Biology subjects

Rubinstein, E.

Publications and source records attributed to Rubinstein, E..

8 recordsLinked to original sources

bioRxiv2026

The intercellular transfer of extracellular vesicles markers CD63, CD9 and CD81 is spatially polarized and restricted to cell vicinity

Extracellular vesicles (EVs) are small lipid structures secreted by cells that originate from the cell surface (typically enriched in the tetraspanin (tspan) CD9) or from multivesicular bodies (typically enriched in the tspan CD63). Current methods for studying EVs involve concentrating and purifying EVs, without providing information about the distance or amount of EVs that may transfer from one cell to another. Here, we developed a coculture assay of human mammary MCF-7 cells to study the transfer of mCherry-CD81 or mCherry-CD9 from "donor" cells to a lawn of "acceptor" cells stained with cell tracker blue or green (CTB/CTG), non-transferrable fluorescent dyes. Using confocal fluorescence microscopy, we observed the presence of spots containing mCherry-CD81 or mCherry-CD9 outside donor cells, concentrated at short distance from donor cells and that overlapped with CTB signal, suggestive of their internalization in acceptor cells. Endogenous CD63, CD81 and CD9 also transferred more efficiently at short distances, even in the presence of a flow, as shown by immunostaining cocultures of wild type and KO CD-63, or -9, or -81 cells with antibodies directed against these tspans. Computation of the (x,y,z) coordinates of tspans-containing spots revealed a double polarized transfer: in (x,y), it distributed along a gradient that started from donor cells and decreased with the distance, and in (z), it was stronger in basal compared to upper planes, a (z) polarization that was affected by syntenin-1 depletion in donor cells. Simultaneous monitoring of CD9/CD81 transfer from into double CD81/CD9 KO cells showed that cells transferred more CD81 spots than of CD9. At the basal level, CD63 and CD81 spots were plasma membrane derived as they almost always contained CD9+, and resembled membranous remnants of migration. However, live cell imaging showed migration independent secretion of EVs in the extracellular space, in upper planes. Altogether, not only is our coculture assay suitable for the direct qualitative and quantitative study of EV-transfer, but it highlighted shared three-dimensional features of EV markers transfer between cells.

cell biology↗

bioRxiv2025

Nano-flow cytometry of single extracellular vesicles reveals subpopulation differences across cell types and pharmacological perturbations

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles released by most cell types, which can transfer signals and cargoes between cells. EVs released by a single donor cell source are increasingly recognized as extremely heterogeneous, in terms of size, intracellular origin, and cargo composition. Analyzing large numbers of EVs at the single vesicle level is therefore the only way to truly decipher their heterogeneity. Here, we developed a reliable pipeline of single EV analysis using a nanoparticle-dedicated flow cytometer, which detects particles and measures their size down to 55 nm in diameter, without the need for vesicle pre-immobilization or fluorescent label. We show that titrating each antibody, eliminating unbound antibodies and using EVs devoid of the analyzed markers as negative controls are required to reliably quantify the proportion of EVs bearing none or any combination of two markers, as well as to measure their sizes. We thus observed, depending on the cell source (human cell lines MDA-MB-231, HeLa, A549), variable proportions of EVs bearing none of the CD9, CD81 and CD63 tetraspanins often used to define EVs, and of single- and double-positive EVs for each of these markers. We also observed CD29 (ITGB1) as a protein detected as frequently on EVs as CD9, while other transmembrane proteins (CD44, SSEA-4, CD98), were detected in a small proportion of EVs, and mostly of relatively large size. Finally, we used this pipeline to uncover differential effects of small molecule drugs on subtypes of EVs, and showed that Homosalate increased the proportion of CD9+/CD63+ EVs while two other drugs, Dipivefrin hydrochloride and Metaraminol bitartrate, instead increased the proportion of CD9-/CD63+ EVs. Overall, nano-flow cytometry allows to reliably quantify proportions of EV subpopulations suggested by bulk analyzes of EV markers, at single EV resolution.

cell biology↗

bioRxiv2022

Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

Tunneling nanotubes (TNTs) are open actin- and membrane-based channels, connecting remote cells and allowing direct transfer of cellular material (e.g. vesicles, mRNAs, protein aggregates) from cytoplasm to cytoplasm. Although they are important especially in pathological conditions (e.g., cancers, neurodegenerative diseases), their precise composition and their regulation were still poorly described. Here, using a biochemical approach allowing to separate TNTs from cell bodies and from extracellular vesicles and particles (EVPs), we obtained the full composition of TNTs compared to EVPs. We then focused to two major components of our proteomic data, the CD9 and CD81 tetraspanins, and further investigated their specific roles in TNT formation and function. We show that these two tetraspanins have distinct non-redundant functions: CD9 participates in stabilizing TNTs, whereas CD81 expression is required to allow the functional transfer of vesicle in the newly formed TNTs, possibly by regulating docking to or fusion with the opposing cell.

cell biology↗

bioRxiv2020

Specificities of exosome versus small ectosome secretion revealed by live intracellulartracking and synchronized extracellular vesicle release of CD9 and CD63

Despite their important and multiple roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized yet. In particular, how and to what extent EVs form either as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (ectosomes) remains unclear. We followed in HeLa cells the intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment and identified transient co-localization both at the plasma membrane (PM) and in endosomes, before they finally segregate. CD9 was more abundantly released in EVs than CD63. However, when forcing expression of CD63 at the PM, by mutating its lysosome-addressing motive, its secretion in EVs was increased. Thus, in HeLa cells, small ectosomes are more prominently released than exosomes. By comparative proteomic analysis, we identified a few surface proteins likely specific of either exosomes (e.g. LAMP1) or ectosomes (e.g. BSG, SLC3A2), based on their known intracellular location in lysosomes or the PM, and on the different effects on their release of Bafilomycin A1, a drug that neutralizes endosomal pH. Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

cell biology↗

bioRxiv2020

Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytic cells.

Sporozoite forms of the malaria parasite Plasmodium are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytic cells. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection. IMPORTANCEMalaria is caused by Plasmodium parasites and remains one of the deadliest parasitic diseases worldwide. The parasite is transmitted by a blood feeding mosquito and first invades the liver for an initial, obligatory and silent round of replication. The liver infection is an attractive target for antimalarial vaccine strategies, however the molecular mechanisms of parasite invasion of hepatocytes remain to be fully elucidated. Two hepatocyte surface proteins are known to be important for parasite entry into hepatocytes, the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1). These receptors constitute independent gateways depending on the Plasmodium species. Here, we identified the structural determinants of SR-B1, an important hepatocyte entry factor for human-infecting P. vivax. This study paves the way toward a better characterization of the molecular interactions underlying the crucial early stages of infection, a pre-requisite for the development of novel malaria vaccine strategies.

microbiology↗

bioRxiv2019

Dynamic regulation of CD45 by tetraspanin CD53

T cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells. We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP. Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.

immunology↗

bioRxiv2019

Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

A disintegrin and metalloprotease 10 (ADAM10) is essential for embryonic development and impacts on diseases such as cancer, Alzheimers and inflammatory diseases. ADAM10 is a molecular scissor that proteolytically cleaves the extracellular region from over 100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors and chemokines. ADAM10 was recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function is critically dependent on a TspanC8 partner. To address this, we generated the first monoclonal antibodies to Tspan15 as a model TspanC8. These were used to show that ADAM10 is the principal Tspan15-interacting protein, that Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Together these findings suggest that ADAM10 exists as an intimate ADAM10/TspanC8 scissor complex.

cell biology↗

bioRxiv2018

HIV-1 specifically traps and depletes CD9 and CD81 tetraspanins during egress

HIV-1 assembly specifically alters both partitioning and dynamics of the tetraspanins CD9 and CD81 forming enriched areas where the virus buds. Importantly the presence of these proteins at exit sites and in viral particles inhibits virus-induced membrane fusion. To get molecular insights into tetraspanins partitioning in this viral context, we correlated nanoscale CD9 mapping obtained by super resolution microscopy to membrane topography probed by Atomic Force Microscopy (AFM). We demonstrated that CD9 is specifically trapped within the nascent viral particles, especially at buds tips, and that Gag mediate CD9 and CD81 depletion from cellular surfaces, even in the absence of Vpu and Nef, resulting from tetraspanins escaping from the plasma membrane during HIV-1 release. In addition, we showed that CD9 is organized as small membrane assemblies of few tens of nanometers that can coalesce upon Gag expression. Our results support a functional redundancy among tetraspanins during HIV release.

microbiology↗