bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–08–11
fourteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP.
  2. Stress pathway outputs are encoded by pH-dependent clustering of kinase components.
  3. Quantification of collective signalling in time-lapse microscopy images.
  4. Thermal stability of bivalent cation/phosphoinositide domains in model membranes.
  5. Akt is a mediator of artery specification during zebrafish development.
  6. PIK3CA mutations enhance the adipogenesis of ADSCs in facial infiltrating lipomatosis through TRPV1.
  7. Targeted Treatments for Vascular Malformations: Current State of the Art.
  8. Single-cell sensor analyses reveal signaling programs enabling Ras-G12C drug resistance.
  9. A unified model for interpretable latent embedding of multi-sample, multi-condition single-cell data.
  10. eIF4E-independent translation is largely eIF3d-dependent.
  11. The origin of novel traits in cancer.
  12. CellTracksColab is a platform that enables compilation, analysis, and exploration of cell tracking data.
  13. Lysosomal TBK1 responds to amino acid availability to relieve Rab7-dependent mTORC1 inhibition.
  14. Nucleocytoplasmic transport senses mechanics independently of cell density in cell monolayers.
  1. Nat Protoc. 2024 Aug 08.
    Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP.
    Andrew Leduc, Luke Khoury, Joshua Cantlon, Saad Khan, Nikolai Slavov.
      Single-cell proteomics by mass spectrometry (MS) allows the quantification of proteins with high specificity and sensitivity. To increase its throughput, we developed nano-proteomic sample preparation (nPOP), a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation using the plexDIA MS multiplexing method, which uses non-isobaric mass tags to barcode peptides from different samples for data-independent acquisition, demonstrates accurate quantification of ~3,000-3,700 proteins per human cell. A separate implementation with isobaric mass tags and prioritized data acquisition demonstrates analysis of 1,827 single cells at a rate of >1,000 single cells per day at a depth of 800-1,200 proteins per human cell. The protocol is implemented by using a cell-dispensing and liquid-handling robot-the CellenONE instrument-and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 d to prepare >3,000 single cells. We provide metrics and software (the QuantQC R package) for quality control and data exploration. QuantQC supports the robust scaling of nPOP to higher plex reagents for achieving reliable and scalable single-cell proteomics.
    DOI:  https://doi.org/10.1038/s41596-024-01033-8
  2. Nat Commun. 2024 Aug 05. 15(1): 6614
    Stress pathway outputs are encoded by pH-dependent clustering of kinase components.
    Yuliia Didan, Milad Ghomlaghi, Lan K Nguyen, Dominic C H Ng.
      Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses.
    DOI:  https://doi.org/10.1038/s41467-024-50638-w
  3. Methods Microsc. 2024 Apr;1(1): 19-30
    Quantification of collective signalling in time-lapse microscopy images.
    Maciej Dobrzyński, Benjamin Grädel, Paolo Armando Gagliardi, Olivier Pertz.
      Live-cell imaging of fluorescent biosensors has demonstrated that space-time correlations in signalling of cell collectives play an important organisational role in morphogenesis, wound healing, regeneration, and maintaining epithelial homeostasis. Here, we demonstrate how to quantify one such phenomenon, namely apoptosis-induced ERK activity waves in the MCF10A epithelium. We present a protocol that starts from raw time-lapse fluorescence microscopy images and, through a sequence of image manipulations, ends with ARCOS, our computational method to detect and quantify collective signalling. We also describe the same workflow in the interactive napari image viewer to quantify collective phenomena for users without prior programming experience. Our approach can be applied to space-time correlations in cells, cell collectives, or communities of multicellular organisms, in 2D and 3D geometries.
    Keywords:  cell signalling; collective phenomena; image analysis; spatial clustering; time-lapse microscopy
    DOI:  https://doi.org/10.1515/mim-2024-0003
  4. Chem Phys Lipids. 2024 Aug 02. pii: S0009-3084(24)00049-5. [Epub ahead of print]264 105424
    Thermal stability of bivalent cation/phosphoinositide domains in model membranes.
    Trevor A Paratore, Greta E Schmidt, Alonzo H Ross, Arne Gericke.
      As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg2+ ions. While PI(4)P shows in the presence of Ca2+ no clustering, PI(4,5)P2 forms with Ca2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P3 with Ca2+ is less than what was observed for PI(4,5)P2, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg2+ or Ca2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca2+ with PI(4,5)P2. Also, in the presence of cholesterol, the interaction of Mg2+ with PI(4,5)P2 remains weak. PI(3,4,5)P3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca2+ and Mg2+ was not influenced by the presence of cholesterol.
    Keywords:  PI(3,4,5)P3; PI(4)P; PI(4,5)P2; PIP2; PIP3; Phosphoinositide
    DOI:  https://doi.org/10.1016/j.chemphyslip.2024.105424
  5. Development. 2024 Aug 05. pii: dev.202727. [Epub ahead of print]
    Akt is a mediator of artery specification during zebrafish development.
    Wenping Zhou, Joey J Ghersi, Emma Ristori, Nicole Semanchik, Andrew Prendergast, Rong Zhang, Paola Carneiro, Gabriel Baldissera, William C Sessa, Stefania Nicoli.
      The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. While vascular endothelial growth factor a (VEGF-A) drives DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B, Akt, in zebrafish by generating a quadruple mutant (aktΔ/Δ), where expression and activity of all akt genes-akt 1, 2, 3a, and 3b are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to reestablish normal arterial specification in aktΔ/Δ. The Akt-loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.
    Keywords:  Akt and Notch signaling; Artery specification; Endothelial cells; Single cell RNA sequencing.; Vascular development; Zebrafish embryo
    DOI:  https://doi.org/10.1242/dev.202727
  6. iScience. 2024 Aug 16. 27(8): 110467
    PIK3CA mutations enhance the adipogenesis of ADSCs in facial infiltrating lipomatosis through TRPV1.
    Hongrui Chen, Bin Sun, Wei Gao, Yajing Qiu, Wei Wei, Yongguo Li, Wei Ye, Haoliang Song, Chen Hua, Xiaoxi Lin.
      Facial infiltrating lipomatosis (FIL) is a congenital disorder. The pathogenesis of FIL is associated with PIK3CA mutations, but the underlying mechanisms remain undetermined. We found that the adipose tissue in FIL demonstrated adipocytes hypertrophy and increased lipid accumulation. All adipose-derived mesenchymal stem cells from FIL (FIL-ADSCs) harbored PIK3CA mutations. Moreover, FIL-ADSCs exhibited a greater capacity for adipogenesis. Knockdown of PIK3CA resulted in a reduction in the adipogenic potential of FIL-ADSCs. Furthermore, WX390, a dual-target PI3K/mTOR inhibitor, was found to impede PIK3CA-mediated adipogenesis both in vivo and in vitro. RNA sequencing (RNA-seq) revealed that the expression of transient receptor potential vanilloid subtype 1 (TRPV1) was upregulated after PI3K pathway inhibition, and overexpression or activation of TRPV1 both inhibited adipogenesis. Our study showed that PIK3CA mutations promoted adipogenesis in FIL-ADSCs and this effect was achieved by suppressing TPRV1. Pathogenesis experiments suggested that WX390 may serve as an agent for the treatment of FIL.
    Keywords:  cell biology; molecular biology; physiology; stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2024.110467
  7. J Thromb Haemost. 2024 Aug 01. pii: S1538-7836(24)00430-6. [Epub ahead of print]
    Targeted Treatments for Vascular Malformations: Current State of the Art.
    Emmanuel Seront, Cedric Hermans, Laurence M Boon, Miikka Vikkula.
      Vascular malformations, which arise from anomalies in angiogenesis, encompass capillary, lymphatic, venous, arteriovenous and mixed malformations, each affecting specific vessel types. Historically, therapeutic options such as sclerotherapy and surgery have shown limited efficacy in complicated malformations. Most vascular malformations stem from hereditary or somatic mutations akin to oncogenic alterations, activating the PI3K-AKT-mTOR, RAS-MAPK-ERK, and G-protein coupled receptor pathways. Recognizing the parallels with oncogenic mutations, we emphasize the potential of targeted molecular inhibitors in the treatment of vascular malformations by repurposing anticancer drugs. This review delves into the recent development and future use of such agents for the management of slow- and fast-flow vascular malformations, including in more specific situations, such as prenatal treatment and the management of associated coagulopathies.
    Keywords:  VASE; antiangiogenic; anticoagulation; sirolimus; thalidomide
    DOI:  https://doi.org/10.1016/j.jtha.2024.07.013
  8. Nat Chem Biol. 2024 Aug 05.
    Single-cell sensor analyses reveal signaling programs enabling Ras-G12C drug resistance.
    Jason Z Zhang, Shao-En Ong, David Baker, Dustin J Maly.
      Clinical resistance to rat sarcoma virus (Ras)-G12C inhibitors is a challenge. A subpopulation of cancer cells has been shown to undergo genomic and transcriptional alterations to facilitate drug resistance but the immediate adaptive effects on Ras signaling in response to these drugs at the single-cell level is not well understood. Here, we used Ras biosensors to profile the activity and signaling environment of endogenous Ras at the single-cell level. We found that a subpopulation of KRas-G12C cells treated with Ras-G12C-guanosine-diphosphate inhibitors underwent adaptive signaling and metabolic changes driven by wild-type Ras at the Golgi and mutant KRas at the mitochondria, respectively. Our Ras biosensors identified major vault protein as a mediator of Ras activation through its scaffolding of Ras signaling pathway components and metabolite channels. Overall, methods including ours that facilitate direct analysis on the single-cell level can report the adaptations that subpopulations of cells adopt in response to cancer therapies, thus providing insight into drug resistance.
    DOI:  https://doi.org/10.1038/s41589-024-01684-4
  9. Nat Commun. 2024 Aug 03. 15(1): 6573
    A unified model for interpretable latent embedding of multi-sample, multi-condition single-cell data.
    Ariel Madrigal, Tianyuan Lu, Larisa M Soto, Hamed S Najafabadi.
      Single-cell analysis across multiple samples and conditions requires quantitative modeling of the interplay between the continuum of cell states and the technical and biological sources of sample-to-sample variability. We introduce GEDI, a generative model that identifies latent space variations in multi-sample, multi-condition single-cell datasets and attributes them to sample-level covariates. GEDI enables cross-sample cell state mapping on par with state-of-the-art integration methods, cluster-free differential gene expression analysis along the continuum of cell states, and machine learning-based prediction of sample characteristics from single-cell data. GEDI can also incorporate gene-level prior knowledge to infer pathway and regulatory network activities in single cells. Finally, GEDI extends all these concepts to previously unexplored modalities that require joint consideration of dual measurements, such as the joint analysis of exon inclusion/exclusion reads to model alternative cassette exon splicing, or spliced/unspliced reads to model the mRNA stability landscapes of single cells.
    DOI:  https://doi.org/10.1038/s41467-024-50963-0
  10. Nat Commun. 2024 Aug 06. 15(1): 6692
    eIF4E-independent translation is largely eIF3d-dependent.
    Mykola Roiuk, Marilena Neff, Aurelio A Teleman.
      Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of 'canonical' translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.
    DOI:  https://doi.org/10.1038/s41467-024-51027-z
  11. Trends Cancer. 2024 Aug 06. pii: S2405-8033(24)00145-6. [Epub ahead of print]
    The origin of novel traits in cancer.
    Steven A Frank, Itai Yanai.
      The traditional view of cancer emphasizes a genes-first process. Novel cancer traits arise by genetic mutations that spread to drive phenotypic change. However, recent data support a phenotypes-first process in which nonheritable cellular variability creates novel traits that later become heritably stabilized by genetic and epigenetic changes. Single-cell measurements reinforce the idea that phenotypes lead genotypes, showing how cancer evolution follows normal developmental plasticity and creates novel traits by recombining parts of different cellular developmental programs. In parallel, studies in evolutionary biology also support a phenotypes-first process driven by developmental plasticity and developmental recombination. These advances in cancer research and evolutionary biology mutually reinforce a revolution in our understanding of how cells and organisms evolve novel traits in response to environmental challenges.
    Keywords:  cancer progression; cellular plasticity; drug resistance; evolution
    DOI:  https://doi.org/10.1016/j.trecan.2024.07.005
  12. PLoS Biol. 2024 Aug 08. 22(8): e3002740
    CellTracksColab is a platform that enables compilation, analysis, and exploration of cell tracking data.
    Estibaliz Gómez-de-Mariscal, Hanna Grobe, Joanna W Pylvänäinen, Laura Xénard, Ricardo Henriques, Jean-Yves Tinevez, Guillaume Jacquemet.
      In life sciences, tracking objects from movies enables researchers to quantify the behavior of single particles, organelles, bacteria, cells, and even whole animals. While numerous tools now allow automated tracking from video, a significant challenge persists in compiling, analyzing, and exploring the large datasets generated by these approaches. Here, we introduce CellTracksColab, a platform tailored to simplify the exploration and analysis of cell tracking data. CellTracksColab facilitates the compiling and analysis of results across multiple fields of view, conditions, and repeats, ensuring a holistic dataset overview. CellTracksColab also harnesses the power of high-dimensional data reduction and clustering, enabling researchers to identify distinct behavioral patterns and trends without bias. Finally, CellTracksColab also includes specialized analysis modules enabling spatial analyses (clustering, proximity to specific regions of interest). We demonstrate CellTracksColab capabilities with 3 use cases, including T cells and cancer cell migration, as well as filopodia dynamics. CellTracksColab is available for the broader scientific community at https://github.com/CellMigrationLab/CellTracksColab.
    DOI:  https://doi.org/10.1371/journal.pbio.3002740
  13. EMBO J. 2024 Aug 05.
    Lysosomal TBK1 responds to amino acid availability to relieve Rab7-dependent mTORC1 inhibition.
    Gabriel Talaia, Amanda Bentley-DeSousa, Shawn M Ferguson.
      Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. This lysosomal TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.
    Keywords:  ALS-FTD; Lysosome; Nutrient Sensing; TBK1; mTORC1
    DOI:  https://doi.org/10.1038/s44318-024-00180-8
  14. J Cell Sci. 2024 Aug 09. pii: jcs.262363. [Epub ahead of print]
    Nucleocytoplasmic transport senses mechanics independently of cell density in cell monolayers.
    Ignasi Granero-Moya, Valeria Venturini, Guillaume Belthier, Bart Groenen, Marc Molina-Jordán, Miguel González-Martín, Xavier Trepat, Jacco van Rheenen, Ion Andreu, Pere Roca-Cusachs.
      Cells sense and respond to mechanical forces through mechanotransduction, which regulates processes in health and disease. In single adhesive cells, mechanotransduction involves the transmission of force from the extracellular matrix to the cell nucleus, where it affects nucleocytoplasmic transport (NCT) and the subsequent nuclear localization of transcriptional regulators such as YAP. However, if and how NCT is mechanosensitive in multicellular systems is unclear. Here, we characterize and use a fluorescent sensor of nucleocytoplasmic transport (Sencyt) and demonstrate that nucleocytoplasmic transport responds to mechanics but not cell density in cell monolayers. Using monolayers of both epithelial and mesenchymal phenotype, we show that NCT is altered in response both to osmotic shocks, and to the inhibition of cell contractility. Further, NCT correlates with the degree of nuclear deformation measured through nuclear solidity, a shape parameter related to nuclear envelope tension. In contrast, YAP but NCT is sensitive to cell density, showing that YAP response to cell-cell contacts is not via a mere mechanical effect of NCT. Our results demonstrate the generality of the mechanical regulation of NCT.
    Keywords:  Cell nucleus; Mechanobiology; Mechanotransduction; Sensor
    DOI:  https://doi.org/10.1242/jcs.262363
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