Biology subjects

Novais-Cruz, M.

Publications and source records attributed to Novais-Cruz, M..

5 recordsLinked to original sources

bioRxiv2025

Megabase-scale detachment of genome-nuclear lamina interactions is required for efficient repair of double stranded breaks

Lamina-associated domains (LADs) are large genomic regions that contact the nuclear lamina (NL). Double-strand breaks (DSBs) in LADs are known to be repaired more slowly and with different pathway preferences compared to other chromatin contexts. However, little is known about the chromatin changes at LADs that occur during DSB repair. Here, we report that a single DSB inside a LAD can cause detachment from the NL over several megabases. This profound spatial rearrangement is transient and reverts within 48 hours. Preventing this detachment slows down repair kinetics and renders repair incomplete, indicating that NL detachment is required for efficient repair of DSBs in LADs. NL detachment is dependent on {psi}H2AX and ATM, while it is antagonized by DNAPKcs activity. Remarkably, {psi}H2AX also antagonizes NL interactions at chromosome ends. Taken together, our data indicate that {psi}H2AX accumulation in LADs induces large scale rewiring of genome-NL interactions, allowing for efficient repair of DSBs.

cell biology↗

bioRxiv2025

Oncogene Silencing via ecDNA Micronucleation

Extrachromosomal DNA (ecDNA) contributes to cancer genome instability by enabling high-copy oncogene amplification, intratumoural heterogeneity and rapid genetic change. Micronuclei (MN) are frequently observed in chromosomally unstable cancers, yet their origins and relevance in ecDNA-driven tumours remain incompletely understood. Here, we investigate the relationship between ecDNA segregation errors and MN formation. We find that ecDNA frequently localizes to MN and represents a prominent source of MN content in ecDNA-positive cancer cells. Mitotic clustering of oncogene-bearing ecDNAs is associated with asymmetric inheritance and mis-segregation into MN. Transfer of oncogenes from ecDNA into MN is accompanied by reduced transcriptional output. Single-MN sequencing shows that individual MN are enriched for ecDNA to a degree that exceeds expectations from stochastic mis-segregation and that in MN oncogenes originating from multiple distinct ecDNAs coalesce. Using live-cell imaging, we observe that cells inheriting ecDNA-positive MN show limited proliferative capacity and an increased likelihood of cell death. In neuroblastoma patients with MYCN-amplified ecDNA, higher frequencies of ecDNA-positive MN at diagnosis are associated with improved event-free and overall survival. Together, these findings link ecDNA mis-segregation to MN formation and reduced cellular fitness, suggesting that ecDNA-positive MN may reflect a state of impaired oncogenic ecDNA function with potential relevance for clinical outcome.

cancer biology↗

bioRxiv2022

Long-term mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells

DNA damage response (DDR) during interphase involves active signalling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy analysis we 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 long-term DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but DDR-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, we show that long-term mitotic DNA damage specifically stabilizes kinetochore-microtubule attachments in cancer cells, causing the missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Overall, these findings unveil that long-term therapeutic DNA damage regimens contribute to genomic instability through a surprising link between the stabilization of kinetochore-microtubule attachments and chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

cell biology↗

bioRxiv2022

Misaligned chromosomes that satisfy the spindle assembly checkpoint are a strong predictor of micronuclei formation in dividing cancer cells

Chromosome alignment to the spindle equator is a hallmark of mitosis that is 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 120 proteins previously implicated in chromosome alignment. Surprisingly, in all cases, cells frequently entered anaphase after a delay with chronically misaligned chromosomes. Using depletion of key proteins as prototypes for defective chromosome alignment, we show that chronically misaligned chromosomes often satisfy the SAC and directly missegregate. In-depth analysis of specific molecular perturbations that prevent proper kinetochore-microtubule attachments revealed that chronically 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, chronically misaligned chromosomes are a strong predictor of micronuclei formation in a cancer cell model of chromosomal instability, but not in normal near-diploid cells. We provide evidence supporting that intrinsic differences in kinetochore-microtubule attachment stability on misaligned chromosomes account for this distinct outcome. Thus, chronically 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.

cell biology↗

bioRxiv2018

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally breaks the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

biophysics↗