Biology subjects

Luini, A.

Publications and source records attributed to Luini, A..

10 recordsLinked to original sources

bioRxiv2026

The COVID-19 RNA vaccine SpikevaxTM transfects human umbilical endothelial cells and induces Spike protein production, inflammation and leukocyte adhesion

Endothelial cell dysfunction plays a key role in the pathogenesis of severe and critical COVID-19, leading to a multi-systemic inflammatory disease, and results from the direct interaction of the SARS-CoV-2 Spike proteins with endothelial cells. COVID-19 RNA vaccines encode a recombinant SARS-CoV-2 Spike protein which undergoes systemic biodistribution. No information is however so far available on the direct effects of COVID-19 RNA vaccines on human endothelial cells. In the present study, we exposed cultured human umbilical venous endothelial cells (HUVEC) to the COVID-19 RNA vaccine SpikevaxTM (Moderna, Inc.), and thereafter we measured the expression of the Spike protein, of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-, of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as the attachment of human leukocytes to HUVEC layers. We also assessed the effects of SpikevaxTM on HUVEC viability. Exposure of HUVEC to the COVID-19 RNA vaccine SpikevaxTM resulted in effective cell transfection, and subsequent production of the Spike protein, which was expressed in the cells and secreted in the culture medium. Spike protein production was accompanied by increased gene expression of IL-6 and TNF- and of ICAM-1 and VCAM-1, as well as by increased attachment of leukocytes to HUVEC monolayers. Exposure to the COVID-19 RNA vaccine SpikevaxTM did not affect HUVEC viability. Our results provide a mechanistic explanation to post-COVID-19 RNA vaccination pathologies resulting from endothelial dysfunction, such as during atherosclerosis, autoimmune inflammation, and systemic inflammatory conditions.

pharmacology and toxicology↗

bioRxiv2026

EFFECT OF A BIORESONANCE DEVICE ON VIABILITY AND METABOLIC ACTIVITY IN HUMAN UMBILICAL VENOUS ENDOTHELIAL CELLS

The present study was aimed at evaluating the effects of the bioresonance (BR)-based device QDOME MINI PERSONAL, produced by the society QMED SWISS SA (Lugano, CH), on the viability and mitochondrial metabolic activity of cultured human umbilical venous endothelial cells (HUVEC). To this end, HUVEC were cultured under standard conditions and exposed for 24 h to an "active" or to a mock BR device, in resting conditions and during treatment with H2O2 at the concentration of 500 M, added at the beginning of the 24 h period. The personnel who performed the experiments, collected, and analysed the data was unaware of which device was "active" and which was mock. At the end of the culture, HUVEC were harvested and evaluated for viability and mitochondrial metabolic activity by means of the Trypan Blue exclusion and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide reduction method, respectively. Under control conditions, viability and mitochondrial metabolic activity were not different in HUVEC exposed to the "active" or to the mock device. HUVEC viability, however, was significantly reduced by exposure to H2O2 in samples exposed to the mock device but not in those exposed to the "active" device. HUVEC mitochondrial metabolic activity was significantly reduced by exposure to H2O2 in both samples exposed to the "active" and to the mock device, however reduction was significantly less in samples exposed to the "active" device. In conclusion, exposure to the BR-based device QDOME MINI PERSONAL, produced by the society QMED SWISS SA (Lugano, CH), prevented the H2O2-induced reduction of viability and reduced the H2O2-induced impairment of mitochondrial metabolic activity in cultured HUVEC.

cell biology↗

bioRxiv2023

The Golgi checkpoint: Golgi unlinking during G2 is required for correct spindle formation and cytokinesis

The decision to enter mitosis requires not only the control of DNA replication but also additional and crucial preparatory steps such as, for example, partial disassembly of the Golgi complex during G2. The Golgi complex is fundamental for the processing and sorting of proteins and lipids in the secretory pathway. It is organized as stacks of cisternae laterally connected by tubules to form a continuous Golgi ribbon. During G2, the Golgi ribbon is unlinked into isolated stacks in preparation for cell division. This structural reorganization is necessary for entry into mitosis, indicating that a "Golgi mitotic checkpoint" controls the correct segregation of this organelle. To understand the physiological significance of the pre-mitotic Golgi unlinking, we devised a strategy to accumulate cells in G2 with an intact Golgi ribbon and then induce entry into mitosis. Here, we show that forcing the entry of cells into mitosis with an intact Golgi ribbon causes remarkable cell division defects, including spindle multipolarity and binucleation, favoring cell transformation. We also find that the cells entering mitosis with an intact Golgi ribbon show reduced levels at the centrosome of the kinase Aurora-A, a pivotal regulator of spindle formation. Overexpression of Aurora-A rescues spindle formation, indicating that the Golgi-dependent Aurora-A recruitment has a crucial role in spindle formation. Thus, our results show that alterations of the pre-mitotic Golgi segregation have profound consequences on the fidelity of the mitotic process, representing potential risk factors for cell transformation and cancer development.

cell biology↗

bioRxiv2022

Calcium flux through ER-TGN contact sites facilitates cargo export

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon a local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains elusive, as the cytosol has a very low steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), whereby the close apposition of the two organelles allows the protein-mediated exchange of molecular species such as lipids. Here we show that the TGN export of Cab45 clients requires the integrity of ER-TGN MCS and IP3R-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real-time. These data show for the first time that ER-TGN MCS facilitates Ca2+ transfer required for SPCA1-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology. SummaryThe current study demonstrates that the trafficking of COMP and LyzC relies on Ca2+ flux between the endoplasmic reticulum (ER) and trans-Golgi Network (TGN). This process requires the activity of IP3 receptors, present in ER membranes, and depends on the integrity of the membrane contact site between these two organelles.

cell biology↗

bioRxiv2022

Prioritization of driver genes in cancer-associated copy number alterations identifies B4GALT5 as a glycooncogene

Cancer is a disease resulting from aberrant communication between cells of a multicellular organism. The glycan coat that surrounds the cells is an important player in cellular communication. While altered cell surface glycans are known biomarkers for cancer, glycan biosynthesis itself has not been considered a potential oncogenic pathway. So, to understand the oncogenic potential of the glycan biosynthetic pathways we have analyzed the copy number alterations (CNA) of genes encoding for glycosylation regulators (glycogenes) in cancer genome datasets and identify novel glyco-oncogenes and glyco-tumor suppressor genes (TSGs). CNA of oncogenes and TSGs is an important cancer-associated genetic alteration that associates with worst prognosis. Nevertheless, identity of the driver genes in the copy number altered segments of the genome remains obscure in most cases. We developed a prioritization pipeline based on bioinformatic and experimental criteria to identify putative driver genes. In addition to correctly identifying several well-established oncogenes/TSGs, this pipeline discerns several novel oncogenes and TSGs, some of which are glycogenes. Further, among glyco-oncogenes there is an enrichment for glycosphingolipid biosynthetic pathway and trans-Golgi associated lysosomal sorting machinery and among glyco-TSGs there is an enrichment for early N-glycan biosynthetic enzymes. As a proof-of-principle we show that one of the identified glycooncogene B4GALT5, encoding a key enzyme in the glycosphingolipid pathway exhibits oncogenic property of promoting increased growth of hepatocellular carcinoma cells. Thus, this study identifies glycosylation pathways with oncoregulatory properties and opens up a new group of enzymes as potential therapeutic targets for cancer.

cancer biology↗

bioRxiv2021

Precision localization of cellular proteins with fluorescent Fab-based probes

Currently, a major technical limitation of microscopy based image analysis is linkage error - a visualization error that is measured by the distance between the cellular protein to be detected and the fluorescence emitter used for detection. With continuously improving resolution of todays (super-resolution) microscopes, the linkage errors can severely hamper the correct interpretation of images and is usually introduced in experiments by the use of standard intracellular staining reagents such as fluorescently labelled antibodies. The linkage error of standard labelled antibodies is caused by the size of the antibody and the random distribution of fluorescent emitters on the antibody surface. Together, these two factors account for a fluorescence displacement of ~40nm when staining proteins by indirect immunofluorescence; and ~20nm when staining with fluorescently coupled primary antibodies. In this study, we describe a class of staining reagents that effectively reduce the linkage error by more than five-fold when compared to conventional staining techniques. These reagents, called Fluo-N-Fabs, consist of an antigen binding fragment of a full-length antibody (Fab/ fragment antigen binding) that is selectively conjugated at the N-terminal amino group with fluorescent organic molecules, thereby reducing the distance between the fluorescent emitter and the protein target of the analysis. Fluo-N-Fabs also exhibit the capability to penetrate tissues and highly crowded cell compartments, thus allowing for the efficient detection of cellular epitopes of interest in a wide range of fixed samples. We believe this class of reagents realize an unmet need in cell biological super resolution imaging studies where the precise localization of the target of interest is crucial for the understanding of complex biological phenomena.

cell biology↗

bioRxiv2020

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity.

The membrane transport apparatus comprises a series of separate membrane bound compartments, or transport stations, that are responsible for the synthesis, processing, transport, sorting and delivery to their final cellular destinations of most transmembrane and soluble lumenal proteins. Over the last decades the membrane transport system has been shown to be extensively regulated both by environmental inputs and by internal homeostatic signalling systems, or control systems, that operate to maintain the homeostasis and optimal functionality of the main transport stations, such as the endoplasmic reticulum and the Golgi, in the face of internal and external perturbations. The trans-Golgi network (TGN) is a major transport and processing station and the main sorting compartment of the transport apparatus. However, the mechanisms that control cargo export and sorting at the TGN have so far remained elusive. Here we focus on the sorting of basolateral cargo proteins and show that these proteins bind to the TGN localized orphan receptor GPRC5A. The cargo-GPRC5A complex triggers the activation of a signaling pathway that involves the G{beta}{gamma} subunits dependent activation of the phospholipase C beta 3 (PLC{beta}3), which inturn induces diacyl glycerol (DAG) production. DAG recruits and activates protein kinase D (PKD) and the phosphorylation of its substrates. This step results in the formation of basolateral carriers for delivery of these cargoes to the basolateral plasma membrane domain. We term this mechanism "ARTG" (AutoRegulation of TGN export). Remarkably, the impairment of ARTG pathway components, and in particular of GPRC5A, causes defects in the polarized organization of epithelial cells.

cell biology↗

bioRxiv2020

Regulated compartmentalization of enzymes in Golgi by GRASP55 controls cellular glycosphingolipid profile and function

Glycans are important regulators of cell and organismal physiology. This requires that the glycan biosynthesis be controlled to achieve specific cellular glycan profiles. Glycans are assembled in the Golgi apparatus on secretory cargoes that traverse it. The mechanisms by which the Golgi apparatus ensures cell- and cargo-specific glycosylation remain obscure. We investigated how the Golgi apparatus regulates glycosylation by studying biosynthesis of glycosphingolipids, glycosylated lipids with critical roles in signalling and differentiation. We identified the Golgi matrix protein GRASP55 as a controller of sphingolipid glycosylation by regulating the compartmentalized localization of key sphingolipid biosynthetic enzymes in the Golgi. GRASP55 controls the localization of the enzymes by binding to them and regulating their entry into peri-Golgi vesicles. Impairing GRASP55-enzyme interaction decompartmentalizes these enzymes, changes the substrate flux across competing glycosylation pathways that results in alteration of the cellular glycosphingolipid profile. This GRASP55 regulated pathway of enzyme compartmentalization allows cells to make cell density-dependent adaptations in glycosphingolipid biosynthesis to suit cell growth needs. Thus, the Golgi apparatus controls the cellular glycan (glycosphingolipid) profile by governing competition between biosynthetic reactions through regulated changes in enzyme compartmentalization.

cell biology↗

bioRxiv2019

Retrograde Transport of Golgi Enzymes by GOLPH3 Across Maturing Cisternae Regulates Glycan Assembly on Sphingolipids and CellGrowth

Glycans are ubiquitous sugar polymers with major biological functions that are assembled by glyco-enzymes onto cargo molecules during their transport through the Golgi complex. How the Golgi determines glycan assembly is poorly understood. By relying on the Golgi cisternal maturation model and using the glyco-enzyme adaptor and oncoprotein GOLPH3 as a molecular tool, we define the first example of how the Golgi controls glycosylation and associated cell functions. GOLPH3, acting as a component of the cisternal maturation mechanism, selectively binds and recycles a subset of glyco-enzymes of the glycosphingolipid synthetic pathway, hinders their escape to the lysosomes and hence increases their levels through a novel lysosomal degradation-regulated mechanism. This enhances the production of specific growth-inducing glycosphingolipids and reprograms the glycosphingolipid pathway to potentiate mitogenic signaling and cell proliferation. These findings unravel unforeseen organizing principles of Golgi-dependent glycosylation and delineate a paradigm for glycan assembly by the Golgi transport mechanisms. Moreover, they indicate a new role of cisternal maturation as a regulator of glycosylation, and outline a novel mechanism of action for GOLPH3-induced proliferation.

cell biology↗

bioRxiv2016

Sphingolipid metabolic flow controls phosphoinositide turnover at thetrans Golgi network

Sphingolipids are membrane lipids, which are globally required for eukaryotic life. Sphingolipid composition varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Thanks to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. Nevertheless, how subcellular sphingolipid levels are maintained in spite of trafficking and metabolic fluxes is only partially understood. Here we describe a homeostatic control circuit that controls sphingolipid levels at the trans Golgi network. Specifically, we show that sphingomyelin production at the trans Golgi network triggers a signalling reaction leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol, and sphingolipid transport to the trans Golgi network, PtdIns(4)P consumption leads to the interruption of this transport in response to excessive sphingomyelin production. Based on this evidence we envisage a model where this homeostatic circuit maintains the sphingolipid composition of trans Golgi network and thus of post-Golgi compartments constant, against instant fluctuations in the sphingolipid biosynthetic flow.

cell biology↗