A new microscopy pipeline for studying the initial stages of nuclear and micronuclear rupture and repair.

Nuclear envelope repair is a fundamental cellular response to stress, especially for cells experiencing frequent nuclear ruptures, such as cancer cells. Moreover, for chromosomally unstable cancer cells, characterized by the presence of micronuclei, the irreversible rupture of these structures constitutes a fundamental step toward cancer progression and therapy resistance. For these reasons, the study of nuclear envelope rupture and repair is of paramount importance. Nonetheless, due to the constraint imposed by the stochastic nature of rupture events, a precise characterization of the initial stage of nuclear repair remains elusive. In this study, we overcame this limitation by developing a new imaging pipeline that deterministically induces rupture while simultaneously imaging fluorescently tagged repair proteins. We provide a detailed step-by-step protocol to implement this method on any confocal microscope and applied it to study the major nuclear repair protein, barrier-to-autointegration factor (BAF). As a proof of principle, we demonstrated two different downstream analysis methods and showed how BAF is differentially recruited at sites of primary and micronuclear rupture. Additionally, we applied this method to study the recruitment at primary nuclei of the inner nuclear membrane protein LEM-domain 2 (LEMD2) and Charged Multivesicular Protein 7 (CHMP7), the scaffolding protein of the endosomal sorting complex required for transport III (ESCRT-III) membrane remodeling complex. The CHMP7-LEMD2 binding is the fundamental step allowing the recruitment of ESCRT-III, which represents the other major nuclear repair mechanism. This demonstrates the method's applicability for investigating protein dynamics at sites of nuclear and micronuclear envelope rupture and paves the way to more time-resolved studies of nuclear envelope repair.

RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability.

Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52's ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.

A p62-dependent rheostat dictates micronuclei catastrophe and chromosome rearrangements.

Chromosomal instability (CIN) generates micronuclei-aberrant extranuclear structures that catalyze the acquisition of complex chromosomal rearrangements present in cancer. Micronuclei are characterized by persistent DNA damage and catastrophic nuclear envelope collapse, which exposes DNA to the cytoplasm. We found that the autophagic receptor p62/SQSTM1 modulates micronuclear stability, influencing chromosome fragmentation and rearrangements. Mechanistically, proximity of micronuclei to mitochondria led to oxidation-driven homo-oligomerization of p62, limiting endosomal sorting complex required for transport (ESCRT)-dependent micronuclear envelope repair by triggering autophagic degradation. We also found that p62 levels correlate with increased chromothripsis across human cancer cell lines and with increased CIN in colorectal tumors. Thus, p62 acts as a regulator of micronuclei and may serve as a prognostic marker for tumors with high CIN.

Micronuclear collapse from oxidative damage.

Chromosome-containing micronuclei are a hallmark of aggressive cancers. Micronuclei frequently undergo irreversible collapse, exposing their enclosed chromatin to the cytosol. Micronuclear rupture catalyzes chromosomal rearrangements, epigenetic abnormalities, and inflammation, yet mechanisms safeguarding micronuclear integrity are poorly understood. In this study, we found that mitochondria-derived reactive oxygen species (ROS) disrupt micronuclei by promoting a noncanonical function of charged multivesicular body protein 7 (CHMP7), a scaffolding protein for the membrane repair complex known as endosomal sorting complex required for transport III (ESCRT-III). ROS retained CHMP7 in micronuclei while disrupting its interaction with other ESCRT-III components. ROS-induced cysteine oxidation stimulated CHMP7 oligomerization and binding to the nuclear membrane protein LEMD2, disrupting micronuclear envelopes. Furthermore, this ROS-CHMP7 pathological axis engendered chromosome shattering known to result from micronuclear rupture. It also mediated micronuclear disintegrity under hypoxic conditions, linking tumor hypoxia with downstream processes driving cancer progression.

Micronuclei and Cancer.

Chromosome-containing micronuclei are a feature of human cancer. Micronuclei arise from chromosome mis-segregation and characterize tumors with elevated rates of chromosomal instability. Although their association with cancer has been long recognized, only recently have we broadened our understanding of the mechanisms that govern micronuclei formation and their role in tumor progression. In this review, we provide a brief historical account of micronuclei, depict the mechanisms underpinning their creation, and illuminate their capacity to propel tumor evolution through genetic, epigenetic, and transcriptional transformations. We also posit the prospect of leveraging micronuclei as biomarkers and therapeutic targets in chromosomally unstable cancers. SIGNIFICANCE: Micronuclei in chromosomally unstable cancer cells serve as pivotal catalysts for cancer progression, instigating transformative genomic, epigenetic, and transcriptional alterations. This comprehensive review not only synthesizes our present comprehension but also outlines a framework for translating this knowledge into pioneering biomarkers and therapeutics, thereby illuminating novel paths for personalized cancer management.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Innate immune signaling drives late cardiac toxicity following DNA-damaging cancer therapies.

Late cardiac toxicity is a potentially lethal complication of cancer therapy, yet the pathogenic mechanism remains largely unknown, and few treatment options exist. Here we report DNA-damaging agents such as radiation and anthracycline chemotherapies inducing delayed cardiac inflammation following therapy due to activation of cGAS- and STING-dependent type I interferon signaling. Genetic ablation of cGAS-STING signaling in mice inhibits DNA damage-induced cardiac inflammation, rescues late cardiac functional decline, and prevents death from cardiac events. Treatment with a STING antagonist suppresses cardiac interferon signaling following DNA-damaging therapies and effectively mitigates cardiac toxicity. These results identify a therapeutically targetable, pathogenic mechanism for one of the most vexing treatment-related toxicities in cancer survivors.

Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression.

Chromosomal instability (CIN) and epigenetic alterations have been implicated in tumor progression and metastasis; yet how these two hallmarks of cancer are related remains poorly understood. By integrating genetic, epigenetic, and functional analyses at the single cell level, we show that progression of uveal melanoma (UM), the most common intraocular primary cancer in adults, is driven by loss of Polycomb Repressive Complex 1 (PRC1) in a subpopulation of tumor cells. This leads to transcriptional de-repression of PRC1-target genes and mitotic chromosome segregation errors. Ensuing CIN leads to the formation of rupture-prone micronuclei, exposing genomic double-stranded DNA (dsDNA) to the cytosol. This provokes tumor cell-intrinsic inflammatory signaling, mediated by aberrant activation of the cGAS-STING pathway. PRC1 inhibition promotes nuclear enlargement, induces a transcriptional response that is associated with significantly worse patient survival and clinical outcomes, and enhances migration that is rescued upon pharmacologic inhibition of CIN or STING. Thus, deregulation of PRC1 can promote tumor progression by inducing CIN and represents an opportunity for early therapeutic intervention.

Measuring Mobility in Chromatin by Intensity-Sorted FCS.

The architectural organization of chromatin can play an important role in genome regulation by affecting the mobility of molecules within its surroundings via binding interactions and molecular crowding. The diffusion of molecules at specific locations in the nucleus can be studied by fluorescence correlation spectroscopy (FCS), a well-established technique based on the analysis of fluorescence intensity fluctuations detected in a confocal observation volume. However, detecting subtle variations of mobility between different chromatin regions remains challenging with currently available FCS methods. Here, we introduce a method that samples multiple positions by slowly scanning the FCS observation volume across the nucleus. Analyzing the data in short time segments, we preserve the high temporal resolution of single-point FCS while probing different nuclear regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a reference, we efficiently sort the FCS segments into different populations and obtain average correlation functions that are associated to different chromatin regions. This sorting and averaging strategy renders the method statistically robust while preserving the observation of intranuclear variations of mobility. Using this approach, we quantified diffusion of monomeric GFP in high versus low chromatin density regions. We found that GFP mobility was reduced in heterochromatin, especially within perinucleolar heterochromatin. Moreover, we found that modulation of chromatin compaction by ATP depletion, or treatment with solutions of different osmolarity, differentially affected the ratio of diffusion in both regions. Then, we used the approach to probe the mobility of estrogen receptor-α in the vicinity of an integrated multicopy prolactin gene array. Finally, we discussed the coupling of this method with stimulated emission depletion FCS for performing FCS at subdiffraction spatial scales.

Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps.

Raster image correlation spectroscopy (RICS) is a powerful method for measuring molecular diffusion in live cells directly from images acquired on a laser scanning microscope. However, RICS only provides single average diffusion coefficients from regions with a lateral size on the order of few micrometers, which means that its spatial resolution is mainly limited to the cellular level. Here we introduce the local RICS (L-RICS), an easy-to-use tool that generates high resolution maps of diffusion coefficients from images acquired on a laser scanning microscope. As an application we show diffusion maps of a green fluorescent protein (GFP) within the nucleus and within the nucleolus of live cells at an effective spatial resolution of 500 nm. We find not only that diffusion in the nucleolus is slowed down compared to diffusion in the nucleoplasm, but also that diffusion in the nucleolus is highly heterogeneous.

Measurement of nanoscale three-dimensional diffusion in the interior of living cells by STED-FCS.

The observation of molecular diffusion at different spatial scales, and in particular below the optical diffraction limit (<200 nm), can reveal details of the subcellular topology and its functional organization. Stimulated-emission depletion microscopy (STED) has been previously combined with fluorescence correlation spectroscopy (FCS) to investigate nanoscale diffusion (STED-FCS). However, stimulated-emission depletion fluorescence correlation spectroscopy has only been used successfully to reveal functional organization in two-dimensional space, such as the plasma membrane, while, an efficient implementation for measurements in three-dimensional space, such as the cellular interior, is still lacking. Here we integrate the STED-FCS method with two analytical approaches, the recent separation of photons by lifetime tuning and the fluorescence lifetime correlation spectroscopy, to simultaneously probe diffusion in three dimensions at different sub-diffraction scales. We demonstrate that this method efficiently provides measurement of the diffusion of EGFP at spatial scales tunable from the diffraction size down to ∼80 nm in the cytoplasm of living cells.The measurement of molecular diffusion at sub-diffraction scales has been achieved in 2D space using STED-FCS, but an implementation for 3D diffusion is lacking. Here the authors present an analytical approach to probe diffusion in 3D space using STED-FCS and measure the diffusion of EGFP at different spatial scales.

G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2.

Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation.