bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–10–16
twenty-two papers selected by
Ralitsa Radostinova Madsen, University College London
  1. AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kβ/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors.
  2. KIF3B promotes a PI3K signaling gradient causing changes in a Shh protein gradient and suppressing polydactyly in mice.
  3. Cellular context alters EGF-induced ERK dynamics and reveals potential crosstalk with GDF-15.
  4. mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.
  5. Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.
  6. AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.
  7. Delving into insulin receptor regulation.
  8. Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.
  9. Up-regulation of the PI3K/AKT and RHO/RAC/PAK signalling pathways in CHK1 inhibitor resistant Eµ-Myc lymphoma cells.
  10. A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1.
  11. scATACpipe: A nextflow pipeline for comprehensive and reproducible analyses of single cell ATAC-seq data.
  12. Mechanistic target of rapamycin signaling in human nervous system development and disease.
  13. Cell growth and the cell cycle: New insights about persistent questions.
  14. A genetically encoded system for oxygen generation in living cells.
  15. Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions.
  16. Cellular barcoding to decipher clonal dynamics in disease.
  17. Spatial epitope barcoding reveals clonal tumor patch behaviors.
  18. An unusual mode of baseline translation adjusts cellular protein synthesis capacity to metabolic needs.
  19. The physiology of alternative splicing.
  20. Neural crest cells hijack pluripotent stem cell factors to realize their developmental potential.
  21. Epidermal growth factor receptor cascade prioritizes the maximization of signal transduction.
  22. Systematic Interrogation of the Temperature Perturbation in the Insulin Signaling Pathway for Optogenetic Stimulation.
  1. Oncogene. 2022 Oct 14.
    AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kβ/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors.
    Shanade Dunn, Cath Eberlein, Jason Yu, Albert Gris-Oliver, Swee Hoe Ong, Urs Yelland, Natalie Cureton, Anna Staniszewska, Robert McEwen, Millie Fox, James Pilling, Philip Hopcroft, Elizabeth A Coker, Patricia Jaaks, Mathew J Garnett, Beverley Isherwood, Violeta Serra, Barry R Davies, Simon T Barry, James T Lynch, Kosuke Yusa.
      The PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kβ inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kβi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kβi resistance through AKT. Conversely deletion of PIK3CA, ERBB2, ERBB3 increased PI3Kβi sensitivity while modulation of RRAGC, LAMTOR1, LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kβi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kβ/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kβi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kβi/AKTi sensitive and resistant breast tumours.
    DOI:  https://doi.org/10.1038/s41388-022-02482-9
  2. Dev Cell. 2022 Oct 10. pii: S1534-5807(22)00637-2. [Epub ahead of print]57(19): 2273-2289.e11
    KIF3B promotes a PI3K signaling gradient causing changes in a Shh protein gradient and suppressing polydactyly in mice.
    Shuo Wang, Yosuke Tanaka, Ying Xu, Sen Takeda, Nobutaka Hirokawa.
      Digit determination in limb buds is driven by a posteriorizing Sonic hedgehog (Shh) protein gradient; however, the mechanism regulating this is unclear. Here, we propose a diffusion-and-trapping hypothesis for Shh gradient formation based on data from the preaxial polydactyly phenotype of KIF3B motor hypomorphic mice. In the limb buds of these mice, a distal-to-proximal gradient of fibroblast growth factor (FGF) and phosphatidylinositol 3-kinase (PI3K) signaling and a posterior-to-anterior gradient of Shh were disorganized. This phenotype was reproduced by transplanting FGF8b-soaked beads. At the subcellular level, KIF3B transported the phosphatase and tensin homolog (PTEN)-like phosphatase Talpid3 to terminate PI3K signaling. High and low PI3K signaling strengths differentially sorted endocytosed Shh toward exosome-like particles and cytonemal punctata, respectively. These results indicate that the Shh-containing particles undergo either the diffusional movement in the periphery or cytonemal trapping in the center and form a spatial gradient along the periphery of developing limb buds.
    Keywords:  FGF signaling; Kinesin; PI3K signaling; Shh gradient; Sonic hedgehog; limb bud morphogenesis; morphogen gradient; polydactyly
    DOI:  https://doi.org/10.1016/j.devcel.2022.09.007
  3. Biomicrofluidics. 2022 Sep;16(5): 054104
    Cellular context alters EGF-induced ERK dynamics and reveals potential crosstalk with GDF-15.
    Harris B Krause, Alexis L Karls, Megan N McClean, Pamela K Kreeger.
      Cellular signaling dynamics are sensitive to differences in ligand identity, levels, and temporal patterns. These signaling patterns are also impacted by the larger context that the cell experiences (i.e., stimuli such as media formulation or substrate stiffness that are constant in an experiment exploring a particular variable but may differ between independent experiments which explore that variable) although the reason for different dynamics is not always obvious. Here, we compared extracellular-regulated kinase (ERK) signaling in response to epidermal growth factor treatment of human mammary epithelial cells cultures in either well culture or a microfluidic device. Using a single-cell ERK kinase translocation reporter, we observed extended ERK activation in well culture and only transient activity in microfluidic culture. The activity in microfluidic culture resembled that of the control condition, suggesting that shear stress led to the early activity and a loss of autocrine factors dampened extended signaling. Through experimental analysis we identified growth differentiation factor-15 as a candidate factor that led to extended ERK activation through a protein kinase C-α/β dependent pathway. Our results demonstrate that context impacts ERK dynamics and that comparison of distinct contexts can be used to elucidate new aspects of the cell signaling network.
    DOI:  https://doi.org/10.1063/5.0114334
  4. Cell Death Dis. 2022 Oct 11. 13(10): 862
    mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.
    Shuo Wan, Yadong Sun, Jiamin Fu, Hongrui Song, Zhiqiang Xiao, Quanli Yang, Sanfeng Wang, Gongwang Yu, Peiran Feng, Wenkai Lv, Liang Luo, Zerong Guan, Feng Liu, Qinghua Zhou, Zhinan Yin, Meixiang Yang.
      The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen. Estrogen acts as an activator of mTOR signaling but its role in vaginal epithelial homeostasis is unknown. We analyzed reproductive tract-specific Rptor or Rictor conditional knockout mice to reveal the role of mTOR signaling in estrogen-dependent vaginal epithelial cell proliferation and differentiation. Loss of Rptor but not Rictor in the vagina resulted in an aberrant proliferation of epithelial cells and failure of keratinized differentiation. As gene expression analysis indicated, several estrogen-mediated genes, including Pgr and Ereg (EGF-like growth factor) were not induced by estrogen in Rptor cKO mouse vagina. Moreover, supplementation of EREG could activate the proliferation and survival of vaginal epithelial cells through YAP1 in the absence of Rptor. Thus, mTORC1 signaling integrates estrogen and growth factor signaling to mediate vaginal epithelial cell proliferation and differentiation, providing new insights into vaginal atrophy treatment for post-menopausal women.
    DOI:  https://doi.org/10.1038/s41419-022-05293-8
  5. Front Oncol. 2022 ;12 852358
    Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.
    Satoshi Asano, Misa Yamasaka, Kairi Ozasa, Kotaro Sakamoto, Atsuko Hayata-Takano, Takanobu Nakazawa, Hitoshi Hashimoto, James A Waschek, Yukio Ago.
      Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Rac-mediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP-VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3.
    Keywords:  GPCR; VIPR2; breast cancer; cell migration; cytoskeleton; phosphatidylinositol signaling
    DOI:  https://doi.org/10.3389/fonc.2022.852358
  6. Cancers (Basel). 2022 Sep 30. pii: 4794. [Epub ahead of print]14(19):
    AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.
    Alice Hui Li Bong, Trinh Hua, Choon Leng So, Amelia A Peters, Mélanie Robitaille, Yin Yi Tan, Sarah J Roberts-Thomson, Gregory R Monteith.
      Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.
    Keywords:  AKT regulation; GCaMP6; ORAI1; PTEN; breast cancer; calcium signaling
    DOI:  https://doi.org/10.3390/cancers14194794
  7. Nat Rev Endocrinol. 2022 Oct 13.
    Delving into insulin receptor regulation.
    Shimona Starling.
      
    DOI:  https://doi.org/10.1038/s41574-022-00765-5
  8. Nat Commun. 2022 Oct 12. 13(1): 6023
    Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.
    Joshua J Rennick, Cameron J Nowell, Colin W Pouton, Angus P R Johnston.
      Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in individual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.
    DOI:  https://doi.org/10.1038/s41467-022-33348-z
  9. Biochem J. 2022 Oct 14. 479(19): 2131-2151
    Up-regulation of the PI3K/AKT and RHO/RAC/PAK signalling pathways in CHK1 inhibitor resistant Eµ-Myc lymphoma cells.
    Jill E Hunter, Amy E Campbell, Scott Kerridge, Callum Fraser, Nicola L Hannaway, Saimir Luli, Iglika Ivanova, Philip J Brownridge, Jonathan Coxhead, Leigh Taylor, Peter Leary, Megan S R Hasoon, Claire E Eyers, Neil D Perkins.
      The development of resistance and the activation of bypass pathway signalling represents a major problem for the clinical application of protein kinase inhibitors. While investigating the effect of either a c-Rel deletion or RelAT505A phosphosite knockin on the Eµ-Myc mouse model of B-cell lymphoma, we discovered that both NF-κB subunit mutations resulted in CHK1 inhibitor resistance, arising from either loss or alteration of CHK1 activity, respectively. However, since Eµ-Myc lymphomas depend on CHK1 activity to cope with high levels of DNA replication stress and consequent genomic instability, it was not clear how these mutant NF-κB subunit lymphomas were able to survive. To understand these survival mechanisms and to identify potential compensatory bypass signalling pathways in these lymphomas, we applied a multi-omics strategy. With c-Rel-/- Eµ-Myc lymphomas we observed high levels of Phosphatidyl-inositol 3-kinase (PI3K) and AKT pathway activation. Moreover, treatment with the PI3K inhibitor Pictilisib (GDC-0941) selectively inhibited the growth of reimplanted c-Rel-/- and RelAT505A, but not wild type (WT) Eµ-Myc lymphomas. We also observed up-regulation of a RHO/RAC pathway gene expression signature in both Eµ-Myc NF-κB subunit mutation models. Further investigation demonstrated activation of the RHO/RAC effector p21-activated kinase (PAK) 2. Here, the PAK inhibitor, PF-3758309 successfully overcame resistance of RelAT505A but not WT lymphomas. These findings demonstrate that up-regulation of multiple bypass pathways occurs in CHK1 inhibitor resistant Eµ-Myc lymphomas. Consequently, drugs targeting these pathways could potentially be used as either second line or combinatorial therapies to aid the successful clinical application of CHK1 inhibitors.
    Keywords:  CHK1 inhibitor; drug resistance; lymphoma; nuclear factor kappaB; p21-activated kinases; protein kinase B
    DOI:  https://doi.org/10.1042/BCJ20220103
  10. Cell Rep. 2022 Oct 11. pii: S2211-1247(22)01325-0. [Epub ahead of print]41(2): 111475
    A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1.
    Lisa Donker, Ronja Houtekamer, Marjolein Vliem, François Sipieter, Helena Canever, Manuel Gómez-González, Miquel Bosch-Padrós, Willem-Jan Pannekoek, Xavier Trepat, Nicolas Borghi, Martijn Gloerich.
      Epithelial cell divisions are coordinated with cell loss to preserve epithelial integrity. However, how epithelia adapt their rate of cell division to changes in cell number, for instance during homeostatic turnover or wounding, is not well understood. Here, we show that epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell-cycle progression. As local cell density increases, tensile forces on E-cadherin adhesions are reduced, which prompts the accumulation of the G2 checkpoint kinase Wee1 and downstream inhibitory phosphorylation of Cdk1. Consequently, dense epithelia contain a pool of cells that are temporarily halted in G2 phase. These cells are readily triggered to divide following epithelial wounding due to the consequent increase in intercellular forces and resulting degradation of Wee1. Our data collectively show that epithelial cell division is controlled by a mechanical G2 checkpoint, which is regulated by cell-density-dependent intercellular forces sensed and transduced by E-cadherin adhesions.
    Keywords:  CP: Cell biology; E-cadherin; G2 checkpoint; adherens junction; cell cycle; cell division; epithelial homeostasis; mechanical forces; mechanotransduction; mitosis
    DOI:  https://doi.org/10.1016/j.celrep.2022.111475
  11. Front Cell Dev Biol. 2022 ;10 981859
    scATACpipe: A nextflow pipeline for comprehensive and reproducible analyses of single cell ATAC-seq data.
    Kai Hu, Haibo Liu, Nathan D Lawson, Lihua Julie Zhu.
      Single cell ATAC-seq (scATAC-seq) has become the most widely used method for profiling open chromatin landscape of heterogeneous cell populations at a single-cell resolution. Although numerous software tools and pipelines have been developed, an easy-to-use, scalable, reproducible, and comprehensive pipeline for scATAC-seq data analyses is still lacking. To fill this gap, we developed scATACpipe, a Nextflow pipeline, for performing comprehensive analyses of scATAC-seq data including extensive quality assessment, preprocessing, dimension reduction, clustering, peak calling, differential accessibility inference, integration with scRNA-seq data, transcription factor activity and footprinting analysis, co-accessibility inference, and cell trajectory prediction. scATACpipe enables users to perform the end-to-end analysis of scATAC-seq data with three sub-workflow options for preprocessing that leverage 10x Genomics Cell Ranger ATAC software, the ultra-fast Chromap procedures, and a set of custom scripts implementing current best practices for scATAC-seq data preprocessing. The pipeline extends the R package ArchR for downstream analysis with added support to any eukaryotic species with an annotated reference genome. Importantly, scATACpipe generates an all-in-one HTML report for the entire analysis and outputs cluster-specific BAM, BED, and BigWig files for visualization in a genome browser. scATACpipe eliminates the need for users to chain different tools together and facilitates reproducible and comprehensive analyses of scATAC-seq data from raw reads to various biological insights with minimal changes of configuration settings for different computing environments or species. By applying it to public datasets, we illustrated the utility, flexibility, versatility, and reliability of our pipeline, and demonstrated that our scATACpipe outperforms other workflows.
    Keywords:  chromatin accessibility; integration of scATAC-seq and scRNA-seq; nextflow; pipeline; scATAC-seq; single cell; trajectory inference; transcription factor activity and footprinting analysis
    DOI:  https://doi.org/10.3389/fcell.2022.981859
  12. Front Mol Neurosci. 2022 ;15 1005631
    Mechanistic target of rapamycin signaling in human nervous system development and disease.
    Marie Girodengo, Sila K Ultanir, Joseph M Bateman.
      Mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that regulates fundamental cellular processes including growth control, autophagy and metabolism. mTOR has key functions in nervous system development and mis-regulation of mTOR signaling causes aberrant neurodevelopment and neurological diseases, collectively called mTORopathies. In this mini review we discuss recent studies that have deepened our understanding of the key roles of the mTOR pathway in human nervous system development and disease. Recent advances in single-cell transcriptomics have been exploited to reveal specific roles for mTOR signaling in human cortical development that may have contributed to the evolutionary divergence from our primate ancestors. Cerebral organoid technology has been utilized to show that mTOR signaling is active in and regulates outer radial glial cells (RGCs), a population of neural stem cells that distinguish the human developing cortex. mTOR signaling has a well-established role in hamartoma syndromes such as tuberous sclerosis complex (TSC) and other mTORopathies. New ultra-sensitive techniques for identification of somatic mTOR pathway mutations have shed light on the neurodevelopmental origin and phenotypic heterogeneity seen in mTORopathy patients. These emerging studies suggest that mTOR signaling may facilitate developmental processes specific to human cortical development but also, when mis-regulated, cause cortical malformations and neurological disease.
    Keywords:  cortex; mTOR; mTORopathy; neuron; organoid; tuberous sclerosis
    DOI:  https://doi.org/10.3389/fnmol.2022.1005631
  13. Bioessays. 2022 Oct 12. e2200150
    Cell growth and the cell cycle: New insights about persistent questions.
    Jan Inge Øvrebø, Yiqin Ma, Bruce A Edgar.
      Before a cell divides into two daughter cells, it typically doubles not only its DNA, but also its mass. Numerous studies in cells ranging from yeast to mammals have shown that cellular growth, stimulated by nutrients and/or growth factor signaling, is a prerequisite for cell cycle progression in most types of cells. The textbook view of growth-regulated cell cycles is that growth signaling activates the transcription of G1 Cyclin genes to induce cell proliferation, and also stimulates anabolic metabolism and cell growth in parallel. However, genetic knockout tests in model organisms indicate that this is not the whole story, and new studies show that additional, "smarter" mechanisms help to coordinate the cell cycle with growth itself. Here we summarize recent advances in this field, and discuss current models in which growth signaling regulates cell proliferation by targeting core cell cycle regulators via non-transcriptional mechanisms.
    Keywords:  E2F; cell cycle; cell growth; cell size; cyclin D; growth factor signaling; metabolism
    DOI:  https://doi.org/10.1002/bies.202200150
  14. Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2207955119
    A genetically encoded system for oxygen generation in living cells.
    Andrew L Markhard, Jason G McCoy, Tsz-Leung To, Vamsi K Mootha.
      Oxygen plays a key role in supporting life on our planet. It is particularly important in higher eukaryotes where it boosts bioenergetics as a thermodynamically favorable terminal electron acceptor and has important roles in cell signaling and development. Many human diseases stem from either insufficient or excessive oxygen. Despite its fundamental importance, we lack methods with which to manipulate the supply of oxygen with high spatiotemporal resolution in cells and in organisms. Here, we introduce a genetic system, SupplemeNtal Oxygen Released from ChLorite (SNORCL), for on-demand local generation of molecular oxygen in living cells, by harnessing prokaryotic chlorite O2-lyase (Cld) enzymes that convert chlorite (ClO2-) into molecular oxygen (O2) and chloride (Cl-). We show that active Cld enzymes can be targeted to either the cytosol or mitochondria of human cells, and that coexpressing a chlorite transporter results in molecular oxygen production inside cells in response to externally added chlorite. This first-generation system allows fine temporal and spatial control of oxygen production, with immediate research applications. In the future, we anticipate that technologies based on SNORCL will have additional widespread applications in research, biotechnology, and medicine.
    Keywords:  Cld; SLC5A5; SNORCL; chlorite; oxygen
    DOI:  https://doi.org/10.1073/pnas.2207955119
  15. Sci Adv. 2022 Oct 14. 8(41): eabp8677
    Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions.
    Feng-Ching Tsai, J Michael Henderson, Zack Jarin, Elena Kremneva, Yosuke Senju, Julien Pernier, Oleg Mikhajlov, John Manzi, Konstantin Kogan, Christophe Le Clainche, Gregory A Voth, Pekka Lappalainen, Patricia Bassereau.
      Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.
    DOI:  https://doi.org/10.1126/sciadv.abp8677
  16. Science. 2022 Oct 14. 378(6616): eabm5874
    Cellular barcoding to decipher clonal dynamics in disease.
    Vijay G Sankaran, Jonathan S Weissman, Leonard I Zon.
      Cellular barcodes are distinct DNA sequences that enable one to track specific cells across time or space. Recent advances in our ability to detect natural or synthetic cellular barcodes, paired with single-cell readouts of cell state, have markedly increased our knowledge of clonal dynamics and genealogies of the cells that compose a variety of tissues and organs. These advances hold promise to redefine our view of human disease. Here, we provide an overview of cellular barcoding approaches, discuss applications to gain new insights into disease mechanisms, and provide an outlook on future applications. We discuss unanticipated insights gained through barcoding in studies of cancer and blood cell production and describe how barcoding can be applied to a growing array of medical fields, particularly with the increasing recognition of clonal contributions in human diseases.
    DOI:  https://doi.org/10.1126/science.abm5874
  17. Cancer Cell. 2022 Oct 12. pii: S1535-6108(22)00443-3. [Epub ahead of print]
    Spatial epitope barcoding reveals clonal tumor patch behaviors.
    Xavier Rovira-Clavé, Alexandros P Drainas, Sizun Jiang, Yunhao Bai, Maya Baron, Bokai Zhu, Alec E Dallas, Myung Chang Lee, Theresa P Chu, Alessandra Holzem, Ramya Ayyagari, Debadrita Bhattacharya, Erin F McCaffrey, Noah F Greenwald, Maxim Markovic, Garry L Coles, Michael Angelo, Michael C Bassik, Julien Sage, Garry P Nolan.
      Intratumoral heterogeneity is a seminal feature of human tumors contributing to tumor progression and response to treatment. Current technologies are still largely unsuitable to accurately track phenotypes and clonal evolution within tumors, especially in response to genetic manipulations. Here, we developed epitopes for imaging using combinatorial tagging (EpicTags), which we coupled to multiplexed ion beam imaging (EpicMIBI) for in situ tracking of barcodes within tissue microenvironments. Using EpicMIBI, we dissected the spatial component of cell lineages and phenotypes in xenograft models of small cell lung cancer. We observed emergent properties from mixed clones leading to the preferential expansion of clonal patches for both neuroendocrine and non-neuroendocrine cancer cell states in these models. In a tumor model harboring a fraction of PTEN-deficient cancer cells, we observed a non-autonomous increase of clonal patch size in PTEN wild-type cancer cells. EpicMIBI facilitates in situ interrogation of cell-intrinsic and cell-extrinsic processes involved in intratumoral heterogeneity.
    Keywords:  MIBI; PTEN; SCLC; multiplex imaging; neuroendocrine; spatial barcoding; tumor heterogeneity
    DOI:  https://doi.org/10.1016/j.ccell.2022.09.014
  18. Cell Rep. 2022 Oct 11. pii: S2211-1247(22)01317-1. [Epub ahead of print]41(2): 111467
    An unusual mode of baseline translation adjusts cellular protein synthesis capacity to metabolic needs.
    Cornelius Schneider, Florian Erhard, Beyenech Binotti, Alexander Buchberger, Jörg Vogel, Utz Fischer.
      In all domains of life, mechanisms exist that adjust translational capacity to nutrient restriction and other growth constraints. The mammalian target of rapamycin (mTOR) regulates the synthesis of ribosomal proteins and translation factors in mammalian cells via phosphorylation of the La-related protein 1 (LARP1). In the present model of starvation-induced translational silencing, LARP1 targets mRNAs carrying a 5' terminal oligopyrimidine (5'TOP) motif to shift these into subpolysomal ribonucleoprotein particles. However, how these mRNAs would be protected from degradation and rapidly made available to restore translation capacity when needed remained enigmatic. Here, to address this, we employ gradient profiling by sequencing (Grad-seq) and monosome footprinting. Challenging the above model, we find that 5'TOP mRNAs, instead of being translationally silenced during starvation, undergo low baseline translation with reduced initiation rates. This mode of regulation ensures a stable 5'TOP mRNA population under starvation and allows fast reversibility of the translational repression.
    Keywords:  5'TOP; CP: Molecular biology; LARP1; TOP mRNAs; TOP response; baseline translation; mRNA; mTORC1; starvation; translation initiation; translation regulation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111467
  19. Nat Rev Mol Cell Biol. 2022 Oct 13.
    The physiology of alternative splicing.
    Luciano E Marasco, Alberto R Kornblihtt.
      Alternative splicing is a substantial contributor to the high complexity of transcriptomes of multicellular eukaryotes. In this Review, we discuss the accumulated evidence that most of this complexity is reflected at the protein level and fundamentally shapes the physiology and pathology of organisms. This notion is supported not only by genome-wide analyses but, mainly, by detailed studies showing that global and gene-specific modulations of alternative splicing regulate highly diverse processes such as tissue-specific and species-specific cell differentiation, thermal regulation, neuron self-avoidance, infrared sensing, the Warburg effect, maintenance of telomere length, cancer and autism spectrum disorders (ASD). We also discuss how mastering the control of alternative splicing paved the way to clinically approved therapies for hereditary diseases.
    DOI:  https://doi.org/10.1038/s41580-022-00545-z
  20. Dev Cell. 2022 Oct 10. pii: S1534-5807(22)00667-0. [Epub ahead of print]57(19): 2249-2250
    Neural crest cells hijack pluripotent stem cell factors to realize their developmental potential.
    Salome Stierli, Lukas Sommer.
      Neural crest cells arise in the neurectoderm of vertebrate embryos, but their developmental potential goes way beyond neurectodermal derivatives. In this issue of Developmental Cell, Hovland et al. reveal that neural crest cells re-employ embryonic stem cell factors in combination with specific transcription factors to enable their broad potential.
    DOI:  https://doi.org/10.1016/j.devcel.2022.09.010
  21. Sci Rep. 2022 Oct 10. 12(1): 16950
    Epidermal growth factor receptor cascade prioritizes the maximization of signal transduction.
    Kaori Kiso-Farnè, Tatsuaki Tsuruyama.
      Many studies have been performed to quantify cell signaling. Cell signaling molecules are phosphorylated in response to extracellular stimuli, with the phosphorylation sequence forming a signal cascade. The information gain during a signal event is given by the logarithm of the phosphorylation molecule ratio. The average information gain can be regarded as the signal transduction quantity (ST), which is identical to the Kullback-Leibler divergence (KLD), a relative entropy. We previously reported that if the total ST value in a given signal cascade is maximized, the ST rate (STR) of each signaling molecule per signal duration (min) approaches a constant value. To experimentally verify this theoretical conclusion, we measured the STR of the epidermal growth factor (EGF)-related cascade in A431 skin cancer cells following stimulation with EGF using antibody microarrays against phosphorylated signal molecules. The results were consistent with those from the theoretical analysis. Thus, signaling transduction systems may adopt a strategy that prioritizes the maximization of ST. Furthermore, signal molecules with similar STRs may form a signal cascade. In conclusion, ST and STR are promising properties for quantitative analysis of signal transduction.
    DOI:  https://doi.org/10.1038/s41598-022-20663-0
  22. Cells. 2022 Oct 05. pii: 3136. [Epub ahead of print]11(19):
    Systematic Interrogation of the Temperature Perturbation in the Insulin Signaling Pathway for Optogenetic Stimulation.
    Qi Dong, Mizuki Endo, Genki Kawamura, Takeaki Ozawa.
      The application of NIR to optogenetic systems is in great demand due to its superior properties enabling in vivo deep tissue penetration. Irradiation of NIR to tissue samples or cells rapidly generates heat locally. The resultant elevation in temperature affects cells at the molecular level because of the activation of the heat shock pathway and ROS generation. Nevertheless, few reports have presented detailed comparisons of the effects of the temperature change rate on signaling pathway biomolecules, especially those of rapid heat changes. Aiming at broadening the understanding of temperature sensitivity, we investigated seven insulin signaling pathway biomolecules (INSR, IRS1, Akt, GSK3β, p70S6K, FoxO1, and ERK1/2) in three cell lines (C2C12, HepG2, and Fao) at temperatures between 25 and 45 °C. The results show that, except for INSR, pAkt(T308), and FoxO1, biomolecules are sensitive to rapid temperature changes at temperatures higher than 42 °C, at which they are significantly phosphorylated. At 25 °C, around a 50% reduction in phosphorylation occurred. Moreover, p70S6K is sensitive over time. It dephosphorylates quickly (5 min) and then phosphorylates over time. Our findings extend the temperature range to 45 °C, while providing additional time course information about the signaling pathway biomolecule response necessary to advance NIR optogenetic research.
    Keywords:  NIR; insulin signaling; optogenetics; temperature
    DOI:  https://doi.org/10.3390/cells11193136
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