Microscopic clusters feature the composition of biochemical tetraspanin-assemblies and constitute building-blocks of tetraspanin enriched domains.

Biochemical approaches revealed that tetraspanins are multi-regulatory proteins forming a web, where they act in tetraspanin-enriched-microdomains (TEMs). A microscopic criterion differentiating between web and TEMs is lacking. Using super-resolution microcopy, we identify co-assemblies between the tetraspanins CD9 and CD81 and CD151 and CD81. CD9 assemblies contain as well the CD9/CD81-interaction partner EWI-2. Moreover, CD9 clusters are proximal to clusters of the CD81-interaction partner CD44 and CD81-/EWI-2-interacting ezrin-radixin-moesin proteins. Assemblies scatter unorganized across the cell membrane; yet, upon EWI-2 elevation, they agglomerate into densely packed arranged-crowds in a process independent from actin dynamics. In conclusion, microscopic clusters are equivalent to biochemical tetraspanin-assemblies, defining in their entirety the tetraspanin web. Cluster-agglomeration enriches tetraspanins, which makes agglomerations to a microscopic complement of TEMs. The microscopic classification of tetraspanin assemblies advances our understanding of this enigmatic protein family, whose members play roles in a plethora of cellular functions, diseases, and pathogen infections.

ADAM17-dependent signaling is required for oncogenic human papillomavirus entry platform assembly.

Oncogenic human papillomaviruses (HPV) are small DNA viruses that infect keratinocytes. After HPV binding to cell surface receptors, a cascade of molecular interactions mediates the infectious cellular internalization of virus particles. Aside from the virus itself, important molecular players involved in virus entry include the tetraspanin CD151 and the epidermal growth factor receptor (EGFR). To date, it is unknown how these components are coordinated in space and time. Here, we studied plasma membrane dynamics of CD151 and EGFR and the HPV16 capsid during the early phase of infection. We find that the proteinase ADAM17 activates the extracellular signal-regulated kinases (ERK1/2) pathway by the shedding of growth factors which triggers the formation of an endocytic entry platform. Infectious endocytic entry platforms carrying virus particles consist of two-fold larger CD151 domains containing the EGFR. Our finding clearly dissects initial virus binding from ADAM17-dependent assembly of a HPV/CD151/EGFR entry platform.

The specificity of homomeric clustering of CD81 is mediated by its δ-loop.

Tetraspanins are cell membrane-scaffolding proteins interacting with one another and a repertoire of interaction partners. Through these interactions, they form extended molecular networks as tetraspanin webs or tetraspanin-enriched microdomains. Microscopic data suggest that these networks contain tetraspanin clusters, with poor overlap between clusters formed by different tetraspanins. Here, we investigate the possibility of targeting tetraspanins CD9 or CD151 to clusters formed by the tetraspanin CD81. We find that the δ-loop from the large extracellular domain of CD81 is sufficient for targeting of CD9/CD151 to CD81 clusters. Moreover, in a pull-down assay, CD9 coprecipitates more CD81 when it carries the CD81 δ-loop. In conclusion, the information for forming homomeric CD81 clusters is encoded in the δ-loop.

Oligomerization of the Tetraspanin CD81 via the Flexibility of Its δ-Loop.

Tetraspanins are master organizers in the plasma membrane, forming tetraspanin-enriched microdomains with one another and other surface molecules. Their rod-shaped structure includes a large extracellular loop (LEL) that plays a pivotal role in tetraspanin network formation. We performed comparative atomistic and coarse-grain molecular-dynamics simulations of the LEL in isolation and full-length CD81, and reproduced LEL flexibility patterns known from wet-lab experiments in which the LEL δ-loop region showed a pronounced flexibility. In a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayer and a plasma membrane environment, the conformational flexibility of the δ-loop initiates CD81-CD81 contacts for oligomerization. Furthermore, in the plasma membrane, CD81-ganglioside bridges arising from preformed glycolipid patches cross-link the complexes. The data suggest that exposing a flexible domain enables binding to interaction partners by circumventing the restriction of orientation and conformational freedom of membrane proteins.

The extracellular δ-domain is essential for the formation of CD81 tetraspanin webs.

CD81 is a ubiquitously expressed member of the tetraspanin family. It forms large molecular platforms, so-called tetraspanin webs that play physiological roles in a variety of cellular functions and are involved in viral and parasite infections. We have investigated which part of the CD81 molecule is required for the formation of domains in the cell membranes of T-cells and hepatocytes. Surprisingly, we find that large CD81 platforms assemble via the short extracellular δ-domain, independent from a strong primary partner binding and from weak interactions mediated by palmitoylation. The δ-domain is also essential for the platforms to function during viral entry. We propose that, instead of stable binary interactions, CD81 interactions via the small δ-domain, possibly involving a dimerization step, play the key role in organizing CD81 into large tetraspanin webs and controlling its function.

Liver sinusoidal endothelial cell-mediated CD8 T cell priming depends on co-inhibitory signal integration over time.

The initiation of adaptive immunity requires cell-to-cell contact between T cells and antigen-presenting cells. Together with immediate TCR signal transduction, the formation of an immune synapse (IS) is one of the earliest events detected during T cell activation. Here, we show that interaction of liver sinusoidal endothelial cells (LSEC) with naive CD8 T cells, which induces CD8 T cells without immediate effector function, is characterized by a multi-focal type IS. The co-inhibitory molecule B7H1, which is pivotal for the development of non-responsive LSEC-primed T cells, did not alter IS structure or TCRß/CD11a cluster size or density, indicating that IS form does not determine the outcome of LSEC-mediated T cell activation. Instead, PD-1 signaling during CD8 T cell priming by LSEC repressed IL-2 production as well as sustained CD25 expression. When acting during the first 24 h of LSEC/CD8 T cell interaction, CD28 co-stimulation inhibited the induction of non-responsive LSEC-primed T cells. However, after more than 36 h of PD-1 signaling, CD28 co-stimulation failed to rescue effector function in LSEC-primed T cells. Together, these data show that during LSEC-mediated T cell priming, integration of co-inhibitory PD-1 signaling over time turns on a program for CD8 T cell development, that cannot be overturned by co-stimulatory signals.

The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication.

Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.

CD81 is essential for the formation of membrane protrusions and regulates Rac1-activation in adhesion-dependent immune cell migration.

CD81 (TAPA-1) is a member of the widely expressed and evolutionary conserved tetraspanin family that forms complexes with a variety of other cell surface receptors and facilitates hepatitis C virus entry. Here, we show that CD81 is specifically required for the formation of lamellipodia in migrating dendritic cells (DCs). Mouse CD81(-/-) DCs, or murine and human CD81 RNA interference knockdown DCs lacked the ability to form actin protrusions, thereby impairing their motility dramatically. Moreover, we observed a selective loss of Rac1 activity in the absence of CD81, the latter of which is exclusively required for integrin-dependent migration on 2-dimensional substrates. Neither integrin affinity for substrate nor the size of basal integrin clusters was affected by CD81 deficiency in adherent DCs. However, the use of total internal reflection fluorescence microscopy revealed an accumulation of integrin clusters above the basal layer in CD81 knockdown cells. Furthermore, ß1- or ß2-integrins, actin, and Rac are strongly colocalized at the leading edge of DCs, but the very fronts of these cells protrude CD81-containing membranes that project outward from the actin-integrin area. Taken together, these data suggest a thus far unappreciated role for CD81 in the mobilization of preformed integrin clusters into the leading edge of migratory DCs on 2-dimensional surfaces.

Specific GC-MS/MS stable-isotope dilution methodology for free 9- and 10-nitro-oleic acid in human plasma challenges previous LC-MS/MS reports.

Nitrated unsaturated fatty acids including nitro-oleic acid (NO(2)-OA) have been measured in human blood samples in their free and esterified forms. Plasma concentrations in healthy subjects have been reported to be of the order of 600 nM for free NO(2)-OA and 300 nM for esterified NO(2)-OA, as measured by LC-MS/MS. In the present article we report a GC-MS/MS method for the specific and accurate quantification of two NO(2)-OA isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, in human plasma using newly prepared, isolated, characterized and standardized (15)N-labeled analogs. This method involves SPE extraction of fatty acids from slightly acidified plasma samples (pH 5), conversion to their pentafluorobenzyl (PFB) esters, isolation by HPLC, solvent extraction from a single HPLC fraction and GC-MS/MS analysis in the electron capture negative-ion chemical ionization (ECNICI) mode. Quantification was performed by selected-reaction monitoring (SRM) of m/z 46 ([NO(2)](-)) and m/z 47 ([(15)NO(2)](-)) produced by collision-induced dissociation (CID) from the parent ions [M-PFB](-) at m/z 326 for endogenous 9-NO(2)-OA and 10-NO(2)-OA and m/z 327 for the internal standards 9-(15)NO(2)-OA and 10-(15)NO(2)-OA. We partially validated the GC-MS/MS method for 9-NO(2)-OA and 10-NO(2)-OA in human plasma and quantified these nitro-oleic species in plasma of 15 healthy volunteers. We identified two isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, in human plasma under physiological conditions and found these nitrated fatty acids at a mean concentration of 1 nM each. This concentration is about 600 times lower than that reported by others using LC-MS/MS. Our GC-MS/MS studies on nitro-oleic acid and 3-nitrotyrosine suggest that the extent of nitration of biomolecules such as unsaturated fatty acids and tyrosine is very low in health. In this article we discuss analytical and biological ramifications potentially associated with measurement of nitrated biomolecules in biological systems.