bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒11‒26
33 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

  1. bioRxiv. 2023 Nov 06. pii: 2023.11.06.565857. [Epub ahead of print]
      Extracellular signal-regulated kinase (ERK) signaling is essential to regulated cell behaviors, including cell proliferation, differentiation, and apoptosis. The influence of cell-cell contacts on ERK signaling is central to epithelial cells, yet few studies have sought to understand the same in cancer cells, particularly with single-cell resolution. To acquire both phenotypic (cell-contact state) and proteomic profile (ERK phosphorylation) on the same HeLa cells, we prepend high-content, whole-cell imaging prior to endpoint cellular-resolution western blot analyses for hundreds of cancer cells cultured on chip. By indexing the phosphorylation level of ERK in each cell or cell-contact cluster to the imaged cell-contact state, we compare ERK signaling between isolated and in-contact cells. We observe attenuated (∼2×) ERK signaling in HeLa cells which are in contact versus isolated. Attenuation is sustained when the HeLa cells are challenged with hyperosmotic stress. The contact-dependent differential ERK-phosphorylation corresponds to the differential EGFR distribution on cell surfaces, suggesting the involvement of EGFRs in contact-inhibited ERK signaling. Our findings show the impact of cell-cell contacts on ERK activation with isolated and in-contact cells, hence providing a new tool into control and scrutiny of cell-cell interactions.
  2. Life Sci Alliance. 2024 Feb;pii: e202302128. [Epub ahead of print]7(2):
      The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9-modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9-modified cells, which could impact cancer research.
  3. NPJ Genom Med. 2023 Nov 24. 8(1): 40
      Somatic PTEN mutations are common and have driver function in some cancer types. However, in colorectal cancers (CRCs), somatic PTEN-inactivating mutations occur at a low frequency (~8-9%), and whether these mutations are actively selected and promote tumor aggressiveness has been controversial. Analysis of genomic data from ~53,000 CRCs indicates that hotspot mutation patterns in PTEN partially reflect DNA-dependent selection pressures, but also suggests a strong selection pressure based on protein function. In microsatellite stable (MSS) tumors, PTEN alterations co-occur with mutations activating BRAF or PI3K, or with TP53 deletions, but not in CRC with microsatellite instability (MSI). Unexpectedly, PTEN deletions are associated with poor survival in MSS CRC, whereas PTEN mutations are associated with improved survival in MSI CRC. These and other data suggest use of PTEN as a prognostic marker is valid in CRC, but such use must consider driver mutation landscape, tumor subtype, and category of PTEN alteration.
  4. Mol Biol Cell. 2023 Nov 22. mbcE23030111
      Loss of cell polarity and disruption of tissue organization are key features of tumorigenesis that are intrinsically linked to spindle orientation. Epithelial tumors are often characterized by spindle orientation defects, but how these defects impact tumor formation driven by common oncogenic mutations is not fully understood. Here, we examine the role of spindle orientation in adult epidermis by deleting a key spindle regulator, LGN, in normal tissue and in a PTEN-deficient mouse model. We report that LGN deficiency in PTEN mutant epidermis leads to a 3-fold increase in the likelihood of developing tumors on the snout, and an over 10-fold increase in tumor burden. In this tissue, loss of LGN alone increases perpendicular and oblique divisions of epidermal basal cells, at the expense of a planar orientation of division. PTEN loss alone does not significantly affect spindle orientation in these cells, but the combined loss of PTEN and LGN fully randomizes basal spindle orientation. A subset of LGN- and PTEN-deficient animals have increased amounts of proliferative spinous cells, which may be associated with tumorigenesis. These results indicate that loss of LGN impacts spindle orientation and accelerates epidermal tumorigenesis in a PTEN-deficient mouse model.
  5. Biochim Biophys Acta Biomembr. 2023 Nov 20. pii: S0005-2736(23)00139-6. [Epub ahead of print] 184257
      Integrin α1β1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α1 integrin cytoplasmic tail directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α1β1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α1β1 integrins on the cell surface, In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α1β1 integrin activity and that the interaction of α1CT with phosphoinositides may contribute to this process.
    Keywords:  AKT; Cytoplasmic tail; FAK; PI(3,4,5)P(3); PI(4,5)P(2); α(1)β(1) integrin
  6. Elife. 2023 Nov 22. pii: e93161. [Epub ahead of print]12
      Biomedical single-cell atlases describe disease at the cellular level. However, analysis of this data commonly focuses on cell-type centric pairwise cross-condition comparisons, disregarding the multicellular nature of disease processes. Here we propose multicellular factor analysis for the unsupervised analysis of samples from cross-condition single-cell atlases and the identification of multicellular programs associated with disease. Our strategy, which repurposes group factor analysis as implemented in multi-omics factor analysis, incorporates the variation of patient samples across cell-types or other tissue-centric features, such as cell compositions or spatial relationships, and enables the joint analysis of multiple patient cohorts, facilitating the integration of atlases. We applied our framework to a collection of acute and chronic human heart failure atlases and described multicellular processes of cardiac remodeling, independent to cellular compositions and their local organization, that were conserved in independent spatial and bulk transcriptomics datasets. In sum, our framework serves as an exploratory tool for unsupervised analysis of cross-condition single-cell atlases and allows for the integration of the measurements of patient cohorts across distinct data modalities.
    Keywords:  computational biology; human; systems biology
  7. Nat Rev Mol Cell Biol. 2023 Nov 24.
      Every cell must satisfy basic requirements for nutrient sensing, utilization and recycling through macromolecular breakdown to coordinate programmes for growth, repair and stress adaptation. The lysosome orchestrates these key functions through the synchronised interplay between hydrolytic enzymes, nutrient transporters and signalling factors, which together enable metabolic coordination with other organelles and regulation of specific gene expression programmes. In this Review, we discuss recent findings on lysosome-dependent signalling pathways, focusing on how the lysosome senses nutrient availability through its physical and functional association with mechanistic target of rapamycin complex 1 (mTORC1) and how, in response, the microphthalmia/transcription factor E (MiT/TFE) transcription factors exert feedback regulation on lysosome biogenesis. We also highlight the emerging interactions of lysosomes with other organelles, which contribute to cellular homeostasis. Lastly, we discuss how lysosome dysfunction contributes to diverse disease pathologies and how inherited mutations that compromise lysosomal hydrolysis, transport or signalling components lead to multi-organ disorders with severe metabolic and neurological impact. A deeper comprehension of lysosomal composition and function, at both the cellular and organismal level, may uncover fundamental insights into human physiology and disease.
  8. J Am Chem Soc. 2023 Nov 22.
      The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargo into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CAE545K), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemically induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry, with significant implications for disease therapy.
  9. Nat Biomed Eng. 2023 Nov 23.
      The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically 'cloak' the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products.
  10. Nat Commun. 2023 11 18. 14(1): 7482
      Benchmarking single-cell RNA-seq (scRNA-seq) and single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) computational tools demands simulators to generate realistic sequencing reads. However, none of the few read simulators aim to mimic real data. To fill this gap, we introduce scReadSim, a single-cell RNA-seq and ATAC-seq read simulator that allows user-specified ground truths and generates synthetic sequencing reads (in a FASTQ or BAM file) by mimicking real data. At both read-sequence and read-count levels, scReadSim mimics real scRNA-seq and scATAC-seq data. Moreover, scReadSim provides ground truths, including unique molecular identifier (UMI) counts for scRNA-seq and open chromatin regions for scATAC-seq. In particular, scReadSim allows users to design cell-type-specific ground-truth open chromatin regions for scATAC-seq data generation. In benchmark applications of scReadSim, we show that UMI-tools achieves the top accuracy in scRNA-seq UMI deduplication, and HMMRATAC and MACS3 achieve the top performance in scATAC-seq peak calling.
  11. Nat Commun. 2023 Nov 21. 14(1): 7579
      Signaling bias is the ability of a receptor to differentially activate downstream signaling pathways in response to different ligands. Bias investigations have been hindered by inconsistent results in different cellular contexts. Here we introduce a methodology to identify and quantify bias in signal transduction across the plasma membrane without contributions from feedback loops and system bias. We apply the methodology to quantify phosphorylation efficiencies and determine absolute bias coefficients. We show that the signaling of epidermal growth factor receptor (EGFR) to EGF and TGFα is biased towards Y1068 and against Y1173 phosphorylation, but has no bias for epiregulin. We further show that the L834R mutation found in non-small-cell lung cancer induces signaling bias as it switches the preferences to Y1173 phosphorylation. The knowledge gained here challenges the current understanding of EGFR signaling in health and disease and opens avenues for the exploration of biased inhibitors as anti-cancer therapies.
  12. Mol Cell. 2023 Nov 14. pii: S1097-2765(23)00903-6. [Epub ahead of print]
      Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.
    Keywords:  2CLC; Integrator; MERVL; RNA catabolism; elongation; endogenous retrovirus; non-coding RNA; stem cell; totipotent-like cells; transcription-associated RNA degradation
  13. Cell Metab. 2023 Nov 11. pii: S1550-4131(23)00385-6. [Epub ahead of print]
      Mammalian target of rapamycin complex 1 (mTORC1) monitors cellular amino acid changes for function, but the molecular mediators of this process remain to be fully defined. Here, we report that depletion of cellular amino acids, either alone or in combination, leads to the ubiquitination of mTOR, which inhibits mTORC1 kinase activity by preventing substrate recruitment. Mechanistically, amino acid depletion causes accumulation of uncharged tRNAs, thereby stimulating GCN2 to phosphorylate FBXO22, which in turn accrues in the cytoplasm and ubiquitinates mTOR at Lys2066 in a K27-linked manner. Accordingly, mutation of mTOR Lys2066 abolished mTOR ubiquitination in response to amino acid depletion, rendering mTOR insensitive to amino acid starvation both in vitro and in vivo. Collectively, these data reveal a novel mechanism of amino acid sensing by mTORC1 via a previously unknown GCN2-FBXO22-mTOR pathway that is uniquely controlled by uncharged tRNAs.
    Keywords:  FBXO22; GCN2; amino acids; mTOR; mTORC1; ubiquitination; uncharged tRNA
  14. Cancer Discov. 2023 Nov 22. OF1
      Pantothetic acid is required for metabolic activity that supports MYC-driven breast tumor growth.
  15. Cell Rep Methods. 2023 Nov 14. pii: S2667-2375(23)00323-5. [Epub ahead of print] 100652
      Glycomics, the comprehensive profiling of all glycan structures in samples, is rapidly expanding to enable insights into physiology and disease mechanisms. However, glycan structure complexity and glycomics data interpretation present challenges, especially for differential expression analysis. Here, we present a framework for differential glycomics expression analysis. Our methodology encompasses specialized and domain-informed methods for data normalization and imputation, glycan motif extraction and quantification, differential expression analysis, motif enrichment analysis, time series analysis, and meta-analytic capabilities, synthesizing results across multiple studies. All methods are integrated into our open-source glycowork package, facilitating performant workflows and user-friendly access. We demonstrate these methods using dedicated simulations and glycomics datasets of N-, O-, lipid-linked, and free glycans. Differential expression tests here focus on human datasets and cancer vs. healthy tissue comparisons. Our rigorous approach allows for robust, reliable, and comprehensive differential expression analyses in glycomics, contributing to advancing glycomics research and its translation to clinical and diagnostic applications.
    Keywords:  CP: systems biology; bioinformatics; carbohydrate; computational biology; glycan; glycomics; statistics
  16. Cell Syst. 2023 Nov 20. pii: S2405-4712(23)00302-2. [Epub ahead of print]
      Hypoxia-induced upregulation of HIF1α triggers adipose tissue dysfunction and insulin resistance in obese patients. HIF1α closely interacts with PPARγ, the master regulator of adipocyte differentiation and lipid accumulation, but there are conflicting results regarding how this interaction controls the excessive lipid accumulation that drives adipocyte dysfunction. To directly address these conflicts, we established a differentiation system that recapitulated prior seemingly opposing observations made across different experimental settings. Using single-cell imaging and coarse-grained mathematical modeling, we show how HIF1α can both promote and repress lipid accumulation during adipogenesis. Our model predicted and our experiments confirmed that the opposing roles of HIF1α are isolated from each other by the positive-feedback-mediated upregulation of PPARγ that drives adipocyte differentiation. Finally, we identify three factors: strength of the differentiation cue, timing of hypoxic perturbation, and strength of HIF1α expression changes that, when considered together, provide an explanation for many of the previous conflicting reports.
    Keywords:  HIF1A; HIF1α; PPARG; PPARγ; adipocyte; adipose tissue; bistable switch; cell differentiation; hypoxia; temporal network isolation
  17. bioRxiv. 2023 Nov 10. pii: 2023.11.09.566272. [Epub ahead of print]
      There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. CRISPR's ease of use and flexibility, coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here we present a new optogenetic CRISPR tool, BLU-VIPR, that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. This simplifies spatiotemporal gene perturbation and works in vivo with cells previously intractable to optogenetic gene editing. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript, allowing for optogenetic gene editing in T lymphocytes in vivo.
  18. Proc Natl Acad Sci U S A. 2023 Nov 28. 120(48): e2314043120
      Hydrogen peroxide (H2O2) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H2O2 can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous "hotspots" of H2O2 generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H2O2 sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H2O2-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H2O2 availability highly specific to individual proteins and protein complexes. These findings suggest that intracellular H2O2 generation is much more localized and functionally differentiated than previously recognized.
    Keywords:  genetically encoded probes; hydrogen peroxide; redox regulation; redox signaling
  19. Nat Protoc. 2023 Nov 21.
      Non-negative matrix factorization (NMF) is an unsupervised learning method well suited to high-throughput biology. However, inferring biological processes from an NMF result still requires additional post hoc statistics and annotation for interpretation of learned features. Here, we introduce a suite of computational tools that implement NMF and provide methods for accurate and clear biological interpretation and analysis. A generalized discussion of NMF covering its benefits, limitations and open questions is followed by four procedures for the Bayesian NMF algorithm Coordinated Gene Activity across Pattern Subsets (CoGAPS). Each procedure will demonstrate NMF analysis to quantify cell state transitions in a public domain single-cell RNA-sequencing dataset. The first demonstrates PyCoGAPS, our new Python implementation that enhances runtime for large datasets, and the second allows its deployment in Docker. The third procedure steps through the same single-cell NMF analysis using our R CoGAPS interface. The fourth introduces a beginner-friendly CoGAPS platform using GenePattern Notebook, aimed at users with a working conceptual knowledge of data analysis but without a basic proficiency in the R or Python programming language. We also constructed a user-facing website to serve as a central repository for information and instructional materials about CoGAPS and its application programming interfaces. The expected timing to setup the packages and conduct a test run is around 15 min, and an additional 30 min to conduct analyses on a precomputed result. The expected runtime on the user's desired dataset can vary from hours to days depending on factors such as dataset size or input parameters.
  20. Trends Genet. 2023 Nov 20. pii: S0168-9525(23)00240-8. [Epub ahead of print]
      Programmable genome-engineering technologies, such as CRISPR (clustered regularly interspaced short palindromic repeats) nucleases and massively parallel CRISPR screens that capitalize on this programmability, have transformed biomedical science. These screens connect genes and noncoding genome elements to disease-relevant phenotypes, but until recently have been limited to individual phenotypes such as growth or fluorescent reporters of gene expression. By pairing massively parallel screens with high-dimensional profiling of single-cell types/states, we can now measure how individual genetic perturbations or combinations of perturbations impact the cellular transcriptome, proteome, and epigenome. We review technologies that pair CRISPR screens with single-cell multiomics and the unique opportunities afforded by extending pooled screens using deep multimodal phenotyping.
    Keywords:  CRISPR; functional genomics; genome engineering; guide RNA capture; multimodal sequencing; multiomics; single-cell sequencing
  21. Commun Biol. 2023 Nov 24. 6(1): 1192
      The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.
  22. Sci Adv. 2023 11 24. 9(47): eadi1867
      Adaptation to low levels of oxygen (hypoxia) is a universal biological feature across metazoans. However, the unique mechanisms how different species sense oxygen deprivation remain unresolved. Here, we functionally characterize a novel long noncoding RNA (lncRNA), LOC105369301, which we termed hypoxia-induced lncRNA for polo-like kinase 1 (PLK1) stabilization (HILPS). HILPS exhibits appreciable basal expression exclusively in a wide variety of human normal and cancer cells and is robustly induced by hypoxia-inducible factor 1α (HIF1α). HILPS binds to PLK1 and sequesters it from proteasomal degradation. Stabilized PLK1 directly phosphorylates HIF1α and enhances its stability, constituting a positive feed-forward circuit that reinforces oxygen sensing by HIF1α. HILPS depletion triggers catastrophic adaptation defect during hypoxia in both normal and cancer cells. These findings introduce a mechanism that underlies the HIF1α identity deeply interconnected with PLK1 integrity and identify the HILPS-PLK1-HIF1α pathway as a unique oxygen-sensing axis in the regulation of human physiological and pathogenic processes.
  23. Nat Protoc. 2023 Nov 23.
      Somatic mutations are the cause of cancer and have been implicated in other, noncancerous diseases and aging. While clonally expanded mutations can be studied by deep sequencing of bulk DNA, very few somatic mutations expand clonally, and most are unique to each cell. We describe a detailed protocol for single-cell whole-genome sequencing to discover and analyze somatic mutations in tissues and organs. The protocol comprises single-cell multiple displacement amplification (SCMDA), which ensures efficiency and high fidelity in amplification, and the SCcaller software tool to call single-nucleotide variations and small insertions and deletions from the sequencing data by filtering out amplification artifacts. With SCMDA and SCcaller at its core, this protocol describes a complete procedure for the comprehensive analysis of somatic mutations in a single cell, covering (1) single-cell or nucleus isolation, (2) single-cell or nucleus whole-genome amplification, (3) library preparation and sequencing, and (4) computational analyses, including alignment, variant calling, and mutation burden estimation. Methods are also provided for mutation annotation, hotspot discovery and signature analysis. The protocol takes 12-15 h from single-cell isolation to library preparation and 3-7 d of data processing. Compared with other single-cell amplification methods or single-molecular sequencing, it provides high genomic coverage, high accuracy in single-nucleotide variation and small insertions and deletion calling from the same single-cell genome, and fewer processing steps. SCMDA and SCcaller require basic experience in molecular biology and bioinformatics. The protocol can be utilized for studying mutagenesis and genome mosaicism in normal and diseased human and animal tissues under various conditions.
  24. FEBS J. 2023 Nov 20.
      Cellular senescence refers to a permanent and stable state of cell cycle exit. This process plays an important role in many cellular functions, including tumor suppression. It was first noted that senescence is associated with increased cell size in the early 1960s; however, how this contributes to permanent cell cycle exit was poorly understood until recently. In this review, we discuss new findings that identify increased cell size as not only a consequence but also a cause of permanent cell cycle exit. We highlight recent insights into how increased cell size alters normal cellular physiology and creates homeostatic imbalances that contribute to senescence induction. Finally, we focus on the potential clinical implications of these findings in the context of cell cycle arrest-causing cancer therapeutics and speculate on how tumor cell size changes may impact outcomes in patients treated with these drugs.
    Keywords:  cell cycle; cell size; senescence
  25. Nat Commun. 2023 Nov 24. 14(1): 7711
      The profiling of multiple molecular layers from the same set of cells has recently become possible. There is thus a growing need for multi-view learning methods able to jointly analyze these data. We here present Multi-Omics Wasserstein inteGrative anaLysIs (Mowgli), a novel method for the integration of paired multi-omics data with any type and number of omics. Of note, Mowgli combines integrative Nonnegative Matrix Factorization and Optimal Transport, enhancing at the same time the clustering performance and interpretability of integrative Nonnegative Matrix Factorization. We apply Mowgli to multiple paired single-cell multi-omics data profiled with 10X Multiome, CITE-seq, and TEA-seq. Our in-depth benchmark demonstrates that Mowgli's performance is competitive with the state-of-the-art in cell clustering and superior to the state-of-the-art once considering biological interpretability. Mowgli is implemented as a Python package seamlessly integrated within the scverse ecosystem and it is available at .
  26. Life Sci Alliance. 2024 Feb;pii: e202302147. [Epub ahead of print]7(2):
      Mitochondria are essential organelles whose dysfunction causes human pathologies that often manifest in a tissue-specific manner. Accordingly, mitochondrial fitness depends on versatile proteomes specialized to meet diverse tissue-specific requirements. Increasing evidence suggests that phosphorylation may play an important role in regulating tissue-specific mitochondrial functions and pathophysiology. Building on recent advances in mass spectrometry (MS)-based proteomics, we here quantitatively profile mitochondrial tissue proteomes along with their matching phosphoproteomes. We isolated mitochondria from mouse heart, skeletal muscle, brown adipose tissue, kidney, liver, brain, and spleen by differential centrifugation followed by separation on Percoll gradients and performed high-resolution MS analysis of the proteomes and phosphoproteomes. This in-depth map substantially quantifies known and predicted mitochondrial proteins and provides a resource of core and tissue-specific mitochondrial proteins ( Predicting kinase substrate associations for different mitochondrial compartments indicates tissue-specific regulation at the phosphoproteome level. Illustrating the functional value of our resource, we reproduce mitochondrial phosphorylation events on dynamin-related protein 1 responsible for its mitochondrial recruitment and fission initiation and describe phosphorylation clusters on MIGA2 linked to mitochondrial fusion.
  27. Curr Biol. 2023 Nov 20. pii: S0960-9822(23)01408-2. [Epub ahead of print]33(22): R1205-R1207
      A new study shows that cell size, in conjunction with specific signaling pathways, controls apoptosis within developing tissues. Cells with smaller sizes and relatively smaller sizes compared to their neighbors exhibit an increased likelihood of undergoing apoptosis. These processes are regulated by the Hippo/YAP and Notch pathways, respectively.
  28. Nat Protoc. 2023 Nov 23.
      Most patients with advanced malignancies are treated with severely toxic, first-line chemotherapies. Personalized treatment strategies have led to improved patient outcomes and could replace one-size-fits-all therapies, yet they need to be tailored by testing of a range of targeted drugs in primary patient cells. Most functional precision medicine studies use simple drug-response metrics, which cannot quantify the selective effects of drugs (i.e., the differential responses of cancer cells and normal cells). We developed a computational method for selective drug-sensitivity scoring (DSS), which enables normalization of the individual patient's responses against normal cell responses. The selective response scoring uses the inhibition of noncancerous cells as a proxy for potential drug toxicity, which can in turn be used to identify effective and safer treatment options. Here, we explain how to apply the selective DSS calculation for guiding precision medicine in patients with leukemia treated across three cancer centers in Europe and the USA; the generic methods are also widely applicable to other malignancies that are amenable to drug testing. The open-source and extendable R-codes provide a robust means to tailor personalized treatment strategies on the basis of increasingly available ex vivo drug-testing data from patients in real-world and clinical trial settings. We also make available drug-response profiles to 527 anticancer compounds tested in 10 healthy bone marrow samples as reference data for selective scoring and de-prioritization of drugs that show broadly toxic effects. The procedure takes <60 min and requires basic skills in R.
  29. Sci Adv. 2023 Nov 24. 9(47): eadg7488
      BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.
  30. Sci Transl Med. 2023 Nov 22. 15(723): eade8460
      Despite their high degree of effectiveness in the management of psychiatric conditions, exposure to antipsychotic drugs, including olanzapine and risperidone, is frequently associated with substantial weight gain and the development of diabetes. Even before weight gain, a rapid rise in circulating leptin concentrations can be observed in most patients taking antipsychotic drugs. To date, the contribution of this hyperleptinemia to weight gain and metabolic deterioration has not been defined. Here, with an established mouse model that recapitulates antipsychotic drug-induced obesity and insulin resistance, we not only confirm that hyperleptinemia occurs before weight gain but also demonstrate that hyperleptinemia contributes directly to the development of obesity and associated metabolic disorders. By suppressing the rise in leptin through the use of a monoclonal leptin-neutralizing antibody, we effectively prevented weight gain, restored glucose tolerance, and preserved adipose tissue and liver function in antipsychotic drug-treated mice. Mechanistically, suppressing excess leptin resolved local tissue and systemic inflammation typically associated with antipsychotic drug treatment. We conclude that hyperleptinemia is a key contributor to antipsychotic drug-associated weight gain and metabolic deterioration. Leptin suppression may be an effective approach to reducing the undesirable side effects of antipsychotic drugs.