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Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. Extracellular vesicles (EVs) play a role in intercellular communication, however the precise biogenesis of different populations of EVs are not clear. Here, the authors follow the intracellular trafficking of two proteins before their secretion in EVs and report the biogenesis and protein markers of EV subtypes: ectosomes budding from the plasma membrane as well as exosomes from late endosomes.

Exosomes, extracellular vesicles (EVs) of endosomal origin, emerge as master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins (TSPNs) and syndecans (SDCs), the latter occurring mainly in proteolytically cleaved form, as membrane-spanning C-terminal fragments of the proteins. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition, and activity, we currently ignore whether these work together to control exosome biology. Here we show that TSPN6, a poorly characterized tetraspanin, acts as a negative regulator of exosome release, supporting the lysosomal degradation of SDC4 and syntenin. We demonstrate that TSPN6 tightly associates with SDC4, the SDC4-TSPN6 association dictating the association of TSPN6 with syntenin and the TSPN6-dependent lysosomal degradation of SDC4-syntenin. TSPN6 also inhibits the shedding of the SDC4 ectodomain, mimicking the effects of matrix metalloproteinase inhibitors. Taken together, our data identify TSPN6 as a regulator of the trafficking and processing of SDC4 and highlight an important physical and functional interconnection between these membrane scaffolds for the production of exosomes. These findings clarify our understanding of the molecular determinants governing EV formation and have potentially broad impact for EV-related biomedicine.

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under‐investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock‐out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P‐1/EWI‐F/PTGFRN and EWI‐2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9‐ and CD81‐deficient cells. In the case of EWI‐2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P‐1 and EWI‐2 by CD9 and CD81.

The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aβ, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.

Background Single particle tracking (SPT) is nowadays one of the most popular technique to probe spatio-temporal dynamics of proteins diffusing within the plasma membrane. Indeed membrane components of eukaryotic cells are very dynamic molecules and can diffuse according to different motion modes. Trajectories are often reconstructed frame-by-frame and dynamic properties often evaluated using mean square displacement (MSD) analysis. However, to get statistically significant results in tracking experiments, analysis of a large number of trajectories is required and new methods facilitating this analysis are still needed. Results In this study we developed a new algorithm based on back-propagation neural network (BPNN) and MSD analysis using a sliding window. The neural network was trained and cross validated with short synthetic trajectories. For simulated and experimental data, the algorithm was shown to accurately discriminate between Brownian, confined and directed diffusion modes within one trajectory, the 3 main of diffusion encountered for proteins diffusing within biological membranes. It does not require a minimum number of observed particle displacements within the trajectory to infer the presence of multiple motion states. The size of the sliding window was small enough to measure local behavior and to detect switches between different diffusion modes for segments as short as 20 frames. It also provides quantitative information from each segment of these trajectories. Besides its ability to detect switches between 3 modes of diffusion, this algorithm is able to analyze simultaneously hundreds of trajectories with a short computational time. Conclusion This new algorithm, implemented in powerful and handy software, provides a new conceptual and versatile tool, to accurately analyze the dynamic behavior of membrane components.

The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and β1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions. In both focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN), kidney injury is characterised by the invasion of glomerular tufts by parietal epithelial cells (PECs). Here Lazareth et al. identify the tetraspanin CD9 as a key regulator of PEC migration, and find its upregulation in FSGS and CGN contributes to kidney injury in both diseases.

Extracellular vesicles (EVs) are thought to mediate intercellular communication by transferring cargoes from donor to acceptor cells. The EV content-delivery process within acceptor cells is still poorly characterized and debated. CD63 and CD9, members of the tetraspanin family, are highly enriched within EV membranes and are respectively enriched within multivesicular bodies/endosomes and at the plasma membrane of the cells. CD63 and CD9 have been suspected to regulate the EV uptake and delivery process. Here we used two independent assays and different cell models (HeLa, MDA-MB-231 and HEK293T cells) to assess the putative role of CD63 and CD9 in the EV delivery process that includes uptake and cargo delivery. Our results suggest that neither CD63, nor CD9 are required for this function. In human cell lines, CD63 and CD9 tetraspanins are knocked-down and shown to not be required for extracellular vesicle uptake and cargo delivery.

Once passed into the bloodstream, bacterial pathogens have a limited time to interact with permissive receptors at the surface of host cells. Neisseria meningitidis has developed an extremely effective strategy allowing it to find its receptors in a few seconds. Here, we report that N. meningitidis type IV pili exploit the physical properties of host cells' plasma membranes to promote the formation of early tubular membrane structures essential for initial bacterial adhesion. These tubular structures, which form before any signaling events in host cells, concentrate and trap multiple plasma membrane-associated proteins in the vicinity of bacteria, thereby facilitating the selection, interaction and activation of specific adhesion and signaling receptors by bacterial ligands present on type IV pili. Our results define an additional paradigm for the recruitment of specific receptors by pathogenic bacteria, which depends on the physical property of bacterial pili to induce the formation of tubular plasma membrane structures enriched in integral plasma membrane receptors. The adhesion of meningococci to endothelial cells relies on type IV pili, which induce the formation of long tubular structures on the host cell membrane. Here, the authors show that the tubular structures accumulate and trap host membrane-associated proteins, which facilitates their interaction with bacterial ligands and the activation of adhesion and signaling receptors.

Skeletal muscle regeneration after injury follows a remarkable sequence of synchronized events. However, the mechanisms regulating the typical organization of the regenerating muscle at different stages remain largely unknown. Here we show that muscle regeneration in mice lacking either CD9 or CD81 is abnormal and characterized by the formation of discrete giant dystrophic myofibres, which form more quickly in the absence of both tetraspanins. We also show that, in myoblasts, these two tetraspanins associate with the immunoglobulin domain molecule CD9P-1 (EWI-F/FPRP), and that grafting of CD9P-1-depleted myoblasts in regenerating muscles also leads to abnormal regeneration. In vitro myotubes lacking CD9P-1 or both CD9 and CD81 fuse with a higher frequency than normal myotubes. Our study unveils a mechanism preventing inappropriate fusion of myotubes that has an important role in the restitution of normal muscle architecture during muscle regeneration. The skeletal muscle has a remarkable capacity to regenerate after injury. Here Charrin et al . reveal that restoration of muscle architecture after injury requires a tight control of muscle cell fusion by the tetraspanin proteins CD9 and CD81 through their interaction with the cell surface protein CD9P-1.

Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, first infect the liver for an initial round of replication before the emergence of pathogenic blood stages. Sporozoites represent attractive targets for antimalarial preventive strategies, yet the mechanisms of parasite entry into hepatocytes remain poorly understood. Here we show that the two main species causing malaria in humans, Plasmodium falciparum and Plasmodium vivax, rely on two distinct host cell surface proteins, CD81 and the Scavenger Receptor BI (SR-BI), respectively, to infect hepatocytes. By contrast, CD81 and SR-BI fulfil redundant functions during infection by the rodent parasite P. berghei. Genetic analysis of sporozoite factors reveals the 6-cysteine domain protein P36 as a major parasite determinant of host cell receptor usage. Our data provide molecular insights into the invasion pathways used by different malaria parasites to infect hepatocytes, and establish a functional link between a sporozoite putative ligand and host cell receptors. Malaria is an infectious disease that affects millions of people around the world and remains a major cause of death, especially in Africa. It is caused by Plasmodium parasites, which are transmitted by mosquitoes to mammals. Once in the mammal, the parasites infect liver cells, where they multiply. Previous studies have suggested that proteins on the surface of the liver cells and on the parasite affect how Plasmodium infects liver cells. Understanding how these proteins enable the parasites to enter the cells may help researchers to develop treatments that interrupt the parasite life cycle and prevent infection. Manzoni et al. have now investigated how different malaria parasite species interact with liver cells. The main parasite species that infect humans are Plasmodium falciparum in Africa and Plasmodium vivax outside Africa. Manzoni et al. found that P. falciparum and P. vivax infect human liver cells by two different routes: P. falciparum interacts with a liver cell protein called CD81, and P. vivax interacts with a liver cell protein called SR-BI. Further experiments that used mutant forms of malaria parasites that infect mice showed that a parasite protein called P36 determines which liver cell protein the parasite will interact with. The next step is to understand how P36 interacts with the liver cell proteins and to identify other parasite proteins that help Plasmodium to invade cells. In the future, such knowledge may help to develop a highly effective malaria vaccine.