Biology subjects

Munoz, F. J.

Publications and source records attributed to Munoz, F. J..

4 recordsLinked to original sources

bioRxiv2026

Piezo1 balances focal and reticular adhesions to enable EGFR clathrin-mediated endocytosis

Cells attach to the extracellular matrix through distinct integrin-mediated adhesive structures, including force-transmitting focal adhesions (FAs) and clathrin-enriched reticular adhesions (RAs). FAs enable mesenchymal cell migration and disassemble at mitotic entry, whereas RAs impede migration, persist during mitosis, and contribute to clathrin-mediated endocytosis (CME) as they disassemble. FAs grow with RhoA contractility, whereas RAs shrink, but the mechanisms coordinating these opposing responses remain unclear. Here, we identify the mechanically activated ion channel Piezo1 as a master regulator of FA/RA balance. Piezo1-dependent calcium influx activates the Src family kinase Fyn, which activates two actin polymerization pathways: FA and stress fiber growth via VAV2-RhoA and RA disassembly via N-WASP-Arp2/3. Inhibition or knockdown of Piezo1, Fyn, or VAV2 decreases FA size and increases RA coverage. Critically, cells lacking Piezo1 fail to internalize ligand-activated EGFR on stiff substrates despite normal CME on soft substrates, establishing an essential role for Piezo1 in EGFR CME mechanoadaption. Our findings reveal Piezo1 as the mechanosensor linking membrane tension to coordinated actin polymerization pathways that co-regulate cell-matrix adhesion and endocytosis. Given that CME contributes to viral entry into host cells and cancer resistance to anti-EGFR antibody therapy, targeting the Piezo1-RA-CME axis may offer novel therapeutic opportunities.

cell biology↗

bioRxiv2025

CLPC2 plays specific roles in CLP complex-mediated regulation of growth, photosynthesis, embryogenesis and response to growth-promoting microbial compounds

In Arabidopsis, exposure to growth-promoting microbial volatile compounds (VCs) enhances CLPC2 levels. This chaperone forms part of the CLP protease complex, which ensures the correct functioning of essential processes in plastids. Previous studies indicated considerable functional redundancy of CLPC2 with its dominant paralogue CLPC1. However, the function and action mechanism of CLPC2 still remain unknown. Here we found that CLPC2-lacking clpc2-2 mutants were unresponsive to microbial VCs, whereas clpc1-1 knockout mutants exhibited a WT-like response to VCs when grown on sucrose-containing medium. Unlike clpc1-1, clpc2-2 plants presented a fully functional photosystem II and lower than WT stomatal conductance. Furthermore, clpc2-2 plants, but not clpc1-1 plants, produced wrinkled seeds with delayed embryonic development and reduced postgerminative establishment rates that resembled those of mutants lacking P and R components of the CLP proteolytic core. Proteomic analyses revealed that knocking out of CLPC2 enhanced the levels of chloroplastic proteins that are essential for growth, embryo development and seedling establishment. These changes differed from those promoted by the lack of CLPC1, but partially resembled those promoted by CLPPR core inactivation. Nearly 40% of the proteins differentially accumulated by the lack of CLPC2 were VC-responsive. Notably, 35S promoter-driven CLPC2 expression promoted changes in the proteome similar to those promoted by the lack of CLPC1. Collectively, our findings highlighted contrasting functional and molecular specificities for CLPC1 and CLPC2, and provided strong evidence that CLPC2 plays specific roles in CLP complex-mediated regulation of plant growth, photosynthesis, embryogenesis, postgerminative seedling establishment and microbial VC responsiveness.

plant biology↗

bioRxiv2025

Network-Based Analysis of Human Astrocytes Links Aging to Neurodegenerative and Cardiovascular Diseases

Astrocytes are central to brain homeostasis, supporting neuronal metabolism, synaptic activity, and the blood-brain barrier. With aging, these glial cells undergo molecular and functional changes that weaken support functions and promote neuroinflammation, contributing to neurodegeneration. Yet the systems-level mechanisms of astrocytic aging remain poorly defined in human models. Because aging also heightens risk for cardiovascular disease, cognitive impairment, type 2 diabetes, and systemic inflammation, clarifying shared astrocytic pathways is critical for understanding brain-body crosstalk. Using an in vitro human astrocyte model exposed to sublethal oxidative stress (10 {micro}M H2O2), we profiled transcriptomic changes and identified differentially expressed genes across antioxidant defences, proteostasis, transcriptional regulation, vesicular trafficking, and inflammatory signalling. We then performed seven network-prioritization analyses on a curated human protein-protein interactome: one seeded with the astrocyte H2O2-responsive genes and six with phenotype-associated gene sets (Alzheimers disease, cardiovascular disease, cognitive impairment, type 2 diabetes, oxidative stress, and inflammation). Intersecting the top 5% scoring genes from each run yielded a 127-gene core shared across all seven, enriched for proteostasis, DNA repair, mitochondrial regulation, and telomere and nuclear envelope maintenance. Structure-guided analyses highlighted vulnerable interfaces, including lamin A/C-lamin B1, -actinin-filamins, 14-3-3 dimers, and aminoacyl-tRNA synthetase assemblies, where pathogenic variants are predicted to destabilize or aberrantly stabilize protein interactions. Structure-based interface predictions also highlight potential interactions between APP-VCP/p97 and p53-14-3-3{zeta} that link proteostasis and stress signalling. Together, these findings define a conserved astrocytic vulnerability network that may couple neurodegeneration with cardiovascular disease and nominate structurally testable targets for biomarkers and interventions.

bioinformatics↗

bioRxiv2025

Phospho-eIF2B-epsilon links stress response-driven translation to amyloidogenesis in Alzheimer's disease

A central etiopathogenic event in Alzheimers disease (AD) is the accumulation of amyloid {beta}-peptide (A{beta}) derived from the amyloidogenic processing of the amyloid precursor protein, a pathway initiated by BACE1. Chronic activation of the Integrated Stress Response is linked to AD, typically through eIF2 phosphorylation, which selectively enhances translation of key stress-responsive mRNAs like ATF4 and BACE1. We investigated a novel regulatory mechanism mediated by Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) a hyperactive kinase in AD, on the translational factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2. The methodology combined cellular and molecular biology approaches (western blot, ELISA, immunofluorescence, and luciferase assays) using pharmacological inhibitors and plasmid transfection in established cell models, along with computational structural modeling (AlphaFold3). Findings were validated in post-mortem human brain tissue, with statistical analyses (t-tests and ANOVA) applied throughout. We treated cells (SH-SY5Y) with a GSK-3{beta} inhibitor (NP031112) or overexpressed a constitutively active GSK-3{beta} mutant (GSK-3{beta}-S9A) to modulate GSK-3{beta} activity. GSK-3{beta} inhibition significantly reduced ATF4 and BACE1 protein levels in SH-SY5Y cells in a dose-dependent manner as we tested by western blot (P<0.001), independent of eIF2 phosphorylation. This effect was translational, as the inhibitor still reduced BACE1 levels when transcription was blocked. Crucially, NP031112 reversed stress-induced eIF2B{varepsilon} phosphorylation at Ser540 (P<0.001). Furthermore, H2O2-induced A{beta}1-42 secretion by SH-SY5Y cells was significantly reversed by GSK-3{beta} inhibition (P<0.001). Luciferase reporter assays using the BACE1 5 untranslated region (5 UTR) confirmed that both eIF2B{varepsilon} silencing and a phosphomimetic mutant eIF2B{varepsilon}-S540E increased BACE1 5 UTR-driven translation (P<0.001), demonstrating that reduced functional eIF2B enhances BACE1 translation. Finally, immunohistofluorescence and western blot analysis of human AD hippocampi showed a significant increase in pS540-eIF2B{varepsilon}, ATF4, and BACE1 levels in AD patients compared to non-demented controls (P<0.001 for all three). Our findings establish a novel GSK-3{beta}-eIF2B{varepsilon}-BACE1 axis that links stress response dysregulation to amyloidogenesis, independent of canonical eIF2 phosphorylation. The phosphorylation of eIF2B{varepsilon} functionally mimics the effects of eIF2 phosphorylation by reducing the available functional eIF2B pool, thereby reprogramming translation to favour the production of stress proteins like ATF4 and BACE1. We proposed eIF2B{varepsilon} phosphorylation is a key etiological mechanism in AD.

neuroscience↗