedicine, Faculdade
de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto,
Portugal
†Current address: Radiation Oncology Division, Geneva University Hospitals (HUG),
Avenue de la Roseraie 53, 1205 Geneva, Switzerland 15
*Correspondence to: maiato@i3s.up.pt
Keywords: mitosis, micronuclei, Aurora B, gradient, error correction, nuclear envelope,
chromosome separation checkpoint, anaphase, phosphorylation 20
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.26.433009doi: bioRxiv preprint
2
Summary
Micronuclei are a hallmark of cancer and other human disorders and have recently been
implicated in chromothripsis, a series of massive genomic rearrangements that may
drive tumor evolution and progression. Here we show that Aurora B kinase mediates a
surveillance mechanism that integrates error correction during anaphase with spatial 5
control of nuclear envelope reformation to protect against micronuclei formation during
human cell division. Using high-resolution live-cell imaging of human cancer and non-
cancer cells we found that anaphase lagging chromosomes are often transient and
rarely formed micronuclei. This strong bias against micronuclei formation relied on a
midzone-based Aurora B phosphorylation gradient that assisted the mechanical 10
transduction of spindle forces at the kinetochore-microtubule interface required for
anaphase error correction, while delaying nuclear envelope reformation on lagging
chromosomes, independently of microtubules. Our results uncover a new layer of
protection against genomic instability and provide a strategy for the rational design of
micronuclei-targeting therapies. 15
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.26.433009doi: bioRxiv preprint
3
Introduction
Micronuclei (MN) are small sized nuclei derived from chromosomes or chromosome
fragments that fail to incorporate into daughter nuclei during cell division (Guo et al.,
2019). MN formation is a widely used biomarker of cancer and inflammation, as well as
several metabolic, reproductive, cardiovascular and neurodegenerative disorders 5
(Fenech et al., 2020). Recently, MN derived from chromosome segregation errors have
drawn exceptional attention due to their causal link with chromothripsis, a series of
massive genomic rearrangements that may drive rapid tumor evolution and account for
acquired drug resistance and oncogene activation (Crasta et al., 2012; Ly and
Cleveland, 2017; Shoshani et al., 2020; Zhang et al., 2015). Chromosome segregation 10
errors during mitosis are normally prevented before anaphase by a correction
mechanism involving the kinase activity of the Chromosomal Passenger Protein (CPC)
Aurora B at centromeres (Lampson and Grishchuk, 2017), under surveillance of the
spindle assembly checkpoint (SAC) that monitors the establishment of kinetochore-
microtubule (KT-MT) attachments (Musacchio, 2015). Despite this, between 0.1-10% of 15
human primary/non-transformed or chromosomally stable cancer cells progress into
anaphase with one or few chromosomes lagging behind due to merotelic attachments
(Bakhoum et al., 2014; Cimini et al., 2002; Thompson and Compton, 2011; Worrall et
al., 2018). Merotelic attachments, in which a single KT is attached to MTs from both
spindle poles (Cimini et al., 2001), evade SAC detection and the resulting lagging 20
chromosomes in anaphase constitute a potential source of MN (Crasta et al., 2012).
However, whether anaphase lagging chromosomes inevitably result in MN remains
controversial, with frequencies ranging from 18-78% reported in the literature (Cohen-
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.26.433009doi: bioRxiv preprint
4
Sharir et al., 2021; Fonseca et al., 2019; Huang et al., 2012; Thompson and Compton,
2011; Worrall et al., 2018). Importantly, despite that chromosomal instability (CIN) is a
hallmark of human cancers (Bakhoum and Cantley, 2018; Hanahan and Weinberg,
2000), MN prevalence in both cancer and non-cancer cells is relatively infrequent
(typically less than 5-10% of the cells) (Bonassi et al., 2011; Jdey et al., 2017), even 5
after induction of massive chromosome segregation errors by experimental abrogation
of the SAC (Cohen-Sharir et al., 2021), implying the existence of surveillance
mechanisms that either prevent MN formation from SAC-invisible mitotic errors or
account for the clearance of micronucleated cells. While mechanisms of MN prevention
from mitotic errors remain unknown, clearance of micronucleated cells involves a p53-10
dependent mechanism that causes cell cycle arrest/apoptosis (Fonseca et al., 2019;
Janssen et al., 2011; Sablina et al., 1998; Thompson and Compton, 2010) and an
innate immune response mediated by cGAS-STING, which is thought to sense cytosolic
DNA on MN with ruptured nuclear envelopes (NEs) (Mackenzie et al., 2017; Santaguida
et al., 2017). Given their potential role in the genesis of MN and the respective 15
implications for human health, here we investigated how human cells deal with SAC-
invisible mitotic errors that result in anaphase lagging chromosomes. Our findings
uncover an active surveillance mechanism operating during anaphase that protects
against MN formation from mitotic errors.
20
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.26.433009doi: bioRxiv preprint
5
Results
Anaphase lagging chromosomes rarely form micronuclei
Many intrinsic factors may influence whether a lagging chromosome results in a MN.
This appears to vary among different chromosomes (Worrall et al., 2018) and depend
on their delay/position relative to the main segregating chromosome mass (Fonseca et 5
al., 2019) or the presence/absence of centromeres (Norppa and Falck, 2003). In
addition, external experimental factors, such as the spatiotemporal resolution of the
microscopy setup/analysis and sample size, may undermine our capacity to accurately
determine the fate of lagging chromosomes in living cells. To mitigate these external
limitations, we used 4D live-cell spinning-disk confocal microscopy covering the entire 10
chromosome set and the mitotic spindle, with 30 sec temporal resolution, to determine
the fate of anaphase lagging chromosomes in human cells. This allowed the
unequivocal identification of all lagging chromosomes, including those of highly transient
nature that normally resolve early in anaphase. We found that 9% of chromosomally
stable (non-transformed) RPE1 cells and 44% of chromosomally unstable (transformed) 15
U2OS cells displayed at least one transient lagging chromosome during anaphase (Fig.
1a, b; Fig. S1a; Movies S1-2). However, only 6% and 14% of the lagging chromosomes
in RPE1 and U2OS cells, respectively, resulted in MN (Fig. 1a, Fig. S1a, c; Movies S1-
2). Thus, lagging chromosomes in both transformed and non-transformed human cells
are often corrected during anaphase, and show a strong bias to re-integrate the main 20
nuclei.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.26.433009doi: bioRxiv preprint