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AbstractForces on the linker of the nucleoskeleton and cytoskeleton (LINC) complex control nuclear mechano-sensing and mechano-adaptation. However, the force transmission dynamics across the LINC complex are not fully understood, mainly because of the lack of imaging tools. We developed a set of genetically-encoded fluorescence resonance energy transfer (FRET)-based nuclear tension sensors (NuTS) that measure tension forces across SUN1/2 proteins in living cells with high sensitivity. SUN2-based NuTS (NuTS2) responded rapidly to mechanical changes in cell contractility and matrix stiffness. Notably, NuTS2 dynamically showed force transmission with high spatiotemporal resolution during cell adhesion, migration and squeeze. We also used NuTS2 to monitor tension force changes as the notochord matures in zebrafish development. NuTS2 detected a gradient tension force that increased from the posterior tail buds to the anterior as vacuoles expanded in notochord cells. Force reduction affected notochord maturation and zebrafish embryo development. Our results provide a biophysical cue for dissecting how force transmission on SUN2 protein regulates embryo development.TeaserNuclear tension sensor (NuTS) was developed for direct force measurement across SUN proteins in living cells and zebrafish.- Book : ()
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2025
AbstractMany G protein-coupled receptors (GPCRs) trigger a second phase of G protein-dependent signaling from internal membranes after agonist-induced endocytosis. However, individual GPCRs differ significantly in their ability to internalize, and it remains unclear if this confers selectivity on cellular signaling through natively coexpressed GPCRs. We addressed this question by examining the activation of the cyclic AMP (cAMP) / cAMP-dependent protein kinase (PKA) pathway by three ligands that stimulate three distinct, endogenously coexpressed GPCRs in HEK293 cells: isoproterenol (Iso) which primarily activates the β2-adrenergic receptor (β2AR), vasoactive intestinal peptide (VIP) which primarily activates the VIP receptor 1 (VIPR1/VPAC1), and 5’-N-ethylcarboxamidoadenosine (NECA) which primarily activates the adenosine 2B receptor (A2BR). Using location-targeted biosensors and a transcriptional reporter, we demonstrate that each ligand triggers a unique cellular signaling profile and that these responses are differentially sensitive to endocytic inhibition. VIP elicited a response that was endocytosis-dependent at every level in the pathway, from upstream global cAMP elevation to downstream activation of nuclear PKA, while Iso elicited a response that was dependent on endocytosis selectively at downstream steps. In contrast, NECA robustly activated the entire cAMP signaling cascade independently of endocytosis, consistent with our observation that human A2BR does not robustly internalize after activation. We conclude that endocytosis indeed sculpts downstream cAMP signaling by GPCRs in a receptor-specific manner. Our results add to the evolving view of compartmentalized signaling in the cAMP / PKA pathway and suggest that differences in GPCR trafficking can encode receptor-specific signaling profiles through a shared signal transduction pathway.- Book : ()
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2025
Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Whereas gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.- Book : ()
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