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Nuclear atypia is one of the hallmarks of cancers. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of nuclear atypia. Tracking the fate of newborn cells exhibiting nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because ~50% of cancers display compromised p53, we explored how multinucleation blocks proliferation. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Multinucleation Associated DNA Damage associated with 53BP1-bodies remains unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. Persistent DNA damage, a DNA replication block, and reduced phospho-Rb, reveal a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity., Hart et al. track newborn single cells by live microscopy after inducing a variety of nuclear atypia by CENP-E inhibitor treatment. They find that that multinucleation, unlike other forms of nuclear atypia, blocks proliferation independently of p53 and is associated with persistent 53BP1 DNA damage foci, thus providing insights into the consequences of multinucleation, often observed in disease states.

Nuclear atypia is one of the earliest hallmarks of cancer progression. How distinct forms of nuclear atypia differently impact cell fate is not understood at the molecular level. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of multinucleation or misshapen nuclei and reveal a significant difference between multinucleation and micronucleation, a catastrophic nuclear atypia known to promote genomic rearrangements and tumour heterogeneity. Tracking the fate of newborn cells exhibiting various nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because compromised p53 is seen in over 50% of cancers, we explored how multinucleation blocks proliferation and promotes quiescence. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Importantly, Multinucleation Associated DNA Damage (MADD) associated 53BP1-bodies remain unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. This persistent MADD signalling blocks the onset of DNA replication and is associated with driving proliferative G1 cells into quiescence, revealing a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

Unlike man-made wheels that are centered and rotated via an axle, the mitotic spindle of a human cell is rotated by external cortical pulling mechanisms. Zulkipli et al. identify MARK2’s role in equatorial spindle centering and astral microtubule length, which in turn control spindle rotation., The plane of cell division is defined by the final position of the mitotic spindle. The spindle is pulled and rotated to the correct position by cortical dynein. However, it is unclear how the spindle’s rotational center is maintained and what the consequences of an equatorially off centered spindle are in human cells. We analyzed spindle movements in 100s of cells exposed to protein depletions or drug treatments and uncovered a novel role for MARK2 in maintaining the spindle at the cell’s geometric center. Following MARK2 depletion, spindles glide along the cell cortex, leading to a failure in identifying the correct division plane. Surprisingly, spindle off centering in MARK2-depleted cells is not caused by excessive pull by dynein. We show that MARK2 modulates mitotic microtubule growth and length and that codepleting mitotic centromere-associated protein (MCAK), a microtubule destabilizer, rescues spindle off centering in MARK2-depleted cells. Thus, we provide the first insight into a spindle-centering mechanism needed for proper spindle rotation and, in turn, the correct division plane in human cells.

Tissue maintenance requires adequate cell proliferation and a directed plane of cell division. Retraction fibres can determine the plane of cell division by directing spindle movements; however, retraction fibre components that direct spindle movements remain unclear. We report MARK2/Par1b kinase as a novel component of actin-rich retraction fibres, important for directed spindle movements. A kinase-dead mutant of MARK2 reveals MARK2’s ability to monitor actin status. MARK2’s localisation at retraction fibres, but not the rest of the cortical membrane or centrosome, is dependent on its kinase activity, highlighting a specialised spatial regulation of MARK2. By subtly perturbing the actin cytoskeleton, we demonstrate MARK2’s role in correcting spindle off-centering, induced by lesions in actin assembly. In addition to this mitotic role, we show MARK2’s post-mitotic role in ensuring normal G1-S progression and cell proliferation. We propose that MARK2 provides a molecular framework to integrate cortical signals and cytoskeletal changes in both mitosis and interphase. Short Summary Coordination of cell proliferation and division is important for tissue maintenance. We report a regulated localisation for MARK2 in mitosis and interphase. We demonstrate its mitotic role in correcting spindle positioning defects and its interphase role in G1-S transition.

Obesity-linked insulin resistance is mechanistically connected to local inflammation of adipose tissue, which produces a metabolic state characterized by oxidative stress and mitochondrial dysfunction. Antioxidants enzymes such as Glutathione S-transferase A4 (GSTA4), peroxiredoxin 3 (Prdx3) and glutathione peroxidase 4 (GPx4) expressions are selectively downregulated in adipose tissue of obese insulin-resistant mice and in human obesity-linked insulin resistance. Also, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 and increased protein carbonylation. In addition, protein carbonylation is implicated as an initiating factor in mitochondrial dysfunction and ER-stress, providing a mechanistic connection between oxidative stress and metabolic disease. Histones are the primary components of chromatin and are notably susceptible to carbonylation because of their long lysine-rich tails. These modifications may have effects on histone code, leading to long lasting implications of human health, including insulin resistance. In this study GSTA4-Prdx3-GPx4-silenced 3T3-L1 adipocytes were evaluated for reactive oxygen species production (ROS), mitochondrial function and histones carbonylation. Downregulation of GSTA4, Prdx3 and GPx4 led to an significant increase in ROS, a significant increase in H3 and H4 histones carbonylation, and mitochondrial dysfunction. These results indicate that a downregulation of antioxidant enzymes in adipocytes leads to increased ROS production, mitochondrial dysfunction and nuclear protein carbonylation, and may contribute to the development of insulin resistance and type 2 diabetes.