bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–07–30
twenty-one papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Alkaline intracellular pH (pHi) increases PI3K kinase activity to promote mTORC1 and mTORC2 signaling and function during growth factor limitation.
  2. Fluctuations of formin binding in the generation of membrane patterns.
  3. Periodicity, mixed-mode oscillations, and multiple timescales in a phosphoinositide-Rho GTPase network.
  4. Social memory in female mice is rapidly modulated by 17β-estradiol through ERK and Akt modulation of synapse formation.
  5. CBF-beta mitigates PI3K-alpha-specific inhibitor killing through PIM1 in PIK3CA mutant gastric cancer.
  6. FilGAP, a GAP for Rac1, down-regulates invadopodia formation in breast cancer cells.
  7. A Role of PI3K/Akt Signaling in Oocyte Maturation and Early Embryo Development.
  8. mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching.
  9. Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement.
  10. Evolutionarily divergent mTOR remodels translatome for tissue regeneration.
  11. Proteome-wide copy-number estimation from transcriptomics.
  12. ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs.
  13. Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.
  14. FISH-Flow to quantify nascent and mature ribosomal RNA in mouse and human cells.
  15. Improved approaches to channel capacity estimation discover compromised GPCR signaling in diverse cancer cells.
  16. FGFR-mediated ERK1/2 signaling contributes to mesendoderm and definitive endoderm formation in vitro.
  17. Simultaneous multi-site editing of individual genomes using retron arrays.
  18. Segmentation metric misinterpretations in bioimage analysis.
  19. Digging deeper into the early steps of cancer progression.
  20. Two-Armed Molecule Flips Switch on Gene Regulation.
  21. Premature aging and reduced cancer incidence associated with near-complete body-wide Myc inactivation.
  1. J Biol Chem. 2023 Jul 26. pii: S0021-9258(23)02125-7. [Epub ahead of print] 105097
    Alkaline intracellular pH (pHi) increases PI3K kinase activity to promote mTORC1 and mTORC2 signaling and function during growth factor limitation.
    Dubek Kazyken, Stephen I Lentz, Maxwell Wadley, Diane C Fingar.
      The conserved protein kinase mTOR (mechanistic target of rapamycin) responds to diverse environmental cues to control cell metabolism and promote cell growth, proliferation, and survival as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. Our prior work demonstrated that an alkaline intracellular pH (pHi) increases mTORC2 activity and cell survival in complete media in part by activating AMPK, a kinase best known to sense energetic stress. It is important to note that an alkaline pHi represents an under-appreciated hallmark of cancer cells that promotes their oncogenic behaviors. In addition, mechanisms that control mTORC1 and mTORC2 signaling and function remain incompletely defined, particularly in response to stress conditions. Here, we demonstrate that an alkaline pHi increases PI3K activity to promote mTORC1 and mTORC2 signaling in the absence of serum growth factors. Alkaline pHi increases mTORC1 activity through PI3K-Akt signaling, which mediates inhibitory phosphorylation of the upstream proteins TSC2 and PRAS40 and dissociates TSC2 from lysosomal membranes, thus enabling Rheb-mediated activation of mTORC1. Thus, we show that an alkaline pHi mimics growth factor-PI3K signaling. Functionally, we also demonstrate that an alkaline pHi increases cap-dependent protein synthesis through inhibitory phosphorylation of 4EBP1 and suppresses apoptosis in a PI3K- and mTOR-dependent manner. We speculate that an alkaline pHi promotes a low, basal level of cell metabolism (e.g., protein synthesis) that enables cancer cells within growing tumors to proliferate and survive despite limiting growth factors and nutrients, in part through elevated PI3K-mTORC1 and/or PI3K-mTORC2 signaling.
    Keywords:  Akt PKB; S6 kinase; apoptosis; cell signaling; eukaryotic translation initiation factor 4E (eIF4E); eukaryotic translation initiation factor 4E-binding protein (eIF4EBP1); pH regulation; phosphatidylinositide 3-kinase (PI 3-kinase); protein synthesis; target of rapamycin (TOR)
    DOI:  https://doi.org/10.1016/j.jbc.2023.105097
  2. Biophys J. 2023 Jul 24. pii: S0006-3495(23)00465-4. [Epub ahead of print]
    Fluctuations of formin binding in the generation of membrane patterns.
    Mary Ecke, Jana Prassler, Günther Gerisch.
      Circular actin waves that propagate on the substrate-attached membrane of Dictyostelium cells separate two distinct membrane domains from each other: an inner territory rich in phosphatidyl-(3,4,5) trisphosphate (PIP3), and an external area decorated with the PIP3-degrading 3-phosphatase PTEN. During wave propagation, the inner territory increases on the expense of the external area. Beyond a size limit, the inner territory becomes unstable, breaking into an inner and an external domain. The sharp boundary between these domains is demarcated by the insertion of an actin wave. During the conversion of inner territory to external area, the state of the membrane fluctuates, as visualized by dynamic landscapes of Formin B binding. Here we analyze the Formin B fluctuations in relation to three markers of the membrane state: activated Ras, PIP3, and PTEN.
    Keywords:  PTEN; Ras; actin waves; formin; phosphatidylinositides
    DOI:  https://doi.org/10.1016/j.bpj.2023.07.014
  3. Cell Rep. 2023 Jul 25. pii: S2211-1247(23)00868-9. [Epub ahead of print]42(8): 112857
    Periodicity, mixed-mode oscillations, and multiple timescales in a phosphoinositide-Rho GTPase network.
    Chee San Tong, X J Xǔ, Min Wu.
      While rhythmic contractile behavior is commonly observed at the cellular cortex, the primary focus has been on excitable or periodic events described by simple activator-delayed inhibitor mechanisms. We show that Rho GTPase activation in nocodazole-treated mitotic cells exhibits both simple oscillations and complex mixed-mode oscillations. Rho oscillations with a 20- to 30-s period are regulated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) via an activator-delayed inhibitor mechanism, while a slow reaction with period of minutes is regulated by phosphatidylinositol 4-kinase via an activator-substrate depletion mechanism. Conversion from simple to complex oscillations can be induced by modulating PIP3 metabolism or altering membrane contact site protein E-Syt1. PTEN depletion results in a period-doubling intermediate, which, like mixed-mode oscillations, is an intermediate state toward chaos. In sum, this system operates at the edge of chaos. Small changes in phosphoinositide metabolism can confer cells with the flexibility to rapidly enter ordered states with different periodicities.
    Keywords:  CP: Cell biology; contractility; membrane contact sites; mitosis; mixed-mode oscillation; phosphoinositides
    DOI:  https://doi.org/10.1016/j.celrep.2023.112857
  4. Proc Natl Acad Sci U S A. 2023 08;120(31): e2300191120
    Social memory in female mice is rapidly modulated by 17β-estradiol through ERK and Akt modulation of synapse formation.
    Paul A S Sheppard, Deepthi Chandramohan, Alanna Lumsden, Daniella Vellone, Matthew C S Denley, Deepak P Srivastava, Elena Choleris.
      Social memory is essential to the functioning of a social animal within a group. Estrogens can affect social memory too quickly for classical genomic mechanisms. Previously, 17β-estradiol (E2) rapidly facilitated short-term social memory and increased nascent synapse formation, these synapses being potentiated following neuronal activity. However, what mechanisms underlie and coordinate the rapid facilitation of social memory and synaptogenesis are unclear. Here, the necessity of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling for rapid facilitation of short-term social memory and synaptogenesis was tested. Mice performed a short-term social memory task or were used as task-naïve controls. ERK and PI3K pathway inhibitors were infused intradorsal hippocampally 5 min before E2 infusion. Forty minutes following intrahippocampal E2 or vehicle administration, tissues were collected for quantification of glutamatergic synapse number in the CA1. Dorsal hippocampal E2 rapid facilitation of short-term social memory depended upon ERK and PI3K pathways. E2 increased glutamatergic synapse number (bassoon puncta positive for GluA1) in task-performing mice but decreased synapse number in task-naïve mice. Critically, ERK signaling was required for synapse formation/elimination in task-performing and task-naïve mice, whereas PI3K inhibition blocked synapse formation only in task-performing mice. While ERK and PI3K are both required for E2 facilitation of short-term social memory and synapse formation, only ERK is required for synapse elimination. This demonstrates previously unknown, bidirectional, rapid actions of E2 on brain and behavior and underscores the importance of estrogen signaling in the brain to social behavior.
    Keywords:  neuroplasticity; social behavior; steroid hormones
    DOI:  https://doi.org/10.1073/pnas.2300191120
  5. Mol Cancer Res. 2023 Jul 26. pii: MCR-23-0034. [Epub ahead of print]
    CBF-beta mitigates PI3K-alpha-specific inhibitor killing through PIM1 in PIK3CA mutant gastric cancer.
    Lyla J Stanland, Hazel X Ang, Jacob P Hoj, Yunqiang Chu, Patrick Tan, Kris C Wood, Micah A Luftig.
      PIK3CA is the second most mutated gene in cancer leading to aberrant PI3K/AKT/mTOR signaling and increased translation, proliferation, and survival. Some 4-25% of gastric cancers display activating PIK3CA mutations including 80% of EBV-associated GCs. Small molecules including pan-PI3K and dual PI3K/mTOR inhibitors have shown moderate success clinically, due to broad on-target/off-tissue effects. Thus, isoform specific and mutant selective inhibitors have been of significant interest. However, drug resistance is a problem and has affected success of new drugs. There has been a concerted effort to define mechanisms of resistance and identify potent combinations in many tumor types, though gastric cancer is comparatively understudied. In this study we identified modulators of the response to the PI3K-alpha-specific inhibitor, BYL719, in PIK3CA mutant GCs. We found that loss of NEDD9 or inhibition of BCL-XL conferred hyper-sensitivity to BYL719, through increased cell cycle arrest and cell death, respectively. Additionally, we discovered that loss of CBFB conferred resistance to BYL719. CBFB loss led to up-regulation of the protein kinase PIM1, which can phosphorylate and activate several overlapping downstream substrates as AKT thereby maintaining pathway activity in the presence of PI3K-alpha inhibition. The addition of a pan-PIM inhibitor re-sensitized resistant cells to BYL719. Our data provide clear mechanistic insights into PI3K-alpha inhibitor response in PIK3CA mutant gastric tumors and can inform future work as mutant selective inhibitors are in development for diverse tumor types. Implications: Loss of either NEDD9 or BCL-XL confers hyper-sensitivity to PI3K-alpha inhibition while loss of CBFB confers resistance through a CBFB/PIM1 signaling axis.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-23-0034
  6. Cell Struct Funct. 2023 Jul 22.
    FilGAP, a GAP for Rac1, down-regulates invadopodia formation in breast cancer cells.
    Koji Saito, Sakino Ozawa, Yosuke Chiba, Ruri Takahashi, Ryoya Ogomori, Kojiro Mukai, Tomohiko Taguchi, Hiroyasu Hatakeyama, Yasutaka Ohta.
      Invadopodia are protrusive structures that mediate the extracellular matrix (ECM) degradation required for tumor invasion and metastasis. Rho small GTPases regulate invadopodia formation, but the molecular mechanisms of how Rho small GTPase activities are regulated at the invadopodia remain unclear. Here we have identified FilGAP, a GTPase-activating protein (GAP) for Rac1, as a negative regulator of invadopodia formation in tumor cells. Depletion of FilGAP in breast cancer cells increased ECM degradation and conversely, overexpression of FilGAP decreased it. FilGAP depletion promoted the formation of invadopodia with ECM degradation. In addition, FilGAP depletion and Rac1 overexpression increased the emergence of invadopodia induced by epidermal growth factor, whereas FilGAP overexpression suppressed it. Overexpression of GAP-deficient FilGAP mutant enhanced invadopodia emergence as well as FilGAP depletion. The pleckstrin-homology (PH) domain of FilGAP binds phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], which is distributed on membranes of the invadopodia. FilGAP localized to invadopodia in breast cancer cells on the ECM, but FilGAP mutant lacking PI(3,4)P2-binding showed low localization. Similarly, the decrease of PI(3,4)P2 production reduced the FilGAP localization. Our results suggest that FilGAP localizes to invadopodia through its PH domain binding to PI(3,4)P2 and down-regulates invadopodia formation by inactivating Rac1, inhibiting ECM degradation in invasive tumor cells.Key Words: Invadopodia, breast carcinoma, Rac1, FilGAP, PI(3,4)P2.
    Keywords:  FilGAP; Invadopodia; PI(3,4)P2; Rac1; breast carcinoma
    DOI:  https://doi.org/10.1247/csf.23032
  7. Cells. 2023 Jul 12. pii: 1830. [Epub ahead of print]12(14):
    A Role of PI3K/Akt Signaling in Oocyte Maturation and Early Embryo Development.
    Jaroslav Kalous, Daria Aleshkina, Martin Anger.
      A serine/threonine-specific protein kinase B (PKB), also known as Akt, is a key factor in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway that regulates cell survival, metabolism and proliferation. Akt phosphorylates many downstream specific substrates, which subsequently control the nuclear envelope breakdown (NEBD), centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. In vertebrates, Akt is also an important player during oogenesis and preimplantation development. In the signaling pathways regulating mRNA translation, Akt is involved in the control of mammalian target of rapamycin complex 1 (mTORC1) and thereby regulates the activity of a translational repressor, the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In this review, we summarize the functions of Akt in mitosis, meiosis and early embryonic development. Additionally, the role of Akt in the regulation of mRNA translation is addressed with respect to the significance of this process during early development.
    Keywords:  Akt kinase; early embryo; mRNA translation; mTORC1; meiosis; mitosis; oocyte; spindle
    DOI:  https://doi.org/10.3390/cells12141830
  8. Cell Rep. 2023 Jul 25. pii: S2211-1247(23)00879-3. [Epub ahead of print]42(8): 112868
    mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching.
    Sejeong Shin, Min-Joon Han, Mark P Jedrychowski, Ziyang Zhang, Kevan M Shokat, David R Plas, Noah Dephoure, Sang-Oh Yoon.
      Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.
    Keywords:  CP: Cell biology; CP: Molecular biology; eIF3D; eIF4E; mRNA translation; mTOR; proteome; proteostasis
    DOI:  https://doi.org/10.1016/j.celrep.2023.112868
  9. Front Cell Dev Biol. 2023 ;11 1195806
    Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement.
    Dhiman Sankar Pal, Yiyan Lin, Huiwang Zhan, Tatsat Banerjee, Jonathan Kuhn, Stephenie Providence, Peter N Devreotes.
      In this article, we provide detailed protocols on using optogenetic dimerizers to acutely perturb activities of guanine nucleotide exchange factors (GEFs) specific to Ras, Rac or Rho small GTPases of the migratory networks in various mammalian and amoeba cell lines. These GEFs are crucial components of signal transduction networks which link upstream G-protein coupled receptors to downstream cytoskeletal components and help cells migrate through their dynamic microenvironment. Conventional approaches to perturb and examine these signaling and cytoskeletal networks, such as gene knockout or overexpression, are protracted which allows networks to readjust through gene expression changes. Moreover, these tools lack spatial resolution to probe the effects of local network activations. To overcome these challenges, blue light-inducible cryptochrome- and LOV domain-based dimerization systems have been recently developed to control signaling or cytoskeletal events in a spatiotemporally precise manner. We illustrate that, within minutes of global membrane recruitment of full-length GEFs or their catalytic domains only, widespread increases or decreases in F-actin rich protrusions and cell size occur, depending on the particular node in the networks targeted. Additionally, we demonstrate localized GEF recruitment as a robust assay system to study local network activation-driven changes in polarity and directed migration. Altogether, these optical tools confirmed GEFs of Ras superfamily GTPases as regulators of cell shape, actin dynamics, and polarity. Furthermore, this optogenetic toolbox may be exploited in perturbing complex signaling interactions in varied physiological contexts including mammalian embryogenesis.
    Keywords:  actin cytoskeleton; cancer metastasis; development; diabetes; growth factor signaling; immunity; neural networks; optogenetics
    DOI:  https://doi.org/10.3389/fcell.2023.1195806
  10. Nature. 2023 Jul 26.
    Evolutionarily divergent mTOR remodels translatome for tissue regeneration.
    Olena Zhulyn, Hannah D Rosenblatt, Leila Shokat, Shizhong Dai, Duygu Kuzuoglu-Öztürk, Zijian Zhang, Davide Ruggero, Kevan M Shokat, Maria Barna.
      An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.
    DOI:  https://doi.org/10.1038/s41586-023-06365-1
  11. bioRxiv. 2023 Jul 11. pii: 2023.07.10.548432. [Epub ahead of print]
    Proteome-wide copy-number estimation from transcriptomics.
    Andrew J Sweatt, Cameron D Griffiths, B Bishal Paudel, Kevin A Janes.
      Protein copy numbers constrain systems-level properties of regulatory networks, but absolute proteomic data remain scarce compared to transcriptomics obtained by RNA sequencing. We addressed this persistent gap by relating mRNA to protein statistically using best-available data from quantitative proteomics-transcriptomics for 4366 genes in 369 cell lines. The approach starts with a central estimate of protein copy number and hierarchically appends mRNA-protein and mRNA-mRNA dependencies to define an optimal gene-specific model that links mRNAs to protein. For dozens of independent cell lines and primary prostate samples, these protein inferences from mRNA outmatch stringent null models, a count-based protein-abundance repository, and empirical protein-to-mRNA ratios. The optimal mRNA-to-protein relationships capture biological processes along with hundreds of known protein-protein interaction complexes, suggesting mechanistic relationships are embedded. We use the method to estimate viral-receptor abundances of CD55-CXADR from human heart transcriptomes and build 1489 systems-biology models of coxsackievirus B3 infection susceptibility. When applied to 796 RNA sequencing profiles of breast cancer from The Cancer Genome Atlas, inferred copy-number estimates collectively reclassify 26% of Luminal A and 29% of Luminal B tumors. Protein-based reassignments strongly involve a pharmacologic target for luminal breast cancer (CDK4) and an α-catenin that is often undetectable at the mRNA level (CTTNA2). Thus, by adopting a gene-centered perspective of mRNA-protein covariation across different biological contexts, we achieve accuracies comparable to the technical reproducibility limits of contemporary proteomics. The collection of gene-specific models is assembled as a web tool for users seeking mRNA-guided predictions of absolute protein abundance ( http://janeslab.shinyapps.io/Pinferna ).
    Significance statement: It is easier to quantify mRNA in cells than it is to quantify protein, but proteins are what execute most cellular functions. Even though protein is synthesized from mRNA in cells, relating a cellular quantity of mRNA to a quantity of protein is challenging. Here, we bring together quantitative measures of mRNA and protein for 4366 genes in 369 different cultured cell types to build equations that predict protein abundance from the abundance of mRNAs expressed. These equations capture facets of biological regulation and work better than existing alternatives that rely on consensus values or ratios. Since mRNA measurements are more widespread than protein, this study makes new analyses possible by protein estimation from mRNA.
    DOI:  https://doi.org/10.1101/2023.07.10.548432
  12. STAR Protoc. 2023 Jul 22. pii: S2666-1667(23)00373-8. [Epub ahead of print]4(3): 102406
    ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs.
    Katarzyna A Ludwik, Narasimha Telugu, Sandra Schommer, Harald Stachelscheid, Sebastian Diecke.
      CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening.
    Keywords:  CRISPR; Molecular Biology; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2023.102406
  13. Proc Natl Acad Sci U S A. 2023 08;120(31): e2302471120
    Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.
    Wooyoung Choi, Hao Wu, Klaus Yserentant, Bo Huang, Yifan Cheng.
      CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.
    Keywords:  CRISPR tagging; cryo-EM; endogenous protein; human cell lines; structure
    DOI:  https://doi.org/10.1073/pnas.2302471120
  14. STAR Protoc. 2023 Jul 22. pii: S2666-1667(23)00430-6. [Epub ahead of print]4(3): 102463
    FISH-Flow to quantify nascent and mature ribosomal RNA in mouse and human cells.
    Charles Antony, Patrick Somers, Erin M Gray, Maxim Pimkin, Vikram R Paralkar.
      FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis. For complete details on the use and execution of this protocol, please refer to Antony et al. (2022).1.
    Keywords:  Flow Cytometry/Mass Cytometry; In Situ Hybridization; Molecular Biology; Single Cell
    DOI:  https://doi.org/10.1016/j.xpro.2023.102463
  15. iScience. 2023 Aug 18. 26(8): 107270
    Improved approaches to channel capacity estimation discover compromised GPCR signaling in diverse cancer cells.
    Alexey Koval, Xin Zhang, Vladimir L Katanaev.
      Intracellular signaling orchestrates an organism's development and functioning and underlies various pathologies, such as cancer, when aberrant. A universal cell signaling characteristic is channel capacity - the measure of how much information a given transmitting system can reliably transduce. Here, we describe improved approaches to quantify GPCR signaling channel capacity in single cells, averaged across cell population. We assess the channel capacity based on distribution of residuals by the cellular response amplitude. We further develop means to handle irregularly responding cancer cells using the integral values of their response to different agonist concentrations. These approaches enabled us to analyze, for the first time, channel capacity in single cancer cells. A universal feature emerging for different cancer cell types is a decreased channel capacity of their GPCR signaling. These findings provide experimental validation to the hypothesis that cancer is an information disease, bearing importance for basic cancer biology and anticancer drug discovery.
    Keywords:  Cancer; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2023.107270
  16. iScience. 2023 Aug 18. 26(8): 107265
    FGFR-mediated ERK1/2 signaling contributes to mesendoderm and definitive endoderm formation in vitro.
    Hwee Hui Lau, Nur Shabrina Amirruddin, Larry Sai Weng Loo, Jun Wei Chan, Elhadi Iich, Vidhya Gomathi Krishnan, Shawn Hoon, Adrian Kee Keong Teo.
      The differentiation of human pluripotent stem cells into the SOX17+ definitive endoderm (DE) germ layer is important for generating tissues for regenerative medicine. Multiple developmental and stem cell studies have demonstrated that Activin/Nodal signaling is the primary driver of definitive endoderm formation. Here, we uncover that the FGF2-FGFR-ERK1/2 signaling contributes to mesendoderm and SOX17+ DE formation. Without ERK1/2 signaling, the Activin/Nodal signaling is insufficient to drive mesendoderm and DE formation. Besides FGF2-FGFR-mediated signaling, IGF1R signaling possibly contributes to the ERK1/2 signaling for DE formation. We identified a temporal relationship between Activin/Nodal-SMAD2 and FGF2-FGFR-ERK1/2 signaling in which Activin/Nodal-SMAD2 participates in the initiation of mesendoderm and DE specification that is followed by increasing activity of FGF2-FGFR-ERK1/2 to facilitate and permit the successful generation of SOX17+ DE. Overall, besides the role of Activin/Nodal signaling for DE formation, our findings shed light on the contribution of ERK1/2 signaling for mesendoderm and DE formation.
    Keywords:  Biological sciences; Developmental biology; Embryology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2023.107265
  17. bioRxiv. 2023 Jul 17. pii: 2023.07.17.549397. [Epub ahead of print]
    Simultaneous multi-site editing of individual genomes using retron arrays.
    Alejandro González-Delgado, Santiago C Lopez, Matías Rojas-Montero, Chloe B Fishman, Seth L Shipman.
      Our understanding of genomics is limited by the scale of our genomic technologies. While libraries of genomic manipulations scaffolded on CRISPR gRNAs have been transformative, these existing approaches are typically multiplexed across genomes. Yet much of the complexity of real genomes is encoded within a genome across sites. Unfortunately, building cells with multiple, non-adjacent precise mutations remains a laborious cycle of editing, isolating an edited cell, and editing again. Here, we describe a technology for precisely modifying multiple sites on a single genome simultaneously. This technology - termed a multitron - is built from a heavily modified retron, in which multiple donor-encoding msds are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization, and metabolic engineering.
    DOI:  https://doi.org/10.1101/2023.07.17.549397
  18. Nat Methods. 2023 Jul 27.
    Segmentation metric misinterpretations in bioimage analysis.
    Dominik Hirling, Ervin Tasnadi, Juan Caicedo, Maria V Caroprese, Rickard Sjögren, Marc Aubreville, Krisztian Koos, Peter Horvath.
      Quantitative evaluation of image segmentation algorithms is crucial in the field of bioimage analysis. The most common assessment scores, however, are often misinterpreted and multiple definitions coexist with the same name. Here we present the ambiguities of evaluation metrics for segmentation algorithms and show how these misinterpretations can alter leaderboards of influential competitions. We also propose guidelines for how the currently existing problems could be tackled.
    DOI:  https://doi.org/10.1038/s41592-023-01942-8
  19. Trends Cancer. 2023 Jul 25. pii: S2405-8033(23)00130-9. [Epub ahead of print]
    Digging deeper into the early steps of cancer progression.
    Jonathan H Sussman, Ben Z Stanger.
      Epigenetically mediated cell signaling is emerging as a core principle of tumor biology. Through single cell multi-omic profiling, Burdziak, Alonso-Curbelo et al. have recently uncovered key epigenetic plasticity programs associated with cellular communication, shedding light on the role of epigenetic cell states during malignant transformation of the inflamed pancreas.
    DOI:  https://doi.org/10.1016/j.trecan.2023.07.003
  20. Cancer Discov. 2023 Jul 27. OF1
    Two-Armed Molecule Flips Switch on Gene Regulation.
      A new class of designer drugs can reprogram gene expression by linking a transcription factor that silences genes involved in cell death with an epigenetic regulator that stimulates gene activity. A prototype molecule showed robust potency in flipping the function of BCL6, a protein that supports lymphoma cell proliferation, offering a promising therapeutic approach to combat cancer cell growth.
    DOI:  https://doi.org/10.1158/2159-8290.CD-NB2023-0053
  21. Cell Rep. 2023 Jul 22. pii: S2211-1247(23)00841-0. [Epub ahead of print]42(8): 112830
    Premature aging and reduced cancer incidence associated with near-complete body-wide Myc inactivation.
    Huabo Wang, Jie Lu, Taylor Stevens, Alexander Roberts, Jordan Mandel, Raghunandan Avula, Bingwei Ma, Yijen Wu, Jinglin Wang, Clinton Van't Land, Toren Finkel, Jerry E Vockley, Merlin Airik, Rannar Airik, Radhika Muzumdar, Zhenwei Gong, Michel S Torbenson, Edward V Prochownik.
      MYC proto-oncogene dysregulation alters metabolism, translation, and other functions in ways that support tumor induction and maintenance. Although Myc+/- mice are healthier and longer-lived than control mice, the long-term ramifications of more complete Myc loss remain unknown. We now describe the chronic consequences of body-wide Myc inactivation initiated postnatally. "MycKO" mice acquire numerous features of premature aging, including altered body composition and habitus, metabolic dysfunction, hepatic steatosis, and dysregulation of gene sets involved in functions that normally deteriorate with aging. Yet, MycKO mice have extended lifespans that correlate with a 3- to 4-fold lower lifetime cancer incidence. Aging tissues from normal mice and humans also downregulate Myc and gradually alter many of the same Myc target gene sets seen in MycKO mice. Normal aging and its associated cancer predisposition are thus highly linked via Myc.
    Keywords:  CP: Cancer; aging
    DOI:  https://doi.org/10.1016/j.celrep.2023.112830
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