We probed the mechanism by which the Parkinson\'s disease-associated protein α-synuclein (α-syn)/SNCA promotes the pathogenesis and progression of melanoma. We found that the human melanoma cell line SK-MEL-28 in which SNCA is knocked out (SNCA-KO) has low levels of tetraspanin CD81, which is a cell-surface protein that promotes invasion, migration, and immune suppression. Analyzing data from the Cancer Genome Atlas, we show that SNCA and CD81 mRNA levels are positively correlated in melanoma; melanoma survival is inversely related to the levels of SNCA and CD81; and SNCA/CD81 are inversely related to the expression of key cytokine genes (IL12A, IL12B, IFN, IFNG, PRF1 and GZMB) for immune activation and immune cell-mediated killing of melanoma cells. We propose that high levels of α-syn and CD81 in melanoma and in immune cells drive invasion and migration and in parallel cause an immunosuppressive microenvironment; these contributing factors lead to aggressive melanomas.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

Tetraspanins, including CD53 and CD81, are four-transmembrane proteins that affect the membrane organization to regulate cellular processes including migration, proliferation and signaling. However, it is unclear how the organizing function of tetraspanins is regulated at the molecular level. Here we investigated whether recently proposed \'open\' and \'closed\' conformations of tetraspanins regulate the nanoscale organization of the plasma membrane of B cells. We generated conformational mutants of CD53 (F44E) and CD81 (4A, E219Q) that represent the \'closed\' and \'open\' conformation, respectively. Surface expression of these CD53 and CD81 mutants was comparable to that of wildtype (WT) protein. Localization of mutant tetraspanins into nanodomains was visualized by super-resolution dSTORM microscopy. Whereas the size of these nanodomains was unaffected by conformation, the clustered fraction of \'closed\' CD53 was higher and of \'open\' CD81 lower than respective WT protein. In addition, knock-out cells lacking CD53 showed an increased likelihood of clustering of its partner CD45. Interestingly, \'closed\' CD53 interacted more with CD45 than WT CD53. Absence of CD81 lowered the cluster size of its partner CD19, and \'closed\' CD81 interacted less with CD19 than WT CD81, but \'open\' CD81 did not affect CD19 interaction. However, none of the tetraspanin conformations made significant impact on the nanoscale organization of their partners CD19 or CD45. Taken together, conformational mutations of CD53 and CD81 differentially affect their nanoscale organization, but not the organization of their partner proteins. This study improves the molecular insight into cell surface nanoscale organization by tetraspanins.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease worldwide. As an extracellular parasite, adhesion to host cells is essential for the development of infection. During attachment, the parasite changes its tear ovoid shape to a flat ameboid form, expanding the contact surface and migrating through tissues. Here, we have identified a novel structure formed at the posterior pole of adherent parasite strains, resembling the previously described uropod, which appears to play a pivotal role as an anchor during the attachment process. Moreover, our research demonstrates that the overexpression of the tetraspanin TSP5 protein (TvTSP5), localized on the parasite\'s cell surface, notably enhances the formation of this posterior anchor structure in adherent strains. Finally, we demonstrate parasites that overexpress TvTSP5 possess an increased ability of the parasite to adhere to host cells, enhanced parasite aggregation and reduced migration on agar plates. Overall, these findings unveil novel proteins and structures involved in the intricate mechanisms of Trichomonas vaginalis interactions with host cells.

Recently tetraspanin CD151 has been identified as an important biological target involved in metastatic processes which include cell adhesion, tumor progression processes, and so forth in different types of cancers, such as breast cancer and glioblastoma. This in Silico study considered 1603 compounds from the Food and Drug Administration database, after performing an ADMET analysis; we selected 853 ligands, which were used for docking analysis. The most promising ligands were selected from docking studies, based on two criteria: (a) showed lowest affinity to the CD151 protein and (b) they interact with the QRD motif, located in the second extracellular loop. Furthermore, we investigate the stability of the protein-ligand complexes through MD simulations as well as free energy MM-PBSA calculations. From these results, loperamide and glipizide were identified as the best evaluated drugs. We suggest an in vitro analysis is needed to confirm our in silico prediction studies.

Membrane remodeling is a fundamental cellular process that is crucial for physiological functions such as signaling, membrane fusion and cell migration. Tetraspanins (TSPANs) are transmembrane proteins of central importance to membrane remodeling events. During these events, TSPANs are known to interact with themselves and other proteins and lipids; however, their mechanism of action in controlling membrane dynamics is not fully understood. Since these proteins span the membrane, membrane properties such as rigidity, curvature and tension can influence their behavior. In this Review, we summarize recent studies that explore the roles of TSPANs in membrane remodeling processes and highlight the unique structural features of TSPANs that mediate their interactions and localization. Further, we emphasize the influence of membrane curvature on TSPAN distribution and membrane domain formation and describe how these behaviors affect cellular functions. This Review provides a comprehensive perspective on the multifaceted function of TSPANs in membrane remodeling processes and can help readers to understand the intricate molecular mechanisms that govern cellular membrane dynamics.

Neurons in the central nervous system release extracellular vesicles (EVs) and exosomes in response to synaptic activity to regulate physiological processes at target neurons. The intercellular transfer of proteins, mRNAs, lipids or metabolites through EVs potentially modulates the structure and function of neurons and circuits. Whereas the biogenesis of EVs, their release from donor cells, and their molecular composition have been studied extensively, the critical factors and mechanisms regulating EV interactions with target cells are incompletely understood. Here, we identified tetraspanin 15 (Tspan15) as a component of tumor susceptibility gene 101 protein (TSG101)- and CD81-positive EV fractions. Tspan15 fluorescent fusion proteins were released from donor cells and interacted with target cells together with the exosomal marker CD63. EVs collected from wildtype cortical neurons (WT-EVs) underwent similar association with target neurons derived from either wildtype (+/+) or Tspan15 knockout (-/-) mice. In contrast, target cell interactions of EVs collected from Tspan15 (-/-) cortical donor neurons (KO-EVs) were significantly impaired, as compared to WT-EVs. Our data suggest that Tspan15 is dispensable at target neuron plasma membranes, but is required at the EV surface to promote EV docking at target neurons.

The transient cellular organelles known as migrasomes, which form during cell migration along retraction fibers, have emerged as a crutial factor in various fundamental cellular processes and pathologies. These membrane vesicles originate from local membrane swellings, encapsulate specific cytoplasmic content, and are eventually released to the extracellular environment or taken up by recipient cells. Migrasome biogenesis entails a sequential membrane remodeling process involving a complex interplay between various molecular factors such as tetraspanin proteins, and mechanical properties like membrane tension and bending rigidity. In this review, we summarize recent studies exploring the mechanism of migrasome formation. We emphasize how physical forces, together with molecular factors, shape migrasome biogenesis, and detail the involvement of migrasomes in various cellular processes and pathologies. A comprehensive understanding of the exact mechanism underlying migrasome formation and the identification of key molecules involved hold promise for advancing their therapeutic and diagnostic applications.

Extracellular vesicles (EVs) are preeminent carriers of biomarkers and have become the subject of intense biomedical research for medical diagnostics using biosensors. To create effective EV-based immunoassays, it is imperative to develop surface chemistry approaches with optimal EV detection targeting transmembrane protein biomarkers that are not affected by cell-to-cell variability. Here, we developed a series of immunoassays for the detection of EVs derived from mouse monocyte cells using surface plasmon resonance (SPR) biosensors. We chemically immobilized antibodies onto mixed self-assembled monolayers of oligo ethylene glycol (OEG) alkanethiolates with carboxylic and hydroxylic terminal groups. The effects of antibody clonality (monoclonal vs polyclonal) and antibody surface coverage in targeting EVs via CD81 tetraspanins were investigated. We determined binding kinetic parameters, establishing trends from steric hindrance effects and epitope recognition properties of antibodies. Our results indicate that a 40% surface coverage of polyclonal antibodies covalently linked onto a mixed SAM with 10% of terminated -COOH groups yields a promising approach for EV detection with a linear range of 1.9 × 108-1.9 × 109 EVs/mL and a limit of detection of 5.9 × 106 EVs/mL. This optimal immunoassay exhibits a 1.92 nM equilibrium dissociation constant for bound EVs, suggesting a high binding affinity when CD81 is targeted. Our study provides important insights into surface chemistry development for EV detection targeted via transmembrane protein biomarkers using antibodies, which has promising applications for disease diagnostics.

The tetraspanin gene family encodes cell-surface proteins that span the membrane 4 times and play critical roles in a wide range of biological processes across numerous organisms. Recent findings highlight the involvement of a tetraspanin of the lepidopteran pest Helicoverpa armigera in resistance to Bacillus thuringiensis Cry insecticidal proteins, which are extensively used in transgenic crops. Thus, a better understanding of lepidopteran tetraspanins is urgently needed. In the current study, genome scanning in 10 lepidopteran species identified a total of 283 sequences encoding potential tetraspanins. Based on conserved cysteine patterns in the large extracellular loop and their phylogenetic relationships, these tetraspanins were classified into 8 subfamilies (TspA to TspH). Six ancestral introns were identified within lepidopteran tetraspanin genes. Tetraspanins in TspA, TspB, TspC, and TspD subfamilies exhibit highly similar gene organization, while tetraspanins in the remaining 4 subfamilies exhibited variation in intron loss and/or gain during evolution. Analysis of chromosomal distribution revealed a lepidopteran-specific cluster of 10 to 11 tetraspanins, likely formed by tandem duplication events. Selective pressure analysis indicated negative selection across all orthologous groups, with ω values ranging between 0.004 and 0.362. However, positive selection was identified at 18 sites within TspB5, TspC5, TspE3, and TspF10. Furthermore, spatiotemporal expression analysis of H. armigera tetraspanins demonstrated variable expression levels across different developmental stages and tissues, suggesting diverse functions of tetraspanin members in this globally important insect pest. Our findings establish a solid foundation for subsequent functional investigations of tetraspanins in lepidopteran species.