bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–05–12
nine papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases.
  2. Reconstructing Signaling Networks Using Biosensor Barcoding.
  3. MOSAIC enables in situ saturation mutagenesis of genes and CRISPR prime editing guide RNA optimization in human cells.
  4. The future of rapid and automated single-cell data analysis using reference mapping.
  5. Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.
  6. Harnessing noncanonical crRNA for highly efficient genome editing.
  7. Continuous distribution of cancer cells in the cell cycle unveiled by AI-segmented imaging of 37,000 HeLa FUCCI cells.
  8. Quantifying cell cycle regulation by tissue crowding.
  9. Tracing embryonic hematopoiesis guides induction of pluripotent stem cells to hematopoietic progenitors.
  1. Elife. 2024 May 07. pii: RP88991. [Epub ahead of print]12
    Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases.
    Benjamin R Duewell, Naomi E Wilson, Gabriela M Bailey, Sarah E Peabody, Scott D Hansen.
      Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
    Keywords:  G-proteins; GPCRs; Rho GTPases; biochemistry; chemical biology; human; phosphatidylinositol phosphate lipids; phosphoinositide 3-kinase; receptor tyrosine kinases
    DOI:  https://doi.org/10.7554/eLife.88991
  2. Methods Mol Biol. 2024 ;2800 189-202
    Reconstructing Signaling Networks Using Biosensor Barcoding.
    Suyang Wang, Wei-Yu Chi, Gabriel Au, Cheng-Chieh Huang, Jr-Ming Yang, Chuan-Hsiang Huang.
      Understanding how signaling networks are regulated offers valuable insights into how cells and organisms react to internal and external stimuli and is crucial for developing novel strategies to treat diseases. To achieve this, it is necessary to delineate the intricate interactions between the nodes in the network, which can be accomplished by measuring the activities of individual nodes under perturbation conditions. To facilitate this, we have recently developed a biosensor barcoding technique that enables massively multiplexed tracking of numerous signaling activities in live cells using genetically encoded fluorescent biosensors. In this chapter, we detail how we employed this method to reconstruct the EGFR signaling network by systematically monitoring the activities of individual nodes under perturbations.
    Keywords:  Deep learning; Epidermal growth factor receptor (EGFR); Genetically encoded fluorescent biosensors; Live cell imaging; Multiplexing; Receptor tyrosine kinases (RTKs); Signaling network; Small-molecule inhibitors
    DOI:  https://doi.org/10.1007/978-1-0716-3834-7_13
  3. bioRxiv. 2024 Apr 26. pii: 2024.04.25.591078. [Epub ahead of print]
    MOSAIC enables in situ saturation mutagenesis of genes and CRISPR prime editing guide RNA optimization in human cells.
    Jonathan Y Hsu, Kin Chung Lam, Justine Shih, Luca Pinello, J Keith Joung.
      CRISPR prime editing offers unprecedented versatility and precision for the installation of genetic edits in situ . Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for In situ Characterization ( MOSAIC ) method, which leverages a non-viral PCR-based prime editing method to enable rapid installation of thousands of defined edits in pooled fashion. We show that MOSAIC can be applied to perform in situ saturation mutagenesis screens of: (1) the BCR-ABL1 fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the IRF1 untranslated region (UTR), re-confirming non-coding regulatory elements involved in transcriptional initiation. Furthermore, we deployed MOSAIC to enable high-throughput, pooled screening of hundreds of systematically designed prime editing guide RNA ( pegRNA ) constructs for a large series of different genomic loci. This rapid screening of >18,000 pegRNA designs identified optimized pegRNAs for 89 different genomic target modifications and revealed the lack of simple predictive rules for pegRNA design, reinforcing the need for experimental optimization now greatly simplified and enabled by MOSAIC. We envision that MOSAIC will accelerate and facilitate the application of CRISPR prime editing for a wide range of high-throughput screens in human and other cell systems.
    DOI:  https://doi.org/10.1101/2024.04.25.591078
  4. Cell. 2024 May 09. pii: S0092-8674(24)00301-5. [Epub ahead of print]187(10): 2343-2358
    The future of rapid and automated single-cell data analysis using reference mapping.
    Mohammad Lotfollahi, Yuhan Hao, Fabian J Theis, Rahul Satija.
      As the number of single-cell datasets continues to grow rapidly, workflows that map new data to well-curated reference atlases offer enormous promise for the biological community. In this perspective, we discuss key computational challenges and opportunities for single-cell reference-mapping algorithms. We discuss how mapping algorithms will enable the integration of diverse datasets across disease states, molecular modalities, genetic perturbations, and diverse species and will eventually replace manual and laborious unsupervised clustering pipelines.
    Keywords:  cross-species comparisons; machine learning; multimodal analysis; reference mapping; single-cell analysis
    DOI:  https://doi.org/10.1016/j.cell.2024.03.009
  5. Nat Commun. 2024 May 10. 15(1): 3931
    Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.
    Lin Li-Bao, Covadonga Díaz-Díaz, Morena Raiola, Rocío Sierra, Susana Temiño, Francisco J Moya, Sandra Rodriguez-Perales, Elisa Santos, Giovanna Giovinazzo, Tore Bleckwehl, Álvaro Rada-Iglesias, Francois Spitz, Miguel Torres.
      MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.
    DOI:  https://doi.org/10.1038/s41467-024-48258-5
  6. Nat Commun. 2024 May 07. 15(1): 3823
    Harnessing noncanonical crRNA for highly efficient genome editing.
    Guanhua Xun, Zhixin Zhu, Nilmani Singh, Jingxia Lu, Piyush K Jain, Huimin Zhao.
      The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.
    DOI:  https://doi.org/10.1038/s41467-024-48012-x
  7. Heliyon. 2024 May 15. 10(9): e30239
    Continuous distribution of cancer cells in the cell cycle unveiled by AI-segmented imaging of 37,000 HeLa FUCCI cells.
    Hamid Cheraghi, Kinga Dóra Kovács, Inna Székács, Robert Horvath, Bálint Szabó.
      Classification of live or fixed cells based on their unlabeled microscopic images would be a powerful tool for cell biology and pathology. For such software, the first step is the generation of a ground truth database that can be used for training and testing AI classification algorithms. The Application of cells expressing fluorescent reporter proteins allows the building of ground truth datasets in a straightforward way. In this study, we present an automated imaging pipeline utilizing the Cellpose algorithm for the precise cell segmentation and measurement of fluorescent cellular intensities across multiple channels. We analyzed the cell cycle of HeLa-FUCCI cells expressing fluorescent red and green reporter proteins at various levels depending on the cell cycle state. To build the dataset, 37,000 fixed cells were automatically scanned using a standard motorized microscope, capturing phase contrast and fluorescent red/green images. The fluorescent pixel intensity of each cell was integrated to calculate the total fluorescence of cells based on cell segmentation in the phase contrast channel. It resulted in a precise intensity value for each cell in both channels. Furthermore, we conducted a comparative analysis of Cellpose 1.0 and Cellpose 2.0 in cell segmentation performance. Cellpose 2.0 demonstrated notable improvements, achieving a significantly reduced false positive rate of 2.7 % and 1.4 % false negative. The cellular fluorescence was visualized in a 2D plot (map) based on the red and green intensities of the FUCCI construct revealing the continuous distribution of cells in the cell cycle. This 2D map enables the selection and potential isolation of single cells in a specific phase. In the corresponding heatmap, two clusters appeared representing cells in the red and green states. Our pipeline allows the high-throughput and accurate measurement of cellular fluorescence providing extensive statistical information on thousands of cells with potential applications in developmental and cancer biology. Furthermore, our method can be used to build ground truth datasets automatically for training and testing AI cell classification. Our automated pipeline can be used to analyze thousands of cells within 2 h after putting the sample onto the microscope.
    Keywords:  Cell cycle analysis; Cell segmentation; Fluorescence imaging; Ground truth datasets
    DOI:  https://doi.org/10.1016/j.heliyon.2024.e30239
  8. Biophys J. 2024 May 06. pii: S0006-3495(24)00317-5. [Epub ahead of print]
    Quantifying cell cycle regulation by tissue crowding.
    Carles Falcó, Daniel J Cohen, José A Carrillo, Ruth E Baker.
      The spatiotemporal coordination and regulation of cell proliferation is fundamental in many aspects of development and tissue maintenance. Cells have the ability to adapt their division rates in response to mechanical constraints, yet we do not fully understand how cell proliferation regulation impacts cell migration phenomena. Here, we present a minimal continuum model of cell migration with cell cycle dynamics, which includes density-dependent effects and hence can account for cell proliferation regulation. By combining minimal mathematical modelling, Bayesian inference, and recent experimental data, we quantify the impact of tissue crowding across different cell cycle stages in epithelial tissue expansion experiments. Our model suggests that cells sense local density and adapt cell cycle progression in response, during G1 and the combined S/G2/M phases, providing an explicit relationship between each cell cycle stage duration and local tissue density, which is consistent with several experimental observations. Finally, we compare our mathematical model predictions to different experiments studying cell cycle regulation and present a quantitative analysis on the impact of density-dependent regulation on cell migration patterns. Our work presents a systematic approach for investigating and analysing cell cycle data, providing mechanistic insights into how individual cells regulate proliferation, based on population-based experimental measurements.
    DOI:  https://doi.org/10.1016/j.bpj.2024.05.003
  9. Dev Cell. 2024 May 06. pii: S1534-5807(24)00226-0. [Epub ahead of print]59(9): 1093-1095
    Tracing embryonic hematopoiesis guides induction of pluripotent stem cells to hematopoietic progenitors.
    Xinjian Mao, Linheng Li.
      In this issue of Developmental Cell, Fowler et al. applied genetic lineage-tracing mouse models to support the notion that artery endothelial cells are the predominant source of hematopoietic stem cells. They leveraged this and developed a method capable of efficiently differentiating human pluripotent stem cells into HLF+HOXA+ hematopoietic progenitors.
    DOI:  https://doi.org/10.1016/j.devcel.2024.04.001
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