bims-ginsta Biomed News
on Genome instability
Issue of 2024–09–22
nineteen papers selected by
Jinrong Hu, National University of Singapore
  1. Artificial kinetochore beads establish a biorientation-like state in the spindle.
  2. Temporal BMP4 effects on mouse embryonic and extraembryonic development.
  3. Distinct mechanisms regulate ventricular and atrial chamber wall formation.
  4. Fasting shapes chromatin architecture through an mTOR/RNA Pol I axis.
  5. Ion-mediated condensation controls the mechanics of mitotic chromosomes.
  6. In vivo DNA replication dynamics unveil aging-dependent replication stress.
  7. Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.
  8. Overactive mitochondrial DNA replication disrupts perinatal cardiac maturation.
  9. Cardiac NAD+ depletion in mice promotes hypertrophic cardiomyopathy and arrhythmias prior to impaired bioenergetics.
  10. Chronic Activation of Tubulin Tyrosination Improves Heart Function.
  11. Systems mapping of bidirectional endosomal transport through the crowded cell.
  12. Structural basis for translational control by the human 48S initiation complex.
  13. Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes.
  14. Reduction of chromosomal instability and inflammation is a common aspect of adaptation to aneuploidy.
  15. Aneuploidy of Specific Chromosomes is Beneficial to Cells Lacking Spindle Checkpoint Protein Bub3.
  16. High-resolution functional mapping of RAD51C by saturation genome editing.
  17. Mechanomemory of pulmonary fibroblasts demonstrates reversibility of transcriptomics and contraction phenotypes.
  18. Plasma membrane curvature regulates the formation of contacts with the endoplasmic reticulum.
  19. Challenges and recommendations for the translation of biomarkers of aging.
  1. Science. 2024 Sep 20. 385(6715): 1366-1375
    Artificial kinetochore beads establish a biorientation-like state in the spindle.
    Kohei Asai, Yuanzhuo Zhou, Osamu Takenouchi, Tomoya S Kitajima.
      Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.
    DOI:  https://doi.org/10.1126/science.adn5428
  2. Nature. 2024 Sep 18.
    Temporal BMP4 effects on mouse embryonic and extraembryonic development.
    Ron Hadas, Hernan Rubinstein, Markus Mittnenzweig, Yoav Mayshar, Raz Ben-Yair, Saifeng Cheng, Alejandro Aguilera-Castrejon, Netta Reines, Ayelet-Hashahar Orenbuch, Aviezer Lifshitz, Dong-Yuan Chen, Michael B Elowitz, Magdalena Zernicka-Goetz, Jacob H Hanna, Amos Tanay, Yonatan Stelzer.
      The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro.
    DOI:  https://doi.org/10.1038/s41586-024-07937-5
  3. Nat Commun. 2024 Sep 17. 15(1): 8159
    Distinct mechanisms regulate ventricular and atrial chamber wall formation.
    Marga Albu, Eileen Affolter, Alessandra Gentile, Yanli Xu, Khrievono Kikhi, Sarah Howard, Carsten Kuenne, Rashmi Priya, Felix Gunawan, Didier Y R Stainier.
      Tissues undergo distinct morphogenetic processes to achieve similarly shaped structures. In the heart, cardiomyocytes in both the ventricle and atrium build internal structures for efficient contraction. Ventricular wall formation (trabeculation) is initiated by cardiomyocyte delamination. How cardiomyocytes build the atrial wall is poorly understood. Using longitudinal imaging in zebrafish, we found that at least 25% of the atrial cardiomyocytes elongate along the long axis of the heart. These cell shape changes result in cell intercalation and convergent thickening, leading to the formation of the internal muscle network. We tested factors important for ventricular trabeculation including Nrg/ErbB and Notch signaling and found no evidence for their role in atrial muscle network formation. Instead, our data suggest that atrial cardiomyocyte elongation is regulated by Yap, which has not been implicated in trabeculation. Altogether, these data indicate that distinct cellular and molecular mechanisms build the internal muscle structures in the atrium and ventricle.
    DOI:  https://doi.org/10.1038/s41467-024-52340-3
  4. Nat Cell Biol. 2024 Sep 19.
    Fasting shapes chromatin architecture through an mTOR/RNA Pol I axis.
    Nada Al-Refaie, Francesco Padovani, Johanna Hornung, Lorenz Pudelko, Francesca Binando, Andrea Del Carmen Fabregat, Qiuxia Zhao, Benjamin D Towbin, Elif Sarinay Cenik, Nicholas Stroustrup, Jan Padeken, Kurt M Schmoller, Daphne S Cabianca.
      Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom, yet how it impacts three-dimensional (3D) genome organization is unknown. Here we show that fasting induces an intestine-specific, reversible and large-scale spatial reorganization of chromatin in Caenorhabditis elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, acting through the regulation of RNA Pol I, but not Pol II nor Pol III, and is accompanied by remodelling of the nucleolus. By uncoupling the 3D genome configuration from the animal's nutritional status, we find that the expression of metabolic and stress-related genes increases when the spatial reorganization of chromatin occurs, showing that the 3D genome might support the transcriptional response in fasted animals. Our work documents a large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients.
    DOI:  https://doi.org/10.1038/s41556-024-01512-w
  5. Nat Mater. 2024 Sep 16.
    Ion-mediated condensation controls the mechanics of mitotic chromosomes.
    Hannes Witt, Janni Harju, Emma M J Chameau, Charlotte M A Bruinsma, Tinka V M Clement, Christian F Nielsen, Ian D Hickson, Erwin J G Peterman, Chase P Broedersz, Gijs J L Wuite.
      During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. How do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? Here we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone depletion and theory to show that chromosomal elasticity originates from the chromatin fibre behaving as a flexible polymer, whereas energy dissipation can be explained by modelling chromatin loops as an entangled polymer solution. Taken together, we show how collective properties of mitotic chromosomes, a biomaterial of incredible complexity, emerge from molecular properties, and how they are controlled by the physico-chemical environment.
    DOI:  https://doi.org/10.1038/s41563-024-01975-0
  6. Cell. 2024 Sep 10. pii: S0092-8674(24)00963-2. [Epub ahead of print]
    In vivo DNA replication dynamics unveil aging-dependent replication stress.
    Giacomo G Rossetti, Noëlle Dommann, Angeliki Karamichali, Vasilis S Dionellis, Ainhoa Asensio Aldave, Tural Yarahmadov, Eddie Rodriguez-Carballo, Adrian Keogh, Daniel Candinas, Deborah Stroka, Thanos D Halazonetis.
      The genome duplication program is affected by multiple factors in vivo, including developmental cues, genotoxic stress, and aging. Here, we monitored DNA replication initiation dynamics in regenerating livers of young and old mice after partial hepatectomy to investigate the impact of aging. In young mice, the origin firing sites were well defined; the majority were located 10-50 kb upstream or downstream of expressed genes, and their position on the genome was conserved in human cells. Old mice displayed the same replication initiation sites, but origin firing was inefficient and accompanied by a replication stress response. Inhibitors of the ATR checkpoint kinase fully restored origin firing efficiency in the old mice but at the expense of an inflammatory response and without significantly enhancing the fraction of hepatocytes entering the cell cycle. These findings unveil aging-dependent replication stress and a crucial role of ATR in mitigating the stress-associated inflammation, a hallmark of aging.
    Keywords:  ATR; DNA replication; DNA replication stress; aging; cryptic DNA damage; liver regeneration; origin of replication; partial hepatectomy
    DOI:  https://doi.org/10.1016/j.cell.2024.08.034
  7. Proc Natl Acad Sci U S A. 2024 Sep 24. 121(39): e2407083121
    Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.
    Stella E Cho, Wei Li, Andrew M Beard, Jonathan A Jackson, Risa Kiernan, Kazunori Hoshino, Adam C Martin, Jianjun Sun.
      Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.
    Keywords:  Rho1; actomyosin contraction; follicle rupture; mechanical force; ovulation
    DOI:  https://doi.org/10.1073/pnas.2407083121
  8. Nat Commun. 2024 Sep 14. 15(1): 8066
    Overactive mitochondrial DNA replication disrupts perinatal cardiac maturation.
    Juan C Landoni, Semin Erkul, Tuomas Laalo, Steffi Goffart, Riikka Kivelä, Karlo Skube, Anni I Nieminen, Sara A Wickström, James Stewart, Anu Suomalainen.
      High mitochondrial DNA (mtDNA) amount has been reported to be beneficial for resistance and recovery of metabolic stress, while increased mtDNA synthesis activity can drive aging signs. The intriguing contrast of these two mtDNA boosting outcomes prompted us to jointly elevate mtDNA amount and frequency of replication in mice. We report that high activity of mtDNA synthesis inhibits perinatal metabolic maturation of the heart. The offspring of the asymptomatic parental lines are born healthy but manifest dilated cardiomyopathy and cardiac collapse during the first days of life. The pathogenesis, further enhanced by mtDNA mutagenesis, involves prenatal upregulation of mitochondrial integrated stress response and the ferroptosis-inducer MESH1, leading to cardiac fibrosis and cardiomyocyte death after birth. Our evidence indicates that the tight control of mtDNA replication is critical for early cardiac homeostasis. Importantly, ferroptosis sensitivity is a potential targetable mechanism for infantile-onset cardiomyopathy, a common manifestation of mitochondrial diseases.
    DOI:  https://doi.org/10.1038/s41467-024-52164-1
  9. Nat Cardiovasc Res. 2024 Sep 18.
    Cardiac NAD+ depletion in mice promotes hypertrophic cardiomyopathy and arrhythmias prior to impaired bioenergetics.
    Khanh V Doan, Timothy S Luongo, Thato T Ts'olo, Won Dong Lee, David W Frederick, Sarmistha Mukherjee, Gabriel K Adzika, Caroline E Perry, Ryan B Gaspar, Nicole Walker, Megan C Blair, Nicole Bye, James G Davis, Corey D Holman, Qingwei Chu, Lin Wang, Joshua D Rabinowitz, Daniel P Kelly, Thomas P Cappola, Kenneth B Margulies, Joseph A Baur.
      Nicotinamide adenine dinucleotide (NAD+) is an essential co-factor in metabolic reactions and co-substrate for signaling enzymes. Failing human hearts display decreased expression of the major NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (Nampt) and lower NAD+ levels, and supplementation with NAD+ precursors is protective in preclinical models. Here we show that Nampt loss in adult cardiomyocytes caused depletion of NAD+ along with marked metabolic derangements, hypertrophic remodeling and sudden cardiac deaths, despite unchanged ejection fraction, endurance and mitochondrial respiratory capacity. These effects were directly attributable to NAD+ loss as all were ameliorated by restoring cardiac NAD+ levels with the NAD+ precursor nicotinamide riboside (NR). Electrocardiograms revealed that loss of myocardial Nampt caused a shortening of QT intervals with spontaneous lethal arrhythmias causing sudden cardiac death. Thus, changes in NAD+ concentration can have a profound influence on cardiac physiology even at levels sufficient to maintain energetics.
    DOI:  https://doi.org/10.1038/s44161-024-00542-9
  10. Circ Res. 2024 Sep 16.
    Chronic Activation of Tubulin Tyrosination Improves Heart Function.
    Niels Pietsch, Christina Y Chen, Svenja Kupsch, Lucas Bacmeister, Birgit Geertz, Marisol Herrera-Rivero, Bente Siebels, Hanna Voß, Elisabeth Krämer, Ingke Braren, Dirk Westermann, Hartmut Schlüter, Giulia Mearini, Saskia Schlossarek, Jolanda van der Velden, Matthew Caporizzo, Diana Lindner, Benjamin L Prosser, Lucie Carrier.
       RATIONALE: Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with the findings that microtubule detyrosination (dTyr-MT) is markedly elevated in heart failure. Acute reduction of dTyr-MT by inhibition of the detyrosinase (VASH [vasohibin]/SVBP [small VASH-binding protein] complex) or activation of the tyrosinase (TTL [tubulin tyrosine ligase]) markedly improved contractility and reduced stiffness in human failing cardiomyocytes and thus posed a new perspective for HCM treatment.
    OBJECTIVE: In this study, we tested the impact of chronic tubulin tyrosination in an HCM mouse model (Mybpc3 knock in), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs).
    METHODS AND RESULTS: Adeno-associated virus serotype 9-mediated TTL transfer was applied in neonatal wild-type rodents, in 3-week-old knock-in mice, and in HCM human induced pluripotent stem cell-derived cardiomyocytes. We show (1) TTL for 6 weeks dose dependently reduced dTyr-MT and improved contractility without affecting cytosolic calcium transients in wild-type cardiomyocytes; (2) TTL for 12 weeks reduced the abundance of dTyr-MT in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in knock-in mice; (3) TTL for 10 days normalized cell area in HCM human induced pluripotent stem cell-derived cardiomyocytes; (4) TTL overexpression activated transcription of tubulins and other cytoskeleton components but did not significantly impact the proteome in knock-in mice; (5) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and wild-type EHTs. RNA sequencing and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-deficient versus TTL-deficient EHTs.
    CONCLUSIONS: This study provides the first proof of concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the nonsarcomeric cytoskeleton in heart disease.
    Keywords:  cardiomyopathy, hypertrophic; induced pluripotent stem cells; microtubules; myocardium; rodentia
    DOI:  https://doi.org/10.1161/CIRCRESAHA.124.324387
  11. Curr Biol. 2024 Sep 09. pii: S0960-9822(24)01138-2. [Epub ahead of print]
    Systems mapping of bidirectional endosomal transport through the crowded cell.
    Marlieke L M Jongsma, Nina Bakker, Lenard M Voortman, Roman I Koning, Erik Bos, Jimmy J L L Akkermans, Lennert Janssen, Jacques Neefjes.
      Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors.
    Keywords:  FYCO1; Lis1; Protrudin; RILP; Rab7; bidirectional transport; endosomes; membrane contact sites; motor proteins; optogenetics
    DOI:  https://doi.org/10.1016/j.cub.2024.08.026
  12. Nat Struct Mol Biol. 2024 Sep 17.
    Structural basis for translational control by the human 48S initiation complex.
    Valentyn Petrychenko, Sung-Hui Yi, David Liedtke, Bee-Zen Peng, Marina V Rodnina, Niels Fischer.
      The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome.
    DOI:  https://doi.org/10.1038/s41594-024-01378-4
  13. Am J Physiol Heart Circ Physiol. 2024 Sep 20.
    Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes.
    Maria Knierim, Thea Bommer, Michael Paulus, Dominic Riedl, Sarah Fink, Arnold Pöppl, Florian Reetz, Peter Wang, Lars Maier, Niels Voigt, Matthias Nahrendorf, Samuel Sossalla, Katrin Streckfuss-Bömeke, Steffen Pabel.
      Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca2+-handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24h or 7d at a physiological rate of 60 bpm on cellular electrophysiology and Ca2+-cycling in late-stage, differentiated hiPSC-CM (>90% troponin+, >60d post differentiation). Electric culture pacing for 7d did not influence cardiomyocyte cell size, apoptosis or generation of reactive oxygen species in differentiated hiPSC-CM compared to 24h pacing. However, epifluorescence measurements revealed that electric pacing for 7d improved systolic Ca2+-transient amplitude and Ca2+-transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca2+-load and SERCA activity. Diastolic Ca2+-leak was not changed in line-scanning confocal microscopy suggesting that the improvement in systolic Ca2+-release was not associated with a higher open probability of RyR2 during diastole. While bulk cytosolic Na+-concentration and NCX activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole-cell voltage-clamp measurements that chronic pacing for 7d led to a decrease in late Na+-current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca2+-handling and modulates the electrophysiology of late-stage, differentiated iPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.
    Keywords:  Calcium; EC-coupling; Electrophysiology; iPSC cardiomyocytes
    DOI:  https://doi.org/10.1152/ajpheart.00536.2024
  14. EMBO Rep. 2024 Sep 18.
    Reduction of chromosomal instability and inflammation is a common aspect of adaptation to aneuploidy.
    Dorine C Hintzen, Michael Schubert, Mar Soto, René H Medema, Jonne A Raaijmakers.
      Aneuploidy, while detrimental to untransformed cells, is notably prevalent in cancer. Aneuploidy is found as an early event during tumorigenesis which indicates that cancer cells have the ability to surmount the initial stress responses associated with aneuploidy, enabling rapid proliferation despite aberrant karyotypes. To generate more insight into key cellular processes and requirements underlying adaptation to aneuploidy, we generated a panel of aneuploid clones in p53-deficient RPE-1 cells and studied their behavior over time. As expected, de novo-generated aneuploid clones initially display reduced fitness, enhanced levels of chromosomal instability (CIN), and an upregulated inflammatory response. Intriguingly, after prolonged culturing, aneuploid clones exhibit increased proliferation rates while maintaining aberrant karyotypes, indicative of an adaptive response to the aneuploid state. Interestingly, all adapted clones display reduced CIN and reduced inflammatory signaling, suggesting that these are common aspects of adaptation to aneuploidy. Collectively, our data suggests that CIN and concomitant inflammation are key processes that require correction to allow for fast proliferation in vitro. Finally, we provide evidence that amplification of oncogenic KRAS can promote adaptation.
    Keywords:  Adaptation; Aneuploidy; Chromosomal Instability; Inflammation
    DOI:  https://doi.org/10.1038/s44319-024-00252-0
  15. bioRxiv. 2024 Sep 03. pii: 2024.09.02.610809. [Epub ahead of print]
    Aneuploidy of Specific Chromosomes is Beneficial to Cells Lacking Spindle Checkpoint Protein Bub3.
    Pallavi Gadgil, Olivia Ballew, Timothy J Sullivan, Soni Lacefield.
      Aneuploidy typically poses challenges for cell survival and growth. However, recent studies have identified exceptions where aneuploidy is beneficial for cells with mutations in certain regulatory genes. Our research reveals that cells lacking the spindle checkpoint gene BUB3 exhibit aneuploidy of select chromosomes. While the spindle checkpoint is not essential in budding yeast, the loss of BUB3 and BUB1 increases the probability of chromosome missegregation compared to wildtype cells. Contrary to the prevailing assumption that the aneuploid cells would be outcompeted due to growth defects, our findings demonstrate that bub3 Δ cells consistently maintained aneuploidy of specific chromosomes over many generations. We investigated whether the persistence of these additional chromosomes in bub3 Δ cells resulted from the beneficial elevated expression of certain genes, or mere tolerance. We identified several genes involved in chromosome segregation and cell cycle regulation that confer an advantage to Bub3-depleted cells. Overall, our results suggest that the upregulation of specific genes through aneuploidy may provide a survival and growth advantage to strains with poor chromosome segregation fidelity.
    AUTHOR SUMMARY: Accurate chromosome segregation is crucial for the proper development of all living organisms. Errors in chromosome segregation can lead to aneuploidy, characterized by an abnormal number of chromosomes, which generally impairs cell survival and growth. However, under certain stress conditions, such as in various cancers, cells with specific mutations and extra copies of advantageous chromosomes exhibit improved survival and proliferation. In our study, we discovered that cells lacking the spindle checkpoint protein Bub3 became aneuploid, retaining specific chromosomes. This finding was unexpected because although bub3 Δ cells have a higher rate of chromosome mis-segregation, they were not thought to maintain an aneuploid karyotype. We investigated whether the increased copy number of specific genes on these acquired chromosomes offered a benefit to Bub3-deficient cells. Our results revealed that several genes involved in chromosome segregation and cell cycle regulation prevented the gain of chromosomes upon Bub3-depletion, suggesting that these genes confer a survival advantage. Overall, our study demonstrates that cells lacking Bub3 selectively retain specific chromosomes to increase the copy number of genes that promote proper chromosome segregation.
    DOI:  https://doi.org/10.1101/2024.09.02.610809
  16. Cell. 2024 Sep 16. pii: S0092-8674(24)00968-1. [Epub ahead of print]
    High-resolution functional mapping of RAD51C by saturation genome editing.
    Rebeca Olvera-León, Fang Zhang, Victoria Offord, Yajie Zhao, Hong Kee Tan, Prashant Gupta, Tuya Pal, Carla Daniela Robles-Espinoza, Fernanda G Arriaga-González, Larissa Satiko Alcantara Sekimoto Matsuyama, Erwan Delage, Ed Dicks, Suzana Ezquina, Charlie F Rowlands, Clare Turnbull, Paul Pharoah, John R B Perry, Maria Jasin, Andrew J Waters, David J Adams.
      Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.
    Keywords:  RAD51C; cancer predisposition; homologous recombination; saturation genome editing
    DOI:  https://doi.org/10.1016/j.cell.2024.08.039
  17. Biomaterials. 2024 Sep 10. pii: S0142-9612(24)00364-8. [Epub ahead of print]314 122830
    Mechanomemory of pulmonary fibroblasts demonstrates reversibility of transcriptomics and contraction phenotypes.
    Caymen M Novak, Jana S Wheat, Samir N Ghadiali, Megan N Ballinger.
      Fibroblasts are cells responsible for producing extracellular matrix (ECM) components, which provides physical support for organs. Although these mesenchymal cells are responsive to mechanical cues in their environment, the permanence of these mechanophenotypes is not well defined. We investigated the mechanomemory of lung fibroblasts and determined how switching culture conditions modulate cell responses and function. Primary murine lung fibroblasts were isolated and cultured on 2D tissue culture plates or within 3D collagen hydrogels and were then passaged within the same or opposite culture condition to assess changes in gene expression, protein production, fibroblast subpopulation, contractile behavior, and traction forces. Compared to fibroblasts isolated on 2D tissue culture plates, fibroblasts within 3D hydrogels exhibited a decreased activation phenotype including reduced contraction profiles, diminished cell traction forces and decreased αSMA gene expression. Cells initially isolated via 2D culture and then cultured in 3D hydrogels exhibited a reversal in activation phenotype as measured by gene expression and contraction profiles. Bulk RNAseq identified groups of genes that exhibit reversible and non-reversable expression patterns. Overall, these findings indicate that lung fibroblasts have a mechanical memory that is altered by culture condition and can be reversible through precondition of cells within a softer 3D microenvironment.
    Keywords:  3D culture; Collagen hydrogel; Extracellular matrix; Fibroblasts; Mechanomemory
    DOI:  https://doi.org/10.1016/j.biomaterials.2024.122830
  18. Nat Cell Biol. 2024 Sep 17.
    Plasma membrane curvature regulates the formation of contacts with the endoplasmic reticulum.
    Yang Yang, Luis A Valencia, Chih-Hao Lu, Melissa L Nakamoto, Ching-Ting Tsai, Chun Liu, Huaxiao Yang, Wei Zhang, Zeinab Jahed, Wan-Ru Lee, Francesca Santoro, Jen Liou, Joseph C Wu, Bianxiao Cui.
      Contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM) play a crucial role in governing calcium regulation and lipid homeostasis. Despite their significance, the factors regulating their spatial distribution on the PM remain elusive. Inspired by observations in cardiomyocytes, where ER-PM contact sites concentrate on tubular PM invaginations known as transverse tubules, we hypothesize that PM curvature plays a role in ER-PM contact formation. Through precise control of PM invaginations, we show that PM curvatures locally induce the formation of ER-PM contacts in cardiomyocytes. Intriguingly, the junctophilin family of ER-PM tethering proteins, specifically expressed in excitable cells, is the key player in this process, whereas the ubiquitously expressed extended synaptotagmin-2 does not show a preference for PM curvature. At the mechanistic level, we find that the low-complexity region (LCR) and membrane occupation and recognition nexus (MORN) motifs of junctophilins can bind independently to the PM, but both the LCR and MORN motifs are required for targeting PM curvatures. By examining the junctophilin interactome, we identify a family of curvature-sensing proteins-Eps15 homology domain-containing proteins-that interact with the MORN_LCR motifs and facilitate the preferential tethering of junctophilins to curved PM. These findings highlight the pivotal role of PM curvature in the formation of ER-PM contacts in cardiomyocytes and unveil a mechanism for the spatial regulation of ER-PM contacts through PM curvature modulation.
    DOI:  https://doi.org/10.1038/s41556-024-01511-x
  19. Nat Aging. 2024 Sep 16.
    Challenges and recommendations for the translation of biomarkers of aging.
    Biomarkers of Aging Consortium
      Biomarkers of aging (BOA) are quantitative parameters that predict biological age and ideally its changes in response to interventions. In recent years, many promising molecular and omic BOA have emerged with an enormous potential for translational geroscience and improving healthspan. However, clinical translation remains limited, in part due to the gap between preclinical research and the application of BOA in clinical research and other translational settings. We surveyed experts in these areas to better understand current challenges for the translation of aging biomarkers. We identified six key barriers to clinical translation and developed guidance for the field to overcome them. Core recommendations include linking BOA to clinically actionable insights, improving affordability and availability to broad populations and validation of biomarkers that are robust and responsive at the level of individuals. Our work provides key insights and practical recommendations to overcome barriers impeding clinical translation of BOA.
    DOI:  https://doi.org/10.1038/s43587-024-00683-3
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