Pascale Bette-Bobillo

Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages

Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.

Exosomal sorting of the cytoplasmic domain of bovine leukemia virus TM Env protein.

Exosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM‐BLV–CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM‐BLV–CD8‐containing exosomes are likely formed from a recycling endosomal/TGN compartment.

Platelet-activating-factor-induced serum-albumin release from rabbit platelets in the absence of calcium

Using a radioimmunoassay for rabbit-serum albumin, platelet-activating-factor-induced serum-albumin release by platelets was monitored under non-aggregating conditions. The four main results from this study are as follows. The EC50 of the release was of the same order of magnitude as the aggregation EC50 in the presence of calcium. The release took place within 2 min and was inhibited by BN 52021, which is a very specific inhibitor of the platelet-activating-factor-aggregating effect. Serum-albumin release was much greater than serotonin release.

Interaction between blood components and a spin-labeled analogue of PAF-acether

The interactions between a spin-labeled analogue of PAF-acether (designated as (0,2)PAF) and different human blood components (platelets, erythrocytes, and serum) have been studied. The rate of spin probe reduction by cytosol provided information about the internalization processes when the hydrolysis rate was also available. Although erythrocyte reactivity is lower than that of platelets, erythrocytes, because of their greater numbers, removed (0,2)PAF from whole blood faster than platelets. Lastly, erythrocytes may be more efficient traps for (0,2)PAF than serum acetylhydrolase. Criteria for extending these results to genuine PAF-acether are also discussed.