bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2025–11–16
27 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Two novel red-FRET ERK bosensors in the 670-720 nm range.
  2. Simultaneous capture of single cell RNA-seq, ATAC-seq, and CRISPR perturbation enables multiomic screens to identify gene regulatory relationships.
  3. Impaired nuclear PTEN function drives macrocephaly, lymphadenopathy and late-onset cancer in PTEN Hamartoma Tumour Syndrome.
  4. Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding.
  5. Linking signaling dynamics and cell fate decisions through single-cell imaging: evidence and challenges.
  6. Bridging single cells to organs: Mesoscale modules as fundamental units of tissue function.
  7. Mutation in IR or IGF1R produces features of long-lived mice while maintaining metabolic health.
  8. C-spine mutations of protein kinase C and Akt as a novel generalizable approach to create stable pseudokinases.
  9. A parts list of promoters and gRNA scaffolds for mammalian genome engineering and molecular recording.
  10. Modeling cell fates across space and time.
  11. The limitations of small molecule and genetic screening in phenotypic drug discovery.
  12. Proteomic sensors for quantitative multiplexed and spatial monitoring of kinase signaling.
  13. Trigonelline activates NRF2 and awakens dormant ovarian follicles to promote pregnancy in aging mice.
  14. PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3KβY962 Complex Assembly to Drive Tumorigenesis.
  15. Universal consensus 3D segmentation of cells from 2D segmented stacks.
  16. Clinical and molecular landscape of paediatric cerebral and spinal cavernous malformations.
  17. iGlucoSnFR2: A genetically encoded fluorescent sensor for measuring intracellular or extracellular glucose in vivo in mouse brain.
  18. Membrane potential modulates ERK activity and cell proliferation in human cells.
  19. Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.
  20. Regulation of receptor tyrosine kinase hetero-interactions.
  21. FDA approves 100th small-molecule kinase inhibitor.
  22. Deciphering the impact of AKT1 pathogenic variants in Juvenile Granulosa Cell Tumors Using a Drosophila model.
  23. A dynamic feedback loop between retrograde sterol transport and TORC2 controls adaptation of the plasma membrane to stress.
  24. Multimodal learning enables chat-based exploration of single-cell data.
  25. Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC.
  26. mTORC1 regulates autophagosomal components recycling through SNX16 phosphorylation.
  27. Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases.
  1. J Biol Eng. 2025 Nov 13. 19(1): 102
    Two novel red-FRET ERK bosensors in the 670-720 nm range.
    Nicholaus L DeCuzzi, Jason Y Hu, Florene Xu, Brayant Rodriguez, Michael Pargett, John G Albeck.
      Cell fate decisions are regulated by intricate signaling networks, with Extracellular signal-Regulated Kinase (ERK) being a central node in the control of cell proliferation and differentiation. ERK typically cooperates with a network of other regulators, making it necessary to study multiple signaling pathways simultaneously at the single-cell level. Many existing fluorescent biosensors for ERK and other pathways have significant spectral overlap, limiting their utility for multiplexing. To address this limitation, we developed two novel red-FRET ERK biosensors, REKAR67 and REKAR76, which operate in the 670-720 nm range using the fluorescent proteins miRFP670nano3 and miRFP720. REKAR67 and REKAR76 differ in fluorophore position, which impacts biosensor characteristics; REKAR67 displayed a higher dynamic range but greater signal variance than REKAR76. In both polyclonal and clonal populations, REKAR67- or REKAR76-expressing cells displayed similar Signal-to-Noise ratios (SNR). Overall, the red-FRET ERK biosensors were highly consistent with existing CFP/YFP biosensors in reporting ERK activity. Both REKAR biosensors expand the available tools for measuring single-cell ERK activity by being spectrally compatible with other CFP/YFP FRET and cpGFP -based biosensors, allowing for multiplexed imaging.
    DOI:  https://doi.org/10.1186/s13036-025-00541-9
  2. Cell Rep Methods. 2025 Nov 10. pii: S2667-2375(25)00258-9. [Epub ahead of print] 101222
    Simultaneous capture of single cell RNA-seq, ATAC-seq, and CRISPR perturbation enables multiomic screens to identify gene regulatory relationships.
    Kaivalya Shevade, Yeqing Angela Yang, Kevin Feng, Karl Mader, Volkan Sevim, Jacob Parsons, Gunisha Arora, Hasnaa Elfawy, Rachel Mace, Scot Federman, Rustam Esanov, Shawn Shafer, Eric D Chow, Laralynne Przybyla.
      Here, we introduce CRISPR and transcriptomics-assay for transposase-accessible chromatin (CAT-ATAC), a technique that adds CRISPR guide RNA (gRNA) capture to the existing 10× Genomics Multiome assay, generating linked transcriptome, chromatin accessibility, and perturbation identity data from the same individual cells. We demonstrate up to 77% capture rate for both arrayed and pooled delivery of lentiviral gRNAs in induced pluripotent stem cells (iPSCs) and cancer cell lines. This capability allows us to construct gene regulatory networks (GRNs) in cells under drug and genetic perturbations. By applying CAT-ATAC, we identified a GRN associated with dasatinib resistance, indirectly activated by the HIC2 gene. Using loss-of-function experiments, we further validated that ZFPM2, a component of the predicted GRN, also contributes to dasatinib resistance. CAT-ATAC can thus be used to generate high-content multidimensional genotype-phenotype maps to reveal gene and cellular interactions and functions.
    Keywords:  CP: biotechnology; CP: cancer biology; CRISPR; guide RNA; multiome; perturb-seq; single cell
    DOI:  https://doi.org/10.1016/j.crmeth.2025.101222
  3. Dis Model Mech. 2025 Nov 11. pii: dmm.052527. [Epub ahead of print]
    Impaired nuclear PTEN function drives macrocephaly, lymphadenopathy and late-onset cancer in PTEN Hamartoma Tumour Syndrome.
    Priyanka Tibarewal, Victoria Rathbone, Sarah E Conduit, Gala Anastasia Electra Classen, Fiona Black, Mohammad Amin Danesh, Georgia Constantinou, Zeinab Asgarian, Koujiro Tohyama, Nisha Kriplani, Virginia Alvarez Garcia, Elizabeth Foxall, Djenat Belarbi, Marie Leverve, Wayne Pearce, Mahreen Adil, Zofia Varyova, Lucia Conde, Adriana Alves, Glenn R Masson, Roger L Williams, Adrienne M Flanagan, Javier Herrero, Isra Ahmed Mohamed, Katerina Stroud, Marc Tischkowitz, Katherine Lachlan, Cheryl L Scudamore, Mark G H Scott, Nicholas R Leslie, Nicoletta Kessaris, Bart Vanhaesebroeck.
      PTEN Hamartoma Tumour Syndrome (PHTS), a rare disease caused by germline heterozygous PTEN mutations, is associated with multi-organ/tissue overgrowth, autism spectrum disorder and increased cancer risk. Phenotypic variability in PHTS is partly due to diverse PTEN mutations and the protein's multifaceted functions. PTEN is primarily a PIP3 phosphatase regulating PI3K/AKT signalling but also maintains chromosomal stability through nuclear functions such as double-strand (ds) DNA damage repair. Here we show that PTEN-R173C, a pathogenic variant frequently found in PHTS and somatic cancer, has elevated PIP3 phosphatase activity that effectively regulates canonical PI3K/AKT signalling. However, PTEN-R173C is unstable and excluded from the nucleus. We generated Pten+/R173C mice which developed few tumours during their lifetime, aligning with normal PI3K/AKT signalling. However, they exhibit lymphoid hyperplasia, macrocephaly and brain abnormalities, associated with impaired nuclear functions of PTEN-R173C, demonstrated by reduced dsDNA damage repair. Integrating PHTS patient data with our mouse-model, we propose that defective nuclear functions of PTEN variants can predict the onset of PHTS phenotypes, and that late-onset cancer in these individuals may arise from secondary genetic alterations, facilitated by compromised dsDNA repair.
    Keywords:  ASD; Cancer; Macrocephaly; PHTS; PI3K; PTEN
    DOI:  https://doi.org/10.1242/dmm.052527
  4. iScience. 2025 Nov 21. 28(11): 113743
    Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding.
    Jhen-Wei Wu, Ming Yang, Chao-Cheng Chen, Gabriel Au, Suyang Wang, Yichu Xu, Gia-Wei Chern, Chuan-Hsiang Huang.
      Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) underpins many genetically encoded fluorescent biosensors for monitoring biochemical activities in live cells. However, the FRET ratio (acceptor-to-donor signal ratio), commonly used as a proxy for FRET efficiency, is highly sensitive to imaging parameters, complicating data interpretation. Using FP-based barcodes, we introduced calibration standards into subsets of cells for normalization of fluorescence signals. Theoretical analysis indicated the need for both high- and low-FRET standards for calibration under different excitation intensities. We validated this prediction using engineered "FRET-ON" and "FRET-OFF" standards, demonstrating that calibrated FRET ratios are independent of imaging conditions. Including donor-only and acceptor-only cells enabled simultaneous determination of FRET efficiency for multiple biosensors. Calibration also restored expected reciprocal changes in donor and acceptor signals, often obscured by imaging fluctuations and photobleaching. Together, our studies introduce a simple strategy for robust, multiplexed FRET biosensor imaging, facilitating cross-experimental and long-term studies.
    Keywords:  Biophysical Chemistry; Biotechnology; Methodology in biological sciences; Sensor
    DOI:  https://doi.org/10.1016/j.isci.2025.113743
  5. Front Cell Dev Biol. 2025 ;13 1656051
    Linking signaling dynamics and cell fate decisions through single-cell imaging: evidence and challenges.
    Fulvio Bonsignore, Sara Pozzi, Erika Aloi, Davide Mazza, Samuel Zambrano.
      Our ever-growing capacity to observe dynamic processes at the single-cell level has highlighted how cells use complex signaling dynamics to provide adequate responses to intra- and extracellular cues. Specifically, there is increasing evidence that signaling dynamics can be functional in determining cell fate decisions. In this work, we provide an overview of the growing body of evidence supporting this idea across diverse biological contexts-including immune responses, reactions to DNA damage and growth factors, and embryonic development. In doing so, we aim to provide a precise conceptualization of what is meant when we say that signaling dynamics can determine cell fate, a unifying view of the methodologies used to sustain this claim and to identify some of the existing gaps in our mechanistic understanding of this process. We believe that the body of work hereby described strongly supports the importance of considering the temporal dimension of signaling when seeking to understand how cellular responses are regulated.
    Keywords:  Hes1; MAPK; NF-kB; cell fate decisions; live-cell microscopy; p53; signaling dynamics
    DOI:  https://doi.org/10.3389/fcell.2025.1656051
  6. Cell. 2025 Nov 13. pii: S0092-8674(25)01151-1. [Epub ahead of print]188(23): 6393-6410
    Bridging single cells to organs: Mesoscale modules as fundamental units of tissue function.
    Yun Chen, Ronald N Germain, Ginger L Hunter, Rajan P Kulkarni, Arthur D Lander, Pedro Lowenstein, Jeremy E Purvis, Harikesh S Wong.
      Recent studies at molecular and genomic scales have enriched our understanding of life's most fundamental building block: the cell. However, bridging the gap between single-cell phenotypes and the emergent functions of tissues and organs remains a formidable challenge. Here, we suggest that the conceptual span from cells to tissues and organs is so large as to warrant intermediate stepping stones. Drawing inspiration from "network motifs"-discrete units of cell-level function that emerge from the interactions of a handful of genes or enzymes-we argue that similarly identifiable units of tissue-level function, which we term "mesoscale modules," emerge from coordinated "interactions" among relatively small numbers of cells and their extracellular milieu. We outline several such modules and propose that a concerted effort to study them will deepen our foundational understanding of tissue and organ functions. By developing these mesoscale insights, we anticipate a more tractable and mechanistic approach to complex human conditions rooted in tissue- and organ-scale dysregulation, including developmental defects, cancer, cardiovascular disease, immune-related disorders, infectious disease, and aging.
    Keywords:  emergent properties; mesoscale modules; network motifs; systems biology; tissue biology
    DOI:  https://doi.org/10.1016/j.cell.2025.10.012
  7. JCI Insight. 2025 Nov 11. pii: e189683. [Epub ahead of print]
    Mutation in IR or IGF1R produces features of long-lived mice while maintaining metabolic health.
    Ulalume Hernández-Arciga, Jun Kyoung Kim, Jacob L Fisher, Alexander Tyshkovskiy, Alibek Moldakozhayev, Catherine Hall, Souvik Ghosh, Yashvandhini Govindaraj, Ian J Sipula, Jake Kastroll, Diana Cooke, Jinping Luo, Jonathan K Alder, Stacey J Sukoff Rizzo, Gene P Ables, Eunhee Choi, Vadim N Gladyshev, Michael J Jurczak, Marc Tatar, Andrey A Parkhitko.
      Insulin/insulin growth factor signaling is a conserved pathway that regulates lifespan. Yet, long-lived loss-of-function mutants often produce insulin-resistance, slow growth, and impair reproduction. Recently, a gain-of-function mutation in the kinase insert domain (KID) of the Drosophila insulin/IGF receptor was seen to dominantly extend lifespan without impairing insulin-sensitivity, growth and reproduction. This substitution occurs within residues conserved in mammalian insulin receptor (IR) and insulin growth factor-1 receptor (IGF-1R). We produced two knock-in mouse strains that carry the homologous KID Arg/Cys substitution in murine IR or IGF-1R, and we replicated these genotypes in human cells. Cells with heterodimer receptors of IR or IGF-1R induce receptor phosphorylation and phospho-Akt when stimulated with insulin or IGF. Heterodimer receptors of IR fully induce pERK but ERK was less phosphorylated in cells with IGF-1R heterodimers. Adults with a single KID allele (producing heterodimer receptors) have normal growth and glucose regulation. At four months, these mice variably display hormonal markers that associate with successful aging counteraction, including elevated adiponectin, FGF21, and reduced leptin and IGF-1. Livers of IGF-1R females show decreased transcriptome-based biological age, which may point toward delayed aging and warrants an actual lifespan experiment. These data suggest that KID mutants may slow mammalian aging while they avoid the complications of insulin resistance.
    Keywords:  Aging; Glucose metabolism; Insulin; Metabolism
    DOI:  https://doi.org/10.1172/jci.insight.189683
  8. J Biol Chem. 2025 Nov 07. pii: S0021-9258(25)02773-5. [Epub ahead of print] 110921
    C-spine mutations of protein kinase C and Akt as a novel generalizable approach to create stable pseudokinases.
    Stefanie J Hodapp, Tiffany H Kao, Jian Wu, Nileeka Balasuriya, Corina E Antal, Susan S Taylor, Alexandra C Newton.
      Protein kinases function not only through their catalytic phospho-transfer activity but also by non-catalytic scaffold mechanisms. Introduction of mutations to inactivate catalysis provides a tool to differentiate between the two; however, kinase-inactivating mutations may alter the structure of the kinase domain and perturb scaffold functions. Here, we developed a strategy that prevents ATP binding, thereby preventing catalysis, while stabilizing the active conformation of the kinase domain. This approach leverages the structural role of ATP in assembling the catalytic spine (C-spine), a hydrophobic core essential for the active conformation. Specifically, we substituted Val or Ala residues proximal to the binding position of the adenosine ring of ATP with Phe in three protein kinase C isozymes (PKCβII, γ, θ) and Akt1. Structural modeling suggests that Phe substitutions at these positions are a surrogate for the adenosine ring of ATP to assemble the C-spine. Live-cell imaging using genetically encoded PKC and Akt activity reporters reveals that C-spine mutations abolish kinase activity. Furthermore, phosphorylation of the hydrophobic motif, an autophosphorylation site, is abolished in C-spine mutants of PKC family members and reduced in C-spine mutants of Akt1, independent of epidermal growth factor (EGF) stimulation. In PKCβII, these C-spine mutations accelerate plasma membrane translocation, consistent with impaired autoinhibition due to the lack of hydrophobic motif phosphorylation. Despite adopting reduced autoinhibition, turnover experiments with PKCθ reveal C-spine mutants do not impair the stability of the full-length PKC. The generation of pseudokinases by C-spine mutations provides a generalizable strategy for elucidating non-catalytic kinase functions.
    Keywords:  Akt; C-spine; kinase-dead; protein kinase C; pseudokinase
    DOI:  https://doi.org/10.1016/j.jbc.2025.110921
  9. Nat Biotechnol. 2025 Nov 11.
    A parts list of promoters and gRNA scaffolds for mammalian genome engineering and molecular recording.
    Troy A McDiarmid, Megan L Taylor, Wei Chen, Florence M Chardon, Junhong Choi, Hanna Liao, Xiaoyi Li, Haedong Kim, Jean-Benoît Lalanne, Tony Li, Jenny F Nathans, Beth K Martin, Jordan Knuth, Alessandro L V Coradini, Jesse M Gray, Sudarshan Pinglay, Jay Shendure.
      A standardized 'parts list' of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a 'ten key' array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits.
    DOI:  https://doi.org/10.1038/s41587-025-02896-2
  10. Nat Methods. 2025 Nov 10.
    Modeling cell fates across space and time.
      
    DOI:  https://doi.org/10.1038/s41592-025-02884-z
  11. Cell Chem Biol. 2025 Nov 12. pii: S2451-9456(25)00345-9. [Epub ahead of print]
    The limitations of small molecule and genetic screening in phenotypic drug discovery.
    Fabien Vincent, Davide Gianni.
      Phenotypic screens carried out with functional genomics or small molecules have led to novel biological insights, revealed previously unknown targets for drug discovery programs, and provided starting points for the development of first-in-class therapies. Despite being valuable research tools, genetic and compound screening also have significant limitations. This perspective aims to shed a light on those limitations and provide mitigation strategies when available, with a goal of helping phenotypic screening practitioners gain an understanding of how and when to best utilize either approach.
    Keywords:  functional genomics; phenotypic drug discovery; phenotypic screening; target identification
    DOI:  https://doi.org/10.1016/j.chembiol.2025.10.008
  12. Nat Commun. 2025 Nov 13. 16(1): 9902
    Proteomic sensors for quantitative multiplexed and spatial monitoring of kinase signaling.
    William J Comstock, Marcos V A S Navarro, Deanna V Maybee, Yiseo Rho, Mateusz Wagner, Khoula Jaber, Yingzheng Wang, Marcus B Smolka.
      Understanding kinase action requires precise quantitative measurements of their activity in vivo. In addition, the ability to capture spatial information of kinase activity is crucial to deconvolute complex signaling networks, interrogate multifaceted kinase actions, and assess drug effects or genetic perturbations. Here we develop a proteomic kinase activity sensor technique (ProKAS) for the analysis of kinase signaling using mass spectrometry. ProKAS is based on a tandem array of peptide sensors with amino acid barcodes that allow multiplexed analysis for spatial, kinetic, and screening applications. We engineered a ProKAS module to simultaneously monitor the activities of the DNA damage response kinases ATR, ATM, and CHK1 in response to genotoxic drugs, while also uncovering differences between these signaling responses in the nucleus, cytosol, and replication factories. Furthermore, we developed an in silico approach for the rational design of specific substrate peptides expandable to other kinases. Overall, ProKAS is a versatile system for systematically and spatially probing kinase action in cells.
    DOI:  https://doi.org/10.1038/s41467-025-65950-2
  13. iScience. 2025 Nov 21. 28(11): 113717
    Trigonelline activates NRF2 and awakens dormant ovarian follicles to promote pregnancy in aging mice.
    Mahboobeh Amoushahi, Emil Hagen Ernst, Anders Heuck, Margit Dueholm, Erik Ernst, Karin Lykke-Hartmann.
      Aging ovaries exhibit increased oxidative stress, contributing to infertility through cellular and hormonal changes. Nuclear factor E2-related factor 2 (NRF2), a key transcription factor, regulates antioxidant responses. This study investigates NRF2 in dormant (primordial) ovarian follicles to determine if NRF2 activation accounts for primordial follicle activation. We show that trigonelline (TRG) transiently activates NRF2, promoting primordial follicle activation in the non-hormonal phase of follicle development. Indeed, TRG enhances egg quality in aged mice. In human ovarian tissues, TRG increased activation of primordial follicles, resulting in more primary and secondary follicles. Mechanistically, TRG induces NRF2 nuclear translocation and upregulates NRF2-responsive genes, including Egf. Elevated epidermal growth factor (EGF) levels activate EGF receptor (EGFR), increasing protein kinase B (AKT) phosphorylation, which leads to FOXO3A nuclear extrusion and primordial follicle activation. These findings demonstrate that transient NRF2 activation is sufficient to initiate primordial follicle activation and follicle growth and development.
    Keywords:  Cell biology; Reproductive medicine
    DOI:  https://doi.org/10.1016/j.isci.2025.113717
  14. Cancer Discov. 2025 Nov 17.
    PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3KβY962 Complex Assembly to Drive Tumorigenesis.
    Shuang Tang, Qian Zhou, Johann S Bergholz, Tao Jiang, Weihua Wang, Renlei Ji, Wendi Huang, Jiachen Wu, Yutong Li, Xiaochun Wan, Xiadi He, Qianchen Jing, Menghong Sun, Jared L Johnson, Tomer M Yaron-Barir, Da-Qiang Li, Bo Dai, Zefeng Wang, Xiaoling Li, Lewis C Cantley, Thomas M Roberts, Jean J Zhao.
      Loss of tumor suppressor PTEN drives cancer progression and therapeutic resistance, yet no targeted therapies exist for PTEN-deficient tumors. Here, we identify a critical druggable mechanism where PTEN-loss induces PI3Kβ phosphorylation for tumorigenesis. Using BioID interactome, we uncovered phosphorylation-dependent PI3Kβ-EPHA2 interaction in PTEN-null cells, driven by p-PI3KβY962. PTEN functions as a tyrosine phosphatase that normally dephosphorylates p-PI3KβY962. In PTEN-deficient contexts, enhanced p-PI3KβY962 forms a complex with EPHA2 and SRC, where both kinases contribute to PI3Kβ phosphorylation, activating oncogenic pERK/c-MYC and pAKT pathways. We developed a selective p-PI3KβY962 antibody detecting p-PI3KβY962 in PTEN-deficient tumors across preclinical models and clinical tumor specimens. Disrupting p-PI3KβY962 suppressed tumor growth in multiple PTEN-null models. Dasatinib, an FDA-approved SRC/EPHA2 inhibitor, effectively reduced p-PI3KβY962 and inhibited tumor progression in PTEN-null but not PTEN-WT tumors. These findings establish p-PI3KβY962 as a druggable target and biomarker for developing targeted therapy in PTEN-deficient cancers beyond conventional PI3K kinase inhibition.
    DOI:  https://doi.org/10.1158/2159-8290.CD-25-1126
  15. Nat Methods. 2025 Nov;22(11): 2386-2399
    Universal consensus 3D segmentation of cells from 2D segmented stacks.
    Felix Y Zhou, Zach Marin, Clarence Yapp, Qiongjing Zou, Benjamin A Nanes, Stephan Daetwyler, Andrew R Jamieson, Md Torikul Islam, Edward Jenkins, Gabriel M Gihana, Jinlong Lin, Hazel M Borges, Bo-Jui Chang, Andrew Weems, Sean J Morrison, Peter K Sorger, Reto Fiolka, Kevin M Dean, Gaudenz Danuser.
      Cell segmentation is the foundation of a wide range of microscopy-based biological studies. Deep learning has revolutionized two-dimensional (2D) cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation and computation. However, three-dimensional (3D) cell segmentation, requiring dense annotation of 2D slices, still poses substantial challenges. Manual labeling of 3D cells to train broadly applicable segmentation models is prohibitive. Even in high-contrast images annotation is ambiguous and time-consuming. Here we develop a theory and toolbox, u-Segment3D, for 2D-to-3D segmentation, compatible with any 2D method generating pixel-based instance cell masks. u-Segment3D translates and enhances 2D instance segmentations to a 3D consensus instance segmentation without training data, as demonstrated on 11 real-life datasets, comprising >70,000 cells, spanning single cells, cell aggregates and tissue. Moreover, u-Segment3D is competitive with native 3D segmentation, even exceeding when cells are crowded and have complex morphologies.
    DOI:  https://doi.org/10.1038/s41592-025-02887-w
  16. Brain Commun. 2025 ;7(6): fcaf408
    Clinical and molecular landscape of paediatric cerebral and spinal cavernous malformations.
    Sandro Benichi, Estelle Balducci, Joseph Benzakoun, Boulos Ghannam, Christian Attieh, Alice Metais, Marie Bourgeois, Lelio Guida, Sophie Kaltenbach, Arnault Tauziede-Espariat, Florence Riant, Patrick Villarese, Coline Lefevre, Charles-Joris Roux, Stéphanie Puget, Olivier Naggara, Catherine Oppenheim, Rima Nabbout, Nathalie Boddaert, Kévin Beccaria, Elisabeth Tournier-Lasserve, Manoëlle Kossorotoff, Pascale Varlet, Vahid Asnafi, Guillaume Canaud, Thomas Blauwblomme.
      We aimed to decipher the natural history of paediatric cerebral and intraspinal medullary cavernous malformations and their genetic background, with an emphasis on the effect of mutational burden on their radiological features and haemorrhagic risk. We retrospectively collected clinical and radiological data and the natural history of the disease during the follow-up of all consecutive patients with cavernous malformations over the last 2 decades at a unique paediatric centre. We created an MRI atlas after lesion segmentation and normalization to perform voxel-based lesion symptom mapping. Targeted germline DNA sequencing of CCM1-3 genes and somatic lesions and targeted sequencing of RAS and PI3 K pathway genes were performed. In total, 257 patients carrying 786 brain and 14 spinal cavernous malformations were analysed. Age at diagnosis was 9.5 ± 5 years. Follow-up data were available for 718 lesions with a mean follow-up of 5.59 ± 3.28 years. Then, 134 brain (80 with focal onset seizures) and 10 spinal cavernous malformations were surgically excised. Furthermore, 95 of the/144 DNA extracted from lesions were successfully sequenced for somatic variants (PIK3CA, MAP3K3, KRIT1 and KRAS). We found an annual haemorrhagic risk of 1.9%, which was increased by type (sporadic or postradiation), location (brainstem or spinal), and radiological parameters (extracapsular haemorrhage, volume, signal, or presence of developmental venous anomaly). Cavernous malformations symptoms and radiological features were highly dependent on brain location. Surgical risk for cortical brain lesions was 0.9%, with 85% international league against epilepsy score grade of 1-2. Furthermore, 62% of operated lesions carried a pathogenic PIK3CA variant. The MAP3K3 somatic variant alone or in association with a PIK3CA variant decreased the annual haemorrhagic risk by 4.8 ± 1.5 (P < 0.05). We confirmed that 96% of cavernous malformations that are satellites of a developmental venous anomaly carried a PIK3CA variant and increased the relative haemorrhagic risk by 3 ± 0.31 (P = 3.8.10-3). We report an annual haemorrhagic risk of 1.9%, which is modulated by radiological features (i.e. extracapsular haemorrhage, volume, signal, and location) and lesion type. Here, we describe the first MRI atlas. Surgery yields excellent general outcomes in supratentorial superficial lesions, unlike deep-seated cavernous malformations, which is associated with high morbidity. Molecular diagnosis of operated lesions highlighted PIK3CA as a factor associated with aggressiveness in both sporadic and familial cavernous malformations. MAP3K3 decreased the risk. Finally, we confirmed that developmental venous anomaly is a radiological landmark of PIK3CA.
    Keywords:  brain mosaicism; brain probability atlas; paediatric intracranial haemorrhage; stroke; vascular malformation
    DOI:  https://doi.org/10.1093/braincomms/fcaf408
  17. Sci Adv. 2025 Nov 14. 11(46): eadz3889
    iGlucoSnFR2: A genetically encoded fluorescent sensor for measuring intracellular or extracellular glucose in vivo in mouse brain.
    Jonathan S Marvin, Philipp Mächler, Chengbo Meng, Tayfun Ates, Ronak H Patel, Raghabendra Adhikari, Monika A Makurath, Zaneta Ku, Daniel Feliciano, Deniz Atasoy, Guohong Cui, David Kleinfeld, Timothy A Brown.
      Continuous glucose monitors have proven invaluable for monitoring blood glucose levels for diabetics, but they are of limited use for observing glucose dynamics at the cellular (or subcellular) level. We have developed a second generation, genetically encoded intensity-based glucose sensing fluorescent reporter (iGlucoSnFR2). We show that when it is targeted to the cytosol, it reports intracellular glucose consumption and gluconeogenesis in cell culture, along with efflux from the endoplasmic reticulum. It outperforms the original iGlucoSnFR in vivo when observed by fiber photometry in mouse brain and reports transient increase in glucose concentration when stimulated by noradrenaline or electrical stimulation. Last, we demonstrate that membrane localized iGlucoSnFR2 can be calibrated in vivo to indicate absolute changes in extracellular glucose concentration in awake mice. We anticipate iGlucoSnFR2 facilitating previously unobservable measurements of glucose dynamics with high spatial and temporal resolution in living mammals and other experimental organisms.
    DOI:  https://doi.org/10.1126/sciadv.adz3889
  18. Elife. 2025 Nov 14. pii: RP101613. [Epub ahead of print]13
    Membrane potential modulates ERK activity and cell proliferation in human cells.
    Mari Sasaki, Masanobu Nakahara, Takuya Hashiguchi, Fumihito Ono.
      Plasma membrane potential has been linked to cell proliferation for over 40 years in vertebrate cells. In this study, we experimentally demonstrated that membrane depolarization promotes mitosis and that this process depends on the voltage-dependent activation of extracellular signal-regulated kinase (ERK) in human cells. Notably, ERK activity showed a clear dependence on the membrane potential, independent of growth factor stimulation. This voltage dependence was observed even near the resting membrane potential, indicating that small shifts in the resting potential can influence proliferative activity. Voltage-dependent ERK activity is derived from the altered dynamics of phosphatidylserine and is not mediated by calcium influx from the extracellular space. These findings suggest that fundamental biological processes such as cell proliferation are regulated by the physicochemical properties of membrane lipids. This study highlights the broader physiological roles of membrane potentials beyond action potentials, which are well-established in neural systems.
    Keywords:  ERK; cell biology; human; human cells; imaging; membrane potential; proliferation
    DOI:  https://doi.org/10.7554/eLife.101613
  19. Stem Cell Reports. 2025 Nov 13. pii: S2213-6711(25)00317-0. [Epub ahead of print] 102713
    Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.
    Sushama Sivakumar, Yihan Wang, Sean C Goetsch, Vrushali Pandit, Lei Wang, Huan Zhao, Anjana Sundarrajan, Daniel Armendariz, Chikara Takeuchi, Minnie Deng, Mpathi Nzima, Wei-Chen Chen, Ashley E Dederich, Lauretta El Hayek, Taosha Gao, Ashlesha Gogate, Kiran Kaur, Hyung Bum Kim, Melissa K McCoy, Hanspeter Niederstrasser, Seiya Oura, Carlos A Pinzon-Arteaga, Menaka Sanghvi, Daniel A Schmitz, Leqian Yu, Yanfeng Zhang, Qinbo Zhou, W Lee Kraus, Lin Xu, Jun Wu, Bruce A Posner, Maria H Chahrour, Gary C Hon, Nikhil V Munshi.
      Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem-cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple single guide RNA (sgRNA) delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.
    Keywords:  CRISPR/Cas9; Perturb-seq; cardiomyocytes; human pluripotent stem cells; neural progenitor cells; single-cell genomics
    DOI:  https://doi.org/10.1016/j.stemcr.2025.102713
  20. Curr Opin Struct Biol. 2025 Nov 12. pii: S0959-440X(25)00205-2. [Epub ahead of print]95 103187
    Regulation of receptor tyrosine kinase hetero-interactions.
    Adam W Smith, Francisco N Barrera.
      Receptor tyrosine kinases (RTKs) control myriads of cellular functions. RTKs are paradigmatic examples of receptors where activity is directly dependent on quaternary structure. In most cases, the monomeric RTK is inactive, and function arises only after a ligand binding event leads the RTK to bind to another copy of itself, activating trans-autophosphorylation of tyrosine residues. Such RTK homodimerization can be accompanied by the formation of homomers of higher stoichiometry. However, RTK monomers can also bind to a second type of RTK, forming heterodimers. RTK heteromerization is believed to result in different signaling than homomerization. Despite its importance, we have a poor understanding of the factors that define if an RTK will form homomers or heteromers. This short review covers recent discoveries on the heteromerization of RTK, in what is called the RTK interactome. We discuss its translational potential, and how ligands and membrane lipids affect heteromer formation.
    DOI:  https://doi.org/10.1016/j.sbi.2025.103187
  21. Nat Rev Drug Discov. 2025 Nov 13.
    FDA approves 100th small-molecule kinase inhibitor.
    Asher Mullard.
      
    DOI:  https://doi.org/10.1038/d41573-025-00188-7
  22. Mol Cell Proteomics. 2025 Nov 12. pii: S1535-9476(25)00565-1. [Epub ahead of print] 101466
    Deciphering the impact of AKT1 pathogenic variants in Juvenile Granulosa Cell Tumors Using a Drosophila model.
    Reiner A Veitia, Laetitia Herman, Bérangère Legois, Sandra Claret, Alain Zider, Anne-Laure Todeschini.
       BACKGROUND: Juvenile-type granulosa cell tumors (JGCTs) manifest during the prepubertal period as precocious pseudo-puberty and/or dysmenorrhea. We have previously identified pathogenic variants in AKT1 in JGCTs. This study aims to understand how these variants affect cellular function at the phenotypic and molecular levels using a Drosophila model.
    METHODS: Transgenic Drosophila models expressing wild-type (WT) AKT1 and four pathogenic variants were created under the control of tissue-specific promoters. Phenotypic effects were studied by assessing Drosophila wings for cell division and growth using wing surface and trichome density and ovarian follicular cells were examined for subcellular localization and morphology. Molecular analyses included Mass Spectrometry (MS) to identify differentially expressed proteins (DEPs) and phospho-peptides, along with RNAseq to characterize transcriptomic changes.
    RESULTS: Wings expressing mutated AKT1 showed increased surface area and reduced trichome density, indicating larger cells. In ovarian follicular cells, WT AKT1 localized primarily to the cytoplasm, while mutated AKT1 variants were associated with the plasma membrane, leading to morphological abnormalities and increased cell size. MS revealed numerous DEPs and phospho-peptides, highlighting changes in pathways such as glycolysis and Rho GTPase signaling. Transcriptomics demonstrated a clear gain-of-function for mutated AKT1 in activating a subset of genes. However, several genes up-regulated by WT AKT1 were less effectively activated by the mutants, indicating a potential loss-of-function in transcriptional regulation for this subset, revealing an unexpected mechanistic complexity.
    CONCLUSIONS: Network analysis of interactions involving DEPs, phosphorylated proteins, and transcription factors suggests these elements mediate the observed proteomic and transcriptional alterations. Taken together, the results underscore the utility of Drosophila models in unraveling the biological relevance of AKT1 pathogenic variants in cancer.
    DOI:  https://doi.org/10.1016/j.mcpro.2025.101466
  23. EMBO J. 2025 Nov 13.
    A dynamic feedback loop between retrograde sterol transport and TORC2 controls adaptation of the plasma membrane to stress.
    Maria G Tettamanti, Paulina Nowak, Beata Kusmider, Jennifer M Kefauver, Vincent Mercier, Aurélien Roux, Robbie Loewith.
      Cells monitor and dynamically regulate the lipid composition and biophysical properties of their plasma membrane (PM). The Target Of Rapamycin complex 2 (TORC2) is a protein kinase that acts as a central regulator of plasma membrane homeostasis, but the mechanisms by which it detects and reacts to membrane stresses are poorly understood. To address this knowledge gap, we characterized a family of amphiphilic molecules that physically perturb plasma membrane organization and in doing so inhibit TORC2 in yeast and mammalian cells. Using fluorescent reporters of various lipids in budding yeast, we show that exposure to these small molecules causes mobilization of PM ergosterol as well as inhibition of TORC2. TORC2 inhibition results in activation of the PM-ER sterol transporters Lam2 and Lam4 and the subsequent rapid removal of accessible ergosterol from the plasma membrane via PM-ER contact sites. This sequence of events, culminating in the reactivation of TORC2, is also observed with several other PM stresses, suggesting that TORC2 acts in a feedback loop to control active sterol levels at the plasma membrane to maintain its homeostasis.
    Keywords:  Membrane Tension; Plasma Membrane Stress; Small Amphipaths; Sterol Transport; TOR Complex 2
    DOI:  https://doi.org/10.1038/s44318-025-00618-7
  24. Nat Biotechnol. 2025 Nov 11.
    Multimodal learning enables chat-based exploration of single-cell data.
    Moritz Schaefer, Peter Peneder, Daniel Malzl, Salvo Danilo Lombardo, Mihaela Peycheva, Jake Burton, Anna Hakobyan, Varun Sharma, Thomas Krausgruber, Celine Sin, Jörg Menche, Eleni M Tomazou, Christoph Bock.
      Single-cell sequencing characterizes biological samples at unprecedented scale and detail, but data interpretation remains challenging. Here, we present CellWhisperer, an artificial intelligence (AI) model and software tool for chat-based interrogation of gene expression. We establish a multimodal embedding of transcriptomes and their textual annotations, using contrastive learning on 1 million RNA sequencing profiles with AI-curated descriptions. This embedding informs a large language model that answers user-provided questions about cells and genes in natural-language chats. We benchmark CellWhisperer's performance for zero-shot prediction of cell types and other biological annotations and demonstrate its use for biological discovery in a meta-analysis of human embryonic development. We integrate a CellWhisperer chat box with the CELLxGENE browser, allowing users to interactively explore gene expression through a combined graphical and chat interface. In summary, CellWhisperer leverages large community-scale data repositories to connect transcriptomes and text, thereby enabling interactive exploration of single-cell RNA-sequencing data with natural-language chats.
    DOI:  https://doi.org/10.1038/s41587-025-02857-9
  25. Cell Death Dis. 2025 Nov 14. 16(1): 829
    Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC.
    Xiaoqiao Yue, Yanping Zhang, Na Zhao, Tao Lang, Guangxin Chen, Qiuhong Xiong, Lei Gao, Wenjing Wang, Ping Li, Changxin Wu.
      Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by inactivating mutations in TSC1 or TSC2 gene, leading to mTORC1 hyperactivation. However, mTORC1-independent mechanisms in this disorder remain poorly understood. In the study, excess glycogen storage was found in Tsc1-/- cells, Tsc1+/- and Tsc1c.2500-2503delAACA mice, as well as in Tsc2-/- cells, Tsc2+/- and Tsc2c.1113delA mice, with more pronounced accumulation in models with TSC2 defects. Mechanistically, the deficiency of TSC1 or TSC2 gene caused redundant uncomplexed-TSC2 or TSC1 protein, respectively. Strikingly, only uncomplexed-TSC1 downregulated the histone demethylase KDM5A, which in turn increased H3K4me3 levels at the METTL3 promoter to enhance its expression. The upregulated m6A "writer" protein METTL3 cooperated with the "reader" protein IGF2BP2 to stabilize GYS2 mRNA, causing the upregulation of GYS2 resulting in the glycogen storage. Thus, our study uncovered a novel mTORC1 independent pathway (TSC1-KDM5A-METTL3-IGF2BP2-GYS2) that underlies the excess glycogen storage, and that synergy of mTORC1-dependent and independent pathways leads to the more pronounced glycogen storage with TSC2 defects compared to those with TSC1 defects, reflecting the more severer clinical phenotypes in TSC patients with TSC2 mutations. Importantly, the restoration of glycogen homeostasis and significant amelioration of liver lesion in TSC2 defect models after the combination treatment of pharmacological inhibitors targeting mTORC1 and METTL3, unveil a potential clinic intervention for TSC patients to whom mTORC1 inhibitors are less effective or even ineffective.
    DOI:  https://doi.org/10.1038/s41419-025-08161-3
  26. Proc Natl Acad Sci U S A. 2025 Nov 18. 122(46): e2511069122
    mTORC1 regulates autophagosomal components recycling through SNX16 phosphorylation.
    Huilin Que, Fengping Liu, Yang Chen, Wenmin Tian, Shuaixin Gao, Catherine C L Wong, Yan Li, Shixuan Wang, Xianbin Meng, Yueguang Rong.
      During autophagy, the contents enclosed within autophagosomes are degraded, while the outer membrane components are recycled from autolysosomes by the recycler complex through the recently discovered autophagosomal components recycling (ACR) process. This recycling is essential for maintaining autophagic activity. However, the molecular machinery and upstream regulatory mechanisms driving this recycling process remain poorly understood. Here, we identify SNX16 as a key component of the recycler complex, which localizes to autolysosomes and is required for ACR. SNX16 functions in ACR by regulating recycler complex formation, facilitating cargo recognition, and mediating the connection between STX17-SNX4-SNX5 and dynein-dynactin complexes. In addition, SNX16-cargo interactions are regulated by two ACR-related small GTPases, Rab32 and Rab38. Importantly, mTORC1 phosphorylates SNX16 to regulate ACR by inhibiting its interactions with STX17 and other recycler components, thus preventing recycler complex formation. Taken together, our findings identify SNX16 as a recycler component and establish a link between mTORC1 and ACR.
    Keywords:  SNX16; autophagosomal components recycling; autophagy
    DOI:  https://doi.org/10.1073/pnas.2511069122
  27. Cell Syst. 2025 Nov 12. pii: S2405-4712(25)00282-0. [Epub ahead of print] 101449
    Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases.
    Maria Chernigovskaya, Khang Lê Quý, Maria Stensland, Sachin Singh, Rowan Nelson, Melih Yilmaz, Konstantinos Kalogeropoulos, Pavel Sinitcyn, Anand Patel, Natalie Castellana, Stefano Bonissone, Stian Foss, Jan Terje Andersen, Geir Kjetil Sandve, Timothy Patrick Jenkins, William S Noble, Tuula A Nyman, Igor Snapkow, Victor Greiff.
      The circulating antibody (Ab) repertoire is crucial for immune protection, holding significant immunological and biotechnological value. While bottom-up mass spectrometry (MS) is widely used for profiling the sequence diversity of circulating Abs (Ab repertoire sequencing [Ab-seq]), it has not been thoroughly benchmarked. We quantified the replicability and robustness of Ab-seq using six monoclonal Ab spike-ins in 70 combinations of concentration and oligoclonality, with and without polyclonal serum immunoglobulin G (IgG) background. Each combination underwent four protease treatments and was analyzed across four experimental and three technical replicates, totaling 3,360 liquid chromatography-tandem MS (LC-MS/MS) runs. We quantified the dependence of Ab-seq identification on Ab sequence, concentration, protease, presence of background IgGs, and bioinformatics methods. Integrating the data from experimental replicates, proteases, and bioinformatics tools enhanced Ab identification. De novo sequencing performed similarly to database-dependent methods at higher Ab concentrations, but de novo Ab reconstruction remains challenging. Our work provides a foundational resource for the field of MS-based Ab profiling. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  Ab-seq, LC-MS/MS; antibody; benchmarking; de novo sequencing
    DOI:  https://doi.org/10.1016/j.cels.2025.101449
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