bims-ginsta Biomed News
on Genome instability
Issue of 2023‒10‒29
33 papers selected by
Jinrong Hu, National University of Singapore

  1. Stem Cell Reports. 2023 Oct 17. pii: S2213-6711(23)00375-2. [Epub ahead of print]
      Three-dimensional (3D) stem cell models of the ovary have the potential to benefit women's reproductive health research. One such model, the reconstituted ovary (rOvary) self-assembles with pluripotent stem cell-derived germ cells creating a 3D ovarian mimic competent to support the differentiation of functional oocytes inside follicles. In this study, we evaluated the cellular composition of the rOvary revealing the capacity to generate multiple follicles surrounded by NR2F2+ stroma cells. However, the rOvary does not develop a surface epithelium, the source of second-wave pre-granulosa cells, or steroidogenic theca. Therefore, the rOvary models represent the self-assembly of activated follicles in a pre-pubertal ovary poised but not yet competent for hormone production.
    Keywords:  follicles; germ cells; iPSCs; ovary; reconstituted ovary; self-assembly
  2. Dev Cell. 2023 Oct 18. pii: S1534-5807(23)00521-X. [Epub ahead of print]
      Mutations in the degradative ubiquitin ligase anaphase-promoting complex (APC) alter neurodevelopment by impairing proteasomal protein clearance, but our understanding of their molecular and cellular pathogenesis remains limited. Here, we employ the proteomic-based discovery of APC substrates in APC mutant mouse brain and human cell lines and identify the chromosome-passenger complex (CPC), topoisomerase 2a (Top2a), and Ki-67 as major chromatin factors targeted by the APC during neuronal differentiation. These substrates accumulate in phosphorylated form, suggesting that they fail to be eliminated after mitosis during terminal differentiation. The accumulation of the CPC kinase Aurora B within constitutive heterochromatin and hyperphosphorylation of its target histone 3 are corrected in the mutant brain by pharmacologic Aurora B inhibition. Surprisingly, the reduction of Ki-67, but not H3S10ph, rescued the function of constitutive heterochromatin in APC mutant neurons. These results expand our understanding of how ubiquitin signaling regulates chromatin during neurodevelopment and identify potential therapeutic targets in APC-related disorders.
    Keywords:  H3S10ph; Ki-67; anaphase-promoting complex; chromatin; chromosome-passenger complex; heterochromatin; neurodevelopment; proteomics/phosphoproteomics; topoisomerase; ubiquitin ligase
  3. Nat Struct Mol Biol. 2023 Oct 23.
      Cohesin forms a proteinaceous ring that is thought to link sister chromatids by entrapping DNA and counteracting the forces generated by the mitotic spindle. Whether individual cohesins encircle both sister DNAs and how cohesin opposes spindle-generated forces remains unknown. Here we perform force measurements on individual yeast cohesin complexes either bound to DNA or holding together two DNAs. By covalently closing the hinge and Smc3Psm3-kleisin interfaces we find that the mechanical stability of the cohesin ring entrapping DNA is determined by the hinge domain. Forces of ~20 pN disengage cohesin at the hinge and release DNA, indicating that ~40 cohesin molecules are sufficient to counteract known spindle forces. Our findings provide a mechanical framework for understanding how cohesin interacts with sister chromatids and opposes the spindle-generated tension during mitosis, with implications for other force-generating chromosomal processes including transcription and DNA replication.
  4. bioRxiv. 2023 Oct 08. pii: 2023.10.07.561364. [Epub ahead of print]
      Dynamic clustering of transcription players, including RNA polymerase II (Pol II), is thought to rely on multivalent interactions of their intrinsically disordered regions, thereby enhancing active transcription. Using the histone locus bodies (HLBs) of Drosophila nurse cells as a model, we find that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or pausing factors disperses these Pol II clusters, leaving HLBs intact. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly spills over throughout the cell cycle. We propose that clustering sequesters numerous poised Pol II molecules near gene promoters to ensure synchronous and efficient gene activation at desired times.
  5. Elife. 2023 Oct 26. pii: e83157. [Epub ahead of print]12
      During human forebrain development, neural progenitor cells (NPCs) in the ventricular zone (VZ) undergo asymmetric cell divisions to produce a self-renewed progenitor cell, maintaining the potential to go through additional rounds of cell divisions, and differentiating daughter cells, populating the developing cortex. Previous work in the embryonic rodent brain suggested that the preferential inheritance of the pre-existing (older) centrosome to the self-renewed progenitor cell is required to maintain stem cell properties, ensuring proper neurogenesis. If asymmetric segregation of centrosomes occurs in NPCs of the developing human brain, which depends on unique molecular regulators and species-specific cellular composition, remains unknown. Using a novel, recombination-induced tag exchange (RITE)-based genetic tool to birthdate and track the segregation of centrosomes over multiple cell divisions in human embryonic stem cell (hESC)-derived regionalized forebrain organoids, we show the preferential inheritance of the older mother centrosome towards self-renewed NPCs. Aberration of asymmetric segregation of centrosomes by genetic manipulation of the centrosomal, microtubule-associated protein Ninein alters fate decisions of NPCs and their maintenance in the VZ of human cortical organoids. Thus, the data described here use a novel genetic approach to birthdate centrosomes in human cells and identify asymmetric inheritance of centrosomes as a mechanism to maintain self-renewal properties and to ensure proper neurogenesis in human NPCs.
    Keywords:  developmental biology; human; regenerative medicine; stem cells
  6. Res Sq. 2023 Oct 05. pii: [Epub ahead of print]
      Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies (PB). During cytokinesis in somatic cells, the midbody (MB) and subsequent assembly of the midbody remnant (MBR), a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the MB and MBR in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic MBs (mMB) and mMB remnants (mMBRs) using mouse oocytes and demonstrate that mMBs have a specialized meiotic mMB cap structure that is orientated toward PBs. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.
  7. Emerg Top Life Sci. 2023 Oct 25. pii: ETLS20230023. [Epub ahead of print]
      Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by their association with copy number variants (CNVs) frequently arise in most tumor types. Over the years, it became clear that a combination of different characteristics underlies the enhanced sensitivity of CFSs to replication stress. As of today, there is a strong evidence that the core fragility regions along CFSs overlap with actively transcribed large genes with delayed replication timing upon replication stress. Recently, the mechanistic basis for CFS instability was further extended to regions which span topologically associated domain (TAD) boundaries, generating a fragility signature composed of replication, transcription and genome organization. The presence of difficult-to-replicate AT-rich repeats was one of the early features suggested to characterize a subgroup of CFSs. These long stretches of AT-dinucleotide have the potential to fold into stable secondary structures which may impede replication fork progression, leaving the region under-replicated. Here, we focus on the molecular mechanisms underlying repeat instability at CFSs and on the proteins involved in the resolution of secondary structure impediments arising along repetitive sequence elements which are essential for the maintenance of genome stability.
    Keywords:  AT-dinucleotide rich sequences; aphidicolin-induced replication stress; common fragile sites; genomic instability; repeat instability; secondary structures
  8. Nat Commun. 2023 Oct 27. 14(1): 6857
      The formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs essential for homology search and strand exchange in DNA double-strand break (DSB) repair is tightly regulated. FIGNL1 AAA+++ ATPase controls RAD51-mediated recombination in human cells. However, its role in gametogenesis remains unsolved. Here, we characterized a germ line-specific conditional knockout (cKO) mouse of FIGNL1. Fignl1 cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on meiotic chromosomes, supporting a positive role of FIGNL1 in homologous recombination at a post-assembly stage of RAD51/DMC1 filaments. Fignl1 cKO spermatocytes also accumulate RAD51/DMC1 on chromosomes in pre-meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. We also showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest an additional role of FIGNL1 in limiting the non-productive assembly of RAD51/DMC1 on native dsDNAs during pre-meiotic S-phase and meiotic prophase I.
  9. J Cell Sci. 2023 Oct 27. pii: jcs.261701. [Epub ahead of print]
      Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here we experimentally manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitosis progression and generating daughter cells with aberrant architecture. In these conditions, F-actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify the S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin and the Leucine-Glycine-Asparagine repeat protein (LGN) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasma membrane-mediated maintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity.
    Keywords:  Cell-cell adhesion; Chromosome segregation; Epithelial identity; Mitotic spindle; Plasma membrane remodelling; Polarised cell divisions
  10. Semin Cell Dev Biol. 2023 Oct 21. pii: S1084-9521(23)00173-8. [Epub ahead of print]
      Centromeres are large structural regions in the genomic DNA, which are essential for accurately transmitting a complete set of chromosomes to daughter cells during cell division. In humans, centromeres consist of highly repetitive α-satellite DNA sequences and unique epigenetic components, forming large proteinaceous structures required for chromosome segregation. Despite their biological importance, there is a growing body of evidence for centromere breakage across the cell cycle, including periods of quiescence. In this review, we provide an up-to-date examination of the distinct centromere environments at different stages of the cell cycle, highlighting their plausible contribution to centromere breakage. Additionally, we explore the implications of these breaks on centromere function, both in terms of negative consequences and potential positive effects.
    Keywords:  Cell Cycle; Centromeres; DNA Damage; DNA Repair; Quiescence
  11. bioRxiv. 2023 Oct 10. pii: 2023.10.08.561452. [Epub ahead of print]
      During development and disease progression, cells are subject to osmotic and mechanical stresses that modulate cell volume, which fundamentally influences cell homeostasis and has been linked to a variety of cellular functions. It is not well understood how the mechanobiological state of cells is programmed by the interplay of intracellular organization and complex extracellular mechanics when stimulated by cell volume modulation. Here, by controlling cell volume via osmotic pressure, we evaluate physical phenotypes (including cell shape, morphodynamics, traction force, and extracellular matrix (ECM) remodeling) and molecular signaling (YAP), and we uncover fundamental transitions in active biophysical states. We demonstrate that volumetric compression shifts the ratiometric balance of Rho GTPase activities, thereby altering mechanosensing and cytoskeletal organization in a reversible manner. Specifically, volumetric compression controls cell spreading, adhesion formation, and YAP nuclear translocation, while maintaining cell contractile activity. Furthermore, we show that on physiologically relevant fibrillar collagen I matrices, which are highly non-elastic, cells exhibit additional modes of cell volume-dependent mechanosensing that are not observable on elastic substrates. Notably, volumetric compression regulates the dynamics of cell-ECM interactions and irreversible ECM remodeling via Rac-directed protrusion dynamics, at both the single-cell level and the multicellular level. Our findings support that cell volume is a master biophysical regulator and reveal its roles in cell mechanical state transition, cell-ECM interactions, and biophysical tissue programming.
  12. Res Sq. 2023 Oct 20. pii: [Epub ahead of print]
      During early development, myosin II mechanically reshapes and folds embryo tissue. A much-studied example is ventral furrow formation in Drosophila , marking the onset of gastrulation. Furrowing is driven by contraction of actomyosin networks on apical cell surfaces, but how the myosin patterning encodes tissue shape is unclear, and elastic models failed to reproduce essential features of experimental cell contraction profiles. The myosin patterning exhibits substantial cell-to-cell fluctuations with pulsatile time-dependence, a striking but unexplained feature of morphogenesis in many organisms. Here, using biophysical modeling we find viscous forces offer the principal resistance to actomyosin-driven apical constriction. In consequence, tissue shape is encoded in the direction-dependent curvature of the myosin patterning which orients an anterior-posterior furrow. Tissue contraction is highly sensitive to cell-to-cell myosin fluctuations, explaining furrowing failure in genetically perturbed embryos whose fluctuations are temporally persistent. In wild-type embryos this disastrous outcome is averted by pulsatile myosin time-dependence, which rescues furrowing by eliminating high frequencies in the fluctuation power spectrum. This low pass filter mechanism may underlie the usage of actomyosin pulsing in diverse morphogenetic processes across many organisms.
  13. bioRxiv. 2023 Oct 21. pii: 2023.10.04.560808. [Epub ahead of print]
      Ectopic expression of OCT4, SOX2, KLF4 and MYC (OSKM) transforms differentiated cells into induced pluripotent stem cells. To refine our mechanistic understanding of reprogramming, especially during the early stages, we profiled chromatin accessibility and gene expression at single-cell resolution across a densely sampled time course of human fibroblast reprogramming. Using neural networks that map DNA sequence to ATAC-seq profiles at base-resolution, we annotated cell-state-specific predictive transcription factor (TF) motif syntax in regulatory elements, inferred affinity- and concentration-dependent dynamics of Tn5-bias corrected TF footprints, linked peaks to putative target genes, and elucidated rewiring of TF-to-gene cis-regulatory networks. Our models reveal that early in reprogramming, OSK, at supraphysiological concentrations, rapidly open transient regulatory elements by occupying non-canonical low-affinity binding sites. As OSK concentration falls, the accessibility of these transient elements decays as a function of motif affinity. We find that these OSK-dependent transient elements sequester the somatic TF AP-1. This redistribution is strongly associated with the silencing of fibroblast-specific genes within individual nuclei. Together, our integrated single-cell resource and models reveal insights into the cis-regulatory code of reprogramming at unprecedented resolution, connect TF stoichiometry and motif syntax to diversification of cell fate trajectories, and provide new perspectives on the dynamics and role of transient regulatory elements in somatic silencing.
  14. Nat Commun. 2023 Oct 27. 14(1): 6850
      hox genes encode a conserved family of homeodomain transcription factors that are essential to determine the identity of body segments during embryogenesis and maintain adult somatic stem cells competent to regenerate organs. In contrast to higher organisms, somatic cells in C. elegans irreversibly exit the cell cycle after completing their cell lineage and the adult soma cannot regenerate. Here, we show that hox gene expression levels in C. elegans determine the temporal competence of somatic cells to proliferate. Down-regulation of the central hox gene lin-39 in dividing vulval cells results in their premature cell cycle exit, whereas constitutive lin-39 expression causes precocious Pn.p cell and sex myoblast divisions and prolongs the proliferative phase of the vulval cells past their normal point of arrest. Furthermore, ectopic expression of hox genes in the quiescent anchor cell re-activates the cell cycle and induces proliferation until young adulthood. Thus, constitutive expression of a single hox transcription factor is sufficient to prolong somatic cell proliferation beyond the restriction imposed by the cell lineage. The down-regulation of hox gene expression in most somatic cells at the end of larval development may be one cause for the absence of cell proliferation in adult C. elegans.
  15. J Cell Biol. 2024 Jan 01. pii: e202106169. [Epub ahead of print]223(1):
      Vinculin is an actin-binding protein (ABP) that strengthens the connection between the actin cytoskeleton and adhesion complexes. It binds to β-catenin/N-cadherin complexes in apical adherens junctions (AJs), which maintain cell-to-cell adhesions, and to talin/integrins in the focal adhesions (FAs) that attach cells to the basal membrane. Here, we demonstrate that β-catenin targets vinculin to the apical AJs and the centrosome in the embryonic neural tube (NT). Suppression of vinculin slows down the basal-to-apical part of interkinetic nuclear migration (BAINM), arrests neural stem cells (NSCs) in the G2 phase of the cell cycle, and ultimately dismantles the apical actin cytoskeleton. In the NSCs, mitosis initiates when an internalized centrosome gathers with the nucleus during BAINM. Notably, our results show that the first centrosome to be internalized is the daughter centrosome, where β-catenin and vinculin accumulate, and that vinculin suppression prevents centrosome internalization. Thus, we propose that vinculin links AJs, the centrosome, and the actin cytoskeleton where actomyosin contraction forces are required.
  16. Nat Commun. 2023 Oct 23. 14(1): 6737
      The molecular mechanisms connecting cellular metabolism with differentiation remain poorly understood. Here, we find that metabolic signals contribute to stem cell differentiation and germline homeostasis during Drosophila melanogaster spermatogenesis. We discovered that external citrate, originating outside the gonad, fuels the production of Acetyl-coenzyme A by germline ATP-citrate lyase (dACLY). We show that this pathway is essential during the final spermatogenic stages, where a high Acetyl-coenzyme A level promotes NatB-dependent N-terminal protein acetylation. Using genetic and biochemical experiments, we establish that N-terminal acetylation shields key target proteins, essential for spermatid differentiation, from proteasomal degradation by the ubiquitin ligase dUBR1. Our work uncovers crosstalk between metabolism and proteome stability that is mediated via protein post-translational modification. We propose that this system coordinates the metabolic state of the organism with gamete production. More broadly, modulation of proteome turnover by circulating metabolites may be a conserved regulatory mechanism to control cell functions.
  17. bioRxiv. 2023 Oct 05. pii: 2023.10.03.560616. [Epub ahead of print]
      Repressive chromatin modifications are thought to compact chromatin to silence transcription. However, it is unclear how chromatin structure changes during silencing and epigenetic memory formation. We measured gene expression and chromatin structure in single cells after recruitment and release of repressors at a reporter gene. Chromatin structure is heterogeneous, with open and compact conformations present in both active and silent states. Recruitment of repressors associated with epigenetic memory produces chromatin compaction across 10-20 kilobases, while reversible silencing does not cause compaction at this scale. Chromatin compaction is inherited, but changes molecularly over time from histone methylation (H3K9me3) to DNA methylation. The level of compaction at the end of silencing quantitatively predicts epigenetic memory weeks later. Similarly, chromatin compaction at the Nanog locus predicts the degree of stem-cell fate commitment. These findings suggest that the chromatin state across tens of kilobases, beyond the gene itself, is important for epigenetic memory formation.
  18. J Clin Invest. 2023 Oct 24. pii: e171430. [Epub ahead of print]
      Reactivation and dysregulation of the mTOR signaling pathway is a hallmark of aging and chronic lung disease, however the impact on microvascular progenitor cells (MVPC), capillary angiostasis and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue resident MVPC present in both adult mouse and human lungs using functional, lineage and transcriptomic analyses. These studies link human and mouse MVPC specific mTORC1 activation to decreased stemness, angiogenic potential, disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation these MVPC adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function, microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.
    Keywords:  Adult stem cells; Endothelial cells; Microcirculation; Stem cells; Vascular Biology
  19. PLoS Biol. 2023 Oct;21(10): e3002339
      Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration:, NCT03393741.
  20. Mol Cell. 2023 Oct 20. pii: S1097-2765(23)00800-6. [Epub ahead of print]
      Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.
    Keywords:  TCA cycle; electron transport chain; glycolysis; lactate; mitochondria; oxidative phosphorylation
  21. Nat Commun. 2023 Oct 23. 14(1): 6720
      Certain archaeal cells possess external proteinaceous sheath, whose structure and organization are both unknown. By cellular cryogenic electron tomography (cryoET), here we have determined sheath organization of the prototypical archaeon, Methanospirillum hungatei. Fitting of Alphafold-predicted model of the sheath protein (SH) monomer into the 7.9 Å-resolution structure reveals that the sheath cylinder consists of axially stacked β-hoops, each of which is comprised of two to six 400 nm-diameter rings of β-strand arches (β-rings). With both similarities to and differences from amyloid cross-β fibril architecture, each β-ring contains two giant β-sheets contributed by ~ 450 SH monomers that entirely encircle the outer circumference of the cell. Tomograms of immature cells suggest models of sheath biogenesis: oligomerization of SH monomers into β-ring precursors after their membrane-proximal cytoplasmic synthesis, followed by translocation through the unplugged end of a dividing cell, and insertion of nascent β-hoops into the immature sheath cylinder at the junction of two daughter cells.
  22. Sci Transl Med. 2023 Oct 25. 15(719): eadg5252
      Effective tissue repair requires coordinated intercellular communication to sense damage, remodel the tissue, and restore function. Here, we dissected the healing response in the intestinal mucosa by mapping intercellular communication at single-cell resolution and integrating with spatial transcriptomics. We demonstrated that a risk variant for Crohn's disease, hepatocyte growth factor activator (HGFAC) Arg509His (R509H), disrupted a damage-sensing pathway connecting the coagulation cascade to growth factors that drive the differentiation of wound-associated epithelial (WAE) cells and production of a localized retinoic acid (RA) gradient to promote fibroblast-mediated tissue remodeling. Specifically, we showed that HGFAC R509H was activated by thrombin protease activity but exhibited impaired proteolytic activation of the growth factor macrophage-stimulating protein (MSP). In Hgfac R509H mice, reduced MSP activation in response to wounding of the colon resulted in impaired WAE cell induction and delayed healing. Through integration of single-cell transcriptomics and spatial transcriptomics, we demonstrated that WAE cells generated RA in a spatially restricted region of the wound site and that mucosal fibroblasts responded to this signal by producing extracellular matrix and growth factors. We further dissected this WAE cell-fibroblast signaling circuit in vitro using a genetically tractable organoid coculture model. Collectively, these studies exploited a genetic perturbation associated with human disease to disrupt a fundamental biological process and then reconstructed a spatially resolved mechanistic model of tissue healing.
  23. Development. 2023 Oct 27. pii: dev.201709. [Epub ahead of print]
      During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate of over 1800 genes and grow 25-fold. Previous work showed that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-hour window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 hours earlier than in wild-type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.
    Keywords:  Cyclin B; Drosophila; Meiosis; RNA; Spermatogenesis; Translation
  24. Development. 2023 Oct 26. pii: dev.201724. [Epub ahead of print]
      Sperm flagellum plays a critical role in male fertility. Here, we generated Ccdc183 knockout (KO) mice using the CRISPR/Cas9 system to reveal the protein function of CCDC183 in spermiogenesis. We demonstrated that the absence of CCDC183 causes male infertility with morphological and motility defects in spermatozoa. Due to the lack of CCDC183, centrioles after elongation of axonemal microtubules do not connect the cell surface and nucleus during spermiogenesis, which causes subsequent loss of cytoplasmic invagination around the flagellum. As a result, the flagellar compartment does not form properly and cytosol-exposed axonemal microtubules collapse during spermiogenesis. In addition, ectopic localization of accessory structures such as the fibrous sheath and outer dense fibers, and abnormal head shape due to abnormal sculpting by the manchette are observed in Ccdc183 KO spermatids. Our results indicate that CCDC183 plays an essential role in cytoplasmic invagination around the flagellum to form functional spermatozoa during spermiogenesis.
    Keywords:  Cytoplasmic invagination; Flagellar formation; Male infertility; Sperm centrioles; Spermiogenesis
  25. bioRxiv. 2023 Oct 10. pii: 2023.10.09.561585. [Epub ahead of print]
      A significant proportion of nascent proteins undergo polyubiquitination on ribosomes in mammalian cells, yet the fate of these proteins remains elusive. The ribosome-associated quality control (RQC) is a mechanism that mediates the ubiquitination of nascent chains on stalled ribosomes. In this study, we find that nascent proteins ubiquitinated on stalled ribosomes by the RQC ligase LTN1 are insufficient for proteasomal degradation. Our biochemical reconstitution studies reveal that ubiquitinated nascent chains are promptly deubiquitinated in the cytosol upon release from stalled ribosomes, as they are no longer associated with LTN1 E3 ligase for continuous ubiquitination to compete with cytosolic deubiquitinases. These deubiquitinated nascent chains can mature into stable proteins. However, if they misfold and expose a degradation signal, cytosolic quality control recognizes them for re-ubiquitination and subsequent proteasomal degradation. Thus, our findings suggest that cycles of ubiquitination and deubiquitination spare foldable nascent proteins while ensuring the degradation of terminally misfolded proteins.
  26. bioRxiv. 2023 Oct 13. pii: 2023.10.10.561736. [Epub ahead of print]
      Endocycling cells grow and repeatedly duplicate their genome without dividing. Cells switch from mitotic cycles to endocycles in response to developmental signals during the growth of specific tissues in a wide range of organisms. The purpose of switching to endocycles, however, remains unclear in many tissues. Additionally, cells can switch to endocycles in response to conditional signals, which can have beneficial or pathological effects on tissues. However, the impact of these unscheduled endocycles on development is underexplored. Here, we use Drosophila ovarian somatic follicle cells as a model to examine the impact of unscheduled endocycles on tissue growth and function. Follicle cells normally switch to endocycles at mid-oogenesis. Inducing follicle cells to prematurely switch to endocycles resulted in lethality of the resulting embryos. Analysis of ovaries with premature follicle cell endocycles revealed aberrant follicular epithelial structure and pleiotropic defects in oocyte growth, developmental gene amplification, and the migration of a special set of follicle cells known as border cells. Overall, these findings reveal how unscheduled endocycles can disrupt tissue growth and function to cause aberrant development.
    Keywords:  Drosophila; border cells; endocycling; epithelium; oogenesis; polyploidy
  27. EMBO Rep. 2023 Oct 27. e57234
      53BP1 acts at the crossroads between DNA repair and p53-mediated stress response. With its interactors p53 and USP28, it is part of the mitotic surveillance (or mitotic stopwatch) pathway (MSP), a sensor that monitors the duration of cell division, promoting p53-dependent cell cycle arrest when a critical time threshold is surpassed. Here, we show that Polo-like kinase 1 (PLK1) activity is essential for the time-dependent release of 53BP1 from kinetochores. PLK1 inhibition, which leads to 53BP1 persistence at kinetochores, prevents cytosolic 53BP1 association with p53 and results in a blunted MSP. Strikingly, the identification of CENP-F as the kinetochore docking partner of 53BP1 enabled us to show that measurement of mitotic timing by the MSP does not take place at kinetochores, as perturbing CENP-F-53BP1 binding had no measurable impact on the MSP. Taken together, we propose that PLK1 supports the MSP by generating a cytosolic pool of 53BP1 and that an unknown cytosolic mechanism enables the measurement of mitotic duration.
    Keywords:  53BP1; PLK1; kinetochore; mitotic stopwatch pathway; mitotic surveillance pathway
  28. Am J Reprod Immunol. 2023 Nov;90(5): e13793
      RESEARCH QUESTION: Decidualization is critical to the establishment of mouse normal pregnancy. The fibroblast-like stromal cells in the process form polyploid multinucleated cells. Aurora kinase B (Aurora B) has previously been shown to regulate polyploidy in various cells. However, whether Aurora B regulates the formation of decidual cell polyploidization and its regulatory mechanisms remain poorly understood.DESIGN: Establish decidualization model of mouse primary endometrial stromal cells in vitro. Construct pseudopregnancy mouse models and delayed-activation mouse models. Detect Aurora B and polyploidization related genes in mouse uteri treated by Aurora B specific inhibitor Barasertib and CPT.
    RESULTS: In this study, we found that Aurora B was strongly expressed in endometrial stromal cells after implantation. Additionally, Aurora B was remarkably up regulated in the stromal cells of oil-induced deciduomoa and in vitro decidualization. As an Aurora B specific inhibitor, Barasertib significantly inhibits the mRNA expression of Prl8a2, a marker of mouse decidualization. Furthermore, the protein levels of p-Plk1, Survivin and p-Cdk1 were inhibited by Barasertib. CPT-induced DNA damage suppressed Aurkb (encodes Aurora B) expression, thus resulting in polyploidization.
    CONCLUSION: Our data shows that Aurora B is expressed in decidual stromal cells of implantation sites and plays a key role for mouse decidualization. The protein of Plk1, Survivn, and Cdk1 may participate in formation of decidual cell polyploidization during mouse decidualization.
    Keywords:  Aurora B; decidualization; polyploidization; uterus
  29. Proc Natl Acad Sci U S A. 2023 Oct 31. 120(44): e2302879120
      Cytoplasmic flows are widely emerging as key functional players in development. In early Drosophila embryos, flows drive the spreading of nuclei across the embryo. Here, we combine hydrodynamic modeling with quantitative imaging to develop a two-fluid model that features an active actomyosin gel and a passive viscous cytosol. Gel contractility is controlled by the cell cycle oscillator, the two fluids being coupled by friction. In addition to recapitulating experimental flow patterns, our model explains observations that remained elusive and makes a series of predictions. First, the model captures the vorticity of cytosolic flows, which highlights deviations from Stokes' flow that were observed experimentally but remained unexplained. Second, the model reveals strong differences in the gel and cytosol motion. In particular, a micron-sized boundary layer is predicted close to the cortex, where the gel slides tangentially while the cytosolic flow cannot slip. Third, the model unveils a mechanism that stabilizes the spreading of nuclei with respect to perturbations of their initial positions. This self-correcting mechanism is argued to be functionally important for proper nuclear spreading. Fourth, we use our model to analyze the effects of flows on the transport of the morphogen Bicoid and the establishment of its gradients. Finally, the model predicts that the flow strength should be reduced if the shape of the domain is more round, which is experimentally confirmed in Drosophila mutants. Thus, our two-fluid model explains flows and nuclear positioning in early Drosophila, while making predictions that suggest novel future experiments.
    Keywords:  Drosophila embryogenesis; actomyosin dynamics; biological fluid dynamics; cytoplasmic flows; multiphase flows
  30. Cell Discov. 2023 Oct 24. 9(1): 105
      Aneuploidy seriously compromises female fertility and increases incidence of birth defects. Rates of aneuploidy in human eggs from even young women are significantly higher than those in other mammals. However, intrinsic genetic factors underlying this high incidence of aneuploidy in human eggs remain largely unknown. Here, we found that ectopic expression of human TUBB8 in mouse oocytes increases rates of aneuploidy by causing kinetochore-microtubule (K-MT) attachment defects. Stretched bivalents in mouse oocytes expressing TUBB8 are under less tension, resulting in continuous phosphorylation status of HEC1 by AURKB/C at late metaphase I that impairs the established correct K-MT attachments. This reduced tension in stretched bivalents likely correlates with decreased recruitment of KIF11 on meiotic spindles. We also found that ectopic expression of TUBB8 without its C-terminal tail decreases aneuploidy rates by reducing erroneous K-MT attachments. Importantly, variants in the C-terminal tail of TUBB8 were identified in patients with recurrent miscarriages. Ectopic expression of an identified TUBB8 variant in mouse oocytes also compromises K-MT attachments and increases aneuploidy rates. In conclusion, our study provides novel understanding for physiological mechanisms of aneuploidy in human eggs as well as for pathophysiological mechanisms involved in recurrent miscarriages.
  31. J Am Chem Soc. 2023 Oct 23.
      In the nucleus, transcriptionally silent genes are sequestered into heterochromatin compartments comprising nucleosomes decorated with histone H3 Lys9 trimethylation and a protein called HP1α. This protein can form liquid-liquid droplets in vitro and potentially organize heterochromatin through a phase separation mechanism that is promoted by phosphorylation. Elucidating the molecular interactions that drive HP1α phase separation and its consequences on nucleosome structure and dynamics has been challenging due to the viscous and heterogeneous nature of such assemblies. Here, we tackle this problem by a combination of solution and solid-state NMR spectroscopy, which allows us to dissect the interactions of phosphorylated HP1α with nucleosomes in the context of phase separation. Our experiments indicate that phosphorylated human HP1α does not cause any major rearrangements to the nucleosome core, in contrast to the yeast homologue Swi6. Instead, HP1α interacts specifically with the methylated H3 tails and slows the dynamics of the H4 tails. Our results shed light on how phosphorylated HP1α proteins may regulate the heterochromatin landscape, while our approach provides an atomic resolution view of a heterogeneous and dynamic biological system regulated by a complex network of interactions and post-translational modifications.
  32. Metabolites. 2023 Oct 17. pii: 1086. [Epub ahead of print]13(10):
      Growing evidence indicates that metabolites and energy metabolism play an active rather than consequential role in regulating cellular fate. Cardiac development requires dramatic metabolic remodeling from relying primarily on glycolysis in pluripotent stem cells (PSCs) to oxidizing a wide array of energy substrates to match the high bioenergetic demands of continuous contraction in the developed heart. However, a detailed analysis of how remodeling of energy metabolism contributes to human cardiac development is lacking. Using dynamic multiple reaction monitoring metabolomics of central carbon metabolism, we evaluated temporal changes in energy metabolism during human PSC 3D cardiac lineage specification. Significant metabolic remodeling occurs during the complete differentiation, yet temporal analysis revealed that most changes occur during transitions from pluripotency to mesoderm (day 1) and mesoderm to early cardiac (day 5), with limited maturation of cardiac metabolism beyond day 5. Real-time metabolic analysis demonstrated that while hPSC cardiomyocytes (hPSC-CM) showed elevated rates of oxidative metabolism compared to PSCs, they still retained high glycolytic rates, confirming an immature metabolic phenotype. These observations support the opportunity to metabolically optimize the differentiation process to support lineage specification and maturation of hPSC-CMs.
    Keywords:  3D cardiomyocyte differentiation; glycolysis; induced pluripotent stem cells; metabolomics; oxidative metabolism