Various studies have demonstrated that the two leaflets of cellular membranes interact, potentially through so-called interdigitation between the fatty acyl groups. While the molecular mechanism ...underlying interleaflet coupling remains to be fully understood, recent results suggest interactions between the very-long-chain sphingolipids in the outer leaflet, and phosphatidylserine PS18:0/18:1 in the inner leaflet, and an important role for cholesterol for these interactions. Here we review the evidence that cross-linking of sphingolipids may result in clustering of phosphatidylserine and transfer of signals to the cytosol. Although much remains to be uncovered, the molecular properties and abundance of PS 18:0/18:1 suggest a unique role for this lipid.
Exosomes are a type of extracellular vesicle released from cells after fusion of multivesicular bodies with the plasma membrane. These vesicles are often enriched in cholesterol, SM, ...glycosphingolipids, and phosphatidylserine. Lipids not only have a structural role in exosomal membranes but also are essential players in exosome formation and release to the extracellular environment. Our knowledge about the importance of lipids in exosome biology is increasing due to recent technological developments in lipidomics and a stronger focus on the biological functions of these molecules. Here, we review the available information about the lipid composition of exosomes. Special attention is given to ether lipids, a relatively unexplored type of lipids involved in membrane trafficking and abundant in some exosomes. Moreover, we discuss how the lipid composition of exosome preparations may provide useful information about their purity. Finally, we discuss the role of phosphoinositides, membrane phospholipids that help to regulate membrane dynamics, in exosome release and how this process may be linked to secretory autophagy. Knowledge about exosome lipid composition is important to understand the biology of these vesicles and to investigate possible medical applications.
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► Studies on the mechanism of interactions between cells and nanoparticles (NPs) must be improved. ► We discuss the possibilities and pitfalls in studies of endocytic pathways ...followed by NPs. ► We evaluate the use of inhibitors, mutated proteins, siRNAs and colocalization experiments in such studies. ► We discuss intracellular transport, recycling to cell exterior, disturbance of cell functions, and metabolism of NPs. ► Knowledge about cellular fate of NPs is essential to develop nanoparticles for clinical use.
During recent years there has been much interest in the use of nanoparticles for
in vitro studies as well as for delivery of drugs and contrast agents in animals and humans. To this end it is necessary to increase our understanding of how these particles are taken up and transported within the cells, and to which extent they are metabolized and secreted. In this review we discuss the possibilities, challenges and pitfalls of studying endocytic pathways involved in cellular uptake of nanoparticles. Thus, the use of pharmacological inhibitors, expression of mutated proteins, use of siRNAs and colocalization experiments in such studies are critically evaluated. Although the main focus is on cellular uptake, also aspects of intracellular transport, recycling of nanoparticles to the cell exterior, disturbance of cellular functions, and metabolism of nanoparticles are discussed.
Several studies have demonstrated interactions between the two leaflets in membrane bilayers and the importance of specific lipid species for such interaction and membrane function. We here discuss ...these investigations with a focus on the sphingolipid and cholesterol-rich lipid membrane domains called lipid rafts, including the small flask-shaped invaginations called caveolae, and the importance of such membrane structures in cell biology and cancer. We discuss the possible interactions between the very long-chain sphingolipids in the outer leaflet of the plasma membrane and the phosphatidylserine species PS 18:0/18:1 in the inner leaflet and the importance of cholesterol for such interactions. We challenge the view that lipid rafts contain a large fraction of lipids with two saturated fatty acyl groups and argue that it is important in future studies of membrane models to use asymmetric membrane bilayers with lipid species commonly found in cellular membranes. We also discuss the need for more quantitative lipidomic studies in order to understand membrane function and structure in general, and the importance of lipid rafts in biological systems. Finally, we discuss cancer-related changes in lipid rafts and lipid composition, with a special focus on changes in glycosphingolipids and the possibility of using lipid therapy for cancer treatment.
Cancer biomarkers are invaluable tools for cancer detection, prognosis, and treatment. Recently, microvesicles have appeared as a novel source for cancer biomarkers. We present here the results from ...a proteomic analysis of microvesicles released to the extracellular environment by the metastatic prostate cancer cell line PC-3. Using nanocapillary liquid chromatography-tandem mass spectrometry 266 proteins were identified with two or more peptide sequences. Further analysis showed that 16% of the proteins were classified as extracellular and that intracellular proteins were annotated in a variety of locations. Concerning biological processes, the proteins found in PC-3 cell-released microvesicles are mainly involved in transport, cell organization and biogenesis, metabolic process, response to stimulus, and regulation of biological processes. Several of the proteins identified (tetraspanins, annexins, Rab proteins, integrins, heat shock proteins, cytoskeletal proteins, 14–3-3 proteins) have previously been found in microvesicles isolated from other sources. However, some of the proteins seem to be more specific to the vesicular population released by the metastatic prostate cancer PC-3 cell line. Among these proteins are the tetraspanin protein CD151 and the glycoprotein CUB domain-containing protein 1. Interestingly, our results show these proteins are promising biomarkers for prostate cancer and therefore candidates for clinical validation studies in biological fluids.
Exosomes are vesicles released from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane. This study aimed to investigate whether the phosphoinositide kinase PIKfyve affects this ...process. Our results show that in PC-3 cells inhibition of PIKfyve by apilimod or depletion by siRNA increased the secretion of the exosomal fraction. Moreover, quantitative electron microscopy analysis showed that cells treated with apilimod contained more MVBs per cell and more intraluminal vesicles per MVB. Interestingly, mass spectrometry analysis revealed a considerable enrichment of autophagy-related proteins (NBR1, p62, LC3, WIPI2) in exosomal fractions released by apilimod-treated cells, a result that was confirmed by immunoblotting. When the exosome preparations were investigated by electron microscopy a small population of p62-labelled electron dense structures was observed together with CD63-containing exosomes. The p62-positive structures were found in less dense fractions than exosomes in density gradients. Inside the cells, p62 and CD63 were found in the same MVB-like organelles. Finally, both the degradation of EGF and long-lived proteins were shown to be reduced by apilimod. In conclusion, inhibition of PIKfyve increases secretion of exosomes and induces secretory autophagy, showing that these pathways are closely linked. We suggest this is due to impaired fusion of lysosomes with both MVBs and autophagosomes, and possibly increased fusion of MVBs with autophagosomes, and that the cells respond by secreting the content of these organelles to maintain cellular homeostasis.
Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumor-specific molecules can be found in exosomes isolated from biological fluids. We have previously ...analyzed the proteome of urinary exosomes by mass spectrometry, and identified proteins differentially expressed in prostate cancer patients compared to healthy males. Since mass spectrometry is so far not commonly used in clinical laboratories, we have here investigated whether antibody-based methods such as Western blot or ELISA can be used to validate the use of the identified proteins as prostate cancer biomarkers. Western blot experiments designed to detect flotillin 2, TMEM256, Rab3B and LAMTOR1 showed that the level of these proteins was higher in urinary exosomes from prostate cancer patients compared to healthy males. Furthermore, a receiver operating characteristic curve of flotillin 2 in samples from 16 controls and 16 patients showed an area under the curve of 0.91, and 88% sensitivity at a threshold set to give 94% specificity. In addition, ELISA-based detection of flotillin 2 and PARK7 showed that the combination of these proteins was able to distinguish prostate cancer patients and healthy controls with 68% sensitivity and 93% specificity. Several promising biomarkers identified by mass spectrometry could not be evaluated by Western blot or ELISA due to their low exosomal amount and/or lack of good antibodies. In conclusion, our results show that several urinary exosomal proteins identified as prostate cancer biomarkers by mass spectrometry have a high diagnostic value also when analyzed by immunology-based methods, thus bringing these biomarkers closer to a potential clinical use.
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Exosomes are released by cells after fusion of multivesicular bodies with the plasma membrane. The molecular mechanism of this process is still unclear. We investigated the role of sphingolipids and ...flotillins, which constitute a raft‐associated family of proteins, in the release of exosomes. Interestingly, our results show that dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol, an inhibitor of glucosylceramide synthase, seemed to affect the composition of exosomes released from PC‐3 cells. However, the inhibition of ceramide formation from the de novo pathway by fumonisin B1 did not affect exosome secretion. Moreover, in contrast to findings obtained with other cell lines published so far, inhibition of neutral sphingomyelinase 2, an enzyme that catalyzes the formation of ceramide from sphingomyelin, did not inhibit the secretion of exosomes in PC‐3 cells. Finally, small interfering RNA‐mediated downregulation of flotillin‐1 and flotillin‐2 did not significantly change the levels of released exosomes as such, but seemed to affect the composition of exosomes. In conclusion, our results reveal the involvement of glycosphingolipids and flotillins in the release of exosomes from PC‐3 cells, and indicate that the role of ceramide in exosome formation may be cell‐dependent.
Exosomes are released by fusion of multivesicular bodies with the plasma membrane. Our results reveal that glycosphingolipids and flotillins affect the exosomes released from PC‐3 cells by changing their composition rather than the level of released exosomes. Furthermore, in PC‐3 cells there is no evidence for involvement of sphingomyelinase in the production of exosomes.
The aim of this study was to identify microRNAs in urinary exosomes that are differently expressed in prostate cancer patients and healthy donors. For this purpose, RNA was extracted from urinary ...exosomes from 20 prostate cancer patients and 9 healthy males and the microRNAs were analyzed by next generation sequencing. Interestingly, 5 microRNAs - miR-196a-5p, miR-34a-5p, miR-143-3p, miR-501-3p and miR-92a-1-5p - were significantly downregulated in exosomes from prostate cancer patients. Furthermore, RT-qPCR analysis of an independent cohort of 28 prostate cancer patients and 19 healthy males confirmed that miR-196a-5p and miR-501-3p were downregulated in prostate cancer samples. These results suggest that specific microRNAs in urinary exosomes might serve as non-invasive biomarkers for prostate cancer. In particular, miR-196a-5p and miR-501-3p are promising biomarkers that need to be further studied in large patient cohorts.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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