Biology subjects

Basto, R.

Publications and source records attributed to Basto, R..

14 recordsLinked to original sources

bioRxiv2026

Profilin 1 maintains cell cycle fidelity to prevent unscheduled genome doubling and polyploidy in cancer

Whole-genome doubling (WGD) represents a major route to genome instability and therapeutic resistance in cancer; yet, the mechanisms enabling genome duplication in p53-proficient cells remain poorly understood. Here, we identify Profilin 1 (PFN1) loss as a driver of WGD through impaired mitotic entry. Using FUCCI live-cell imaging and single-cell genomic profiling, we show that PFN1-deficient cells bypass mitosis and undergo endoreplication, generating tetraploid cells. Rather than undergoing stable arrest after mitotic bypass, these genome-doubled cells retain proliferative capacity and proceed through aberrant mitotic divisions, thereby amplifying genomic instability. Proteomic analyses reveal coordinated attenuation of late cell-cycle programs, including downregulation of key mitotic regulators such as CDK1, PLK1 and CKS2, consistent with impaired G2/M transition. Despite accumulating polyploidy, PFN1-knockout cells fail to activate an effective p53 tetraploidy checkpoint and display increased nuclear MDM2, promoting cell-cycle arrest evasion and chemotherapeutic resistance. We supported clinical relevance by an orthotopic osteosarcoma xenograft model, in which PFN1-deficient SaOS2 cells showed enhanced metastatic dissemination, and by pan-cancer TCGA analyses confirming a recurrent association between PFN1 loss and WGD. Together, these findings identify Profilin 1 as a safeguard of cell-cycle fidelity whose loss enables genome doubling, cellular plasticity and therapy tolerance.

cancer biology↗

bioRxiv2025

A novel mechanism for centrosome expulsion ensures metabolic activity in polyploid cells

Programmed polyploidy is often linked to increase cell size to support enhanced metabolism, barrier function or regeneration. In certain polyploid cells, the cytoskeleton is drastically remodeled-most notably by eliminating centrosomes. However, the purpose and mechanisms underlying centrosome elimination have remained unclear. We investigated this question in Drosophila acentrosomal salivary glands (SGs), a physiological polyploid model where cells reach high chromosome content through endoreplication. Using genetic tools, live imaging approaches, super-resolution microscopy combined with tissue clearing and electron microscopy, our study uncovers a novel centrosome elimination pathway in vivo. This process requires non-muscle myosin II (MyoII) activity and the macroautophagic machinery to drive centrosome release into the lumen of the salivary glands via autolysosomal exocytosis. Failure to eliminate centrosomes disrupts the mitochondria network, impairing respiration and ATP production. Our findings reveal a previously unknown mechanism that removes centrosomes through the secretory autophagy pathway to protect mitochondrial function and support the high metabolic demands of polyploid cells.

cell biology↗

bioRxiv2025

Brain biomechanics governs mitotic fidelity of embryonic neural progenitors

Accurate chromosome segregation is essential to maintain genetic stability and prevent the onset of diseases such as developmental disorders, infertility, and cancer. While many intrinsic factors and processes involved in mitosis have been extensively characterized, less is known about how extrinsic factors and tissue properties contribute to mitotic fidelity. In this study, using both in vivo and ex vivo systems, in combination with pharmacological perturbations and high-resolution microscopy, we investigated mitosis in apical radial glial (aRG) cells--the primary neural progenitors in the developing mammalian brain. We found that the high cell density typical of early neurogenic stages enhances microtubule polymerization rates from the spindle poles, thereby influencing chromosome segregation. Mechanistically, our results indicate that cortical actin and elevated cortical tension function as sensors of biomechanical stress during mitosis. These findings identify biomechanics as a threat to mitotic fidelity in the embryonic brain.

cell biology↗

bioRxiv2025

Mitotic slippage causes nuclear instability in polyploid cells

Whole-genome duplication (WGD), leading to polyploidy can arise in physiological and pathological contexts1-5. WGD can occur via non-canonical cell cycles such as mitotic slippage, cytokinesis failure or endoreplication1,3. Whether the routes to WGD influence the behaviour of the resulting polyploid cells remains unclear. Here, we compared these routes under both physiological and non-physiological conditions. Remarkably, only mitotic slippage led to widespread nuclear abnormalities defined by highly variable nuclear deformations that we termed nuclear instability. Mechanistically, we found that these nuclei were softer - due to high levels of histone 3 phosphorylation in G1 altering chromatin compaction - and thus more vulnerable to microtubule-driven deformations. The resulting nuclear instability leads to local nuclear reorganisation and changes in 3D genome organisation impacting ultimately gene expression. Importantly, we observed similar nuclear instability in megakaryocytes, which are physiological polyploid cells that we show here to be generated by mitotic slippage, providing a molecular mechanism for their atypical nuclear architecture6,7. In striking contrast, nuclear shape was stable in different physiological polyploid cells generated by cytokinesis failure and endoreplication. Overall, our findings highlight that the route towards WGD matters and that mitotic slippage uniquely destabilizes nuclear architecture, with implications for both physiology and disease.

cell biology↗

bioRxiv2025

A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation

Unscheduled whole genome duplication (WGD), also described as unscheduled or non-physiological polyploidy, can lead to genetic instability and is commonly observed in human cancers. WGD generates DNA damage due to scaling defects between replication factors and DNA content. As a result, DNA damage repair mechanisms are thought to be critical for ensuring cell viability and proliferation under these conditions. In this study, we explored the role of homologous recombination and Holliday junction resolution in non-physiological polyploidy in vivo. Using Drosophila genetics and high-resolution imaging, we identified a key and surprising role for Gen/Gen1 nuclease. Our findings revealed that loss-of-function and overexpression of Gen have opposing effects, delaying or accelerating the proliferation of polyploid cells, respectively. These changes ultimately impact cell proliferation, nuclear asynchrony and mitotic DNA damage levels. Surprisingly, our findings show that this effect is unrelated with the expected Gens function in DNA damage repair. Instead, Gen seems to influence polyploid DNA replication rates. This work identifies a novel function for Gen nuclease and provides new insights into the cellular and molecular requirements of non-physiological polyploidy.

cell biology↗

bioRxiv2023

Centrosome amplification primes for apoptosis and favors the response to chemotherapy in ovarian cancer beyond multipolar divisions

Centrosome amplification is a feature of cancer cells associated with chromosome instability and invasiveness. Enhancing chromosome instability and subsequent cancer cell death via centrosome unclustering and multipolar divisions is an aimed-for therapeutic approach. Here we show that centrosome amplification favors responses to conventional chemotherapy independently of multipolar divisions and chromosome instability. We perform single-cell live imaging of chemotherapy responses in epithelial ovarian cancer cell lines and observe increased cell death when centrosome amplification is induced. By correlating cell fate with mitotic behaviors, we show that enhanced cell death occurs independently of chromosome instability. We identify that cells with centrosome amplification are primed for apoptosis. We show they are dependent on the apoptotic inhibitor BCL-XL, and that this is not a consequence of mitotic stresses associated with centrosome amplification. Given the multiple mechanisms that promote chemotherapy responses in cells with centrosome amplification, we assess such a relationship in an epithelial ovarian cancer patient cohort. We show that high centrosome numbers associate with improved chemotherapy responses and longer overall survival. Our work identifies apoptotic priming as a clinically relevant consequence of centrosome amplification, expanding our understanding of this pleiotropic cancer cell feature.

cell biology↗

bioRxiv2021

Mechanisms of genetic instability in a single S-phase following whole genome doubling

Diploid and stable karyotypes are associated with health and fitness in animals. In contrast, whole genome duplications (WGDs) - doubling full chromosome content - are linked to genetic instability (GIN) and frequently found in human cancers 1-3. It has been established that WGDs fuel chromosome instability through abnormal mitosis 4-8, however, the immediate consequences of tetraploidy in the first interphase are not known. This is an essential question because single WGD events such as cytokinesis failure can promote tumorigenesis 9. Here, we found that newly born tetraploid human cells undergo high rates of DNA damage during DNA replication in the first S-phase. Using DNA combing and single cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we found that these defects result from the lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.

cell biology↗

bioRxiv2021

Illuminati, a novel form of gene expression plasticity in Drosophila neural stem cells

With the aim of developing a genetic instability (GI) sensor in vivo we used the well-established Gal80/Gal4-UAS system combined with a visual GFP marker in Drosophila. We generated a collection of 25 Drosophila lines carrying GAL80 transgenes in different locations in all major chromosomes (X, Y, II, and III). We found low rates of GFP cells in epithelial tissues such as wing discs. In contrast, in larval brains, GFP positive clusters containing neural stem cells- also called neuroblasts (NBs)- and their offspring, were highly frequent. Using genetic and imaging-based approaches, we show that GFP NBs do not result from aneuploidy or mutations in the GAL80 gene, but rather by stochastic repression of GAL80 expression. We named this novel type of gene expression instability Illuminati. Importantly, Illuminati frequency is influenced by environmental and stress conditions. Further, we found that once established, Illuminati can be propagated over many cell cycles.

developmental biology↗

bioRxiv2020

Drosophila neural stem cells show a unique dynamic pattern of gene expression that is influenced by environmental factors

With the aim of developing a sensor for chromosome loss in vivo, we used the well-established GAL4/GAL80 system combined with a visual GFP marker in Drosophila. We show a low frequency of green cells in most Drosophila tissues, suggesting low aneuploidy levels. Unexpectedly, in the brain, GFP positive cells are more frequent, but in this case, they do not represent chromosome loss. Using genetic manipulations, RNA FISH and time-lapse microscopy, we uncovered a dynamic and reversible silencing of GAL80 that occurs in Drosophila neural stem cells. Further, we showed that this novel gene expression regulation is influenced by environmental changes such as temperature variations or food composition. These results have important implications for the Drosophila community, namely the possible interpretation of false positive cells in clonal experiments. Additionally, they also highlight a level of mosaicism and plasticity in the brain, consistent with possible epigenetic regulation of fly chromosomes, which is different from other organs and tissues.

cell biology↗

bioRxiv2020

Microvilli-derived Extracellular Vesicles Govern Morphogenesis in Drosophila wing epithelium

The regulation and coordination of developmental processes involves the secretion of morphogens and membrane carriers, including extracellular vesicles, which facilitate their transport over long distance. The long-range activity of the Hedgehog morphogen is conveyed by extracellular vesicles. However, the site and the molecular basis of their biogenesis remains unknown. By combining fluorescence and electron microscopy combined with genetics and cell biology approaches, we investigated the origin and the cellular mechanisms underlying extracellular vesicle biogenesis, and their contribution to Drosophila wing disc development, exploiting Hedgehog as a long-range morphogen. We show that microvilli of Drosophila wing disc epithelium are the site of generation of small extracellular vesicles that transport Hedgehog across the tissue. This process requires the Prominin-like protein, whose activity, together with interacting cytoskeleton components and lipids, is critical for maintaining microvilli integrity and function in secretion. Our results provide the first evidence that microvilli-derived extracellular vesicles contribute to Hedgehog long-range signaling activity highlighting their physiological significance in tissue development in vivo.

cell biology↗

bioRxiv2019

Centrosome amplification favours survival and impairs ovarian cancer progression

Centrosome amplification, the presence of more than two centrosomes in a cell is a common feature of most human cancer cell lines. However, little is known about centrosome numbers of human cancers and whether amplification or other numerical aberrations are frequently present. To address this question, we have analyzed a large cohort of human epithelial ovarian cancers (EOCs) from 100 patients. Using state-of-the-art microscopy, we have determined the Centrosome-Nucleus Index (CNI) of each tumor. We found that EOCs show infrequent centrosome amplifications. Strikingly, the large majority of these tumors presented low CNIs. We show that low CNI tumors are enriched in the mesenchymal subgroup and correlate with poor patient survival. Our findings highlight a novel paradigm linking low centrosome number with highly aggressive behavior in ovarian cancers and show that the CNI signature may be used to stratify ovarian cancers.

cancer biology↗

bioRxiv2019

CELL CYCLE ASYNCHRONY GENERATES DNA DAMAGE AT MITOTIC ENTRY IN POLYPLOID CELLS

Polyploidy arises from the gain of complete chromosomes sets [1] and is known to promote cancer genome evolution. Recent evidence suggests that a large proportion of human tumours experience whole genome duplications (WGDs), which might favour the generation of highly abnormal karyotypes within a short time frame, rather than in a stepwise manner [2-6]. However, the molecular mechanisms linking whole genome duplication to genetic instability remain poorly understood. Further, possible mechanisms responsible for rapid genome reshuffling have not been described yet. Using repeated cytokinesis failure to induce polyploidization of Drosophila neural stem cells (NSCs, also called neuroblasts - NBs), we investigated the consequences of polyploidy in vivo. Here, we show that polyploid NSCs accumulate high levels of chromosome instability. Surprisingly, we found that DNA damage is generated in a subset of nuclei of polyploid NBs during mitosis, in an asymmetric manner. Importantly, our observations in flies were confirmed in mouse NSCs (mNSCs) after acute cytokinesis inhibition. Interestingly, DNA damage occurs in nuclei that were not ready to enter mitosis but were forced to do so when exposed to the mitotic environment of neighbouring nuclei within the same cell. Additionally, we found that polyploid cells are cell cycle asynchronous and forcing cell cycle synchronization is sufficient to lower the levels of DNA damage generated during mitosis. Overall, this work supports a model in which DNA damage at mitotic entry can generate a mutated genetic landscape that contributes to the onset of genetic instability.

cell biology↗

bioRxiv2019

Chromosomes function as a barrier to mitotic spindle bipolarity in polyploid cells

Whole genome duplications (WGDs) are found in a variety of tumors and are associated with chromosomal instability (CIN) and poor prognosis [1,2]. When induced experimentally, through cytokinesis failure, polyploid cells generate tumors [3]. Cytokinesis failure results in the accumulation of double DNA content, but also of cytoplasmic organelles, such as centrosomes, which are the major microtubule (MT) organizing centers of animal cells. Importantly, even if there is a correlation between polyploidy and CIN [4], the underlying mechanisms generating error-prone mitosis in cells with extra DNA and extra centrosomes are not known. When considering polyploid mitosis, it is essential to take into account the increase in MT nucleation due to the presence of extra centrosomes and extra DNA. The presence of supernumerary centrosomes in a cell, centrosome amplification [5], is associated with mitotic spindle multipolarity and CIN [6-9]. Importantly, additional MTs can be nucleated from the chromatin (chromatin mediated pathway-CMP) or from pre-existing MTs-through the Augmin pathway. We hypothesized that the increase in DNA and centrosome content in a cell could lead to an increased MT mass, which might account for abnormal mitosis described in polyploid cells [4, 10, 11, 12]. Using genetics, live imaging and modeling approaches, we investigated the mechanisms establishing multipolarity in vivo in polyploid cells. We found that MT nucleation from the centrosomes is the major contributor to multipolarity, while other pathways seem to play minor roles. Unexpectedly, we found that even if Ncd/HSET, plays an essential role in promoting centrosome clustering in early mitosis, the increase in chromosome mass associated with cytokinesis failure functions as a barrier to centrosome clustering into two main poles. Our work provides a mechanistic link between polyploidy and the generation of CIN.

cell biology↗

bioRxiv2018

Gene editing can generate fragile bivalents in mouse oocytes

Mouse female meiotic spindles assemble from acentriolar MTOCs (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOCs biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master MTOC regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.

cell biology↗