Mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

DNA damage response (DDR) during interphase involves active signaling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy, it was found that mitotic cells exposed to several cancer chemotherapy compounds acquire and signal DNA damage, regardless of how they interact with DNA. In-depth analysis upon DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but ataxia telangiectasia mutated-independent, mitotic delay. This delay was due to the presence of misaligned chromosomes that ultimately satisfy the SAC and missegregate, leading to micronuclei formation. Mechanistically, it is shown that mitotic DNA damage causes missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Importantly, with the exception of DNA damage induced by etoposide-a topoisomerase II inhibitor-this outcome was independent of a general effect on kinetochore microtubule stability. Colony formation assays in pan-cancer cell line models revealed that mitotic DNA damage causes distinct cytotoxic effects, depending on the nature and extent of the damage. Overall, these findings unveil and raise awareness that therapeutic DNA damage regimens may contribute to genomic instability through a surprising link with chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells.

Chromosome alignment to the spindle equator is a hallmark of mitosis thought to promote chromosome segregation fidelity in metazoans. Yet chromosome alignment is only indirectly supervised by the spindle assembly checkpoint (SAC) as a byproduct of chromosome bi-orientation, and the consequences of defective chromosome alignment remain unclear. Here, we investigated how human cells respond to chromosome alignment defects of distinct molecular nature by following the fate of live HeLa cells after RNAi-mediated depletion of 125 proteins previously implicated in chromosome alignment. We confirmed chromosome alignment defects upon depletion of 108/125 proteins. Surprisingly, in all confirmed cases, depleted cells frequently entered anaphase after a delay with misaligned chromosomes. Using depletion of prototype proteins resulting in defective chromosome alignment, we show that misaligned chromosomes often satisfy the SAC and directly missegregate without lagging behind in anaphase. In-depth analysis of specific molecular perturbations that prevent proper kinetochore-microtubule attachments revealed that misaligned chromosomes that missegregate frequently result in micronuclei. Higher-resolution live-cell imaging indicated that, contrary to most anaphase lagging chromosomes that correct and reintegrate the main nuclei, misaligned chromosomes are a strong predictor of micronuclei formation in a cancer cell model of chromosomal instability, but not in non-transformed near-diploid cells. We provide evidence supporting that intrinsic differences in kinetochore-microtubule attachment stability on misaligned chromosomes account for this distinct outcome. Thus, misaligned chromosomes that satisfy the SAC may represent a previously overlooked mechanism driving chromosomal/genomic instability during cancer cell division, and we unveil genetic conditions predisposing for these events.

Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam.

Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in x-y imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.

Mitotic progression, arrest, exit or death relies on centromere structural integrity, rather than de novo transcription.

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain a robust spindle assembly checkpoint (SAC) response. Here, we used live-cell imaging of human cells, RNA-seq and qPCR to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC signaling and cell fate. However, we were unable to detect significant changes in mitotic transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on Aurora B centromeric localization, SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, mitotic progression, arrest, exit or death is determined by centromere structural integrity, rather than de novo transcription.