Biology subjects

Morone, D.

Publications and source records attributed to Morone, D..

8 recordsLinked to original sources

bioRxiv2026

A reversible temperature block captures pre-lysosomal intermediates of starvation-induced ERphagy

The selective lysosomal turnover of endoplasmic reticulum (ER) portions, termed ERphagy, removes aged, excess or damaged ER domains. The intermediates forming between receptor activation and lysosomal delivery are short-lived and remain poorly resolved. Here we identify a reversible temperature block at 25{degree sign}C that arrests ERphagy before lysosomal delivery. At this temperature, nutrient restriction still drives focal accumulation of the endogenously Halo-tagged ERphagy receptor TEX264 (puncta formation), segregation of ER cargo, remodelling of the ER membrane, whereas delivery of this material to degradative endolysosomes is strongly reduced. Rewarming above 27{degrees} restores lysosomal delivery, showing that the accumulated eTEX264-Halo puncta correspond to pre-lysosomal intermediates of the ERphagy program. Their formation requires ULK1/2 activity, the ATG7-dependent conjugation machinery, ATG9A, ATG13 and FIP200, coupling receptor clustering to autophagosome biogenesis. Consistently, expansion microscopy and electron tomography reveal curved, cradle-like TEX264-positive ER domains containing ER cargo, co-localizing with autophagy markers and lying adjacent to p62-cargo condensates.

cell biology↗

bioRxiv2026

Epstein Barr virus exploits ER stress and TMX4 dependent nuclear envelope remodeling to enable capsid egress

Herpesvirus capsids assemble within the nucleoplasm of infected cells in highly ordered icosahedral structures with a diameter of about 100nm. Despite the small distance between outer and inner nuclear membrane, which is fixed between the 25 and the 50nm by disulfide bonded LINC complexes, the capsid particles cross the barrier and are delivered into the cytoplasm, where viral particle assembly continues. How Epstein-Barr virus (EBV) overcomes the spatial constraints imposed by the narrow perinuclear space for nuclear egress of the viral capsids remains unclear. Here, we show that EBV exploits an ER-stress-responsive nuclear envelope (NE) remodeling pathway to promote capsid egress. Induction of EBV lytic replication activates the IRE1 branch of the unfolded protein response and triggers TMX4-dependent remodeling of the NE. Inhibition of IRE1 signaling or depletion of TMX4 prevents efficient redistribution of viral capsid proteins from the nucleus to the cytoplasm, causes accumulation of unused viral glycoprotein GP350 in Golgi-derived membranes, and markedly reduces production of infectious viral particles. Thus, EBV hijacks a host NE adaptation pathway to overcome a fundamental physical barrier during viral maturation.

cell biology↗

bioRxiv2026

GRK-dependent ACKR3 endocytosis and chemokine scavenging is independent of receptor phosphorylation and beta-arrestin

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting {beta}-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and {beta}-arrestin interaction. However, the respective roles of {beta}-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)-based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with {beta}-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in {beta}-arrestin-deficient ({Delta}{beta}arr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. {beta}-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired {beta}-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in {Delta}GRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G{beta}{gamma} was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of {beta}-arrestins but requires GRKs. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

cell biology↗

bioRxiv2025

ER-phagy and mitophagy programs in human CD4+ memory T cells before and after activation

T lymphocytes can remain in a resting state for very long times. Encountering their cognate antigen triggers their activation, leading to an increase in cell size, proliferation, enhanced protein production, and secretion of signaling molecules. This activation and clonal expansion phase is followed by a contraction phase, with a reduction in cell size, eventually leading to cell death or progression to resting (memory) cells. Here, we show that human resting primary CD4+ memory T cells degrade endoplasmic reticulum (ER) and mitochondrial portions via ER-phagy and mitophagy, respectively. The temporary interruption of ER and mitochondria turnover coupled with a transient induction of the unfolded protein response results in the enlargement of the ER and cellular volume. Cell growth is stabilized and then reversed during the clonal expansion and contraction phases by the resumption of a catabolic phase characterized by the reactivation of ER-phagy and outer mitochondrial membrane (OMM)-phagy. Collectively, our data show that the maintenance and the activity of human CD4+ memory T cells rely on finely tuned execution of anabolic and catabolic programs that regulate mass and activity of the ER and mitochondria.

cell biology↗

bioRxiv2022

SEC62 and TMX4 control asymmetric autophagy of the nuclear envelope upon LINC complex disassembly

The endoplasmic reticulum (ER) is a dynamic organelle of nucleated cells that produces proteins, lipids and oligosaccharides. The volume and the activities of the ER are adapted to cellular needs. They are increased upon induction of unfolded protein responses (UPR 1) and are reduced upon activation of ER-phagy programs 2. A specialized domain of the ER, the nuclear envelope (NE), protects the cell genome with two juxtaposed lipid bilayers, the inner and outer nuclear membranes (INM and ONM). SUN proteins in the INM form disulfide-bonded Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes with NESPRIN proteins in the ONM. These complexes set and maintain the width of the periplasmic space (PS), a continuum of the ER lumen, below the 50 nm 3-5 and in yeast prevent transmission of ER volume variations to the PS 6,7. Here we report that expansion of the mammalian ER upon homeostatic perturbations is transmitted to the NE, where the ONM forms large bulges. The process is reverted on recovery of ER homeostasis, by asymmetric vesiculation and autophagic clearance of ONM portions. Remodeling of the mammalian NE requires TMX4-driven reduction of the intermolecular disulfide bond stabilizing LINC complexes, the LC3 lipidation machinery, and the autophagy receptor SEC62, identified here as the first mammalian nucleo-phagy receptor.

cell biology↗

bioRxiv2022

ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies

Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing 2P mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.

immunology↗

bioRxiv2021

IL-1α secreted by subcapsular sinus macrophages promotes melanoma metastasis in the sentinel lymph node by upregulating STAT3 signaling in the tumor.

During melanoma metastasization, tumor cells originated in the skin migrate via lymphatic vessels to the sentinel lymph node (sLN) in a process that facilitates their spread across the body. Here, we characterized the innate inflammatory responses to melanoma metastasis in the sLN. For this purpose, we confirmed the migration of fluorescent metastatic melanoma cells to the sLN and we characterized the inflammatory response in the metastatic microenvironment. We found that macrophages located in the subcapsular sinus (SSM), produce pro-tumoral IL-1 after recognition of tumor antigens. Moreover, we confirmed that the administration of an anti-IL-1 depleting antibody reduced metastasis. Conversely, the administration of recombinant IL-1 accelerated the lymphatic spreading of the tumor. Additionally, the elimination of the macrophages significantly reduced the progression of the metastatic spread. To understand the mechanism of action of IL-1 in the context of the lymph node microenvironment, we applied single-cell RNA sequencing to dissected metastases obtained from animals treated with an anti-IL-1 blocking antibody. Amongst the different pathways affected, we identified STAT3 as one of the main targets of IL-1 signaling in metastatic cells. Moreover, we found that the anti-IL-1 anti-tumoral effect was not mediated by lymphocytes, as IL-1R1 KO mice did not show any improvement in metastasis growth. Finally, we found a synergistic anti-metastatic effect of the combination of IL-1 blocking and the STAT3 inhibitor (STAT3i) stattic. In summary, we described a new mechanism by which SSM support melanoma metastasis, highlighting a new target for immunotherapy.

immunology↗

bioRxiv2020

Extracellular Vesicles Hijack the Autophagic Pathway to Induce Tau Accumulation in Endolysosomes

Clinical progression of tauopathies is reflected by the transcellular propagation of pathogenic Tau seeds with the possible involvement of extracellular vesicles as transport vectors. However, the mechanism regulating extracellular vesicle cargo delivery to recipient cells is poorly understood. We established a cell model for investigating extracellular vesicle-delivery of membranes and proteins. In this model, extracellular vesicles are readily internalized and accumulate in endolysosomes. For the first time, we show that in this acidic compartment of recipient cells, extracellular vesicle-delivered Tau seeds cause the accumulation and abnormal folding of normal Tau by a process that requires the participation of autophagy. Endolysomes represent thus a cross-road where Tau seeds released from extracellular vesicles propagate on cellular Tau on its route for autophagy-mediated degradation, ultimately driving its accumulation, endolysosomal stress and cytotoxicity. Whilst, autophagy stimulation is considered as a viable solution to protect neurons from harmful cytosolic protein inclusions, our data suggest that this approach may favour the aberrant accumulation of neurodegeneration-associated proteins induced by exogenous pathogenic protein forms, with possible implications in the spreading of the disease.

cell biology↗