In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Background: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. Aims: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. Methods: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. Results: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by coimmunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. Conclusion: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level.

In a search for molecular markers of progression in prostate cancer by means of di erential display, we have identi®ed a new gene, which we have designated PTOV1. Semiquantitative RT ± PCR has established that nine out of 11 tumors overexpress PTOV1 at levels signi®cantly higher than benign prostatic hyperplasia or normal prostate tissue. The human PTOV1 protein consists almost entirely of two repeated blocks of homology of 151 and 147 amino acids, joined by a short linker peptide, and is encoded by a 12-exon gene localized in chromosome 19q13.3. A Drosophila melanogaster PTOV1 homolog also contains two tandemly arranged PTOV blocks. A second gene, PTOV2, was identi®ed in humans and Drosophila, coding for proteins with a single PTOV homology block and unrelated amino-and carboxyl-terminal extensions. A 1.8-Kb PTOV1 transcript was detected abundantly in normal human brain, heart, skeletal muscle, kidney and liver, and at low levels in normal prostate. Immunocytochemical analysis and expression of chimeric GFP-PTOV1 proteins in cultured cells showed a predominantly perinuclear localization of PTOV1. In normal prostate tissue and in prostate adenomas, PTOV1 was undetectable or expressed at low levels, whereas nine out of 11 prostate adenocarcinomas showed a strong immunoreactivity, with a focal distribution in areas of carcinoma and prostatic intraepithelial neoplasia. Therefore, PTOV1 is a previously unknown gene, overexpressed in early and late stages of prostate cancer. The PTOV homology block represents a new class of conserved sequence blocks present in human, rodent and¯y proteins. Oncogene (2001) 20, 1455 ± 1464.

We have isolated and sequenced cDNAs for corticosteroid binding globulin (CBG) Corticosteroid binding globulin (CBG) is the major transport protein for glucocorticoids in the blood of almost all vertebrate species (1), and >90% of the cortisol in human plasma is bound by this protein (2). The remaining fraction is distributed more evenly between albumin and the pool of nonprotein-bound or "free" steroid that is generally assumed to be biologically active (2, 3). In humans, CBG is an acidic, -58-kDa glycoprotein (4-6) comprising five N-linked oligosaccharide chains (7) that collectively represent -23% of the molecule by mass (6, 7). The binding site for natural glucocorticoids appears to be a hydrophobic pocket containing one of two cysteine residues that have been identified by amino acid composition analyses (8)(9)(10)(11). Apart from this information, and the identification of eight residues at the NH2 terminus of human CBG (5, 11), there is virtually no information about its primary structure or the location of its steroid binding site.Like many other plasma transport proteins, CBG is produced and secreted by hepatocytes (12), but has also been identified in a number of glucocorticoid responsive cells (2, 13), and may even interact directly with the plasma membranes of some cells (14,15). The objectives of this study were, therefore, to predict the amino acid sequence of human CBG from a cDNA and to determine whether tissues other than the liver possess the capacity to produce this protein. § METHODS cDNA Cloning. A monospecific rabbit antiserum for human CBG (6) was initially used to screen a Xgtll human liver cDNA library that was kindly provided by S. L. C. Woo (Baylor College of Medicine, Houston). The screening method was based on the technique described by Young and Davis (16), with the exception that peroxidase-labeled protein A was used to detect antibody-antigen complexes in the presence of the chromogenic substrate 4-chloro-1-naphthol. The recombinant phage isolated in this way were used to prepare plate lysates using NZC top agar (GIBCO). The phage were harvested and purified, and the cDNA inserts were excised and inserted into the EcoRI site of pBR322 according to Maniatis et al. (17). Plasmids containing CBG cDNAs were used to transform competent Escherichia coli (strain MM 294), and transformants were propagated in Luria broth in the presence of ampicillin and chloramphenicol to amplify the plasmid (17). Plasmids were isolated by the alkaline lysis method and purified using benzoylated-naphthoylated-DEAE cellulose (Sigma) according to Gamper et al. (18). The cDNAs were routinely excised from the plasmid and purified by polyacrylamide gel electrophoresis, prior to nick-translation with 32P-labeled dCTP (17).In an attempt to isolate a full-length CBG cDNA, the radiolabeled cDNA was employed to rescreen the library. Nitrocellulose filters (Schleicher & Schuell; BA85, 0.45-,um pore size) were used to transfer DNA and were hybridized with 2 x 106 dpm of the CBG cDNA probe per ml, in the pr...