Biology subjects

Saleppico, R.

Publications and source records attributed to Saleppico, R..

5 recordsLinked to original sources

bioRxiv2023

An optogenetic method for the controlled release of single molecules

We developed a system for optogenetic release of single molecules in live cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for the controlled delivery of functional proteins to cytosol and plasma membrane in amounts compatible with single molecule imaging, greatly simplifying access to single molecule microscopy of any protein in live cells. Furthermore, we could reconstitute cellular functions such as ion conductance by delivering BK and VRAC ion channels to the plasma membrane. Finally, we could induce NF-kB signaling in T-Lymphoblasts stimulated by IL-1 by controlled release of a signaling protein that had been knocked-out in the same cells. We observed light induced formation of functional inflammatory signaling complexes that could trigger IKK phosphorylation in single cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single molecule level.

biophysics↗

bioRxiv2023

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space

Fibroblast Growth Factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by PI(4,5)P2-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the protein-protein interaction interface through which FGF2 binds to the 1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we provide insights into a FGF2 dimerization interface that brings C95 residues in close proximity, resulting in disulfide bridged FGF2 dimers. We propose a mechanism by which they bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95 bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

biochemistry↗

bioRxiv2021

Glypican-1 drives unconventional secretion of Fibroblast Growth Factor 2

Fibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is transported into the extracellular space by an unconventional secretory mechanism. Cell surface heparan sulfate proteoglycans are known to play an essential role in this process. Unexpectedly, we found that among the diverse sub-classes consisting of syndecans, perlecans, glypicans and others, Glypican-1 (GPC1) is both the principle and rate-limiting factor that drives unconventional secretion of FGF2. By contrast, we demonstrate GPC1 to be dispensable for FGF2 signaling into cells. We provide first insights into the structural basis for GPC1-dependent FGF2 secretion, identifying disaccharides with N-linked sulfate groups to be enriched in the heparan sulfate chains of GPC1 to which FGF2 binds with high affinity. Our findings have broad implications for the role of GPC1 as a key molecule in tumor progression.

biochemistry↗

bioRxiv2021

Cholesterol promotes both head group visibility and clustering of PI(4,5)P2 driving unconventional secretion of Fibroblast Growth Factor 2

Fibroblast Growth Factor 2 (FGF2) is a cell survival factor involved in tumor-induced angiogenesis. FGF2 is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further reveal cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers in a fully reconstituted system. Based on extensive atomistic molecular dynamics simulations and membrane tension experiments, we propose cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 towards the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

biophysics↗

bioRxiv2019

The α1 subunit of the Na,K-ATPase acts upstream of PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2 from tumor cells

Fibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is exported from cells by an unconventional secretory pathway. This process is based on direct translocation of FGF2 across the plasma membrane. FGF2 membrane translocation depends on PI(4,5)P2-induced formation of membrane-inserted FGF2 oligomers followed by extracellular trapping of FGF2 at the outer leaflet mediated by cell surface heparan sulfate proteoglycans. Beyond the well-characterized core mechanism of FGF2 membrane translocation, the Na,K-ATPase has been proposed to play a so far unknown role in unconventional secretion of FGF2. Here, we define a direct physical interaction of FGF2 with a subdomain of the cytoplasmic part of the 1 subunit of the Na,K-ATPase. Employing NMR spectroscopy and molecular dynamics simulations, we identified two lysine residues on the molecular surface of FGF2 that are shown to be essential for its interaction with 1. In intact cells, the corresponding lysine-to-glutamate variants of FGF2 were characterized by inefficient secretion and reduced recruitment to the inner plasma membrane leaflet as shown by single molecule TIRF microscopy. Our findings suggest that 1 acts upstream of PI(4,5)P2 facilitating efficient membrane translocation of FGF2 to the cell surface of tumor cells.

biochemistry↗