Ariel Savina

Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells. Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells. Multivesicular bodies (MVBs) 1The abbreviations used are: MVBs, multivesicular bodies; MON, monensin; Tf, transferrin; TfR, Tf receptor; AM, acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 2-APB, 2-aminoethoxy-diphenylborate; N-Rh-Pe, N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine; PBS, phosphate-buffered saline; PM, plasma membrane, AchE, acetylcholinesterase; TG, thapsigargin; IP3, inositol 1,4,5-triphosphate. are endocytic organelles that contain small internal vesicles generated from inward budding of the limiting membrane. In antigen-presenting cells, the fusion of these MVBs with the plasma membrane leads to the release of internal vesicles into the extracellular space (1Clayton A. Court J. Navabi H. Adams M. Mason M.D. Hobot J.A. Newman G.R. Jasani B. J. Immunol. Methods. 2001; 247: 163-174Crossref PubMed Scopus (425) Google Scholar). The released vesicles, termed exosomes (for a review see Refs. 2Stoorvogel W. Kleijmeer M.J. Geuze H.J. Raposo G. Traffic. 2002; 3: 321-330Crossref PubMed Scopus (659) Google Scholar and 3Théry C. Zitvogel L. Amigorena S. Nat. Immunol. 2002; 2: 569-579Crossref Scopus (3835) Google Scholar), were initially described in reticulocyte maturation, where their function was to discard plasma membrane proteins that were no longer necessary, such as the transferrin receptor (4Johnstone R.M. Mathew A. Mason A.B. Teng K. J. Cell. Physiol. 1991; 147: 27-33Crossref PubMed Scopus (214) Google Scholar, 5Harding C. Heuser J. Stahl P. J. Cell Biol. 1983; 97: 329-339Crossref PubMed Scopus (1140) Google Scholar, 6Harding C. Heuser J. Stahl P. Eur. J. Cell Biol. 1984; 35: 256-263PubMed Google Scholar). Although other plasma membrane proteins (e.g. acetylcholinesterase) are secreted via exosomes, these small vesicles are devoid of both cytosolic proteins and proteins associated with other intracellular organelles, indicating that only a select group of macromolecules is shed via this pathway. Exosomes are also secreted by other cell types such as activated platelets, which may function in signaling/adhesion, thus having a role at sites of vascular injury (7Heijnen H.F. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar, 8Denzer K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar). Exosomes from cytotoxic T cells and B lymphocytes may be involved in targeting molecules for cell death (9Peters P.J. Geuze H.J. Van der Donk H.A. Slot J.W. Griffith J.M. Stam N.J. Clevers H.C. Borst J. Eur. J. Immunol. 1989; 19: 1469-1475Crossref PubMed Scopus (190) Google Scholar) or antigen presentation (10Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2517) Google Scholar, 11Zitvogel L. Regnault A. Lozier A. Wolfers J. Flament C. Tenza D. Ricciardi-Castagnoli P. Raposo G. Amigorena S. Nat. Med. 1998; 4: 594-600Crossref PubMed Scopus (1704) Google Scholar). Despite the diverse extracellular functions that are carried out by exosomes, very little is known about the molecular machinery involved in either the formation of the MVBs or in the exosome secretory process. We have recently shown that in K562 cells, a human erythroleukemia cell line, overexpression of Rab11 regulates the exosome pathway (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Interestingly, treatment of green fluorescent protein-Rab11-transfected cells with the ionophore monensin (MON) generated large MVBs decorated with Rab11 and labeled with a fluorescent lipid that accumulates in exosomes. MON, a membrane-permeable Na+ ionophore that mediates an antiporter activity exchanging Na+ ions with H+ ions (13Pressman B.C. Ann. Rev. Biochem. 1976; 45: 501-530Crossref PubMed Scopus (1457) Google Scholar), acts on acidic intracellular organelles such as endosomes and lysosomes, causing swelling of these vesicles. MON is also known to induce Ca2+ entry by reversed activity of the Na+/Ca2+ exchanger (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). A rise in intracellular Ca2+ concentration, a universal intracellular signal (for a review see Refs. 17Cullen P.J. Lockyer P.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 339-348Crossref PubMed Scopus (304) Google Scholar and 18Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Crossref PubMed Scopus (678) Google Scholar), is necessary to induce regulated secretion in most cell types (reviewed in Refs.19Gerber S.H. Sudhof T.C. Diabetes. 2002; 51: S3-S11Crossref PubMed Google Scholar and 20Wasle B. Edwardson J.M. Cell. Signal. 2002; 14: 191-197Crossref PubMed Scopus (49) Google Scholar). During regulated exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane in a tightly controlled Ca2+-triggered reaction. In endocrine cells, secretory granules contain large amounts of Ca2+ ions, and it has been suggested that the high intragranular Ca2+ concentration is needed to sustain optimal exocytosis (21Scheenen W.J. Wollheim C.B. Pozzan T. Fasolato C. J. Biol. Chem. 1998; 273: 19002-19008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because MON generates large MVBs in K562 cells, the aim of the present study was to MON exosome release and Ca2+ is involved in this process. Our results indicate that both MON treatment and a rise in intracellular Ca2+ markedly exosome secretion. Furthermore, the MON-stimulated exosome release was a Ca2+-dependent process. Interestingly, we have also observed that MON the of Ca2+ in the MVBs, suggesting that Ca2+ might be involved in the secretory To a physiological signal might the Ca2+-dependent exosome cells were with transferrin Our results indicate that Tf stimulates exosome release in a Ca2+-dependent cell and were from ester and were from Fluo3-AM, and were from N-(lissamine rhodamine B was from and were from and were from other were from or Cell a human erythroleukemia cell line, was in with and Exosome were from of K562 The were on at for to the cells, and at for to the Exosomes were from the by at for The exosome was in a large of and in of of of released exosomes was by the activity of an that is to these vesicles (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). activity was a described J. 16: PubMed Scopus Google Scholar). of the exosome were in of and with and in a of The was carried out in at and the in at was The the activity at of an exosomes were by the of the by of the were in for at on and to an membrane. The were for in and and with with or with were with and The were an enhanced and by MVBs with the and for fluorescent was into the plasma membrane as described J. M. G. D. Eur. J. Cell Biol. Google Scholar). an of the in was and in was with a into The was to the cells, which were for at this the was and the cells were with to the of and labeled cells were for as described and with were on and by In the cells were with by for at labeling with the fluorescent lipid. were observed these cells were an with the to and to were with a and the were with a and with the of were in the presence of for at were to the extracellular and in cells were from were in the of MON or by MON were in a the of MVBs and Exosome cells are human cells that exosomes R.M. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar), the small internal vesicles released into the extracellular by fusion of MVBs with the plasma membrane has been shown by that treatment of K562 cells with the ionophore MON the formation of MVBs J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar, A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). the by which these large MVBs are formed and the effect of MON on exosome release have been To into these MVBs in K562 cells were labeled with the fluorescent lipid and have that this lipid is via and to the accumulates in exosomes that are secreted into the extracellular (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar, M. P. D. J. Cell Sci. PubMed Google Scholar). The lipid was to the at and cells were and at for in the or the presence of shown in MON treatment caused the formation of large MVBs labeled by the fluorescent lipid. In a of the MVBs formed is with the internal vesicles labeled with the fluorescent lipid Because fusion of the MVBs with the results in the release of exosomes, we the effect of MON on the release of exosomes from K562 cells. Exosomes are in proteins such as the transferrin receptor and R.M. A. Teng K. Blood. 1989; PubMed Google Scholar). exosomes were in the by the activity of the of and was determined by as described (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Exosomes were from the extracellular with of MON and by the of the proteins and by shown in MON a increase in exosome release in a A increase was observed by in the the activity of which was at MON in cell were observed as by for which a concentration of MON was used in the of the this concentration, cells were also for by the with of was observed In the of exosomes released was also by the fluorescent lipid this lipid accumulates in intracellular vesicles that are secreted into the extracellular as exosomes. MON also the release of exosomes labeled with the fluorescent lipid the results indicate that MON only generates large MVBs also the secretion of the internal vesicles termed exosomes. A in the Exosome has been shown that MON, a Na+ increase cytosolic Ca2+ by the Na+/Ca2+ (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). to in our the enhanced exosome release by MON was to an increase in intracellular Ca2+, we MON the intracellular Ca2+ concentration in K562 cells. this cells were for at with the intracellular Ca2+ concentration was by for of the of that was an Ca2+ and a rise in intracellular Ca2+ that was the The Ca2+ rise was by the of the intracellular Ca2+ chelator Interestingly, in the presence of the extracellular Ca2+ chelator MON the which was to Ca2+ release from intracellular no increase was indicating that the is a of Ca2+ from the extracellular The results that the increase in exosome release might be to a Ca2+-dependent To this we the MON effect on exosome release also be by Ca2+ To the Ca2+ present in the extracellular cells were for in the presence of these the Ca2+ concentration was as with the was used to intracellular Ca2+, this is a membrane-permeable that Ca2+. The released exosomes were from the and by activity as shown in both and the release of exosomes. the increase was completely by the Ca2+ and no were observed both were The that Ca2+ from the extracellular and also from intracellular is for the exosome secretion. Ca2+ in exosome release was the Ca2+ ionophore shown in with the Ca2+ ionophore stimulated exosome secretion to a as were observed both were the secretory effect of the Ca2+ ionophore was by the or is known that MON acts on acidic by the vesicle in a Ca2+ into the K. J. J. J. Cell Sci. 2001; 115: Google Scholar). We the effect of known to the of the and the R. T. A. 2001; PubMed Scopus Google Scholar). has shown that these may also intracellular Ca2+ from acidic Biochem. 1998; PubMed Scopus Google Scholar, R. Cell Calcium. 2001; PubMed Scopus Google Scholar). We have that intracellular Ca2+ in a to MON shown in stimulated the release of exosomes, to a were observed by the of with also the release of exosomes and were by extracellular Ca2+ with the chelator indicating that these via a a by indicate the of intracellular Ca2+ S. S. J. R. Biochem. J. 1996; PubMed Scopus Google for a review see Refs. A. A. Cell Calcium. 1996; 19: PubMed Scopus Google Scholar and R. 2001; PubMed Scopus Google is a that accumulates in the where cytosolic the the fluorescent membrane it has been shown used at of this is of also in intracellular and be used as an for intracellular Ca2+ A. P. B. Physiol. Rev. 1999; PubMed Scopus Google Scholar). were with for at to the MVBs were labeled with the fluorescent lipid as and the cells were with the for at shown in the large MVBs by MON treatment were labeled by Fluo3-AM, indicating that Ca2+ accumulates in these intracellular Ca2+ was also present in the large MVBs formed by The presence of the MVBs calcium in both the of the MVBs was markedly indicating that a is involved in the of the MVBs formed by MON or by and a in the of is that a of the present in the of the P.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), by a Ca2+ from other Ca2+ as as from the extracellular leads to a and increase in the concentration of treatment of K562 with this stimulated exosome secretion in a as MON, and this effect was also by a role for Ca2+ in the exosome secretory pathway and the of a Ca2+ in the process. Because stimulated exosome we were in the large MVBs by MON were also formed by treatment with shown in generated large MVBs the formation of the structures that were with that an increase in cytosolic Ca2+ is by to the MVBs, to exosome secretion. The a role in the of Ca2+ from intracellular (for a review see Refs. Cell. Full Text PDF PubMed Scopus Google Scholar and T. 2001; PubMed Scopus Google Scholar). The for the inositol a of Ca2+ for the of intracellular Ca2+ The increase in the of in the of Ca2+ present in the and the release of Ca2+ into the A. 1991; 51: PubMed Scopus Google Scholar). To this of might be involved in the exosome cells were with 2-APB, a membrane-permeable of Ca2+ that exosome results were with a of indicating that a Ca2+ rise by the of an receptor is critical for the MON-stimulated exosome the formation of the large MVBs by MON was completely by was a in the of the MVBs with the vesicles generated by MON in the of in the cells was the of the to vesicles that the release of Ca2+ via the Ca2+ at in to the of the The of the MVBs with by results indicate that Ca2+ is for of the MVBs, these structures are formed in the presence of Ca2+ to in the the of the large MVBs was only by that of Ca2+ is involved in the Ca2+ rise and the of Ca2+ in the is known that the by MON cytosolic Ca2+ by the Na+/Ca2+ the activity of a Na+/Ca2+ exchanger to be critical for the MON we an of the and Na+/Ca2+ shown in the exosome release and also completely the MON-stimulated secretion of exosomes. the formation of the MVBs generated by was completely by In the was by an of a Ca2+ results are with the that of a Na+/Ca2+ exchanger is a for the Ca2+ rise in the that leads to exosome secretion and the formation of the MVBs with Ca2+ generated by Exosome in a the results indicate that Ca2+ is a in the exosome release process. we were in a physiological stimulus might also exosome secretion in a Ca2+-dependent K562 is a human erythroleukemia cell line that high of TfR, it has been shown that of Tf to receptor intracellular Ca2+ concentration J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar), Tf might be a for exosome secretion. we Tf intracellular Ca2+ in K562 cells. this cells were with as described and human Tf was to the described for other cell types J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar), Tf an increase in Ca2+ that was for the We Tf to the exosome secretion. were for in the presence of human Tf, and exosomes were from the as described shown in Tf stimulated exosome an effect that was by or indicating that was a Ca2+-dependent process. Furthermore, the exosome release was also by 2-APB, suggesting that Ca2+ in this process. is in with a that the of to cells the of C.B. C. J.M. J. 2002; PubMed Scopus Google Scholar). In this study we have shown that the ionophore MON induces the formation of large MVBs and stimulates the release of the internal vesicles exosomes. has been shown that MON induces secretion from cells (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, A. H. Biochem. 35: PubMed Scopus Google Scholar). The release of regulated secretory granules is known to be we present that the MON-stimulated exosome secretion in K562 cells is a The application of MON generated a of Ca2+ that was on both an extracellular and intracellular Ca2+ The role for Ca2+ on exosome release was by the of the Ca2+ ionophore results were with known to the of such as and an of the stimulated exosome release via a Ca2+-dependent the effect was by We have that intracellular Ca2+ in a as are in with indicating that a Ca2+ release in the Biochem. 1998; PubMed Scopus Google Scholar). it has been shown that cytosolic Ca2+ by intracellular Ca2+ in cells R. Cell Calcium. 2001; PubMed Scopus Google Scholar). our results indicate that Ca2+ is a in the exosome release process. We that this is a on their exosomes play in physiological K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar). activated release exosomes at sites of vascular injury where may have a function (7Heijnen H.F. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar). cells also exosomes that molecules as for our that Ca2+ regulates exosome release that a signal is involved in the of cells to release these small vesicles at the The of MON to that Ca2+ an role in exosome secretion. has been that MON acts on acidic intracellular organelles such as endosomes and lysosomes, exchanging H+ for Na+ and causing swelling of these vesicles by Cell. 1983; Full Text PDF PubMed Scopus Google Scholar). it be that a might be involved in the of the large MVBs formed MON our results indicate that the formation of the endosomes is a this was completely by the Ca2+ chelator is to that the formation of these endosomes is only to swelling of the vesicles also the of from other implying fusion is that intracellular on Ca2+ A. Cell Biol. 1999; PubMed Scopus Google Scholar) it is that Ca2+ might be for the fusion involved in the of the are to this MON, as a Na+ a of Na+ the cells of H+ to the extracellular The increase in intracellular Na+ the Na+/Ca2+ exchanger in a to an increase in cytosolic Ca2+ X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). Our are in with a requirement for an of the were completely by an of the and Na+/Ca2+ Our are also with the that Na+ entry by MON the of IP3, which in releases Ca2+ from intracellular the of these might the of Ca2+ at the plasma membrane that induces a Ca2+ rise with the requirement for extracellular Ca2+ in our in this indicate that are involved in MON-stimulated exosome indicating that a Ca2+ rise by the of is critical for this the formation of the MVBs was only and completely by of the suggesting that an might be involved in the of these large Interestingly, we have observed that the large MVBs generated by are with Ca2+. is that the MON-stimulated activity might be at the plasma membrane, it is also that a effect on intracellular it has been shown that MON induces the secretion of by the membrane C. J. Biol. 1991; PubMed Scopus Google Scholar). and K. J. J. J. Cell Sci. 2001; 115: Google Scholar) have that contain high concentration of Ca2+ and function as an intracellular Ca2+ have shown that changes in in the of Ca2+ out of into the via calcium or Our results indicate a Ca2+ is involved in the release of exosomes, of this either the formation of the MVBs or the of Ca2+ the large it is that of Ca2+ present in intracellular might be for the a Ca2+ present in A. G. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to the plasma membrane Ca2+ is A Ca2+ in the of J. Physiol. Cell Physiol. 2000; PubMed Google Scholar) with from the and the Ca2+ has also been secretory pathway to be a to the and is to be for the function of the secretory pathway. Interestingly, we have recently shown that MVBs in K562 cells are at in by membrane from (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Furthermore, it has been shown that endocytic vesicles from a Ca2+ that Ca2+ into the lumen of the vesicles M. B. J. J. Biochem. Mol. Biol. 35: Google Scholar). it be that a Ca2+ is involved in the formation of the MVBs and the of the Ca2+. possibility is that the Ca2+ present the MVBs is a critical role in the exosome secretory process. A role for Ca2+ in secretory and intracellular fusion has been Fusion of endosomes to the release of Ca2+ for fusion to S. M.J. J. Cell Sci. 2001; PubMed Google Scholar). in cells it has been shown that Ca2+ from granules exocytosis (21Scheenen W.J. Wollheim C.B. Pozzan T. Fasolato C. J. Biol. Chem. 1998; 273: 19002-19008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). the presence of on and secretory granules has been suggesting that these organelles a Ca2+ the secretory H. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). we it is known that MON the the release of Ca2+. it is that the release of Ca2+ from the MVBs at the may play an role in the fusion our results indicate that Ca2+ is a critical in the MON-stimulated exosome are to the Ca2+ in this process. we have indicating that Tf intracellular calcium in K562 cells and stimulated exosome release in a Ca2+-dependent manner, suggesting that the secretion of exosomes is physiological Our results are with that the of transferrin to receptor intracellular Ca2+ and stimulates receptor in cells J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar). transferrin was stimulated by Ca2+ in cells Traffic. 2002; 3: PubMed Scopus (13) Google Scholar). Our results that a of the machinery involved in exosome secretion is regulated by Ca2+. such as and have been in where function as Ca2+ Biochem. J. 2002; PubMed Google Scholar). Tf, by increasing intracellular Ca2+, may critical of the Interestingly, in a it has been shown that the of to receptor via a the of and proteins known to be involved in C.B. C. J.M. J. 2002; PubMed Scopus Google Scholar). In the to function as a signal that only also via the exosome and Ca2+ is of the of this pathway by Tf is to that exosomes released to from cells are the of the R.M. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar). have been in K. J. PubMed Scopus Google Scholar). the physiological role of the J. J. 2002; PubMed Scopus Google Scholar) has been our that Tf stimulates exosome release as a the of TfR, to that this is a to the of We Amigorena and for critical of this We also for