bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–10–13
seventeen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Spatial and functional separation of mTORC1 signalling in response to different amino acid sources.
  2. AKT-mediated phosphorylation of TSC2 controls stimulus- and tissue-specific mTORC1 signaling and organ growth.
  3. Scalable, compressed phenotypic screening using pooled perturbations.
  4. Non-Hotspot PIK3CA Variants Have Higher Variant Allele Frequency and are More Common in Syndromic Vascular Malformations.
  5. Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth.
  6. kallisto, bustools and kb-python for quantifying bulk, single-cell and single-nucleus RNA-seq.
  7. Tracking and Measuring Local Protein Synthesis In Vivo.
  8. Long-term breast cancer response to CDK4/6 inhibition defined by TP53-mediated geroconversion.
  9. A Genetically Encoded Fluorescent Biosensor for Intracellular Measurement of Malonyl-CoA.
  10. The fate of pyruvate dictates cell growth by modulating cellular redox potential.
  11. Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView.
  12. Experimental variables determine the outcome of RAS-RAS interactions.
  13. Deep learning pipeline for automated cell profiling from cyclic imaging.
  14. Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells.
  15. Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap.
  16. Developmental regulation of endothelial-to-hematopoietic transition from induced pluripotent stem cells.
  17. Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.
  1. Nat Cell Biol. 2024 Oct 09.
    Spatial and functional separation of mTORC1 signalling in response to different amino acid sources.
    Stephanie A Fernandes, Danai-Dimitra Angelidaki, Julian Nüchel, Jiyoung Pan, Peter Gollwitzer, Yoav Elkis, Filippo Artoni, Sabine Wilhelm, Marija Kovacevic-Sarmiento, Constantinos Demetriades.
      Amino acid (AA) availability is a robust determinant of cell growth through controlling mechanistic/mammalian target of rapamycin complex 1 (mTORC1) activity. According to the predominant model in the field, AA sufficiency drives the recruitment and activation of mTORC1 on the lysosomal surface by the heterodimeric Rag GTPases, from where it coordinates the majority of cellular processes. Importantly, however, the teleonomy of the proposed lysosomal regulation of mTORC1 and where mTORC1 acts on its effector proteins remain enigmatic. Here, by using multiple pharmacological and genetic means to perturb the lysosomal AA-sensing and protein recycling machineries, we describe the spatial separation of mTORC1 regulation and downstream functions in mammalian cells, with lysosomal and non-lysosomal mTORC1 phosphorylating distinct substrates in response to different AA sources. Moreover, we reveal that a fraction of mTOR localizes at lysosomes owing to basal lysosomal proteolysis that locally supplies new AAs, even in cells grown in the presence of extracellular nutrients, whereas cytoplasmic mTORC1 is regulated by exogenous AAs. Overall, our study substantially expands our knowledge about the topology of mTORC1 regulation by AAs and hints at the existence of distinct, Rag- and lysosome-independent mechanisms that control its activity at other subcellular locations. Given the importance of mTORC1 signalling and AA sensing for human ageing and disease, our findings will probably pave the way towards the identification of function-specific mTORC1 regulators and thus highlight more effective targets for drug discovery against conditions with dysregulated mTORC1 activity in the future.
    DOI:  https://doi.org/10.1038/s41556-024-01523-7
  2. bioRxiv. 2024 Sep 23. pii: 2024.09.23.614519. [Epub ahead of print]
    AKT-mediated phosphorylation of TSC2 controls stimulus- and tissue-specific mTORC1 signaling and organ growth.
    Yann Cormerais, Samuel C Lapp, Krystle C Kalafut, Madi Y Cissé, Jong Shin, Benjamin Stefadu, Jean Personnaz, Sandra Schrotter, Angelica D'Amore, Emma R Martin, Catherine L Salussolia, Mustafa Sahin, Suchithra Menon, Vanessa Byles, Brendan D Manning.
      Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.
    DOI:  https://doi.org/10.1101/2024.09.23.614519
  3. Nat Biotechnol. 2024 Oct 07.
    Scalable, compressed phenotypic screening using pooled perturbations.
    Nuo Liu, Walaa E Kattan, Benjamin E Mead, Conner Kummerlowe, Thomas Cheng, Sarah Ingabire, Jaime H Cheah, Christian K Soule, Anita Vrcic, Jane K McIninch, Sergio Triana, Manuel Guzman, Tyler T Dao, Joshua M Peters, Kristen E Lowder, Lorin Crawford, Ava P Amini, Paul C Blainey, William C Hahn, Brian Cleary, Bryan Bryson, Peter S Winter, Srivatsan Raghavan, Alex K Shalek.
      High-throughput phenotypic screens using biochemical perturbations and high-content readouts are constrained by limitations of scale. To address this, we establish a method of pooling exogenous perturbations followed by computational deconvolution to reduce required sample size, labor and cost. We demonstrate the increased efficiency of compressed experimental designs compared to conventional approaches through benchmarking with a bioactive small-molecule library and a high-content imaging readout. We then apply compressed screening in two biological discovery campaigns. In the first, we use early-passage pancreatic cancer organoids to map transcriptional responses to a library of recombinant tumor microenvironment protein ligands, uncovering reproducible phenotypic shifts induced by specific ligands distinct from canonical reference signatures and correlated with clinical outcome. In the second, we identify the pleotropic modulatory effects of a chemical compound library with known mechanisms of action on primary human peripheral blood mononuclear cell immune responses. In sum, our approach empowers phenotypic screens with information-rich readouts to advance drug discovery efforts and basic biological inquiry.
    DOI:  https://doi.org/10.1038/s41587-024-02403-z
  4. Am J Med Genet A. 2024 Oct 07. e63883
    Non-Hotspot PIK3CA Variants Have Higher Variant Allele Frequency and are More Common in Syndromic Vascular Malformations.
    Themis-Areti A Andreoti, Massimo Maiolo, Aleksandra Tuleja, Yvonne Döring, André Schaller, Erik Vassella, Laurence M Boon, Iris Baumgartner, Sarah M Bernhard, Christiane Zweier, Miikka Vikkula, Jochen Rössler.
      PIK3CA variants are known to cause vascular malformations. We were interested in studying the phenotypic spectrum, the location within the PIK3CA gene, and the variant allele frequency (VAF) of somatic PI3KCA variants in vascular malformations. Clinical data of consecutive patients with extracranial/extraspinal vascular malformations were collected in the context of the VASCOM cohort (2008-2022, n = 558). Starting October 2020, biopsy samples were tested with the TSO500 gene panel (Illumina). All consenting patients with PIK3CA variants were included in this study. Eighty-nine patients had available genetic results by June 2022. PIK3CA variants (n = 25) were found in 16 simple/combined (nonsyndromic) vascular malformations and in nine vascular malformations associated with other anomalies (syndromic). Four hotspot variants in exons 9 and 20 (c.1624G>A, c.1633G>A, c.3140A>G, c.3140A>T) were identified in 16/25 patients (VAF 0.9%-9.7%). Six non-hotspot variants (c.328_330del, c.323_337del, c.353G>A, c.1258T>C, c.3132T>A, c.3195_3203delinsT) were detected in nine patients (VAF 3.6%-31.7%). Non-hotspot variants were more frequent in syndromic than nonsyndromic vascular malformations (p = 0.0034) and exhibited a higher VAF than hotspot variants (p = 0.0253). Our study contributes to the growing body of knowledge of the genetic background in vascular malformations. Further studies will enrich the ever-growing list of pathogenic PIK3CA variants associated with vascular malformations.
    Keywords:   PIK3CA ; PI3K; pathogenic variant; somatic mutation; vascular malformation
    DOI:  https://doi.org/10.1002/ajmg.a.63883
  5. Nat Commun. 2024 Oct 06. 15(1): 8660
    Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth.
    Apeksha Shapeti, Jorge Barrasa-Fano, Abdel Rahman Abdel Fattah, Janne de Jong, José Antonio Sanz-Herrera, Mylène Pezet, Said Assou, Emilie de Vet, Seyed Ali Elahi, Adrian Ranga, Eva Faurobert, Hans Van Oosterwyck.
      Force-driven cellular interactions are crucial for cancer cell invasion but remain underexplored in vascular abnormalities. Cerebral cavernous malformations (CCM), a vascular abnormality characterized by leaky vessels, involves CCM mutant cells recruiting wild-type endothelial cells to form and expand mosaic lesions. The mechanisms behind this recruitment remain poorly understood. Here, we use an in-vitro model of angiogenic invasion with traction force microscopy to reveal that hyper-angiogenic Ccm2-silenced endothelial cells enhance angiogenic invasion of neighboring wild-type cells through force and extracellular matrix-guided mechanisms. We demonstrate that mechanically hyperactive CCM2-silenced tips guide wild-type cells by transmitting pulling forces and by creating paths in the matrix, in a ROCKs-dependent manner. This is associated with reinforcement of β1 integrin and actin cytoskeleton in wild-type cells. Further, wild-type cells are reprogrammed into stalk cells and activate matrisome and DNA replication programs, thereby initiating proliferation. Our findings reveal how CCM2 mutants hijack wild-type cell functions to fuel lesion growth, providing insight into the etiology of vascular malformations. By integrating biophysical and molecular techniques, we offer tools for studying cell mechanics in tissue heterogeneity and disease progression.
    DOI:  https://doi.org/10.1038/s41467-024-52866-6
  6. Nat Protoc. 2024 Oct 10.
    kallisto, bustools and kb-python for quantifying bulk, single-cell and single-nucleus RNA-seq.
    Delaney K Sullivan, Kyung Hoi Joseph Min, Kristján Eldjárn Hjörleifsson, Laura Luebbert, Guillaume Holley, Lambda Moses, Johan Gustafsson, Nicolas L Bray, Harold Pimentel, A Sina Booeshaghi, Páll Melsted, Lior Pachter.
      The term 'RNA-seq' refers to a collection of assays based on sequencing experiments that involve quantifying RNA species from bulk tissue, single cells or single nuclei. The kallisto, bustools and kb-python programs are free, open-source software tools for performing this analysis that together can produce gene expression quantification from raw sequencing reads. The quantifications can be individualized for multiple cells, multiple samples or both. Additionally, these tools allow gene expression values to be classified as originating from nascent RNA species or mature RNA species, making this workflow amenable to both cell-based and nucleus-based assays. This protocol describes in detail how to use kallisto and bustools in conjunction with a wrapper, kb-python, to preprocess RNA-seq data. Execution of this protocol requires basic familiarity with a command line environment. With this protocol, quantification of a moderately sized RNA-seq dataset can be completed within minutes.
    DOI:  https://doi.org/10.1038/s41596-024-01057-0
  7. Development. 2024 Oct 07. pii: dev.202908. [Epub ahead of print]
    Tracking and Measuring Local Protein Synthesis In Vivo.
    Ibrahim Kays, Brian E Chen.
      Detecting when and how much a protein molecule is synthesized is important for understanding cell function, but current methods either cannot be performed in vivo or have poor temporal resolution. Here, we developed a technique to detect and quantify subcellular protein synthesis events in real time in vivo. This Protein Translation Reporting (PTR) technique uses a genetic tag that produces a stoichiometric ratio of a small peptide portion of a split fluorescent protein and the protein of interest during protein synthesis. We show that the split fluorescent protein peptide can generate fluorescence within milliseconds upon binding the larger portion of the fluorescent protein, and that the fluorescence intensity is directly proportional to the number of molecules of the protein of interest synthesized. Using PTR, we tracked and measured protein synthesis events in single cells over time in vivo. We use different color split fluorescent proteins to detect multiple genes or alleles in single cells simultaneously. We also split a photoswitchable fluorescent protein to photoconvert the reconstituted fluorescent protein to a different channel to continually reset the time of detection of synthesis events.
    Keywords:  live imaging; local translation; protein quantification; protein synthesis
    DOI:  https://doi.org/10.1242/dev.202908
  8. Cancer Cell. 2024 Oct 04. pii: S1535-6108(24)00357-X. [Epub ahead of print]
    Long-term breast cancer response to CDK4/6 inhibition defined by TP53-mediated geroconversion.
    Rei Kudo, Anton Safonov, Catherine Jones, Enrico Moiso, Jonathan R Dry, Hong Shao, Sharanya Nag, Edaise M da Silva, Selma Yeni Yildirim, Qing Li, Elizabeth O'Connell, Payal Patel, Marie Will, Atsushi Fushimi, Marimar Benitez, Martina Bradic, Li Fan, Harikrishna Nakshatri, Dhivya R Sudhan, Christopher R Denz, Jorge S Reis-Filho, Shom Goel, Andrew Koff, Britta Weigelt, Qamar J Khan, Pedram Razavi, Sarat Chandarlapaty.
      Inhibition of CDK4/6 kinases has led to improved outcomes in breast cancer. Nevertheless, only a minority of patients experience long-term disease control. Using a large, clinically annotated cohort of patients with metastatic hormone receptor-positive (HR+) breast cancer, we identify TP53 loss (27.6%) and MDM2 amplification (6.4%) to be associated with lack of long-term disease control. Human breast cancer models reveal that p53 loss does not alter CDK4/6 activity or G1 blockade but instead promotes drug-insensitive p130 phosphorylation by CDK2. The persistence of phospho-p130 prevents DREAM complex assembly, enabling cell-cycle re-entry and tumor progression. Inhibitors of CDK2 can overcome p53 loss, leading to geroconversion and manifestation of senescence phenotypes. Complete inhibition of both CDK4/6 and CDK2 kinases appears to be necessary to facilitate long-term response across genomically diverse HR+ breast cancers.
    Keywords:  CDK2; CDK4/6; breast cancer; cell cycle; cyclin dependent kinase; drug resistance; p53; quiescence; senescence
    DOI:  https://doi.org/10.1016/j.ccell.2024.09.009
  9. bioRxiv. 2024 Sep 28. pii: 2024.09.27.615526. [Epub ahead of print]
    A Genetically Encoded Fluorescent Biosensor for Intracellular Measurement of Malonyl-CoA.
    Brodie L Ranzau, Tiffany D Robinson, Jack M Scully, Edmund D Kapelczack, Teagan S Dean, Tara TeSlaa, Danielle L Schmitt.
      Malonyl-CoA is the essential building block of fatty acids and regulates cell function through protein malonylation and allosteric regulation of signaling networks. Accordingly, the production and use of malonyl-CoA is finely tuned by the cellular energy status. Most studies of malonyl-CoA dynamics rely on bulk approaches that take only a snapshot of the average metabolic state of a population of cells, missing out on dynamic changes in malonyl-CoA and fatty acid biosynthesis that could be occurring within a single cell. To overcome this limitation, we have developed a genetically encoded fluorescent protein-based biosensor for malonyl-CoA that can be used to capture malonyl-CoA dynamics in single cells. This biosensor, termed Malibu (malonyl-CoA i ntracellular biosensor to understand dynamics), exhibits an excitation-ratiometric change in response to malonyl-CoA binding. We first used Malibu to monitor malonyl-CoA dynamics during inhibition of fatty acid biosynthesis using cerulenin in E. coli , observing an increase in Malibu response in a time- and dose-dependent manner. In HeLa cells, we used Malibu to monitor the impact of fatty acid biosynthesis inhibition on malonyl-CoA dynamics in single cells, finding that two inhibitors of fatty acid biosynthesis, cerulenin and orlistat, which inhibit different steps of fatty acid biosynthesis, increase malonyl-CoA levels. Altogether, we have developed a new genetically encoded biosensor for malonyl-CoA, which can be used to sensitively study malonyl-CoA dynamics in single cells, providing an unparalleled view into fatty acid biosynthesis.
    DOI:  https://doi.org/10.1101/2024.09.27.615526
  10. bioRxiv. 2024 Sep 24. pii: 2024.09.23.614588. [Epub ahead of print]
    The fate of pyruvate dictates cell growth by modulating cellular redox potential.
    Ashish G Toshniwal, Geanette Lam, Alex J Bott, Ahmad A Cluntun, Rachel Skabelund, Hyuck-Jin Nam, Dona R Wisidagama, Carl S Thummel, Jared Rutter.
      Pyruvate occupies a central node in carbohydrate metabolism such that how it is produced and consumed can optimize a cell for energy production or biosynthetic capacity. This has been primarily studied in proliferating cells, but observations from the post-mitotic Drosophila fat body led us to hypothesize that pyruvate fate might dictate the rapid cell growth observed in this organ during development. Indeed, we demonstrate that augmented mitochondrial pyruvate import prevented cell growth in fat body cells in vivo as well as in cultured mammalian hepatocytes and human hepatocyte-derived cells in vitro . This effect on cell size was caused by an increase in the NADH/NAD + ratio, which rewired metabolism toward gluconeogenesis and suppressed the biomass-supporting glycolytic pathway. Amino acid synthesis was decreased, and the resulting loss of protein synthesis prevented cell growth. Surprisingly, this all occurred in the face of activated pro-growth signaling pathways, including mTORC1, Myc, and PI3K/Akt. These observations highlight the evolutionarily conserved role of pyruvate metabolism in setting the balance between energy extraction and biomass production in specialized post-mitotic cells.
    DOI:  https://doi.org/10.1101/2024.09.23.614588
  11. Nat Biotechnol. 2024 Oct 07.
    Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView.
    Takamasa Kudo, Ana M Meireles, Reuben Moncada, Yushu Chen, Ping Wu, Joshua Gould, Xiaoyu Hu, Opher Kornfeld, Rajiv Jesudason, Conrad Foo, Burkhard Höckendorf, Hector Corrada Bravo, Jason P Town, Runmin Wei, Antonio Rios, Vineethkrishna Chandrasekar, Melanie Heinlein, Amy S Chuong, Shuangyi Cai, Cherry Sakura Lu, Paula Coelho, Monika Mis, Cemre Celen, Noelyn Kljavin, Jian Jiang, David Richmond, Pratiksha Thakore, Elia Benito-Gutiérrez, Kathryn Geiger-Schuller, Jose Sergio Hleap, Nobuhiko Kayagaki, Felipe de Sousa E Melo, Lisa McGinnis, Bo Li, Avtar Singh, Levi Garraway, Orit Rozenblatt-Rosen, Aviv Regev, Eric Lubeck.
      Optical pooled screening (OPS) is a scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture due to limitations associated with in situ sequencing of perturbation barcodes. Here, we develop PerturbView, an OPS technology that leverages in vitro transcription to amplify barcodes before in situ sequencing, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in induced pluripotent stem cell-derived neurons, primary immune cells and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF-κB signaling. Furthermore, we combine PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in situ screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.
    DOI:  https://doi.org/10.1038/s41587-024-02391-0
  12. J Biol Chem. 2024 Oct 05. pii: S0021-9258(24)02361-5. [Epub ahead of print] 107859
    Experimental variables determine the outcome of RAS-RAS interactions.
    Zhiwei Zhou, Tra Ly Nguyen, Xingxiao Li, Christel Poujol, Ewa Berlinska, Sandra Vietti Michelina, Jonas N Kapp, Andreas Plückthun, Monte M Winslow, Chiara Ambrogio, Yibing Shan, David Santamaría, Kenneth D Westover.
      RAS clustering at the cell membrane is critical to activate signaling in cells, but whether this clustering is mediated exclusively by its c-terminal hypervariable region, receives contributions from the G-domain of RAS, and/or is influenced by secondary effectors has been intensely debated. Reports that G-domain mutations do not modulate RAS-RAS interactions, have led some to question the validity of previous experiments that indicate the G-domain plays a role in RAS clustering/interactions. Here we reconcile these findings by clarifying the impact of experimental variables, such as protein expression levels, cellular context, RAS zygosity, and secondary effector interactions on RAS clustering. Lack of control over these variables impact the results using G-domain mutations across various assay systems and can lead to unsound conclusions.
    Keywords:  D154Q; G-domain; KRAS; RAS clustering; membrane complex
    DOI:  https://doi.org/10.1016/j.jbc.2024.107859
  13. Sci Rep. 2024 10 09. 14(1): 23600
    Deep learning pipeline for automated cell profiling from cyclic imaging.
    Christian Landeros, Juhyun Oh, Ralph Weissleder, Hakho Lee.
      Cyclic fluorescence microscopy enables multiple targets to be detected simultaneously. This, in turn, has deepened our understanding of tissue composition, cell-to-cell interactions, and cell signaling. Unfortunately, analysis of these datasets can be time-prohibitive due to the sheer volume of data. In this paper, we present CycloNET, a computational pipeline tailored for analyzing raw fluorescent images obtained through cyclic immunofluorescence. The automated pipeline pre-processes raw image files, quickly corrects for translation errors between imaging cycles, and leverages a pre-trained neural network to segment individual cells and generate single-cell molecular profiles. We applied CycloNET to a dataset of 22 human samples from head and neck squamous cell carcinoma patients and trained a neural network to segment immune cells. CycloNET efficiently processed a large-scale dataset (17 fields of view per cycle and 13 staining cycles per specimen) in 10 min, delivering insights at the single-cell resolution and facilitating the identification of rare immune cell clusters. We expect that this rapid pipeline will serve as a powerful tool to understand complex biological systems at the cellular level, with the potential to facilitate breakthroughs in areas such as developmental biology, disease pathology, and personalized medicine.
    Keywords:  Cell segmentation; Cyclic microscopy; Imaging analysis; Machine learning; Software
    DOI:  https://doi.org/10.1038/s41598-024-74597-w
  14. bioRxiv. 2024 Sep 07. pii: 2024.09.07.611779. [Epub ahead of print]
    Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells.
    Kaitlin P McCreery, Aki Stubb, Rebecca Stephens, Nadezda A Fursova, Andrew Cook, Kai Kruse, Anja Michelbach, Leah C Biggs, Adib Keikhosravi, Sonja Nykanen, Christel Hyden-Granskog, Jizhong Zou, Jan-Wilm Lackmann, Carien M Niessen, Sanna Vuoristo, Yekaterina A Miroshnikova, Sara A Wickstrom.
      Acquisition of specific cell shapes and morphologies is a central component of cell fate transitions. Although signaling circuits and gene regulatory networks that regulate pluripotent stem cell differentiation have been intensely studied, how these networks are integrated in space and time with morphological transitions and mechanical deformations to control state transitions remains a fundamental open question. Here, we focus on two distinct models of pluripotency, primed pluripotent stem cells and pre-implantation inner cell mass cells of human embryos to discover that cell fate transitions associate with rapid changes in nuclear shape and volume which collectively alter the nuclear mechanophenotype. Mechanistic studies in human induced pluripotent stem cells further reveal that these phenotypical changes and the associated active fluctuations of the nuclear envelope arise from growth factor signaling-controlled changes in chromatin mechanics and cytoskeletal confinement. These collective mechano-osmotic changes trigger global transcriptional repression and a condensation-prone environment that primes chromatin for a cell fate transition by attenuating repression of differentiation genes. However, while this mechano-osmotic chromatin priming has the potential to accelerate fate transitions and differentiation, sustained biochemical signals are required for robust induction of specific lineages. Our findings uncover a critical mechanochemical feedback mechanism that integrates nuclear mechanics, shape and volume with biochemical signaling and chromatin state to control cell fate transition dynamics.
    DOI:  https://doi.org/10.1101/2024.09.07.611779
  15. Nat Biotechnol. 2024 Oct 07.
    Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap.
    Jiacheng Gu, Abhishek Iyer, Ben Wesley, Angelo Taglialatela, Giuseppe Leuzzi, Sho Hangai, Aubrianna Decker, Ruoyu Gu, Naomi Klickstein, Yuanlong Shuai, Kristina Jankovic, Lucy Parker-Burns, Yinuo Jin, Jia Yi Zhang, Justin Hong, Xiang Niu, Jonathon A Costa, Mikael G Pezet, Jacqueline Chou, Hans-Willem Snoeck, Dan A Landau, Elham Azizi, Edmond M Chan, Alberto Ciccia, Jellert T Gaublomme.
      Unlike sequencing-based methods, which require cell lysis, optical pooled genetic screens enable investigation of spatial phenotypes, including cell morphology, protein subcellular localization, cell-cell interactions and tissue organization, in response to targeted CRISPR perturbations. Here we report a multimodal optical pooled CRISPR screening method, which we call CRISPRmap. CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection. Barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency. CRISPRmap enables in situ barcode readout in cell types and contexts that were elusive to conventional optical pooled screening, including cultured primary cells, embryonic stem cells, induced pluripotent stem cells, derived neurons and in vivo cells in a tissue context. We conducted a screen in a breast cancer cell line of the effects of DNA damage repair gene variants on cellular responses to commonly used cancer therapies, and we show that optical phenotyping pinpoints likely pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance.
    DOI:  https://doi.org/10.1038/s41587-024-02386-x
  16. bioRxiv. 2024 Sep 24. pii: 2024.09.24.612755. [Epub ahead of print]
    Developmental regulation of endothelial-to-hematopoietic transition from induced pluripotent stem cells.
    Rachel Wellington, Xiaoyi Cheng, Clyde A Campbell, Cole Trapnell, Raquel Espin-Palazon, Brandon Hadland, Sergei Doulatov.
      Hematopoietic stem cells (HSCs) arise in embryogenesis from a specialized hemogenic endothelium (HE). In this process, HE cells undergo a unique fate change termed endothelial-to-hematopoietic transition, or EHT. While induced pluripotent stem cells (iPSCs) give rise to HE with robust hemogenic potential, the generation of bona fide HSCs from iPSCs remains a challenge. Here, we map single cell dynamics of EHT during embryoid body differentiation from iPSCs and integrate it with human embryo datasets to identify key transcriptional differences between in vitro and in vivo cell states. We further map ligand-receptor interactions associated with differential expression of developmental programs in the iPSC system. We found that the expression of endothelial genes was incompletely repressed during iPSC EHT. Elevated FGF signaling by FGF23, an endothelial pathway ligand, was associated with differential gene expression between in vitro and in vivo EHT. Chemical inhibition of FGF signaling during EHT increased HSPC generation in the zebrafish, while an FGF agonist had the opposite effect. Consistently, chemical inhibition of FGF signaling increased hematopoietic output from iPSCs. In summary, we map the dynamics of EHT from iPSCs at single cell resolution and identify ligand-receptor interactions that can be modulated to improve iPSC differentiation protocols. We show, as proof of principle, that chemical inhibition of FGF signaling during EHT improves hematopoietic output in zebrafish and the iPSC system.
    DOI:  https://doi.org/10.1101/2024.09.24.612755
  17. bioRxiv. 2024 Sep 23. pii: 2024.09.23.614499. [Epub ahead of print]
    Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.
    Josh P Graham, Jose Gabriel Castro, Lisette C Werba, Luke C Fardone, Kevin P Francis, Anand Ramamurthi, Michael Layden, Helen O McCarthy, Tomas Gonzalez-Fernandez.
      CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.
    DOI:  https://doi.org/10.1101/2024.09.23.614499
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