Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.     

Multivesicular bodies and autophagy in erythrocyte maturation

During reticulocyte maturation, hematopoietic progenitors undergo numerous changes to reach the final functional stage which concludes with the release of reticulocytes and erythrocytes into circulation. During this process some proteins, which are not required in the mature stage, are sequestered in the internal vesicles present in multivesicular bodies (MVBs). These small vesicles are known as exosomes because they are released into the extracellular medium by fusion of the MVB with the plasma membrane. Interestingly, during this maturation process some organelles, such as mitochondria and endoplasmic reticulum, are wrapped in double membrane vacuoles and degraded via autophagy. We have demonstrated in human leukemic K562 cells a role for calcium and Rab11 in the biogenesis of MVBs and exosome release. Here we discuss evidence indicating that K562 cells present a high basal level of autophagy, and that there is an association between MVBs and autophagosomes, suggesting a role for the autophagic pathway in the maturation process of this cell type.     

Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes)

Vesicles are released during the in vitro culture of sheep reticulocytes which can be harvested by centrifugation at 100,000 X g for 90 min. These vesicles contain a number of activities, characteristic of the reticulocyte plasma membrane, which are known to diminish or disappear upon reticulocyte maturation. The activities include acetylcholinesterase, cytochalasin B binding (glucose transporter) nucleoside binding (i.e. nucleoside transporter), Na+-independent amino acid transport, and the transferrin receptor. Enzymes of cytosolic origin are not detectable or are present at low activity in the vesicles. Cultures of whole blood, mature red cells, or white cells do not yield comparable levels of these activities, supporting the conclusion that the activities arise from the reticulocytes. In addition, the lipid composition of the vesicles shows the high sphingomyelin content characteristic of sheep red cell plasma membranes, but not white cell or platelet membranes, also consistent with the conclusion that the vesicles are of reticulocyte origin. It is suggested that vesicle externalization may be a mechanism for shedding of specific membrane functions which are known to diminish during maturation of reticulocytes to erythrocytes.     

In vitro acylation of the transferrin receptor

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.     

Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach.

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.     

Multivesicular Bodies and Autophagy in Erythrocyte Maturation

During reticulocyte maturation, hematopoietic progenitors undergo numerous changes to reach the final functional stage which concludes with the release of reticulocytes and erythrocytes into circulation. During this process some proteins, which are not required in the mature stage, are sequestered in the internal vesicles present in multivesicular bodies (MVBs). These small vesicles are known as exosomes because they are released into the extracellular medium by fusion of the MVB with the plasma membrane. Interestingly, during this maturation process some organelles, such as mitochondria and endoplasmic reticulum, are wrapped in double membrane vacuoles and degraded via autophagy. We have demonstrated in human leukemic K562 cells a role for calcium and Rab11 in the biogenesis of MVBs and exosome release. Here we discuss evidence indicating that K562 cells present a high basal level of autophagy, and that there is an association between MVBs and autophagosomes, suggesting a role for the autophagic pathway in the maturation process of this cell type.

Addendum to:

Exosome secretion and red cell maturation: Exploring molecular components involved in the docking and fusion of multivesicular bodies in K562 cells.

Fader CM, Savina A, Sánchez D, Colombo MI. Blood Cells Mol Dis 2005; 35:153-7.

and

Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner.

Savina A, Fader CM, Damiani MT, Colombo MI. Traffic 2005; 6:131-43.     

Exosomes from Plasmodium yoelii-infected reticulocytes protect mice from lethal infections

Exosomes are 30-100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. Here, we describe the isolation and characterization of exosomes from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain. Importantly, proteomic analysis revealed the presence of parasite proteins in these vesicles. Moreover, immunization of mice with purified exosomes elicited IgG antibodies capable of recognizing P. yoelii-infected red blood cells. Furthermore, lethal challenge of immunized mice with the normocyte-prone lethal P. yoelii 17XL strain caused a significant attenuation in the course of parasitaemia, increased survival time, and altered the cell tropism to reticulocytes. These results were obtained also when the exosomes were isolated from a P. yoelii-infected reticulocyte culture indicating that reticulocyte-derived exosomes carry antigens and are involved in immune modulation. Moreover, inclusion of CpG ODN 1826 in exosome immunizations elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. To our knowledge, this is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the modulation of anti-malaria responses.     

Intracellular localization of newly synthesized transferrin receptors in the peripheral sheep reticulocyte

In this paper, we provide evidence for an incompletely glycosylated transferrin receptor (TfR) which is not transported to the plasma membrane in the sheep reticulocyte. Cleveland peptide maps of the native (preexisting) TfR and [35S]methionine-labeled TfR were different. If the receptors were deglycosylated before mapping, the peptides were identical. There was preferential binding of the [35S]TfR to Con A-Sepharose, indicating the existence of a higher density of high mannose chains on the 35S-labeled TfR. Moreover, when total [3H]mannose-labeled glycopeptides from reticulocytes were separated on a column of Bio-Gel P6, the [3H]mannose was associated with endoglycosidase H-sensitive high mannose or hybrid oligosaccharides, but not with complex sugars. After Percoll density gradient centrifugation, the [35S]TfR peaked in a fraction which separated from the bulk of the native TfR. The transmembrane glycoproteins, Band 3 and mature glycophorins, are not synthesized in the sheep reticulocyte. It appears that the reticulocyte, at this stage of red cell development, has lost the vesicles and/or proteins which are required to transport proteins from the site of translation to the cell surface.     

Cleavage of the transferrin receptor by human granulocytes: Preferential proteolysis of the exosome-bound TFR

In contrast with other mammalian granulocytes, human granulocytes rapidly cleave the transferrin receptor (TFR) from sheep exosomes. Proteolysis of TFR from exosomes is more rapid and more extensive than that from the sheep reticulocyte cell surface itself, although little difference in cleavage is seen when immunoprecipitates or when immobilized, solubilized receptors from the two sources are compared. Circulating exosomes but not the plasma membrane fraction from seven species of immature red cells or erythropoietic cells show the presence of a peptide of ∼18 kD recognized by an antibody to the cytoplasmic domain of the TFR. This 18 kD peptide is virtually absent from the corresponding cellular plasma membranes including human reticulocyte membranes. Taken together, the data are consistent with the conclusion that the exosomes released to the circulation from maturing red cells are the principal source of the soluble, circulating, truncated TFR. The granulocyte protease appears to be present on the cell surface and not released into the medium after short (30 min) periods of incubation at 37°C. The protease is inhibited by PMSF but only at high (1 mM) concentrations. Using sheep exosome bound-TFR as substrate, human granulocytes exceed other granulocytes in their capacity to cleave TFR, suggesting that this may be a key factor for the prominent amount of circulating, soluble receptor found in human sera during periods of elevated reticulocyte levels. Human exosomes, unlike those from other species, contain little native size TFR. Truncated receptor containing the cytoplasmic domain being predominant in human exosomes. © 1996 Wiley-Liss, Inc.     

Diacylglycerol kinase α regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of Fas ligand-containing exosomes in T lymphocytes

Multivesicular bodies (MVBs) are endocytic compartments that contain intraluminal vesicles formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these vesicles contain pro-apoptotic Fas ligand (FasL), which may be secreted as 'lethal exosomes' upon fusion of MVBs with the plasma membrane. Diacylglycerol kinase α (DGKα) regulate the secretion of exosomes, but it is unclear how this control is mediated. T-lymphocyte activation increases the number of MVBs that contain FasL. DGKα is recruited to MVBs and to exosomes in which it has a double function. DGKα kinase activity exerts a negative role in the formation of mature MVBs, as we demonstrate by the use of an inhibitor. Downmodulation of DGKα protein resulted in inhibition of both the polarisation of MVBs towards immune synapse and exosome secretion. The subcellular location of DGKα together with its complex role in the formation and polarised traffic of MVBs support the notion that DGKα is a key regulator of the polarised secretion of exosomes.     

Transferrin receptors during rabbit reticulocyte maturation.

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

Isolation and characterization of RNA-containing exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen, eradication of established tumors in mice, inhibition and activation of natural killer cells, promotion of differentiation into T regulatory cells, stimulation of T cell proliferation and induction of T cell apoptosis. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.     

Exosomal transfer of proteins and RNAs at synapses in the nervous system

Background  

Many cell types have been reported to secrete small vesicles called exosomes, that are derived from multivesicular bodies and that can also form from endocytic-like lipid raft domains of the plasma membrane. Secretory exosomes contain a characteristic composition of proteins, and a recent report indicates that mast cell exosomes harbor a variety of mRNAs and microRNAs as well. Exosomes express cell recognition molecules on their surface that facilitate their selective targeting and uptake into recipient cells.     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

Exosome Secretion: The Art of Reutilizing Nonrecycled Proteins?

Multivesicular bodies contain membrane vesicles which either undergo lysosomal digestion or are released in the extracellular environment as exosomes. Evidence is accumulating that supports a physiological role for exosomes in, for example, antigen presentation or removal of transferrin receptor during reticulocyte development. Here, inspired by observations on exosomal release from reticulocytes, we discuss the potential involvement of the so-called ESCRT mechanism in the entrapment of both lysosomal and exosomal cargo within the intralumenal vesicles of multivesicular bodies. We propose that this mechanism operates at different sites in the endocytic itinerary in different cells, thereby providing a tool for directional sorting. We also explore the possibility that the efficiency of sorting of molecules into exosomes increases when the recycling kinetics of molecules decreases, exosomal sorting being favored by intermolecular interactions occurring within lipid domains, or with protein webs, that slow lateral mobility. These considerations are mirrored in the context of current knowledge on the mechanism of protein sorting for degradation in lysosomes, and the hijacking of such mechanisms by some retroviruses for particle budding.     

Transferrin receptors during rabbit reticulocyte maturation

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

Hepatocytes and reticulocytes have different mechanisms for the uptake of iron from transferrin

The uptake of iron from transferrin by isolated rat hepatocytes and rat reticulocytes has been compared. The results show the following. 1) Reticulocytes and hepatocytes express plasma membrane NADH:ferricyanide oxidoreductase activity. The activity, expressed per 10(6) cells, is approximately 60-fold higher in the hepatocyte than in the reticulocyte. 2) Hepatocyte plasma membrane NADH:ferricyanide oxidoreductase activity and uptake of iron from transferrin are stimulated by low oxygen concentration and inhibited by iodoacetate. In reticulocytes, similar changes are seen in NADH:ferricyanide oxidoreductase activity, but not on iron uptake. 3) Ferricyanide inhibits the uptake of iron from transferrin by hepatocytes, but has no effect on iron uptake by reticulocytes. 4) Perturbants of endocytosis and endosomal acidification have no inhibitory effect on hepatocyte iron uptake, but inhibit reticulocyte iron uptake. 5) Hydrophilic iron chelators effectively inhibit hepatocyte iron uptake, but have no effect on reticulocyte iron uptake. Hydrophobic iron chelators generally inhibit both hepatocyte and reticulocyte iron uptake. 6) Divalent metal cations with ionic radii similar to or less than the ferrous iron ion are effective inhibitors of hepatocyte iron uptake with no effect on reticulocyte iron uptake. The results are compatible with hepatocyte uptake of iron from transferrin by a reductive process at the cell surface and reticulocyte iron uptake by receptor-mediated endocytosis.     

Calcium chelators induce association with the detergent-insoluble cytoskeleton and functional inactivation of the transferrin receptor in reticulocytes

Incubation of reticulocytes with EDTA, EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), but not with desferrioxamine B, at temperatures above 20 degrees C resulted in the loss of their ability to take up iron in a temperature-, time- and concentration-dependent manner. No inhibition of transferrin or iron uptake occurred if the incubations were performed at 20 degrees C or below. At higher temperatures, the inhibition was attributable to loss of functional transferrin receptors, not to altered affinity or endocytosis of the remaining receptors. The changes could not be reversed by washing the cells and reincubation in the presence of Ca2+, Mg2+ or Zn2+. However, they could be completely prevented by performing the initial incubation with chelators in the presence of diferric transferrin and partly prevented by the use of apotransferrin. Incubation with the chelators resulted in much less reduction in the ability of the cells to bind anti-transferrin receptor immunoglobulin than transferrin. The fate of the receptor was studied by polyacrylamide gel electrophoresis of reticulocyte membrane proteins before and after extraction with Triton X-100, and by immunological staining of Western blots for the transferrin receptor. Treatment of the cells with EDTA led to a loss of the ability of Triton X-100 to solubilize the receptor and its retention in the Triton-insoluble cytoskeletal matrix of the cells. It is concluded that incubation of reticulocytes with the chelators at temperatures above 20 degrees C causes an altered interaction of the transferrin receptor with the cytoskeleton. This change, which is probably due to chelation of Ca2+ in the cell membrane, is accompanied by an irreversible loss of the receptor's ability to bind transferrin.     

Exosomes released during reticulocyte maturation bind to fibronectin via integrin alpha4beta1.

Exosomes are vesicles formed in the endosomal compartment and released in the extracellular medium during reticulocyte maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, e.g. acetylcholinesterase and transferrin receptor. We show here that integrin alpha4beta1, present on the surface of erythroid precursors but absent from the mature red cell membrane, is at least partly cleared from the reticulocyte plasma membrane by the exosomal pathway. Using flow cytometry, we found that the alpha4 subunit disappears from the reticulocyte surface during in vitro maturation. Two different monoclonal antibodies (B-5G10 and HP 2/1) were used to demonstrate the presence of the alpha4 chain on the exosome surface. Moreover, membrane acetylcholinesterase and lumenal peroxidase-like (i.e. hemoglobin) enzymatic activities were assayed to demonstrate exosome binding to plates coated with increasing fibronectin (FN) concentrations. This interaction was dependent on divalent cations (MnCl2 > MgCl2 > CaCl2). Similarly, vesicles bound to plates coated with the chymotryptic 40 K fragment (FN-40) containing the heparin-binding region of FN. This binding was inhibited by exosome preincubation with fibronectin CS1 peptide and with a monoclonal antibody (HP 2/1) against the integrin alpha4-chain, confirming an alpha4beta1-induced interaction. The importance of the exosome clearance function is highlighted here, since the presence of VLA-4 on reticulocytes often leads to blood circulation complications in some diseases. Moreover, the presence of alpha4beta1 on the exosome surface, by allowing binding to endothelial cells through vascular cell adhesion molecule 1 (VCAM-1), might confer another physiological function to the secreted vesicles.     

Characterization and localization of the transferrin receptor on rat reticulocytes

1. A further characterization and localization of the membrane receptor for transferrin on rat reticulocytes is described. PAGE studies with a purified membrane complex B2, from which the functional role in transferrin binding and iron uptake has been shown previously, showed that the transferrin receptor is localized on a membrane protein with a mol. wt of approximately 70-80.10(3). 2. Selective solubilization of the rat reticulocyte membrane has shown that this receptor protein belongs to one of the minor integral membrane polypeptides, embedded in the lipid bilayer of the membrane. 3. Proteolipid complexes, glycolipids and sialoglycoproteins of the rat reticulocyte membrane play no direct role in the binding capacity of the receptor.