Biology subjects

Grumati, P.

Publications and source records attributed to Grumati, P..

10 recordsLinked to original sources

bioRxiv2026

LC3C-ATG4D regulatory axis supports coronavirus replication organelle formation independently of canonical autophagy

Coronaviruses hijack host membranes to assemble ER-derived double-membrane vesicles (DMVs) that shield viral RNA replication from the cell intrinsic surveillance. Although DMVs morphologically resemble autophagosomes, whether and how autophagy factors actively support their biogenesis has remained elusive. Here, we identify a non-canonical requirement for the autophagy protein LC3C in {beta}-coronavirus replication. Loss of LC3s impaired viral RNA replication, whereas genetic ablation of ATG7 did not, indicating that canonical ATG7-dependent lipidation is dispensable in this context. Reconstitution experiments showed that only LC3C substantially restored replication in LC3-deficient cells and that LC3C phospho-mutants, differing in accessibility to ATG4-mediated processing, displayed distinct proviral activities. Additionally, ATG4D, the main protease responsible for maintaining the LC3 non-lipidated pool, is selectively required for viral replication. Both ATG4D and LC3s depletion triggers formation of aberrant DMV-like structures and potently suppresses SARS-CoV-2 replication. Ultrastructural analysis of nsp3-nsp4-induced membranes showed that depletion of LC3s or ATG4 proteases altered DMV abundance and morphology, supporting a role for the LC3C-ATG4D axis in replication organelle biogenesis. These data establish that {beta}-coronaviruses repurpose ATG4D-driven LC3C de-lipidation for non-canonical LC3 recruitment to replication organelles, identifying the lipidation state of LC3 as a molecular determinant of replication organelle biogenesis and efficient viral replication. HighlightsThe manuscript shows that {beta}-coronavirus replication depends on LC3 proteins and particularly on LC3C in reconstitution experiments, that this dependency is independent of ATG7-mediated lipidation, and that ATG4D promotes efficient replication and replication organelle morphology. Together, the data support a model in which a non-canonical LC3C-ATG4D pathway contributes to DMV biogenesis and viral RNA replication.

cell biology↗

bioRxiv2025

Mechanical Cues Regulate Cargo Sorting and Export at the Golgi

The secretory pathway is a sophisticated endomembrane machinery designed to transport and deliver proteins and lipids to intracellular organelles and the extracellular space. While the molecular components of the secretory pathway are well understood, less is known about their regulation, especially by mechanical cues. Here, we report that substrate stiffness stimulates conventional secretion. We have unravelled a molecular pathway that links a mechanical cue through Src and FAK kinases to promote the trafficking of secretory proteins out of the Golgi apparatus and prevent their post-Golgi lysosomal degradation. Phosphoproteomic analysis revealed the Golgi-specific Brefeldin A resistance factor 1 (GBF1) as a key downstream mechano-responsive regulator, whose phosphorylation state orchestrates post-Golgi cargo sorting, directing proteins either toward secretion or to lysosomes. Finally, we identified AMPK as a stiffness-dependent upstream regulator of GBF1 phosphorylation. Together, our data reveal a molecular regulatory loop in which matrix stiffness positively regulates cellular secretion via the Src-FAK-AMPK-GBF1 axis, which can have relevant medical implications in conditions like cancer and fibrosis and their treatment.

cell biology↗

bioRxiv2025

Ubiquitin-dependent degradation of p21Waf1/Cip1 is mediated by AMBRA1 to limit DNA replication stress

The cell cycle is a fundamental process that orchestrates the events that lead to cell replication and division. AMBRA1 is a scaffold factor that interacts with the E3 ubiquitin ligase CRL4-DDB1 complex to regulate the stability of cyclin D-type proteins, key regulators of the G1/S phase transition. However, how cells complete S-phase and coordinate the speed of DNA synthesis is still to be fully elucidated. Here we demonstrate that AMBRA1 affects the turnover of p21Waf1/Cip1 and p27Kip1, by coupling the CRL4-DDB1 complex directly to these proteins. In the absence of AMBRA1, the increased stability of p21Waf1/Cip1, rather than p27Kip1, resulted in the accumulation of replication stress by leaving under-replicated DNA during the S phase. We also found that AMBRA1-depleted cells with consequent high p21Waf1/Cip1 are sensitive to the inhibition of lagging-strand DNA synthesis. Of clinical relevance, low levels of AMBRA1 in Sonic-Hedgehog medulloblastoma correlate with a worse prognosis, suggesting that AMBRA1 expression levels could be an informative criterion for patient stratification and treatment.

molecular biology↗

bioRxiv2025

Autophagy selectively clears ER in inflammation-induced muscle atrophy

Skeletal muscle atrophy is a pathological condition characterized by the progressive loss of muscle mass and function, driven by factors such as disuse, inflammation, and aging. While the ubiquitin-proteasome system is established as the central mediator of myofibrillar protein degradation, the role of autophagy in selective protein turnover remains largely unexplored. To address this, we employed a quantitative, time-resolved analysis of protein synthesis and degradation in C2C12 myotubes undergoing TNF--induced atrophy, using dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (dynamic SILAC) coupled with LC-MS/MS. Our data challenges the classical view of atrophy as a uniform, degradation-centric process. Instead, we reveal temporally distinct patterns of selective protein turnover, including differential degradation of myofibrillar, ribosomal, and endoplasmic reticulum (ER)-resident proteins. Early atrophy is characterized by suppressed short-term protein synthesis, increased ubiquitin-ligase expression, proteasomal activation, and ribosome turnover. In contrast, late atrophy features proteasome-dependent myofibrillar protein degradation, selective synthesis of mitochondrial ribosomes and cytoplasmic ribosome degradation, indicative of metabolic adaptation. Moreover, we identify a temporal shift in autophagic selectivity: from ER homeostasis maintenance to a stress-induced ER-degradation program. Notably, inhibition of autophagy during atrophy leads to accumulation of ER-phagy receptors Tex264 and Calcoco1, implicating ER-phagy as a key contributor to atrophic remodeling, underscoring an underexplored regulatory mechanism in muscle proteostasis. By elucidating the role of autophagy in degradation of the ER, this study opens new avenues for therapeutic interventions targeting proteostasis regulation in inflammation-induced muscle-wasting disorders, ultimately contributing to a more refined understanding of muscle atrophy beyond proteasomal degradation.

molecular biology↗

bioRxiv2025

Feeder-free culture of naive human pluripotent stem cells retaining embryonic, extraembryonic and blastoid generation potential

Conventional human pluripotent stem cells (hPSCs) are widely used to study early embryonic development, generate somatic cells, and model diseases, with differentiation potential aligned to a post-implantation epiblast identity. In the past decade, naive hPSCs, representing a pre-implantation stage, have been derived. Naive hPSCs efficiently differentiate towards embryonic and extraembryonic lineages such as trophectoderm, primitive endoderm, and extraembryonic mesoderm, and also self-organize into blastocyst-like structures called blastoids. However, their culture typically relies on mouse embryonic fibroblasts (MEFs), which are variable, resource-intensive, and can confound analyses. We report the long-term maintenance of naive hPSCs in a feeder-free, serum-coated system. We successfully expanded for up to 25 passages 8 different naive hPSCs lines across 5 laboratories. Growth rate, clonogenicity, and gene expression profiles on serum coating were comparable to MEF-based cultures, but serum coating eliminated fibroblast contamination. Naive hPSCs cultured on serum exhibited more efficient germ layer specification, retained trophectoderm potential and high blastoid formation efficiency. Exome sequencing revealed fewer mutations in serum-cultured cells, and mass spectrometry identified extracellular matrix proteins such as vitronectin, fibronectin, and collagens in the serum coating. Overall, serum coating offers a scalable, cost-effective and therefore widely applicable alternative for naive hPSC culture, maintaining developmental potential, reducing DNA mutations, and eliminating MEF-related confounding factors. We believe serum coating will expand the use of naive hPSCs to large-scale studies and facilitate the investigation of mechanistic insights into developmental and disease modelling.

cell biology↗

bioRxiv2024

MACanalyzeR: scRNA-seq Analysis Tool Reveals PPARγHI Lipid-Associated Macrophages Facilitate Thermogenic Expansion in BAT

Macrophages in brown adipose tissue (BAT) play a complex role in regulating its activity. However, the role of macrophages in regulating BAT activation/deactivation has not yet been comprehensively characterized. To elucidate this, we developed MACanalyzeR, a scRNAseq-based tool specifically designed to explore the macrophage features at molecular and metabolic level. MACanalyzeR was applied in scRNA-seq datasets obtained from BAT with thermogenic loss (db/db mice) and activation (High Fat Diet, HFD). Our computational approach revealed that macrophages accumulating in BAT upon these conditions resemble lipid-associated macrophages (LAMs) with foaming-like features. BAT LAMs also show a significant enrichment of genes associated with mitochondria and lysosomes. Interestingly, LAMs identified in BAT from HFD mice positively correlate with thermogenic genes and exhibit an enrichment in PPAR{gamma} signaling pathway, with an activated mitochondrial metabolism. Cell dynamic strategy, revealed that LAM with high Pparg expression levels (PpargHIGH) progressively accumulate during skeletal muscle regeneration, suggesting a potential role for this LAM subcluster in maintaining tissue homeostasis. Our findings suggest PpargHIGH LAMs as a subclass of macrophages potentially contributing in preserving tissue homeostasis associated with high energy demand conditions such as thermogenic and regenerative stimuli.

physiology↗

bioRxiv2024

Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling

Endosomes have emerged as major signaling hubs where different internalized ligand-receptor complexes are integrated and the outcome of signaling pathways are organized to regulate the strength and specificity of signal transduction events. Ezrin, a major membrane-actin linker that assembles and coordinates macromolecular signaling complexes at membranes, has emerged recently as an important regulator of lysosomal function. Here, we report that endosomal-localized EGFR/Ezrin complex interacts with and triggers the inhibition of the Tuberous Sclerosis Complex (TSC complex) in response to EGF stimuli. This is regulated through activation of the AKT signaling pathway. Loss of Ezrin was not sufficient to repress TSC complex by EGF and culminated in translocation of TSC complex to lysosomes triggering suppression of mTORC1 signaling. Overexpression of constitutively active EZRINT567D is sufficient to relocalize TSC complex to the endosomes and reactivate mTORC1. Our findings identify EZRIN as a critical regulator of autophagy via TSC complex in response to EGF stimuli and establish the central role of early endosomal signaling in the regulation of mTORC1. Consistently, Medaka fish deficient for Ezrin exhibit defective endo-lysosomal pathway, attributable to the compromised EGFR/AKT signaling, ultimately leading to retinal degeneration. Our data identify a pivotal mechanism of endo-lysosomal signaling involving Ezrin and its associated EGFR/TSC complex, which are essential for retinal function.

cell biology↗

bioRxiv2023

3D bioprinting directly affects proteomic signature and myogenic maturation in muscle pericytes-derived human myo-substitute.

Skeletal muscle tissue engineering (SMTE) has recently emerged to address major clinical challenges such as volumetric muscle loss. Here, we report a rotary wet-spinning (RoWS) biofabrication technique for producing human myo-substitutes with biomimetic architectures and functions. We show how the proposed technique may be used to establish a well-tailored, anisotropic microenvironment that promotes exceptional myogenic differentiation of human skeletal muscle-derived pericytes (hPeri). Using high-resolution mass spectrometry-based proteomics with the integration of literature-derived signaling networks, we uncovered that i) 3D biomimetic matrix environment (PEG-Fibrinogen) confers a lower mitogenicity microenvironment compared to standard 2D cultures, favoring the formation of contractile-competent bundles of pericytes-derived myotubes in an anchoring-independent 3D state, and ii) the bioprinting method promotes an upregulation of muscle matrix structural protein besides increasing contractile machinery proteins with respect to 3D bulk cultures. Finally, in vivo investigations demonstrate that the 3D bioprinted myo-substitute is fully compatible with the host ablated muscular tissue, exhibiting myo-substitute engraftment and muscle regeneration in a mouse VML model. Overall, the results show that 3D bioprinting has a superior capability for controlling the myogenic differentiation process on a macroscale and, with future refining, may have the potential to be translated into clinical practice.

bioengineering↗

bioRxiv2023

Function of ERphagy receptors is regulated via phosphorylation-dependent ubiquitination pathways

Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and essential to maintain cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a critical role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyzed phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy upon mTOR (mechanistic target of rapamycin) inhibition. An unbiased screen of kinase inhibitors revealed that CK2 is essential for FAM134B- and FAM134C-driven ER-phagy after mTOR inhibition. Furthermore, we provide evidence that ER-phagy receptors are regulated by ubiquitination events and that treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Using super-resolution microscopy, we show that CK2 activity is essential for the formation of high-density FAM134B and FAM134C clusters. In addition, dense clustering of FAM134B and FAM134C requires phosphorylation-dependent ubiquitination of FAM134B and FAM134C. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of FAM134B and FAM134C phosphosites prevents ubiquitination of FAM134 proteins, formation of high-density clusters, as well as Torin1-induced ER-phagy flux. Therefore, we propose that CK2-dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.

cell biology↗

bioRxiv2020

Combinatorial CRISPR screening reveals functional buffering in autophagy

Functional genomics studies in model organisms and human cell lines provided important insights into gene functions and their context-dependent role in genetic circuits. However, our functional understanding of many of these genes and how they combinatorically regulate key biological processes, remains limited. To enable the SpCas9-dependent mapping of gene-gene interactions in human cells, we established 3Cs multiplexing for the generation of combinatorial gRNA libraries in a distribution-unbiased manner and demonstrate its robust performance. The optimal number for combinatorial hit calling was 16 gRNA pairs and the skew of a librarys distribution was identified as a critical parameter dictating experimental scale and data quality. Our approach enabled us to investigate 247,032 gRNA-pairs targeting 12,736 gene-interactions in human autophagy. We identified novel genes essential for autophagy and provide experimental evidence that gene-associated categories of phenotypic strengths exist in autophagy. Furthermore, circuits of autophagy gene interactions reveal redundant nodes driven by paralog genes. Our combinatorial 3Cs approach is broadly suitable to investigate unexpected gene-interaction phenotypes in unperturbed and diseased cell contexts.

cell biology↗