Background: Mitochondrial dysfunction is a leading contributor to the decline in oocyte quality associated with maternal aging. Prior investigations of mitochondrial function in the ovarian follicle have largely treated the mitochondrial pool as a homogeneous population, reporting aggregate values that may obscure biologically meaningful differences between distinct mitochondrial subpopulations. The present study addresses this limitation by characterizing mitochondrial subpopulation dynamics in oocytes and cumulus granulosa cells at single-organelle resolution using fluorescence-activated mitochondria sorting (FAMS). Results: Analysis of the aggregate mitochondrial population in mouse oocytes revealed no significant age-associated differences in mitochondrial DNA copy number or membrane potential, a result that would previously have been interpreted as evidence of minimal age-related mitochondrial change. Subpopulation analysis revealed this conclusion to be incomplete: aged oocytes showed significantly elevated mitochondrial DNA copy number specifically within the high membrane potential and small mitochondrial subpopulations, with no significant differences in the low membrane potential or large subpopulations. NMN supplementation normalized mitochondrial DNA copy number in the high membrane potential and small subpopulations toward young levels while producing an opposing effect in large mitochondria, demonstrating subpopulation-specific rather than uniform rejuvenation. In cumulus cells, significant age-associated changes were detectable at the aggregate level, including a reduction in mitochondrial DNA copy number and an elevation in membrane potential, and subpopulation analysis further resolved these findings. The age-associated reduction in cumulus cell mitochondrial DNA copy number was driven predominantly by the high membrane potential subpopulation. NMN supplementation exerted opposing effects on small and large cumulus cell mitochondrial subpopulations, increasing mitochondrial DNA copy number above both young and aged levels in small mitochondria while further reducing it below aged levels in large mitochondria. Conclusions: Viewing the mitochondrial pool as a heterogeneous mixture of functionally distinct subpopulations rather than a uniform population reveals age-associated alterations in oocytes and cumulus cells that are undetectable by aggregate analysis. NMN supplementation exerts subpopulation-specific effects in both cell types, identifying specific mitochondrial subtypes as more precise targets for future mechanistic investigation of age-associated infertility than the mitochondrial pool considered in aggregate. ### Competing Interest Statement D.C.W. and J.L.T. declare interest in intellectual property described in U.S. Patent 8,642,329, U.S. Patent 8,647,869, U.S. Patent 9,150,830, and U.S. Patent 10,525,086. Hannah Sheehan is the CEO and CSO of SauveBio, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. U.S. National Science Foundation, 2227756, DGE-1938052
Cancer displays remarkable robustness, exemplified by its ability to develop resistance to virtually every therapy. Resistance has traditionally been explained by clonal selection of pre-existing mutations, but there is now abundant evidence for resistance by non-genetic pathways including signals from normal stromal and immune cells. It is largely unclear why normal cells help cancer cells overcome treatment. We propose that physiological circuits responsible for tissue homeostasis can explain why cells cooperate to produce pathological resistance to therapy. To show this, we construct mathematical models of physiological dynamics. We then simulate cancer treatments within the context of a functioning tissue. We find that classic examples of resistance to therapy can be explained by homeostatic feedback regulation - including BRAF inhibitors in melanoma and anti-angiogenic therapy. The homeostatic theory of resistance (HTOR) reframes resistance as a byproduct of tissue robustness, rather than solely tumor-specific adaptation. Finally, we analyze two large-scale single-cell RNAseq databases of normal and cancer samples: the Tabula Sapiens1 and the Curated Cancer Atlas2. We show that in multiple cancers (breast, colon, kidney, liver, lung, ovary, prostate, and skin), malignant cells preserve their tissue-specific homeostatic cell-signaling. We thus expect the robust feedback loops from healthy tissues to play a role in cancer. ### Competing Interest Statement The authors have declared no competing interest.
Prasugrel is a prodrug, widely used in antiplatelet strategy for secondary prevention after acute coronary syndrome. The metabolism of prasugrel leads to the formation of the Prasugrel Active Metabolite (PAM), an irreversible P2Y12 receptor antagonist. Its mode of binding has not yet been fully established, although it is known that it binds covalently to P2Y12 by forming a disulfide bridge with cysteines and its sulfur moiety. PAM is a molecule with two chiral centers, resulting in four stereoisomers which appear to be stereoselective upon binding. A combination of different molecular modeling methods, such as molecular dynamics, ensemble docking, and Density Functional Theory (DFT), were used to rationalize these differences in antagonism observed in vitro and to elucidate the mode of binding of PAM to P2Y12. PAM is found to bind to the closed P2Y12 conformation in a preferential way. Although the four stereoisomers have comparable affinity, the location of the RS stereoisomer makes the formation of a disulfide bond with cysteines more favorable, particularly with cysteine 175. Compared to the RR stereoisomer, the RS stereoisomer interacts less deeply with the P2Y12 receptor, interacting in particular with the second and third extracellular loops, explaining the competition observed with cangrelor and an intermediate metabolite of prasugrel. Furthermore, DFT calculations have shown that the formation of a disulfide bridge is energetically more favorable with the RS stereoisomer than with the RR stereoisomer. The physical interactions and chemical reaction between the RS stereoisomer and the P2Y12 receptor are key factors in explaining the stereoselective binding of PAM to P2Y12. ### Competing Interest Statement The authors have declared no competing interest.
Low- to moderate-intensity ultrasound (US) technologies are increasingly being used to non-invasively modulate biological function in both clinical and laboratory settings. Realizing the full potential of these approaches requires a detailed mechanistic understanding of how ultrasound interacts with living cells. Here, we developed a well-controlled experimental platform to expose adherent cells to ultrasound stimulation while monitoring cellular activation via calcium imaging. We show that cell activation is dependent on cell type and identify NIH3T3 fibroblasts as a particularly robust responder. Our findings indicate that acoustic streaming is the primary mechanism underlying ultrasound-induced activation in our in vitro experiments. Surprisingly, the investigation of calcium dynamics revealed that the observed cytoplasmic calcium elevation originates predominantly from intracellular stores rather than extracellular influx, with membrane ion channels not contributing directly to the response. Notably, the biomechanical property of the cell-cortex emerges as a critical determinant of the cells' sensitivity to ultrasound. Overall, our results provide clear evidence that the underlying mechanistic response involves external and internal factors that modulate the ultrasound-cell interaction and highlight important mechanistic considerations for ultrasound-based strategies aimed at cellular stimulation. ### Competing Interest Statement The authors have declared no competing interest.
Vascular remodeling within the developing fetus and placenta is essential for supporting the growth and function of emerging tissues and organs. Pericytes (PCs) play a central role in stabilizing and maturing microvascular networks by extending along endothelial cells (ECs) and reinforcing vessel integrity. In the placenta, as in other organs, PC-EC communication is mediated in part by platelet-derived growth factor-BB (PDGF-BB) signaling, which governs PC differentiation, proliferation, migration, and survival, ultimately enabling their recruitment and retention along capillaries. In this study, we identified progressive PC investment along feto-placental capillaries in both murine and human tissues across gestation, supported by morphological and molecular evidence. Placental PCs displayed phenotypic heterogeneity comparable to that observed in the brain and heart, suggesting conserved diversity across organ systems. In addition to characterizing PC dynamics, we examined the expression of recently identified soluble PDGF Receptor-β (sPDGFRβ) isoforms. These variants were detected at the protein and transcript levels in mouse and human placentas, as well as in a murine trophoblast-embryonic stem cell (TESC) differentiation model that recapitulates aspects of early placental vascular development. Within this model, sPDGFRβ expression was independent of ADAM10 activity and exogenous growth factors during early vessel formation but was markedly upregulated during hypoxia. To assess how elevated sPDGFRβ might influence PDGF-BB signaling, we exposed TESC-derived vascular networks to excess PDGF-BB with or without a sPDGFRβ mimetic. PDGF-BB alone reduced full-length PDGFRβ levels while increasing receptor phosphorylation, consistent with known ligand-induced regulatory mechanisms. Inclusion of the sPDGFRβ mimetic shifted these responses toward baseline, suggesting a potential modulatory or feedback role for soluble receptor variants. Together, these findings demonstrate that PCs are progressively recruited to placental capillaries and exhibit diverse phenotypes during development, and that soluble PDGFRβ isoforms may modulate PDGF-BB signaling in a manner sensitive to oxygen tension. Understanding these mechanisms provides insight into the regulation of placental vascular maturation and may inform strategies to improve human health by targeting disorders rooted in impaired placental development. ### Competing Interest Statement The authors have declared no competing interest. The Seale Foundation
Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset. ### Competing Interest Statement The authors have declared no competing interest.
Intercellular communication is governed by the spatiotemporal dynamics of protein complexes at the cell-cell interface. However, conventional static interaction models fail to incorporate key physical constraints, such as steric hindrance, spatial compartmentalization, and dimensionality reduction that regulate complex assembly in vivo. To bridge the gap between static network topology and dynamic systems biology, we developed a multi-scale computational framework. We first identified a highly conserved, Fibroblast Growth Factor Receptor 1 (FGFR1)-centered cell adhesion and signaling motif by analyzing a diverse set of human cell–cell interfaces. We then constructed a multi-layer spatial stochastic simulator to recapitulate and interrogate the dynamic behavior of this network motif at cell-cell interfaces. Atomic-resolution structural models of the protein complexes within the motif were further generated using AlphaFold to define interaction rules for the stochastic simulations by categorizing binding interfaces. Our results show that the structural arrangement of cell-cell adhesion complexes controls how FGFR1 receptors cluster at the cell–cell interface, effectively dividing the membrane into distinct functional microdomains. Competition from decoy receptors further regulates this process by capturing receptors before they can participate in signaling. Even small changes in binding affinity can therefore alter receptor organization and disrupt normal signal transduction, which may contribute to human disease. By integrating macro-scale interactomics, atomic-level structural bioinformatics, and mesoscale stochastic modeling, this study reveals how structural interaction rules, combined with spatial constraints, shape the formation and function of intercellular signaling networks. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, https://ror.org/04q48ey07, R01GM122804 United States-Israel Binational Science Foundation, https://ror.org/00j8z2m73, 2023336
Directional persistence is essential for efficient immune cell migration in tissues, yet how cytoskeletal systems stabilize migration in complex three-dimensional environments remains unclear. Using intravital subcellular microscopy and quantitative analysis of membrane dynamics, we identify two spatially distinct architectures of non-muscle myosin II (NMII) that coordinate protrusion dynamics during neutrophil migration. In vivo and in collagen matrices, NMII assembles at the leading edge into lattice-like structures that are structurally and functionally distinct from rear contractile actomyosin bundles. Protrusion-resolved analyses reveal that directional persistence correlates strongly with protrusion lifetime and sustained NMII engagement, with rear NMII load showing the strongest association with protrusion persistence. Strikingly, directional migration is not determined by the abundance of favorable protrusions but by their temporal organization during migration. Pharmacological perturbations that redistribute NMII activity disrupt this temporal organization and alter migration trajectories. Together, these findings reveal that spatially distinct NMII architectures coordinate protrusion dynamics across time to stabilize directional migration in complex environments. Highlights ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, National Cancer Institute, Center for Cancer Research Intramural Research Program, ZIA BC 011682
Rxiv mechanobio