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The Journal of Cell Biology Mar 2020The intraluminal vesicles (ILVs) of endosomes mediate the delivery of activated signaling receptors and other proteins to lysosomes for degradation, but they also...
The intraluminal vesicles (ILVs) of endosomes mediate the delivery of activated signaling receptors and other proteins to lysosomes for degradation, but they also modulate intercellular communication when secreted as exosomes. The formation of ILVs requires four complexes, ESCRT-0, -I, -II, and -III, with ESCRT-0, -I, and -II presumably involved in cargo sorting and ESCRT-III in membrane deformation and fission. Here, we report that an active form of the ESCRT-associated protein ALIX efficiently recruits ESCRT-III proteins to endosomes. This recruitment occurs independently of other ESCRTs but requires lysobisphosphatidic acid (LBPA) in vivo, and can be reconstituted on supported bilayers in vitro. Our data indicate that this ALIX- and ESCRT-III-dependent pathway promotes the sorting and delivery of tetraspanins to exosomes. We conclude that ALIX provides an additional pathway of ILV formation, secondary to the canonical pathway, and that this pathway controls the targeting of exosomal proteins.
Topics: Calcium-Binding Proteins; Cell Cycle Proteins; Endosomal Sorting Complexes Required for Transport; Endosomes; Exosomes; HeLa Cells; Humans; Lysophospholipids; Monoglycerides; Protein Transport; Tetraspanin 28; Tetraspanin 29; Tetraspanin 30; Tetraspanins; Time Factors
PubMed: 32049272
DOI: 10.1083/jcb.201904113 -
Nature Communications Feb 2023Migrasomes are newly discovered cell organelles forming by local swelling of retraction fibers. The migrasome formation critically depends on tetraspanin proteins...
Migrasomes are newly discovered cell organelles forming by local swelling of retraction fibers. The migrasome formation critically depends on tetraspanin proteins present in the retraction fiber membranes and is modulated by the membrane tension and bending rigidity. It remained unknown how and in which time sequence these factors are involved in migrasome nucleation, growth, and stabilization, and what are the possible intermediate stages of migrasome biogenesis. Here using live cell imaging and a biomimetic system for migrasomes and retraction fibers, we reveal that migrasome formation is a two-stage process. At the first stage, which in biomimetic system is mediated by membrane tension, local swellings largely devoid of tetraspanin 4 form on the retraction fibers. At the second stage, tetraspanin 4 molecules migrate toward and onto these swellings, which grow up to several microns in size and transform into migrasomes. This tetraspanin 4 recruitment to the swellings is essential for migrasome growth and stabilization. Based on these findings we propose that the major role of tetraspanin proteins is in stabilizing the migrasome structure, while the migrasome nucleation and initial growth stages can be driven by membrane mechanical stresses.
Topics: Tetraspanin 28; Tetraspanins; Cell Membrane; Stress, Mechanical
PubMed: 36823145
DOI: 10.1038/s41467-023-36596-9 -
Cell Research Apr 2022TSPAN family of proteins are generally considered to assemble as multimeric complexes on the plasma membrane. Our previous work uncovered that TSPAN8 can translocate...
TSPAN family of proteins are generally considered to assemble as multimeric complexes on the plasma membrane. Our previous work uncovered that TSPAN8 can translocate into the nucleus as a membrane-free form, a process that requires TSPAN8 palmitoylation and association with cholesterol to promote its extraction from the plasma membrane and subsequent binding with 14-3-3θ and importin-β. However, what upstream signal(s) regulate(s) the nuclear translocation of TSPAN8, the potential function of TSPAN8 in the nucleus, and the underlying molecular mechanisms all remain unclear. Here, we demonstrate that, epidermal growth factor receptor (EGFR) signaling induces TSPAN8 nuclear translocation by activating the kinase AKT, which in turn directly phosphorylates TSPAN8 at Ser129, an event essential for its binding with 14-3-3θ and importin ß1. In the nucleus, phosphorylated TSPAN8 interacts with STAT3 to enhance its chromatin occupancy and therefore regulates transcription of downstream cancer-promoting genes, such as MYC, BCL2, MMP9, etc. The EGFR-AKT-TSPAN8-STAT3 axis was found to be hyperactivated in multiple human cancers, and associated with aggressive phenotype and dismal prognosis. We further developed a humanized monoclonal antibody hT8 that specifically recognizes the large extracellular loop of TSPAN8 (TSPAN8-LEL), thus being able to block the extraction of TSPAN8 from the plasma membrane and consequently its nuclear localization. Importantly, both in vitro and in vivo studies demonstrated an antitumor effect of hT8. Collectively, we discovered an unconventional function of TSPAN8 and dissected the underlying molecular mechanisms, which not only showcase a new layer of biological complexity of traditional membrane proteins, but also shed light on TSPAN8 as a novel therapeutic target for refractory cancers.
Topics: Cell Line, Tumor; ErbB Receptors; Humans; Neoplasms; STAT3 Transcription Factor; Signal Transduction; Tetraspanins
PubMed: 35197608
DOI: 10.1038/s41422-022-00628-8 -
Cell Jun 2018Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However,...
Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.
Topics: Animals; Argonaute Proteins; Cell Cycle; Gene Expression Regulation; Helminth Proteins; Planarians; Pluripotent Stem Cells; Principal Component Analysis; RNA Interference; RNA, Double-Stranded; RNA, Helminth; Regeneration; Sequence Analysis, RNA; Single-Cell Analysis; Tetraspanins; Whole-Body Irradiation
PubMed: 29906446
DOI: 10.1016/j.cell.2018.05.006 -
Journal of Extracellular Vesicles Aug 2023The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7...
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.
Topics: Cell Movement; Extracellular Vesicles; Proteomics; Tetraspanin 28; Humans; MCF-7 Cells; Tetraspanin 29; Tetraspanin 30
PubMed: 37525398
DOI: 10.1002/jev2.12352 -
Current Biology : CB Mar 2020Lang and Hochheimer introduce the physiological and pathological functions of tetraspanins.
Lang and Hochheimer introduce the physiological and pathological functions of tetraspanins.
Topics: Humans; Tetraspanins
PubMed: 32155418
DOI: 10.1016/j.cub.2020.01.007 -
Journal of Extracellular Vesicles May 2022The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few...
BACKGROUND
The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum.
METHOD
Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis.
RESULTS
This study shows that a higher number of CD9 EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63 EVs were enriched in serum, while CD81 vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation.
CONCLUSION
These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail.
Topics: Blood Platelets; Edetic Acid; Extracellular Vesicles; Humans; Mass Spectrometry; Tetraspanins
PubMed: 35524458
DOI: 10.1002/jev2.12213 -
Cell Adhesion & Migration 2015Tetraspanins are a superfamily of small transmembrane proteins that are expressed in almost all eukaryotic cells. Through interacting with one another and with other... (Review)
Review
Tetraspanins are a superfamily of small transmembrane proteins that are expressed in almost all eukaryotic cells. Through interacting with one another and with other membrane and intracellular proteins, tetraspanins regulate a wide range of proteins such as integrins, cell surface receptors, and signaling molecules, and thereby engage in diverse cellular processes ranging from cell adhesion and migration to proliferation and differentiation. In particular, tetraspanins modulate the function of proteins involved in all determining factors of cell migration including cell-cell adhesion, cell-ECM adhesion, cytoskeletal protrusion/contraction, and proteolytic ECM remodeling. We herein provide a brief overview of collective in vitro and in vivo studies of tetraspanins to illustrate their regulatory functions in the migration and trafficking of cancer cells, vascular endothelial cells, skin cells (keratinocytes and fibroblasts), and leukocytes. We also discuss the involvement of tetraspanins in various pathologic and remedial processes that rely on cell migration and their potential value as targets for therapeutic intervention.
Topics: Animals; Cell Adhesion; Cell Movement; Cytoskeleton; Humans; Neoplasms; Proteolysis; Tetraspanins
PubMed: 26091149
DOI: 10.1080/19336918.2015.1005465 -
Frontiers in Immunology 2021Tetraspanin family of proteins participates in numerous fundamental signaling pathways involved in viral transmission, virus-specific immunity, and virus-mediated... (Review)
Review
Tetraspanin family of proteins participates in numerous fundamental signaling pathways involved in viral transmission, virus-specific immunity, and virus-mediated vesicular trafficking. Studies in the identification of novel therapeutic candidates and strategies to target West Nile virus, dengue and Zika viruses are highly warranted due to the failure in development of vaccines. Recent evidences have shown that the widely distributed tetraspanin proteins may provide a platform for the development of novel therapeutic approaches. In this review, we discuss the diversified and important functions of tetraspanins in exosome/extracellular vesicle biology, virus-host interactions, virus-mediated vesicular trafficking, modulation of immune mechanism(s), and their possible role(s) in host antiviral defense mechanism(s) through interactions with noncoding RNAs. We also highlight the role of tetraspanins in the development of novel therapeutics to target arthropod-borne flaviviral diseases.
Topics: Exosomes; Extracellular Vesicles; Flavivirus Infections; Host Microbial Interactions; Humans; Immunomodulation; RNA, Untranslated; Tetraspanins
PubMed: 33968023
DOI: 10.3389/fimmu.2021.630571 -
Frontiers in Immunology 2018Metastasis is the ultimate consequence of cancer progression and the cause of patients' death across different cancer types. Patients with initial diagnosis of distant... (Review)
Review
Metastasis is the ultimate consequence of cancer progression and the cause of patients' death across different cancer types. Patients with initial diagnosis of distant disease have a worst 5-year survival compared to patients with localized disease. Therapies that target primary tumors fail to eradicate distant dissemination of cancer. Recently, immunotherapies have improved the survival of patients with metastatic disease, such as melanoma and lung cancer. However, only a fraction of patients responds to immunotherapy modalities that target the host immune system. The need to identify new druggable targets that inhibit or prevent metastasis is, therefore, much needed. Tetraspanins have emerged as key players in regulating cell migration, invasion, and metastasis. By serving as molecular adaptors that cluster adhesion receptors, signaling molecules, and cell surface receptors; tetraspanins are involved in all steps of the metastatic cascade. They regulate cell proliferation, participate in EMT transition, modulate integrin-mediated cell adhesion, and participate in angiogenesis and invasion processes. Tetraspanins have also been shown to modulate metastasis indirectly through exosomes and by regulating cellular interactions in the immune system. Importantly, targeting individual tetraspanin with antibodies has impacted tumor progression. This review will focus on the contribution of tetraspanins to the metastatic process and their potential as therapeutic tumor targets.
Topics: Animals; Antineoplastic Agents, Immunological; Binding Sites; Biomarkers, Tumor; Cell Movement; Disease Progression; Humans; Immunotherapy; Molecular Targeted Therapy; Neoplasm Metastasis; Neoplasms; Prognosis; Protein Binding; Tetraspanins; Translational Research, Biomedical
PubMed: 29946318
DOI: 10.3389/fimmu.2018.01277