Harboring four transmembrane domains in their structural hallmark, Tetraspanins (Tspans) are a family of glycoproteins with pivotal functions in a variety of biological and cellular processes. Through interacting laterally with each other or specific membrane proteins, Tspans organize tetraspanin-enriched microdomains (TEMs), modulating cellular signaling, adhesion, fusion, and proliferation. An abundance of evidence has identified the multiple functions in the progression of cancer as well as the underlying molecular mechanisms. Recently, plenty of studies have focused on the utilities of Tspans by pathogens for infection, especially the infection of viruses. The expression of Tspans correlates with the phase of viral infection, the type of virus, and targeted therapies. In particular, perturbations of Tspans in host cells can affect viral attachment, intracellular trafficking, translation, virus assembly, and release. In this review, we summarize and provide a historical overview of the discovery and characterization of various kinds of virus infection and highlight their diversity and complexity, along with the virus life cycle. Furthermore, we examined the current understanding of how various Tspans are involved in the regulatory mechanisms underlying viral infection. This review aims to offer a comprehensive understanding of the targeting of Tspans for therapeutic intervention in infections caused by diverse pathogens.

Exosomes are a class of extracellular vesicles that encompass a diverse array of bioactive molecules, including proteins, lipids, mRNA, and microRNA(miRNA). Virtually all cell types release exosomes under both physiological and pathological conditions. In addition to electrical and chemical signals, exosomes are an alternative route of signaling between cells in the brain. In the brain, they are involved in processes such as synaptic plasticity, neuronal stress response, intercellular communication, and neurogenesis. A number of studies have shown that exosomes regulate the occurrence and development of depression by participating in the regulation of hypothalamic-pituitary-adrenal axis, brain-derived neurotrophic factor, immune inflammatory response and other mechanisms, showing that they may become potential biological agents for the diagnosis and treatment of depression. In addition, exosomes have the ability to easily cross the blood-brain barrier, making them ideal drug or molecular delivery tools for the central nervous system. Engineered exosomes have good brain targeting ability, and their research in central nervous system diseases has begun to emerge. However, the molecular pathways involved in the pathogenesis of depression remain unknown, and further studies are needed to fully understand the role of exosomes in the development or improvement of depression. Therefore, in this review, we mainly focus on the diagnostic performance and therapeutic effect of exosomes in depression, and explore the advantages of exosomes as biomarkers and gene delivery vectors for depression.

Exposure to microgravity has become the primary factor threatening astronauts' health. Immunity is dysregulated rapidly following spaceflight, and reactivation of latent virus has been observed. However, systematic studies and molecular mechanisms of the adverse impact of microgravity on the antiviral immune response are still elusive. As a member of the Tetraspanins family, CD82 participates in the biological process between the cell and the trafficking of cellular transmembrane proteins that interact with many viruses. Herein, we established a ground-based zebrafish model of microgravity to research the embryogenesis processes under space microgravity. In this study, we cloned and validated the open reading frame (ORF) sequence of CD82 homolog and explored the expression profile of CD82 homolog in various zebrafish tissues. Overexpression of zebrafish CD82a increased host IFN1 and vig1 transcription during the spring viremia of carp virus (SVCV) infection. Furthermore, a significant down-regulation of gene expression, including IFN1 and vig1, caused by knockdown of CD82a was observed. Additionally, the knockdown of CD82a significantly upregulated the SVCV replication in the EPC cells. Accordingly, CD82a positively induced the cellular antiviral responses triggered by the SVCV. Moreover, the mRNA level of CD82 homolog was upregulated in vivo and in vitro under simulated microgravity. Collectively, our studies further implied that the high expression of CD82a could modulate innate antiviral immunity in an IFN-independent manner during microgravity conditions.

Extracellular vesicles (EVs) play a vital role in numerous biological processes. Proteomic research of EVs is crucial for understanding their functions and potential therapeutic implications. Despite many sample preparation protocols for mass spectrometry-based proteomics of EVs being described, the variability in protein extraction across different protocols has not been extensively investigated. Moreover, given the inherent heterogeneity of EVs, it is vital to conduct a thorough evaluation of normalization methods. Here, we present a comprehensive comparison of three widely used lysis agents─sodium dodecyl sulfate (SDS), urea, and sodium deoxycholate (SDC)─for protein extraction from EVs. We also assess the impact of different normalization strategies on protein quantification, which is crucial for ensuring reliable results. Our results show that method-dependent differences in protein recovery were observed, particularly for membrane-associated proteins. We also find that common normalization strategies, such as urine creatinine and EV markers, did not significantly stabilize protein quantification, indicating that these methods are not universally applicable as normalization standards. Our work thereby provides a reference for the selection of MS sample preparation and normalization strategies for a given EV proteomics project.

Cancer is the second-highest cause of death worldwide. Accordingly, finding new cancer treatments is of great interest to researchers. The current platforms to fight cancer such as chemotherapy, radiotherapy, and surgery are limited in efficacy, especially in the metastatic setting. In this war against cancer, the immune system is a powerful ally, but tumor cells often outsmart it through alternative pathways. Cluster of differentiation 47 (CD47), a protein that normally prevents healthy cells from being attacked by immune cells, is often overexpressed on cancer cells. This makes CD47 a prime target for immunotherapy. Blocking of CD47 has the potential to unleash the immune system's cell populations-such as myeloid cells, macrophages, and T cells-to allow the immune system to discover and destroy cancer cells more successfully. In this review, we aimed to provide the latest information and findings about the roles of CD47 in the regulation of various cellular pathways and, thus, the importance of CD47 as a potential target in cancer therapy.

Tetraspanins are a family of 33 proteins in mammals believed to play a crucial role in the compartmentalization of various associated proteins within cells and membranes. Recent studies have elucidated the structure of several tetraspanin members, revealing that while the four transmembrane domains typically adopt a cone-shaped configuration in crystals, other conformations are also possible. This cone-shaped structure may explain why tetraspanins are often enriched in curved and tubular cellular structures, such as microvilli, tunneling nanotubes, retraction fibers, or at the site of virus budding, and may contribute to the formation or maintenance of these structures. Tetraspanins have also been detected on midbody remnants and migrasomes, as well as on extracellular vesicles (EVs), for which CD9, CD81, and CD63 are widely used as markers. Although their impact on certain membrane structures and their ability to regulate the function and trafficking of associated proteins would suggest a potential role of tetraspanins either in EV formation or in regulating their protein composition, or both, efforts to characterize these roles have been complicated by conflicting results. In line with the interaction of certain tetraspanins with cholesterol, two recent studies have suggested that the presence or organization of oxysterols and cholesterol in EVs may be regulated by Tspan6 and CD63, respectively, paving the way for further research on the influence of tetraspanins on the lipid composition of EVs.

Migrasomes are a newly discovered type of extracellular vesicle (EV) formed during cell migration, playing a pivotal role in intercellular communication. These vesicles are generated by retracting fibers of migrating cells and encapsulate various molecules, such as proteins, lipids, and RNA, allowing the transfer of biochemical signals to neighboring cells. Current evidence suggests that migrasomes are involved in a wide range of physiological processes such as embryogenesis, angiogenesis, immune modulation, and mitochondrial quality control. Moreover, migrasomes are implicated in pathological conditions, including cancer metastasis, cardiovascular diseases, and viral infections. To fully understand their significance, it is critical to first explore the molecular mechanisms underlying their formation and function. Recent studies have shed light on the biogenesis, release, and biological properties of migrasomes, all of which are key to understanding their role in cell-to-cell communication. In this review, we provide an up-to-date summary of migrasome biogenesis, release, characterization, and their biological activities in intercellular communication, while also proposing potential new functions for these vesicles.

The Drosophila genus is ideal for studying genome evolution due to its relatively simple chromosome structure and small genome size, with rearrangements mainly restricted to within chromosome arms, such as Muller elements. However, work on the rapidly evolving repetitive genomic regions, composed of transposons and tandem repeats, have been hampered by the lack of genus-wide chromosome-level assemblies.

Integrating long-read genomic sequencing and chromosome capture technology, here we produce and annotate 30 chromosome-level genome assemblies within the Drosophila genus. Based on this dataset, we reveal the evolutionary dynamics of genome rearrangements across the Drosophila phylogeny, including the identification of genomic regions that show comparatively high structural stability throughout evolution. Moreover, within the ananassae subgroup, we uncover the emergence of new chromosome conformations and the rapid expansion of novel satellite DNA sequence families, which form large and continuous pericentromeric domains with higher-order repeat structures that are reminiscent of those observed in the human and Arabidopsis genomes.

These chromosome-level genome assemblies present a valuable resource for future research, the power of which is demonstrated by our analysis of genome rearrangements and chromosome evolution. In addition, based on our findings, we propose the ananassae subgroup as an ideal model system for studying the evolution of centromere structure.

Previous studies showed that tetraspanins activate integrins, but the mechanism of this action is unclear. We previously showed that the extracellular-2 (EC2) domains of CD9, CD81, and CD151 bind to the classical RGD-binding site (site 1) of integrin αvβ3, suggesting that they are integrin ligands. We showed that several inflammatory cytokines (e.g., CX3CL1, CXCL12, CCL5, and CD40L) bind to the allosteric site (site 2) of integrins, which is distinct from site 1, and activate integrins (allosteric activation). 25-hydroxycholesterol, a major inflammatory lipid mediator, is known to bind to site 2 and induce inflammatory signals, suggesting that site 2 plays a role in inflammatory signaling. We hypothesized that the EC2 domains activate integrins by binding to site 2. Here we describe that docking simulation predicted that CD81 EC2 binds to site 2 of αvβ3 and more strongly to site 2 of α5β1. Peptide from site 2 bound to isolated EC2 domains, suggesting that the EC2 domains bind to site 2. The EC2 domains only weakly activated αvβ3 but more efficiently activated cell surface integrins α5β1 and α4β1 on the cell surface. These results are consistent with the previous findings that these tetraspanins preferentially interact with β1 integrins. The integrin activation by the EC2 domains was increased at low EC2 concentrations and reduced as EC2 concentrations increased (biphasic), which is consistent with the findings that the EC2 domains bind to two sites (site 1 and 2). We propose that the EC2 binding to site 2 is a novel target for drug discovery.

Extracellular vesicles (EVs) convey complex signals between cells that can be used to promote neuronal plasticity and neurological recovery in brain disease models. These EV signals are multimodal and context-dependent, making them unique therapeutic principles. This review analyzes how EVs released from various cell sources control neuronal metabolic function, neuronal survival and plasticity. Preferential sites of EV communication in the brain are interfaces between pre- and postsynaptic neurons at synapses, between astrocytes and neurons at plasma membranes or tripartite synapses, between oligodendrocytes and neurons at axons, between microglial cells/macrophages and neurons, and between cerebral microvascular cells and neurons. At each of these interfaces, EVs support mitochondrial function and cell metabolism under physiological conditions and orchestrate neuronal survival and plasticity in response to brain injury. In the injured brain, the promotion of neuronal survival and plasticity by EVs is tightly linked with EV actions on mitochondrial function, cell metabolism, oxidative stress and immune responses. Via the stabilization of cell metabolism and immune balance, neuronal plasticity responses are activated and functional neurological recovery is induced. As such, EV lay the ground for neuronal plasticity.

Spermatogenesis is driven by dramatic changes in chromatin regulation, gene transcription, and protein expression. To assess the mechanistic bases for these developmental changes, we utilized multiomic single-cell/nucleus RNA sequencing (sc/snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to identify chromatin changes associated with transcription in adult mouse and rat testes. We characterized the relationships between the transcriptomes and chromatin of both species, including the divergent expression of Id4 in spermatogonial stem cells between species. Promoter accessibility and gene expression showed the greatest association during meiosis in both species. We mapped the cross-species conservation of putative regulatory regions for key spermatogenic genes, including Cd9 and Spam1, and investigated correlations and disconnects in chromatin accessibility, gene expression, and protein expression via antibody-derived tags. Using a gene regulatory network (GRN) model, we identified 40 core regulons conserved between mouse and rat germ cells, highlighting the relevance of chromatin-related factors in regulating the transcription of canonical genes across spermatogenesis.

Tetraspanins (Tspans) are transmembrane proteins that coordinate life cycle steps of viruses from distinct families. Here, we identify the human Tspan10 and Tspan15, both members of the TspanC8 subfamily, as replication factors for alphavirus Venezuelan equine encephalitis virus (VEEV) in astrocytoma cells. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of TspanC8 interactor a disintegrin and metalloproteinase 10 (ADAM10) reduced VEEV infection. Silencing of Tspan10, Tspan15, and ADAM10 did not affect VEEV entry but diminished viral genome replication. We report that Tspan10 is important for VEEV infection of several cell lines, while silencing of Tspan15 diminishes infection with several alphaviruses, but not flaviviruses, in astrocytoma cells. Conversely, we demonstrate that siRNA-mediated silencing of Tspan14, another member of the TspanC8 family, enhances infection with lentiviral pseudoparticles harbouring the envelope proteins of VEEV, identifying it as a restriction factor for VEEV entry. Silencing of ADAM10/Tspan15 substrate neuronal (N)-cadherin reduced VEEV infectivity, suggesting potential roles of ADAM10 substrates in VEEV infection. In sum, our study identifies three TspanC8s and ADAM10 as important modulators of VEEV infectivity.

Intravascular schistosomes have an unusual outer, heptalaminate (seven-layered) covering consisting of not one but two lipid bilayers. Here, we present an updated model of the molecular composition of these bilayers in Schistosoma mansoni that places most identified proteins in the outer, and not the inner, membrane. Here, enzymes would have access to their recently described (non-membrane-permeable) substrates. By contrast, nutrient transporter proteins must be in both membranes to facilitate uptake into the worm's inner tissues. Ectoenzyme activities displayed by living worms suggest the presence on their outer surface of several noncanonically extracellular proteins. The advantages of having a double-bilayered covering may relate to impeding host immunological attack and/or to the worm's ability to acquire selected host molecules onto their exterior.

Exosomes play an important role in regulating physiological processes and mediating the systemic dissemination of various types of cancer. We investigated the association of exosomal tetraspanins CD9, CD63, and CD81 in patients with ovarian cancer (OC).

We measured the plasma tetraspanins CD9, CD63, and CD81 by enzyme-linked immunosorbent assay in 91 patients who underwent treatment for OC between April 2018 and March 2024. Additionally, we analyzed clinical pathologic factors, chemotherapy response, and prognosis.

In terms of stages, CD63 expression was significantly higher in patients with stage IV compared to those with stage I OC (p = 0.003). In terms of histological type, CD63 expression was significantly higher in high-grade serous carcinoma (HGSC) than in clear cell carcinoma (CCC) with OC (p = 0.009). Furthermore, CD63 levels were significantly higher in advanced-stage, HGSC than in patients with early-stage, non-HGSC and early-stage, HGSC OC (p = 0.045 and p = 0.002, respectively). In the Neoadjuvant chemotherapy (NAC) of 12 patients with OC assessed as having either a partial response (PR) or complete response (CR), CD63 was significantly decreased (p = 0.043), whereas perforin was significantly increased (p = 0.001). In the NAC of 16 patients with OC, CD63 of the response rate to chemotherapy tended to differ between the progressive disease (PD) and PR/CR groups (p = 0.056). A moderate inverse correlation was observed between CD63 and perforin levels (R = 0.638, R2 = 0.428, p = 0.008).

CD63 could be a potential biomarker for all types of OC patients.

CD63, a member of the tetraspanins, is involved in cell movement, adhesion, immune response. Nevertheless, the role of CD63 in combating pathogen infections in invertebrates remains largely unclear. Tremor disease, whose pathogen is Spiroplasma eriocheiris, is one of the most prevalent illnesses affecting Eriocheir sinensis. EsCD63 is 1474 bp, with a 756 bp open reading frame that encodes for 252 amino acids. The qPCR data demonstrated that gills showed significant levels of transcription for EsCD63, followed by hemocytes, hepatopancreas, intestines and nerves, while showing low levels of transcription in the heart and muscles. After infection with S. eriocheiris, an obvious drop in the transcription level of EsCD63 was observed. Both the amount of S. eriocheiris copies in hemocytes and the mortality of E. sinensis significantly increased after the injection of chemically synthesized EsCD63 siRNA and stimulation with S. eriocheiris. After EsCD63 interference, the phagocytosis of hemocytes to S. eriocheiris, the apoptosis of hemocytes, and reactive oxygen species level of hemocytes were all decreased significantly, by laser scanning confocal microscopy and flow cytometry analysis. Meanwhile, the mitochondrial membrane potential of hemocytes was increased after EsCD63 interference. These findings indicated that EsCD63 was crucial for E. sinensis immunity and defense mechanisms against infection of S. eriocheiris.

The CAP (cysteine-rich secretory proteins, antigen-5, and pathogenesis-related) proteins are widely expressed and have been implicated to play diverse roles ranging from mammalian reproduction to plant immune response. Increasing evidence supports a role of CAP proteins in lipid binding. The Caenorhabditis elegans CAP protein LON-1 is known to regulate body size and bone morphogenetic protein (BMP) signaling. LON-1 is a secreted protein with a conserved CAP domain and a C-terminal unstructured domain with no homology to other proteins. In this study, we report that the C-terminal domain of LON-1 is dispensable for its function. Instead, key conserved residues located in the CAP domain are critical for LON-1 function in vivo. We further showed that LON-1 is capable of binding sterol, but not fatty acid, in vitro, and that certain key residues implicated in LON-1 function in vivo are also important for LON-1 sterol binding in vitro. These findings suggest a role of LON-1 in regulating body size and BMP signaling via sterol binding.

To characterize the gingival crevicular fluid (GCF) and plasma extracellular vesicles (EVs) and explore their proteomic cargo in healthy pregnant women compared to those with gestational diabetes mellitus (GDM) and periodontitis.

One-hundred and four pregnant women were recruited at 24-30 gestation weeks. GDM was diagnosed by an oral glucose tolerance test. GCF and plasma samples were obtained to isolate EVs and characterized by nanoparticle tracking, immunoassays, electron microscopy and mass spectrometry.

Of the recruits,17.3% women were healthy, 50% had periodontitis and 32.7% had both GDM and periodontitis. Probing depth, clinical attachment loss and bleeding on probing were more severe in GDM and periodontitis pregnancies (p < 0.0001). Additionally, this group showed an increase concentration of total, small and large GCF-EVs (p = 0.0015, p = 0.0011 and p = 0.0008, respectively), with decreased expression of CD9, CD81 and CD81/CD63 ratio (p = 0.0461, p = 0.0164 and p = 0.0005, respectively). No differences were observed in plasmatic EVs concentration or markers expression. Proteomic analysis of GCF-EVs showed peptides of both host and bacterial origin. Gene ontology analysis revealed that proteins of GCF-EVs participate in immune inflammatory responses, glucose metabolism and insulin response mechanisms.

GCF-EVs were increased in both GDM and periodontitis, and their proteomic cargo suggest their involvement in immune inflammatory response, glucose metabolism and insulin pathways during pregnancy.

Tetraspanins are key players in various physiological and pathological processes, including malignancy, immune response, fertilization, and infectious disease. Affinity ligands targeting the interactions between tetraspanins and partner proteins are promising for modulating downstream signaling pathways, thus emerging as attractive candidates for interfering related biological functions. Due to the involvement in vesicle biogenesis and cargo trafficking, tetraspanins are also regarded as exosome markers, and become molecular targets for drug loading and delivery. Given the rapid development in these areas, this minireview focuses on recent advances in design and engineering of affinity binders toward tetraspanins including CD63, CD81, and CD9. Their mechanism of actions in modulating protein interactions at cell interfaces and treatment of malignant diseases are discussed. Strategies for constructing exosome-based drug delivery platforms are also reviewed, with emphasis on the important roles of tetraspanins and the affinity ligands. Finally, challenges and future development of tetraspanin-targeting therapy and exosomal drug delivery platforms are also discussed.

Oral diseases such as dental caries, periodontitis, and oral cancer are prevalent and present significant challenges to global public health. Although these diseases are typically treated through procedures like dental preparation and resin filling, scaling and root planning, or surgical excision, these interventions are often not entirely effective, and postoperative drug therapy is usually required. Traditional drug treatments, however, are limited by factors such as poor drug penetration, significant side effects, and the development of drug resistance. As a result, there is a growing need for novel drug delivery systems that can enhance therapeutic efficacy, reduce side effects, and improve treatment outcomes. In recent years, drug-loaded vesicles, such as liposomes, polymersomes, and extracellular vesicles (EVs), have emerged as promising drug delivery platforms due to their high drug encapsulation efficiency, controlled release properties, and excellent biocompatibility. This review provides an in-depth examination of the characteristics, advantages, and limitations of liposomes, polymersomes, and extracellular vesicles in the context of oral disease treatment. It further explores the reasons for their advantages and limitations and discusses the specific applications, development prospects, and strategies for optimizing these vesicle-based systems for improved clinical outcomes.

Tetraspanins (TETs) are integral membrane proteins, characterized by four transmembrane domains and a unique signature motif in their large extracellular loop. They form dynamic supramolecular complexes called tetraspanin-enriched microdomains (TEMs), through interactions with partner proteins. In plants, TETs are involved in development, reproduction and immune responses, but their role in defining abiotic stress responses is largely underexplored. We focused on OsTET5, which is differentially expressed under various abiotic stresses and localizes to both plasma membrane and endoplasmic reticulum. Using overexpression and underexpression transgenic lines we demonstrate that OsTET5 contributes to salinity and drought stress tolerance in rice. OsTET5 can interact with itself in yeast, suggesting homomer formation. Immunoblotting of native PAGE of microsomal fraction enriched from OsTET5-Myc transgenic rice lines revealed multimeric complexes containing OsTET5, suggesting the potential formation of TEM complexes. Transcriptome analysis, coupled with quantitative PCR-based validation, of OsTET5-altered transgenic lines unveiled the differential expression patterns of several stress-responsive genes, as well as those coding for transporters under salt stress. Notably, OsTET5 plays a crucial role in maintaining the ionic equilibrium during salinity stress, particularly by preserving an elevated potassium-to-sodium (K+/Na+) ratio. OsTET5 also regulates reactive oxygen species homeostasis, primarily by modulating the gene expression and activities of antioxidant pathway enzymes and proline accumulation. Our comprehensive investigation underscores the multifaceted role of OsTET5 in rice, accentuating its significance in developmental processes and abiotic stress tolerance. These findings open new avenues for potential strategies aimed at enhancing stress resilience and making valuable contributions to global food security.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder marked by progressive muscle wasting leading to premature mortality1,2. Discovery of the DMD gene encoding dystrophin both revealed the cause of DMD and helped identify a family of at least ten dystrophin-associated proteins at the muscle cell membrane, collectively forming the dystrophin-glycoprotein complex (DGC)3-9. The DGC links the extracellular matrix to the cytoskeleton, but, despite its importance, its molecular architecture has remained elusive. Here we determined the native cryo-electron microscopy structure of rabbit DGC and conducted biochemical analyses to reveal its intricate molecular configuration. An unexpected β-helix comprising β-, γ- and δ-sarcoglycan forms an extracellular platform that interacts with α-dystroglycan, β-dystroglycan and α-sarcoglycan, allowing α-dystroglycan to contact the extracellular matrix. In the membrane, sarcospan anchors β-dystroglycan to the β-, γ- and δ-sarcoglycan trimer, while in the cytoplasm, β-dystroglycan's juxtamembrane fragment binds dystrophin's ZZ domain. Through these interactions, the DGC links laminin 2 to intracellular actin. Additionally, dystrophin's WW domain, along with its EF-hand 1 domain, interacts with α-dystrobrevin. A disease-causing mutation mapping to the WW domain weakens this interaction, as confirmed by deletion of the WW domain in biochemical assays. Our findings rationalize more than 110 mutations affecting single residues associated with various muscular dystrophy subtypes and contribute to ongoing therapeutic developments, including protein restoration, upregulation of compensatory genes and gene replacement.

Among the analytes circulating in body fluids, microRNAs, a type of non-coding RNA and known to exist 2655 in primates, have attracted attention as a novel biomarker for cancer screening. MicroRNAs are signaling molecules with important gene expression regulatory functions that can simultaneously control many gene functions and multiple different pathways in living organisms. These microRNAs are transported in extracellular vesicles (EVs), which are lipid bilayers with 50-150 nm in diameter, and are used as communication tools between cells. Furthermore, the EVs that carry these microRNAs circulate in the bloodstream and have other important implications for understanding the pathogenesis and diagnosis of breast cancer. The greatest benefit from cancer screening is the reduction in breast cancer mortality rate through early detection. Other benefits include reduced incidence of breast cancer, improved quality of life, prognosis prediction, contribution to personalized medicine, and relative healthcare cost containment. This paper outlines the latest developments in liquid biopsy for breast cancer, especially focusing on microRNA and EV diagnostics.

Human CD81 and CD9 are members of the tetraspanin family of proteins characterized by a canonical structure of four transmembrane domains and two extracellular loop domains. Tetraspanins are known as molecular facilitators, which assemble and organize cell surface receptors and partner molecules forming clusters known as tetraspanin-enriched microdomains. They have been implicated to play various biological roles including an involvement in infections with microbial pathogens. Here, we demonstrate an important role of CD81 for the invasion of epithelial cells by Salmonella enterica. We show that the overexpression of CD81 in HepG2 cells enhances invasion of various typhoidal and non-typhoidal Salmonella serovars. Deletion of CD81 by CRISPR/Cas9 in intestinal epithelial cells (C2BBe1 and HT29-MTX-E12) reduces S. Typhimurium invasion. In addition, the effect of human CD81 is species-specific as only human but not rat CD81 facilitates Salmonella invasion. Finally, immunofluorescence microscopy and proximity ligation assay revealed that both human tetraspanins CD81 and CD9 are recruited to the entry site of S. Typhimurium during invasion but not during adhesion to the host cell surface. Overall, we demonstrate that the human tetraspanin CD81 facilitates Salmonella invasion into epithelial host cells.

There is little information about transmembrane protein 35B (TMEM35B) expression in glioma, and its functions in glioma remains no clue.

Immunohistochemistry was used to measure TMEM35B expression levels and CCK8 and Transwell assays were analyzed the proliferative and migratory and invasive.

TMEM35B protein was significantly higher in gliomas and correlated with a higher tumor TNM stages. Receiver operating characteristic curve analysis revealed that TMEM35B had high diagnostic value in distinguishing among glioma, normal tissues, tumor stages III+IV, and I+II. Additionally, TMEM35B knockdown inhibited the proliferative, migratory, and invasive capacities of glioma cells.

TMEM35B expression is upregulated in gliomas, and its knockdown hinders tumor progression, highlighting the protein as a potential biomarker for glioma diagnosis.

Liver fluke infection is a major risk for cholangiocarcinoma (CCA). It has been established that the Asian liver flukes, Clonorchis sinensis and Opisthorchis viverrini secrete growth factors, digestive enzymes, and extracellular vesicles (EVs) which contribute to abnormal cell development in the bile ducts where the worms reside. These secretions - combined with aberrant inflammation and repeated cycles of chronic wounding at the site of parasite attachment and grazing on the epithelium - promote biliary hyperplasia and fibrosis and ultimately malignant transformation. Application of post-genomic and gene-editing tools to the study of liver fluke immunobiology and pathogenesis has accelerated the discovery of essential virulence factors to which targeted therapies and diagnostics can be directed.

The important role of extracellular vesicles, which are considered key mediators of intercellular communication under physiological and pathological conditions, in various cellular processes, including those crucial for mammalian reproduction, has been increasingly studied. Tetraspanins, including CD63, are widely used as markers of extracellular vesicles, but they may also play a role in their biogenesis, cargo selection, cell targeting, and uptake. This study aimed to map the journey of the extracellular vesicle protein tetraspanin CD63 from the testis through the epididymis into mature bull sperm via an approach that included immunohistochemistry (immunofluorescence and immunoperoxidase staining), Western blot analysis, and immunoprecipitation analysis. We described the presence of CD63 in bull testicular and epididymal tissues, extracellular vesicles produced in these organs and spermatozoa during epididymal transit and after ejaculation. In addition, we revealed the nonuniform distribution of potential CD63 partners, such as CD9, integrin αV and syntenin-1, in the sperm head and tail and in extracellular vesicles. These findings contribute to understanding the complex mechanisms underlying sperm maturation and point to the possible involvement of tetraspanins and their associated partners, either as part of extracellular vesicles or sperm membranes, in these processes.

Migrasomes, vesicular structures discovered in migrating cells, arise from the junctions or tips of retraction fibers, and gradually grow to microscale vesicles. Migrasomes have garnered attention for their role in intercellular communication and potential therapeutic implications. This review presents an overview of recent advances in migrasome biology, covering the mechanisms of migrasome biogenesis, essential physiological roles, and their association with various diseases, alongside potential therapeutic applications. Furthermore, we share our perspectives on potential future directions in the study of migrasomes and highlight the challenges that remain in this developing area of research.

Dectin-1 is a C-type lectin-receptor involved in sensing fungi by innate immune cells. Encoded by the Clec7a gene, Dectin-1 exists in two major splice isoforms, Dectin-1a and 1b, which differ in the presence of a membrane-proximal stalk domain. As reported previously, this domain determines degradative routes for Dectin-1a and 1b leading to the generation of a stable N-terminal fragment exclusively from Dectin-1a. Here, we narrow down the responsible part of the stalk and demonstrate the stabilisation of the Dectin-1a N-terminal fragment in tetraspanin-enriched microdomains. C57BL/6 and BALB/c mice show divergent Dectin-1 isoform expression patterns, which are caused by a single nucleotide polymorphism in exon 3 of the Clec7a gene, leading to a non-sense Dectin-1a mRNA in C57BL/6 mice. Using backcrossing, we generated mice with the C57BL/6 Clec7a allele on a BALB/c background and compared these to the parental strains. Expression of the C57BL/6 allele leads to the exclusive presence of the Dectin-1b protein. Furthermore, it was associated with higher Dectin-1 mRNA expression, but less Dectin-1 at the cell surface according to flow cytometry. In neutrophils, this altered ROS production induced by Dectin-1 model ligands, while cellular responses in macrophages and dendritic cells were not significantly influenced by the Dectin-1 isoform pattern.

Extracellular vesicles (EVs) are emerging as key diagnostic biomarkers due to their widespread presence in body fluids and the proteins on their surfaces, which reflect the identity and condition of their parent cells. Research has focused on detecting EVs with biosensors that target individual transmembrane proteins (TMPs) like tetraspanins. However, due to TMP heterogeneity and the formation of tetraspanin-enriched microdomains (TEMs), cotargeting multiple TMPs is a promising strategy for enhancing EV detection. In this work, we introduce a dual-antibody surface functionalization approach using surface plasmon resonance (SPR) biosensors to cotarget tetraspanins on EVs derived from mouse macrophages. The expression of EV tetraspanin markers followed the trend of CD9 > CD63 > CD81, which was consistent with the EV detection targeting their nontetraspanin partners, exhibiting LFA-1 > ICAM-1 > VCAM-1, and suggesting a differential role of tetraspanins with their associated TMPs. Cotargeting EV tetraspanins via CD81/CD63, CD81/CD9, and CD63/CD9 dual monoclonal antibody surfaces resulted in higher EV detection compared to predictions based on binding with two monoclonal antibodies against tetraspanins without cotargeting. Furthermore, the optimization of dual monoclonal antibody surface ratios to improve cotargeting effect yielded a statistically significant enhancement in the sensitivity of EV detection. These findings underscore the importance of TEMs in designing EV-based biosensing platforms to achieve optimized sensitivity in EV detection.

Exocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task that is prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on Fiji/ImageJ2 software. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable for studying distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNARE protein reporters. Assessment of performance on synthetic data shows that ExoJ is a robust tool that is capable of correctly identifying exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.

Tetraspanins (TSPANs) are a family of highly conserved proteins present in a wide variety of eukaryotes. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native-PAGE analysis showed that these TSPANs form several complexes of 120-250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases, while repression of tspan4 led to a four-fold increase in the activity of cysteine proteases in crude extracellular vesicles (EVs) fraction. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.

During wound healing, fibroblast to myofibroblast transition is required for wound contraction and remodeling. While hypoxia is an important biophysical factor in wound microenvironment, the exact regulatory mechanism underlying hypoxia and fibroblast-to-myofibroblast transition remains unclear. We previously found that tetraspanin CD9 plays an important role in oxygen sensing and wound healing. Herein, we investigated the effects of physiological hypoxia on fibroblast-to-myofibroblast transition and the biological function and mechanism of CD9 in it.

Human skin fibroblasts (HSF) and mouse dermis wounds model were established under physiological hypoxia (2% O2). The cell viability and contractility of HSF under hypoxia were evaluated by CCK8 and collagen gel retraction, respectively. The expression and distribution of fibroblast-to-myofibroblast transition markers and CD9 in HSF were detected by Western blotting and immunofluorescence. CD9 slicing and overexpressing HSFs were constructed to determine the role of CD9 by small interfering RNA and recombinant adenovirus vector. The association of TβR2 and TβR1 was measured by immunoprecipitation to explore the regulatory mechanism. Additionally, further validation was conducted on mouse dermis wounds model through histological analysis.

Enhanced fibroblast-to-myofibroblast transition and upregulated CD9 expression was observed under hypoxia in vitro and in vivo. Besides, reversal of fibroblast-to-myofibroblast transition under hypoxia was observed when silencing CD9, suggesting that CD9 played a key role in this hypoxia-induced transition. Moreover, hypoxia increased fibroblast-to-myofibroblast transition by activating TGF-β1/Smad2/3 signaling, especially increased interaction of TβR2 and TβR1. Ultimately, CD9 was determined to directly affect TβR1-TβR2 association in hypoxic fibroblast.

Collectively, these findings suggest that CD9 promotes TβR2-TβR1 association, thus driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.

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 the cytoplasm to the 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 on 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 vesicles in the newly formed TNTs, possibly by regulating docking to or fusion with the opposing cell.

Migrasomes, newly identified organelles, play crucial roles in intercellular communication, contributing to organ development and angiogenesis. These vesicles, forming on retraction fibers of migrating cells, showcase a sophisticated architecture. Recent research reveals that migrasome biogenesis is a complicated and highly regulated process. This review summarizes the mechanisms governing migrasome formation, proposing a model in which biogenesis is understood through the lens of membrane microdomain assembly. It underscores the critical interplay between biochemistry and biophysics. The biogenesis unfolds in three distinct stages: nucleation, maturation, and expansion, each characterized by unique morphological, biochemical, and biophysical features. We also explore the broader implications of migrasome research in membrane biology and outline key unanswered questions that represent important directions for future investigation.

Sepsis following burn trauma is a global complication with high mortality, with ∼60% of burn patient deaths resulting from infectious complications. Diagnosing sepsis is complicated by confounding clinical manifestations of the burn injury, and current biomarkers lack the sensitivity and specificity required for prompt treatment. There is a strong rationale to assess circulating extracellular vesicles (EVs) from patient liquid biopsy as sepsis biomarkers due to their release by pathogens from bacterial biofilms and roles in the subsequent immune response. This study applies Raman spectroscopy to patient plasma-derived EVs for rapid, sensitive, and specific detection of sepsis in burn patients, achieving 97.5% sensitivity and 90.0% specificity. Furthermore, spectral differences between septic and non-septic burn patient EVs could be traced to specific glycoconjugates of bacterial strains associated with sepsis morbidity. This work illustrates the potential application of EVs as biomarkers in clinical burn trauma care and establishes Raman analysis as a fast, label-free method to specifically identify features of bacterial EVs relevant to infection amongst the host background.

TSPAN7 is an important factor in tumor progression. However, the precise function of TSPAN7 and its role in pan-cancer are not clear.

Based on Xinhua cohort incorporating 370 patients with kidney neoplasm, we conducted differential expression analysis by immunohistochemistry between tumor and normal tissues, and explored correlations of TSPAN7 with patients' survival. Subsequently, we conducted a pan-cancer study, and successively employed differential expression analysis, competing endogenous RNA (ceRNA) analysis, protein-protein interaction (PPI) analysis, correlation analysis of TSPAN7 with clinical characteristics, tumor purity, tumor genomics, tumor immunity, and drug sensitivity. Last but not least, gene set enrichment analysis was applied to identify enriched pathways of TSPAN7.

In Xinhua cohort, TSPAN7 expression was significantly up-regulated (P-value = 0.0019) in tumor tissues of kidney neoplasm patients. High TSPAN7 expression was associated with decreases in overall survival (OS) (P-value = 0.009) and progression-free survival (P-value = 0.009), and it was further revealed as an independent risk factor for OS (P-value = 0.0326, HR = 5.66, 95%CI = 1.155-27.8). In pan-cancer analysis, TSPAN7 expression was down-regulated in most tumors, and it was associated with patients' survival, tumor purity, tumor genomics, tumor immunity, and drug sensitivity. The ceRNA network and PPI network of TSPAN7 were also constructed. Last but not least, the top five enriched pathways of TSPAN7 in various tumors were identified.

TSPAN7 served as a promising biomarker of various tumors, especially kidney neoplasms, and it was closely associated with tumor purity, tumor genomics, tumor immunology, and drug sensitivity in pan-cancer level.

Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis.

Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors.

Our findings define integrin α2β1 as an early marker of OC differentiation.

Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different receptors on the plasma membrane. Most tetraspanins are highly glycosylated at their large extracellular loop; however, little is known about the function of tetraspanin glycosylation in immune cells. In this study we investigated the effects of glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants of CD37 and CD53 and analyzed their localization, nanoscale plasma membrane organization, and partner protein interaction capacity. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas surface expression of CD53 was unaffected by its glycosylation. Single-molecule dSTORM microscopy revealed that the nanoscale organization of CD53 was not dependent on glycosylation. CD37 interaction with its partner proteins CD53 and CD20 was affected by glycosylation in a localization-dependent way, whereas its interaction with IL-6Rα was independent of glycosylation. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45, CD20, and, to a lesser extent CD37. Together, our data show that glycosylation affects the interaction capacity of immune-specific tetraspanins CD37 and CD53, which adds another layer of regulation to immune membrane organization.

In recent decades, the pursuit of drug delivery systems has led to the development of numerous synthetic options aimed at enhancing drug efficacy while minimizing side effects. However, the practical application of these systems is often hindered by challenges such as inefficiency, cytotoxicity, and immunogenicity. Extracellular vesicles, natural carriers for drugs, emerge as promising alternatives with distinct advantages over synthetic carriers. Notably, EVs exhibit biocompatibility, low immunogenicity, and inherent tissue-targeting capabilities, thus opening new avenues for drug delivery strategies. This review provides an overview of EVs, including their biogenesis and absorption mechanisms. Additionally, we explore the current research efforts focusing on harnessing their potential as drug carriers, encompassing aspects such as purification techniques, drug loading, and bioengineering for targeted delivery. Finally, we discuss the existing challenges and future prospects of EVs as therapeutic agents in clinical settings. This comprehensive analysis aims to shed light on the potential of EVs as versatile and effective tools for drug delivery, particularly in the realm of cancer therapy.

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.

This study aims to investigate the role of RNF149 and tetraspanin CD63 in lipopolysaccharide/Toll-like receptor 4 (LPS/TLR4) signal transduction. TNF-α was assessed using enzyme-linked immunosorbent assay. The distribution of TLR4 was examined through flow cytometry after CD63 knockdown. Real-time polymerase chain reaction was used to analyze the expression of the target genes RNF149 and CD63 under different conditions. Western blotting was employed to detect gene expression, while immunoprecipitation and confocal microscopy were used to evaluate protein interactions. Transcriptome array data from stimulated monocytes (GSE7547) was obtained from GEO and subjected to bioinformatic analysis. It is suggested that CD63 may serve as a substrate of RNF149, with RNF149 capable of directly interacting with CD63. RNF149 degrades CD63 through covalent modification of CD63 at lysine 29 of the ubiquitin monomer, leading to the formation of a multiubiquitin chain. Both RNF149 and CD63 interact with TLR4, with CD63 promoting LPS/TLR4 signaling and RNF149 inhibits it. CD63 does not impact the distribution of TLR4 on the cell surface and does not directly interact with TIRAP, IRAK4, or TRAF6, but does interact with Myd88.RNF149 plays a negative regulatory role in LPS/TLR4 signal transduction by mediating ubiquitination-induced CD63 degradation.

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.