bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–07–02
24 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. An optogenetic-phosphoproteomic study reveals dynamic Akt1 signaling profiles in endothelial cells.
  2. Multiple Inositol Polyphosphate Phosphatase Compartmentalization Separates Inositol Phosphate Metabolism from Inositol Lipid Signaling.
  3. Reprogramming tumour-associated macrophages to outcompete cancer cells.
  4. A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on macropinosomes and phagosomes.
  5. Single-cell proteomics: quantifying post-transcriptional regulation during development with mass-spectrometry.
  6. Targeted treatment of severe vascular malformations harboring PIK3CA and TEK mutations with alpelisib is highly effective with limited toxicity.
  7. Cell-type specific defects in PTEN-mutant cortical organoids converge on abnormal circuit activity.
  8. MultiVI: deep generative model for the integration of multimodal data.
  9. Identification of XAF1 as an endogenous AKT inhibitor.
  10. mTORC2-NDRG1-CDC42 axis couples fasting to mitochondrial fission.
  11. Epigenetic Control of Cell Potency and Fate Determination during Mammalian Gastrulation.
  12. A biologist's guide to planning and performing quantitative bioimaging experiments.
  13. Exploring the potential of in vivo reprogramming for studying embryonic development, tissue regeneration, and organismal aging.
  14. High purity and integrity RNA from human cell lines stored in liquid nitrogen for over 20 years.
  15. Epidemiology of the disorders of the Pik3ca-related overgrowth spectrum (Pros).
  16. Spatially resolved single-cell translatomics at molecular resolution.
  17. Base editor screens for in situ mutational scanning at scale.
  18. MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq with Image-Based Phenotypic Screening in 2D and 3D Cell Models.
  19. Cell facilitation promotes growth and survival under drug pressure in breast cancer.
  20. Gene regulatory network inference in the era of single-cell multi-omics.
  21. DCATS: differential composition analysis for flexible single-cell experimental designs.
  22. A model of the post-implantation human embryo derived from pluripotent stem cells.
  23. Dynamic mapping of proteome trafficking within and between living cells by TransitID.
  24. Self-patterning of human stem cells into post-implantation lineages.
  1. Nat Commun. 2023 06 26. 14(1): 3803
    An optogenetic-phosphoproteomic study reveals dynamic Akt1 signaling profiles in endothelial cells.
    Wenping Zhou, Wenxue Li, Shisheng Wang, Barbora Salovska, Zhenyi Hu, Bo Tao, Yi Di, Ujwal Punyamurtula, Benjamin E Turk, William C Sessa, Yansheng Liu.
      The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.
    DOI:  https://doi.org/10.1038/s41467-023-39514-1
  2. Biomolecules. 2023 05 24. pii: 885. [Epub ahead of print]13(6):
    Multiple Inositol Polyphosphate Phosphatase Compartmentalization Separates Inositol Phosphate Metabolism from Inositol Lipid Signaling.
    Jia Yu, Barbara Leibiger, Shao-Nian Yang, Stephen B Shears, Ingo B Leibiger, Per-Olof Berggren, Christopher J Barker.
      Multiple inositol polyphosphate phosphatase (MINPP1) is an enigmatic enzyme that is responsible for the metabolism of inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6 pentakisphosphate (Ins(1,3,4,5,6)P5 in mammalian cells, despite being restricted to the confines of the ER. The reason for this compartmentalization is unclear. In our previous studies in the insulin-secreting HIT cell line, we expressed MINPP1 in the cytosol to artificially reduce the concentration of these higher inositol phosphates. Undocumented at the time, we noted cytosolic MINPP1 expression reduced cell growth. We were struck by the similarities in substrate preference between a number of different enzymes that are able to metabolize both inositol phosphates and lipids, notably IPMK and PTEN. MINPP1 was first characterized as a phosphatase that could remove the 3-phosphate from inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). This molecule shares strong structural homology with the major product of the growth-promoting Phosphatidyl 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PTEN can degrade both this lipid and Ins(1,3,4,5)P4. Because of this similar substrate preference, we postulated that the cytosolic version of MINPP1 (cyt-MINPP1) may not only attack inositol polyphosphates but also PtdIns(3,4,5)P3, a key signal in mitogenesis. Our experiments show that expression of cyt-MINPP1 in HIT cells lowers the concentration of PtdIns(3,4,5)P3. We conclude this reflects a direct effect of MINPP1 upon the lipid because cyt-MINPP1 actively dephosphorylates synthetic, di(C4:0)PtdIns(3,4,5)P3 in vitro. These data illustrate the importance of MINPP1's confinement to the ER whereby important aspects of inositol phosphate metabolism and inositol lipid signaling can be separately regulated and give one important clarification for MINPP1's ER seclusion.
    Keywords:  PIP3; inositol lipid; inositol phosphate; multiple inositol polyphosphate phosphatase (MINPP1); pancreatic beta cell; phosphatidylinositol 3,4,5-trisphosphate
    DOI:  https://doi.org/10.3390/biom13060885
  3. Nature. 2023 Jun 28.
    Reprogramming tumour-associated macrophages to outcompete cancer cells.
    Xian Zhang, Shun Li, Isha Malik, Mytrang H Do, Liangliang Ji, Chun Chou, Wei Shi, Kristelle J Capistrano, Jing Zhang, Ting-Wei Hsu, Briana G Nixon, Ke Xu, Xinxin Wang, Andrea Ballabio, Laura S Schmidt, W Marston Linehan, Ming O Li.
      In metazoan organisms, cell competition acts as a quality control mechanism to eliminate unfit cells in favour of their more robust neighbours1,2. This mechanism has the potential to be maladapted, promoting the selection of aggressive cancer cells3-6. Tumours are metabolically active and are populated by stroma cells7,8, but how environmental factors affect cancer cell competition remains largely unknown. Here we show that tumour-associated macrophages (TAMs) can be dietarily or genetically reprogrammed to outcompete MYC-overexpressing cancer cells. In a mouse model of breast cancer, MYC overexpression resulted in an mTORC1-dependent 'winner' cancer cell state. A low-protein diet inhibited mTORC1 signalling in cancer cells and reduced tumour growth, owing unexpectedly to activation of the transcription factors TFEB and TFE3 and mTORC1 in TAMs. Diet-derived cytosolic amino acids are sensed by Rag GTPases through the GTPase-activating proteins GATOR1 and FLCN to control Rag GTPase effectors including TFEB and TFE39-14. Depletion of GATOR1 in TAMs suppressed the activation of TFEB, TFE3 and mTORC1 under the low-protein diet condition, causing accelerated tumour growth; conversely, depletion of FLCN or Rag GTPases in TAMs activated TFEB, TFE3 and mTORC1 under the normal protein diet condition, causing decelerated tumour growth. Furthermore, mTORC1 hyperactivation in TAMs and cancer cells and their competitive fitness were dependent on the endolysosomal engulfment regulator PIKfyve. Thus, noncanonical engulfment-mediated Rag GTPase-independent mTORC1 signalling in TAMs controls competition between TAMs and cancer cells, which defines a novel innate immune tumour suppression pathway that could be targeted for cancer therapy.
    DOI:  https://doi.org/10.1038/s41586-023-06256-5
  4. J Cell Biol. 2023 Sep 04. pii: e202209077. [Epub ahead of print]222(9):
    A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on macropinosomes and phagosomes.
    James H Vines, Hannes Maib, Catherine M Buckley, Aurelie Gueho, Zhou Zhu, Thierry Soldati, David H Murray, Jason S King.
      Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.
    DOI:  https://doi.org/10.1083/jcb.202209077
  5. Development. 2023 Jul 01. pii: dev201492. [Epub ahead of print]150(13):
    Single-cell proteomics: quantifying post-transcriptional regulation during development with mass-spectrometry.
    Nikolai Slavov.
      Many developmental processes are regulated post-transcriptionally. Such post-transcriptional regulatory mechanisms can now be analyzed by robust single-cell mass spectrometry methods that allow accurate quantification of proteins and their modification in single cells. These methods can enable quantitative exploration of protein synthesis and degradation mechanisms that contribute to developmental cell fate specification. Furthermore, they may support functional analysis of protein conformations and activities in single cells, and thus link protein functions to developmental processes. This Spotlight provides an accessible introduction to single-cell mass spectrometry methods and suggests initial biological questions that are ripe for investigation.
    Keywords:  Post-transcriptional regulation; Protein degradation; Protein synthesis; Proteomics
    DOI:  https://doi.org/10.1242/dev.201492
  6. Sci Rep. 2023 06 28. 13(1): 10499
    Targeted treatment of severe vascular malformations harboring PIK3CA and TEK mutations with alpelisib is highly effective with limited toxicity.
    Martin Sterba, Petra Pokorna, Renata Faberova, Blanka Pinkova, Jarmila Skotakova, Anna Seehofnerova, Jan Blatny, Lucia Janigova, Olga Koskova, Hana Palova, Michal Mahdal, Lukas Pazourek, Petr Jabandziev, Ondrej Slaby, Peter Mudry, Jaroslav Sterba.
      This was a prospective cohort study of eighteen patients with large and debilitating vascular malformations with one or more major systemic complications. In all patients, we discovered activating alterations in either TEK or PIK3CA. Based on these findings, targeted treatment using the PI3K inhibitor alpelisib was started with regular check-ups, therapy duration varied from 6 to 31 months. In all patients, marked improvement in quality of life was observed. We observed radiological improvement in fourteen patients (two of them being on combination with either propranolol or sirolimus), stable disease in 2 patients. For 2 patients, an MRI scan was not available as they were shortly on treatment, however, a clinically visible response in size reduction or structure regression, together with pain relief was observed. In patients with elevated D-dimer levels before alpelisib administration, a major improvement was noted, suggesting its biomarker role. We observed overall very good tolerance of the treatment, documenting a single patient with grade 3 hyperglycemia. Patients with size reduction were offered local therapies wherever possible. Our report presents a promising approach for the treatment of VMs harboring different targetable TEK and PIK3CA gene mutations with a low toxicity profile and high efficacy.
    DOI:  https://doi.org/10.1038/s41598-023-37468-4
  7. Hum Mol Genet. 2023 Jun 29. pii: ddad107. [Epub ahead of print]
    Cell-type specific defects in PTEN-mutant cortical organoids converge on abnormal circuit activity.
    Martina Pigoni, Ana Uzquiano, Bruna Paulsen, Amanda Kedaigle, Sung Min Yang, Panagiotis Symvoulidis, Xian Adiconis, Silvia Velasco, Rafaela Sartore, Kwanho Kim, Ashley Tucewicz, Sarah Yoshimi Tropp, Kalliopi Tsafou, Xin Jin, Lindy Barrett, Fei Chen, Ed Boyden, Aviv Regev, Joshua Z Levin, Paola Arlotta.
      De novo heterozygous loss-of-function mutations in PTEN are strongly associated with Autism spectrum disorders (ASD); however, it is unclear how heterozygous mutations in this gene affects different cell types during human brain development, and how these effects vary across individuals. Here, we used human cortical organoids from different donors to identify cell-type-specific developmental events that are affected by heterozygous mutations in PTEN. We profiled individual organoids by single-cell RNA-seq, proteomics, and spatial transcriptomics, and revealed abnormalities in developmental timing in human outer radial glia progenitors and deep layer cortical projection neurons, which varied with the donor genetic background. Calcium imaging in intact organoids showed that both accelerated and delayed neuronal development phenotypes resulted in similar abnormal activity of local circuits, irrespective of genetic background. The work reveals donor-dependent, cell-type specific developmental phenotypes of PTEN heterozygosity that later converge on disrupted neuronal activity.
    DOI:  https://doi.org/10.1093/hmg/ddad107
  8. Nat Methods. 2023 Jun 29.
    MultiVI: deep generative model for the integration of multimodal data.
    Tal Ashuach, Mariano I Gabitto, Rohan V Koodli, Giuseppe-Antonio Saldi, Michael I Jordan, Nir Yosef.
      Jointly profiling the transcriptome, chromatin accessibility and other molecular properties of single cells offers a powerful way to study cellular diversity. Here we present MultiVI, a probabilistic model to analyze such multiomic data and leverage it to enhance single-modality datasets. MultiVI creates a joint representation that allows an analysis of all modalities included in the multiomic input data, even for cells for which one or more modalities are missing. It is available at scvi-tools.org .
    DOI:  https://doi.org/10.1038/s41592-023-01909-9
  9. Cell Rep. 2023 Jun 27. pii: S2211-1247(23)00701-5. [Epub ahead of print]42(7): 112690
    Identification of XAF1 as an endogenous AKT inhibitor.
    Min Chen, Kangjunjie Wang, Ying Han, Shukun Yan, Huairui Yuan, Qiuli Liu, Long Li, Ni Li, Hongwen Zhu, Dayun Lu, Kaihua Wang, Fen Liu, Dakui Luo, Yuxue Zhang, Jun Jiang, Dali Li, Lei Zhang, Hongbin Ji, Hu Zhou, Yong Chen, Jun Qin, Daming Gao.
      AKT kinase is a key regulator in cell metabolism and survival, and its activation is strictly modulated. Herein, we identify XAF1 (XIAP-associated factor) as a direct interacting protein of AKT1, which strongly binds the N-terminal region of AKT1 to block its K63-linked poly-ubiquitination and subsequent activation. Consistently, Xaf1 knockout causes AKT activation in mouse muscle and fat tissues and reduces body weight gain and insulin resistance induced by high-fat diet. Pathologically, XAF1 expression is low and anti-correlated with the phosphorylated p-T308-AKT signal in prostate cancer samples, and Xaf1 knockout stimulates the p-T308-AKT signal to accelerate spontaneous prostate tumorigenesis in mice with Pten heterozygous loss. And ectopic expression of wild-type XAF1, but not the cancer-derived P277L mutant, inhibits orthotopic tumorigenesis. We further identify Forkhead box O 1 (FOXO1) as a transcriptional regulator of XAF1, thus forming a negative feedback loop between AKT1 and XAF1. These results reveal an important intrinsic regulatory mechanism of AKT signaling.
    Keywords:  AKT; CP: Cell biology; FOXO1; XAF1; metabolism; phosphorylation; prostate cancer; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2023.112690
  10. Nat Cell Biol. 2023 Jun 29.
    mTORC2-NDRG1-CDC42 axis couples fasting to mitochondrial fission.
    Nuria Martinez-Lopez, Pamela Mattar, Miriam Toledo, Henrietta Bains, Manu Kalyani, Marie Louise Aoun, Mridul Sharma, Laura Beth J McIntire, Leslie Gunther-Cummins, Frank P Macaluso, Jennifer T Aguilan, Simone Sidoli, Mathieu Bourdenx, Rajat Singh.
      Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, RictorKO, NDRG1Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.
    DOI:  https://doi.org/10.1038/s41556-023-01163-3
  11. Genes (Basel). 2023 May 25. pii: 1143. [Epub ahead of print]14(6):
    Epigenetic Control of Cell Potency and Fate Determination during Mammalian Gastrulation.
    Adrienne E Sullivan.
      Pluripotent embryonic stem cells have a unique and characteristic epigenetic profile, which is critical for differentiation to all embryonic germ lineages. When stem cells exit the pluripotent state and commit to lineage-specific identities during the process of gastrulation in early embryogenesis, extensive epigenetic remodelling mediates both the switch in cellular programme and the loss of potential to adopt alternative lineage programmes. However, it remains to be understood how the stem cell epigenetic profile encodes pluripotency, or how dynamic epigenetic regulation helps to direct cell fate specification. Recent advances in stem cell culture techniques, cellular reprogramming, and single-cell technologies that can quantitatively profile epigenetic marks have led to significant insights into these questions, which are important for understanding both embryonic development and cell fate engineering. This review provides an overview of key concepts and highlights exciting new advances in the field.
    Keywords:  embryonic development; embryonic stem cells; epigenetic remodelling; gene regulation; lineage specification
    DOI:  https://doi.org/10.3390/genes14061143
  12. PLoS Biol. 2023 06;21(6): e3002167
    A biologist's guide to planning and performing quantitative bioimaging experiments.
    Rebecca A Senft, Barbara Diaz-Rohrer, Pina Colarusso, Lucy Swift, Nasim Jamali, Helena Jambor, Thomas Pengo, Craig Brideau, Paula Montero Llopis, Virginie Uhlmann, Jason Kirk, Kevin Andrew Gonzales, Peter Bankhead, Edward L Evans, Kevin W Eliceiri, Beth A Cimini.
      Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently.
    DOI:  https://doi.org/10.1371/journal.pbio.3002167
  13. Curr Opin Genet Dev. 2023 Jun 23. pii: S0959-437X(23)00047-3. [Epub ahead of print]81 102067
    Exploring the potential of in vivo reprogramming for studying embryonic development, tissue regeneration, and organismal aging.
    Sho Ohta, Yasuhiro Yamada.
      Forced expression of a specific set of transcription factors can reprogram terminally differentiated cells and convert them into induced pluripotent stem cells that correspond to cells in the inner cell mass of the developing embryo. It is now recognized that the scope of the reprogramming factors extends far beyond the stem cell biology. Studies using mouse models demonstrated that the induction of the reprogramming factors promotes cellular reprogramming in vivo. Closer inspection of these mice has revealed that expression of the reprogramming factors results in unique consequences that are not seen when cells are reprogrammed ex vivo, and can provide insights into development, tissue regeneration, cancer, and aging.
    DOI:  https://doi.org/10.1016/j.gde.2023.102067
  14. Drug Dev Res. 2023 Jun 29.
    High purity and integrity RNA from human cell lines stored in liquid nitrogen for over 20 years.
    Irena Voinsky, Elad Shapira, David Gurwitz.
      Biobanks are a key resource for obtaining human cell lines for biomedical research, including for drug development projects. Such projects often include comparative RNA-sequencing of large panels of human cell lines from individuals affected by certain disorders and healthy controls, or from individuals with different drug response phenotypes. RNA extractions are typically done from growing cell cultures, a process that may take several weeks. However, maintaining large numbers of cell lines in parallel increases the project workload. Here, we show that extracting RNAs directly from frozen vials of human cell lines stored for over 20 years in a liquid nitrogen freezer yields RNAs with the high purity and integrity parameters that conform to those required for optimal RNA-sequencing and are closely similar to those obtained for RNAs extracted from growing human cell lines.
    Keywords:  RNA integrity number (RIN); RNA-sequencing (RNA-seq); lymphoblastoid cell lines (LCLs)
    DOI:  https://doi.org/10.1002/ddr.22090
  15. Eur J Hum Genet. 2023 Jun 26.
    Epidemiology of the disorders of the Pik3ca-related overgrowth spectrum (Pros).
    Giuseppe Reynolds, Simona Cardaropoli, Diana Carli, Maria Luca, Andrea Gazzin, Paola Coppo, Roberta La Selva, Marilidia Piglionica, Rosanna Bagnulo, Antonella Turchiano, Carlotta Ranieri, Nicoletta Resta, Alessandro Mussa.
      PIK3CA pathogenic variants are responsible for a group of overgrowth syndromes, collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). These gain-of-function variants arise postzygotically, and, according to time of onset, kind of embryonal tissue affected and regional body extension, give rise to heterogeneous phenotypes. PROS rarity and heterogeneity hamper the correct estimation of its epidemiology. Our work represents the first attempt to define the prevalence of PROS according to the established diagnostic criteria and molecular analysis and based on solid demographic data. We assessed the prevalence in Piedmont Region (Italy), including in the study all participants diagnosed with PROS born there from 1998 to 2021. The search identified 37 cases of PROS born across the 25-year period, providing a prevalence of 1:22,313 live births. Molecular analysis was positive in 81.0% of participants. Taking into account the cases with a detected variant in PIK3CA (n = 30), prevalence of molecularly positive PROS was 1:27,519.
    DOI:  https://doi.org/10.1038/s41431-023-01414-9
  16. Science. 2023 Jun 30. 380(6652): eadd3067
    Spatially resolved single-cell translatomics at molecular resolution.
    Hu Zeng, Jiahao Huang, Jingyi Ren, Connie Kangni Wang, Zefang Tang, Haowen Zhou, Yiming Zhou, Hailing Shi, Abhishek Aditham, Xin Sui, Hongyu Chen, Jennifer A Lo, Xiao Wang.
      The precise control of messenger RNA (mRNA) translation is a crucial step in posttranscriptional gene regulation of cellular physiology. However, it remains a challenge to systematically study mRNA translation at the transcriptomic scale with spatial and single-cell resolution. Here, we report the development of ribosome-bound mRNA mapping (RIBOmap), a highly multiplexed three-dimensional in situ profiling method to detect cellular translatome. RIBOmap profiling of 981 genes in HeLa cells revealed cell cycle-dependent translational control and colocalized translation of functional gene modules. We mapped 5413 genes in mouse brain tissues, yielding spatially resolved single-cell translatomic profiles for 119,173 cells and revealing cell type-specific and brain region-specific translational regulation, including translation remodeling during oligodendrocyte maturation. Our method detected widespread patterns of localized translation in neuronal and glial cells in intact brain tissue networks.
    DOI:  https://doi.org/10.1126/science.add3067
  17. Mol Cell. 2023 Jun 23. pii: S1097-2765(23)00431-8. [Epub ahead of print]
    Base editor screens for in situ mutational scanning at scale.
    Nicholas Z Lue, Brian B Liau.
      A fundamental challenge in biology is understanding the molecular details of protein function. How mutations alter protein activity, regulation, and response to drugs is of critical importance to human health. Recent years have seen the emergence of pooled base editor screens for in situ mutational scanning: the interrogation of protein sequence-function relationships by directly perturbing endogenous proteins in live cells. These studies have revealed the effects of disease-associated mutations, discovered novel drug resistance mechanisms, and generated biochemical insights into protein function. Here, we discuss how this "base editor scanning" approach has been applied to diverse biological questions, compare it with alternative techniques, and describe the emerging challenges that must be addressed to maximize its utility. Given its broad applicability toward profiling mutations across the proteome, base editor scanning promises to revolutionize the investigation of proteins in their native contexts.
    Keywords:  CRISPR; base editing; base editor screen; drug resistance; functional genomics; genome editing; mutational scanning; variant classification
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.009
  18. Methods Mol Biol. 2023 ;2691 279-325
    MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq with Image-Based Phenotypic Screening in 2D and 3D Cell Models.
    Xiang Mark Li, David Yoannidis, Susanne Ramm, Jennii Luu, Gisela Mir Arnau, Timothy Semple, Kaylene J Simpson.
      Transcriptomic profiling has fundamentally influenced our understanding of cancer pathophysiology and response to therapeutic intervention and has become a relatively routine approach. However, standard protocols are usually low-throughput, single-plex assays and costs are still quite prohibitive. With the evolving complexity of in vitro cell model systems, there is a need for resource-efficient high-throughput approaches that can support detailed time-course analytics, accommodate limited sample availability, and provide the capacity to correlate phenotype to genotype at scale. MAC-seq (multiplexed analysis of cells) is a low-cost, ultrahigh-throughput RNA-seq workflow in plate format to measure cell perturbations and is compatible with high-throughput imaging. Here we describe the steps to perform MAC-seq in 384-well format and apply it to 2D and 3D cell cultures. On average, our experimental conditions identified over ten thousand expressed genes per well when sequenced to a depth of one million reads. We discuss technical aspects, make suggestions on experimental design, and document critical operational procedures. Our protocol highlights the potential to couple MAC-seq with high-throughput screening applications including cell phenotyping using high-content cell imaging.
    Keywords:  3D cell models; High-content imaging; High-throughput screening; Multiplexing; RNA-seq
    DOI:  https://doi.org/10.1007/978-1-0716-3331-1_22
  19. Nat Commun. 2023 Jun 29. 14(1): 3851
    Cell facilitation promotes growth and survival under drug pressure in breast cancer.
    Rena Emond, Jason I Griffiths, Vince Kornél Grolmusz, Aritro Nath, Jinfeng Chen, Eric F Medina, Rachel S Sousa, Timothy Synold, Frederick R Adler, Andrea H Bild.
      The interplay of positive and negative interactions between drug-sensitive and resistant cells influences the effectiveness of treatment in heterogeneous cancer cell populations. Here, we study interactions between estrogen receptor-positive breast cancer cell lineages that are sensitive and resistant to ribociclib-induced cyclin-dependent kinase 4 and 6 (CDK4/6) inhibition. In mono- and coculture, we find that sensitive cells grow and compete more effectively in the absence of treatment. During treatment with ribociclib, sensitive cells survive and proliferate better when grown together with resistant cells than when grown in monoculture, termed facilitation in ecology. Molecular, protein, and genomic analyses show that resistant cells increase metabolism and production of estradiol, a highly active estrogen metabolite, and increase estrogen signaling in sensitive cells to promote facilitation in coculture. Adding estradiol in monoculture provides sensitive cells with increased resistance to therapy and cancels facilitation in coculture. Under partial inhibition of estrogen signaling through low-dose endocrine therapy, estradiol supplied by resistant cells facilitates sensitive cell growth. However, a more complete blockade of estrogen signaling, through higher-dose endocrine therapy, diminished the facilitative growth of sensitive cells. Mathematical modeling quantifies the strength of competition and facilitation during CDK4/6 inhibition and predicts that blocking facilitation has the potential to control both resistant and sensitive cancer cell populations and inhibit the emergence of a refractory population during cell cycle therapy.
    DOI:  https://doi.org/10.1038/s41467-023-39242-6
  20. Nat Rev Genet. 2023 Jun 26.
    Gene regulatory network inference in the era of single-cell multi-omics.
    Pau Badia-I-Mompel, Lorna Wessels, Sophia Müller-Dott, Rémi Trimbour, Ricardo O Ramirez Flores, Ricard Argelaguet, Julio Saez-Rodriguez.
      The interplay between chromatin, transcription factors and genes generates complex regulatory circuits that can be represented as gene regulatory networks (GRNs). The study of GRNs is useful to understand how cellular identity is established, maintained and disrupted in disease. GRNs can be inferred from experimental data - historically, bulk omics data - and/or from the literature. The advent of single-cell multi-omics technologies has led to the development of novel computational methods that leverage genomic, transcriptomic and chromatin accessibility information to infer GRNs at an unprecedented resolution. Here, we review the key principles of inferring GRNs that encompass transcription factor-gene interactions from transcriptomics and chromatin accessibility data. We focus on the comparison and classification of methods that use single-cell multimodal data. We highlight challenges in GRN inference, in particular with respect to benchmarking, and potential further developments using additional data modalities.
    DOI:  https://doi.org/10.1038/s41576-023-00618-5
  21. Genome Biol. 2023 Jun 26. 24(1): 151
    DCATS: differential composition analysis for flexible single-cell experimental designs.
    Xinyi Lin, Chuen Chau, Kun Ma, Yuanhua Huang, Joshua W K Ho.
      Differential composition analysis - the identification of cell types that have statistically significant changes in abundance between multiple experimental conditions - is one of the most common tasks in single cell omic data analysis. However, it remains challenging to perform differential composition analysis in the presence of flexible experimental designs and uncertainty in cell type assignment. Here, we introduce a statistical model and an open source R package, DCATS, for differential composition analysis based on a beta-binomial regression framework that addresses these challenges. Our empirical evaluation shows that DCATS consistently maintains high sensitivity and specificity compared to state-of-the-art methods.
    DOI:  https://doi.org/10.1186/s13059-023-02980-3
  22. Nature. 2023 Jun 27.
    A model of the post-implantation human embryo derived from pluripotent stem cells.
    Bailey A T Weatherbee, Carlos W Gantner, Lisa K Iwamoto-Stohl, Riza M Daza, Nobuhiko Hamazaki, Jay Shendure, Magdalena Zernicka-Goetz.
      The human embryo undergoes morphogenetic transformations following implantation into the uterus, yet our knowledge of this crucial stage is limited by the inability to observe the embryo in vivo. Stem cell-derived models of the embryo are important tools to interrogate developmental events and tissue-tissue crosstalk during these stages1. Here, we establish a model of the human post-implantation embryo, a human embryoid, comprised of embryonic and extraembryonic tissues. We combine two types of extraembryonic-like cells generated by transcription factor overexpression with wildtype embryonic stem cells and promote their self-organization into structures that mimic several aspects of the post-implantation human embryo. These self-organized aggregates contain a pluripotent epiblast-like domain surrounded by extraembryonic-like tissues. Our functional studies demonstrate that the epiblast-like domain robustly differentiates to amnion, extraembryonic mesenchyme, and primordial germ cell-like cells in response to BMP cues. In addition, we identify an inhibitory role for SOX17 in the specification of anterior hypoblast-like cells2. Modulation of the subpopulations in the hypoblast-like compartment demonstrated that extraembryonic-like cells impact epiblast-like domain differentiation, highlighting functional tissue-tissue crosstalk. In conclusion, we present a modular, tractable, integrated3 model of the human embryo that will allow us to probe key questions of human post-implantation development, a critical window when significant numbers of pregnancies fail.
    DOI:  https://doi.org/10.1038/s41586-023-06368-y
  23. Cell. 2023 Jun 23. pii: S0092-8674(23)00596-2. [Epub ahead of print]
    Dynamic mapping of proteome trafficking within and between living cells by TransitID.
    Wei Qin, Joleen S Cheah, Charles Xu, James Messing, Brian D Freibaum, Steven Boeynaems, J Paul Taylor, Namrata D Udeshi, Steven A Carr, Alice Y Ting.
      The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
    Keywords:  JUN; intercellular signaling; membraneless organelles; protein trafficking; proximity labeling; spatial proteomics; stress granules; tumor-associated macrophages
    DOI:  https://doi.org/10.1016/j.cell.2023.05.044
  24. Nature. 2023 Jun 27.
    Self-patterning of human stem cells into post-implantation lineages.
    Monique Pedroza, Seher Ipek Gassaloglu, Nicolas Dias, Liangwen Zhong, Tien-Chi Jason Hou, Helene Kretzmer, Zachary D Smith, Berna Sozen.
      Investigating human development is a significant scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here, we show that human pluripotent stem cells can be triggered to self-organise into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Importantly, our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators, and can undergo symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the peri-gastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie-stage16 (CS) 4 to CS7, offering a reproducible, tractable, and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.
    DOI:  https://doi.org/10.1038/s41586-023-06354-4
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