Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding

Endocytosis and intracellular processing of transferrin (Tf) and Tf receptors were examined in rat reticulocytes. Subcellular fractionation revealed that Tf enters a non-lysosomal endocytic compartment with a density between those of plasma membrane and lysosomes. After 20 min of uptake at (37 degrees C) 35 to 40% of cell-associated Tf was contained in this intermediate-density compartment. To test the fidelity of colloidal gold-Tf (AuTf) as a probe for Tf processing, reticulocytes were fractionated after uptake of 131I-Tf and 125I-AuTf. The subcellular distributions of the two ligands were indistinguishable by this method, a result suggesting that AuTf is processed similarly to Tf. Electron microscopy revealed that AuTf entered multivesicular endosomes (MVEs) as well as various small vesicles and tubular structures. In addition MVE exocytosis was observed with discharge of inclusion vesicles and associated AuTf. AuTf was bound to the outside of these vesicles both before and after exocytosis. These data suggest that Tf receptors are shed from developing reticulocytes by incorporation into the limiting membrane of inclusion vesicles, followed by discharge of these vesicles by MVE exocytosis. As further evidence of this process, we isolated inclusion vesicles after their discharge and found them to contain Tf receptors. Moreover, the rate of Tf receptor shedding by inclusion vesicle discharge matches Tf receptor loss rates closely enough to suggest that this is the primary path of receptor loss during reticulocyte development.     

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.     

The Jeanne Manery-Fisher Memorial Lecture 1991. Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins.

The transferrin receptor is a member of a group of reticulocyte surface proteins that disappear from the membranes of reticulocytes as the cells mature to the erythrocyte stage. The selective loss of membrane proteins appears to be preceded by the formation of multivesicular bodies (MVBs). At the reticulocyte stage, many species of mammalian red cells including man, and one nucleated avian species (chicken), contain these intracellular structures in both natural and induced anemias. Also characteristic of blood containing reticulocytes is the presence of circulating vesicles (exosomes), which contain proteins and lipids characteristic of the plasma membrane. These exosomes appear to arise from the contents of the MVBs, after the fusion of MVBs with the plasma membrane. The proteins in the exosomes are those frequently lost during red cell maturation (e.g., transferrin receptor). The major transmembrane proteins (such as the anion transporter) are fully retained into the mature red cell, indicating a highly selective mechanism of recognition of a specific group of proteins. The exosomes are largely devoid of soluble proteins and proteins associated with lysozomes or mitochondria. A speculative model is proposed which addresses the questions of the maturation-induced structural changes in a class of membrane proteins, their recognition and selective loss involving exosome formation, and the release of exosomes to the circulation.     

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     

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.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.     

Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.     

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.     

Fractionation analysis of the endosomal compartment during rat reticulocyte maturation

A subcellular fractionation procedure was developed to isolate the different endosomal compartments present during reticulocyte maturation. After reticulocyte lysis and removal of excess haemoglobin by gel chromatography, membrane vesicles were separated over a discontinuous sucrose gradient (10-40%). Two fractions were isolated: P1 at the 25-35% sucrose interface and P2 at the 17-25% sucrose interface. These fractions were morphologically characterized by electron microscopy and the distribution of endocytic markers in the fractions was detected by Western blot. Moreover, this fractionation technique was used to study the effect of 3-methyladenine (3-MA), an autophagy inhibitor, on reticulocyte maturation. The presence of 3-MA during in vitro maturation of reticulocytes induced a decrease in exosome secretion, as measured by the amount of transferrin receptor (TfR) released in the extracellular medium. The subcellular fractionation results suggested that multivesicular endosome formation from early endosomes is the step affected by 3-MA.     

Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes

We have used combinations of subcellular fractionation, specific cytochemical tracers, and quantitative immunoadsorption to determine when, where, and in which intracellular structure internalized asialoglycoproteins (ASGPs) are segregated from their receptor. All membrane vesicles containing the receptor (R+ vesicles) were quantitatively immunoadsorbed from crude microsomes with Staphylococcus aureus cells and affinity-purified anti-ASGP receptor. Using this assay, we varied the time and temperature of exposure of perfused livers to 125I-asialoorosomucoid (125I-ASOR) and followed the movement of ligand from R+ to R- vesicles. After 2.5 min at 37 degrees C, 98% of the internalized ligand could be immunoadsorbed and thus was in R+ vesicles. Over the next 12 min of continuous 37 degrees C perfusion with 125I-ASOR, an increasing fraction of the ligand was not immunoadsorbed and therefore was present in R- vesicles. A maximum of 30% of the ligand could be found in R- vesicles (14-44 min). When livers were maintained at 16 degrees C, ligand was internalized but remained in R+ vesicles. Furthermore, ligand accumulating in R- vesicles at 37 degrees C remained there when livers were cooled to 16 degrees C. R- endosomes could be separated from R+ endosomes by flotation on sucrose density gradients and visualized by the presence of sequestered ASOR-horseradish peroxidase (ASOR-HRP). These structures resembled those labeled by ASOR-HRP in situ: R+ vesicles were relatively dense (1.12 g/cc), frequently tubular or spherical and small (100-nm diam), corresponding to the peripheral and internal tubular endosomes; R- structures were of lower density (1.09 g/cc), large (400-nm diam), and resembled internal multivesicular endosomes (MVEs). Endocytosed ASOR-HRP was found in both the peripheral and internal tubular endosomes in situ under conditions where 95% of the ligand was present in R+ vesicles by immunoadsorption, whereas MVEs containing ASOR-HRP were predominant in situ when ligand was found in R- vesicles and were often in continuity with the tubular internal endosomes. All of these results suggest that complete segregation of ligand and receptor occurs after arrival in the Golgi-lysosome region of the hepatocyte and that MVEs are R- and represent the final prelysosomal compartment.     

Morphological analysis of ligand uptake and processing: the role of multivesicular endosomes and CURL in receptor-ligand processing

The receptor-mediated endocytosis and intracellular processing of transferrin and mannose receptor ligands were investigated in bone marrow-derived macrophages, fibroblasts and reticulocytes. Mannosylated bovine serum albumin (BSA) conjugated to colloidal gold (Au-man-BSA) or colloidal gold-transferrin (AuTf) were used to trace ligand processing in these cells. These ligands appeared to be processed by mechanisms similar to those observed previously with other mannose receptor and galactose receptor ligand probes. After uptake via coated pits and coated vesicles, Au-man-BSA appeared in small uncoated vesicles and tubular structures and was transferred to large, sometimes multivesicular endosomes (MVEs), which sometimes had arm-like protrusions reminiscent of CURL (compartment of uncoupling of receptor and ligand) [10, 11]. Initially these structures became increasingly multivesicular, but during longer incubations the inclusion vesicles appeared to disintegrate to leave a denser, amorphous lumen. Inclusion vesicle disintegration may result from the introduction of lysosomal enzymes into these structures. These results suggest a model for differential receptor-ligand and ligand-ligand sorting. As suggested [10, 11] membrane constituents may be recycled to the plasma membrane from the arms of CURL. Receptor-bound ligands, such as transferrin, would also recycle. The luminal contents, including dissociated ligands, other soluble proteins and inclusion vesicles (containing some membrane proteins), would target to lysosomes. This would result in the lysosomal degradation of any membrane proteins that were incorporated in the inclusion vesicle membranes.     

Receptor-mediated endocytosis of transferrin and ferritin by guinea-pig reticulocytes. Uptake by a common endocytic pathway

Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.     

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.     

Sphingolipids and glycoproteins are differentially trafficked to the Chlamydia trachomatis inclusion

Chlamydia trachomatis is an obligate intracellular pathogen that multiples within the confines of a membrane-bound vacuole called an inclusion. Approximately 40-50% of the sphingomyelin synthesized from exogenously added NBD-ceramide is specifically transported from the Golgi apparatus to the chlamydial inclusion (Hackstadt, T., M.A. Scidmore, and D.D. Rockey. 1995. Proc. Natl. Acad. Sci. USA. 92: 4877- 4881). Given this major disruption of a cellular exocytic pathway and the similarities between glycolipid and glycoprotein exocytosis, we wished to determine whether the processing and trafficking of glycoproteins through the Golgi apparatus to the plasma membrane in chlamydia-infected cells was also disrupted. We analyzed the processing of several model glycoproteins including vesicular stomatitis virus G- protein, transferrin receptor, and human histocompatibility leukocyte class I antigen. In infected cells, the posttranslational processing and trafficking of these specific proteins through the Golgi apparatus and subsequent transport to the plasma membrane was not significantly impaired, nor were these glycoproteins found associated with the chlamydial inclusion membrane. Studies of receptor recycling from endocytic vesicles employing fluorescently and HRP-tagged transferrin and anti-transferrin receptor antibody revealed an increased local concentration of transferrin and transferrin receptor around but never within the chlamydial inclusion. However, Scatchard analysis failed to show either an increased intracellular accumulation of transferrin receptor or a decreased number of plasma membrane receptors in infected cells. Furthermore, the rate of exocytosis from the recycling endosomes to the plasma membrane was not altered in chlamydia-infected cells. Thus, although C. trachomatis disrupts the exocytosis of sphingolipids and the Golgi apparatus appears physically distorted, glycosylation and exocytosis of representative secreted and endocytosed proteins are not disrupted. These results suggest the existence of a previously unrecognized sorting of sphingolipids and glycoproteins in C. trachomatis-infected cells.     

Degradation of AP2 during reticulocyte maturation enhances binding of hsc70 and Alix to a common site on TFR for sorting into exosomes

Reticulocytes release small membrane vesicles termed exosomes during their maturation in erythrocytes. The transferrin receptor (TfR) is completely lost from the red cell surface by its segregation in the secreted vesicles where it interacts with the heat shock cognate 70 kDa protein (hsc70). We have now determined a region of the TfR that can potentially interact with hsc70. The peptide P1 (YTRFSLARQV) from the TfR cytosolic domain: (i) binds to hsc70 (ii) with an increased affinity in oxidative conditions, (iii) competes for binding of an unfolded protein to hsc70, and (iv) inhibits the interaction of hsc70 with a recombinant protein corresponding to the cytosolic domain of the receptor. This peptide encompasses the internalization motif (YTRF) of the receptor, and accordingly an affinity column made with the immobilized peptide retains hsc70 and also the AP2 adaptor complex. On the other hand, we show that AP2 is degraded by the proteasome system during reticulocyte maturation and that the presence of the proteasome inhibitor during in vitro red cell maturation inhibits AP2 degradation and specifically decreases TfR secretion via exosomes. Finally, coimmunoprecipitation of Alix with the exosomal TfR, and binding of P1 peptide to the Alix homolog PalA suggest that Alix also interacts with the YTRF motif and contributes to exosomal TfR sorting.     

High mobility of vesicles supports continuous exocytosis at a ribbon synapse

BACKGROUND: Most synapses release neurotransmitter as transient pulses, but ribbon synapses of sensory neurons support continuous exocytosis in response to maintained stimulation. We have investigated how the movement and retrieval of vesicles might contribute to continuous exocytosis at the ribbon synapse of retinal bipolar cells. RESULTS: Using a combination of total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, we found that the great majority of vesicles within 50-120 nm of the plasma membrane move in a random fashion with an effective diffusion coefficient of approximately 1.5 x 10(-2) microm(2) s(-1). Using confocal microscopy, we found that vesicles are similarly mobile across the whole terminal and that this motion is not altered by calcium influx or the actin cytoskeleton. We calculated that the cytoplasmic reservoir of approximately 300,000 vesicles would generate about 900 vesicle collisions/s against ribbons and 28,000 collisions/s against the surface membrane. The efficient resupply of vesicles to ribbons was confirmed by electron microscopy. A 1 min depolarization, releasing 500-1000 vesicles/s, caused a 70% reduction in the number of vesicles docked at the active zone without reducing the number of vesicles attached to ribbons or remote areas of the plasma membrane. These sites were not repopulated by retrieved vesicles because 80-90% of the recycled membrane was taken up into cisternae that pinched off from the surface. CONCLUSIONS: These results indicate that the random motion of cytoplasmic vesicles provides an efficient supply to the ribbon and plasma membrane and allows the maintenance of high rates of exocytosis without an equally rapid recycling of vesicles. The selective depletion of vesicles docked under ribbons suggests that the transfer of vesicles to the active zone limits the rate of exocytosis during maintained stimulation.     

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.     

Correlation between plasma membrane surface area and transferrin secretion rate in isolated hepatocytes

It is generally considered that in exocytosis the size of the secreting cells does not increase when the membranes of exocytosis vesicles fuse with the plasma membrane. As the factors involved in the regulation of this phenomenon are poorly understood, we thought it worthwhile to investigate the relationship between the plasma membrane surface area and secretory activity. Isolated rat hepatocytes were prepared by liver collagenase perfusion. Secretion of the plasma protein, transferrin (Tf) was detected at the single cell level with specific anti-rat transferrin antibodies using the reverse hemolytic plaque test. Hepatocyte surface and hemolytic ring surface areas were calculated from diameters of hepatocyte and hemolytic plaque measured after 5h of incubation. A highly significant correlation was established between the plaque-forming hepatocyte surface areas and the corresponding hemolytic surface areas. This result was confirmed using an automatic image analysis method. Two-month-old rats were compared to 4-month-old rats. We observed that the ratio of the quantity of transferrin secreted by hepatocytes to the hepatocyte surface area was constant for a given incubation time, whatever the size of the hepatocytes. These results suggest that the plasma membrane surface area of hepatocytes may constitute a limiting factor in Tf secretion.     

Member-associated changes during erythropoiesis. On the mechanism of maturation of reticulocytes to erythrocytes

The mature mammalian erythrocyte has a unique membranoskeleton, the spectrin-actin complex, which is responsible for many of the unusual membrane properties of the erythrocyte. Previous studies have shown that in successive stages of differentiation of the erythropoietic series leading to the mature erythrocyte there is a progressive increase in the density of spectrin associated with the membranes of these cells. An important stage of this progression occurs during the enucleation of the late erythroblast to produce the incipient reticulocyte, when all of the spectrin of the former cell is sequestered to the membrane of the reticulocyte. The reticulocyte itself, however, does not exhibit a fully formed membranoskeleton. In particular, the in vitro binding of multivalent ligands to specific membrane receptors on the reticulocyte was shown to cause a clustering of some fractions of these ligand-receptor complexes into special mobile domains on the cell surface. These domains of clustered ligand-receptor complexes became invaginated and endocytosed as small vesicles. By immunoelectron microscopic experiments, these invaginations and endocytosed vesicles were found to be specifically free of spectrin on their cytoplasmic surfaces. These earlier findings then raised the possibility that the maturation of reticulocytes to mature erythrocytes in vivo might involve a progressive loss of reticulocyte membrane free of spectrin, thereby producing a still more concentrated spectrin-actin membranoskeleton in the erythrocyte than in the reticulocyte. This proposal is tested experimentally in this paper. In vivo reticulocytes were observed in ultrathin frozen sections of spleens from rabbits rendered anemic by phenylhydrazine treatment. These sections were indirectly immunolabeled with ferritin-antibody reagents directed to rabbit spectrin. Most reticulocytes in a section had one or more surface invaginations and one or more intra-cellular vesicles that were devoid of spectrin labeling. The erythrocytes in the same sections did not exhibit these features, and their membranes were everywhere uniformly labeled for spectrin. Spectrin-free surface invaginations and intracellular vesicle were also observed with reticulocytes within normal rabbit spleens. Based on these results, a scheme for membrane remodeling during reticulocyte maturation in vivo is proposed.     

Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons

Intact neurons in cultures of fetal rodent spinal cord explants show stimulation-dependent uptake of horseradish peroxidase (HRP) into many small vesicles and occasional tubules and multivesicular bodies (MVB) at presynaptic terminals. Presynaptic terminals were allowed to take up HRP during 1 h of strychnine-enhanced stimulation of synaptic transmitter release and then "chased" in tracer-free medium either with strychnine or with 10 mM Mg++ which depresses transmitter release. Tracer-containing vesicles are lost from terminals under both chase conditions; the loss is more rapid (4-8 h) with strychnine than with 10 mM Mg++ (8-16 h). There is a parallel decrease in the numbers of labeled MVB's at terminals. Loss of tracer with 10 mM Mg++ does not appear to be due to the membrane rearrangements (exocytosis coupled to endocytosis) that presumably lead to initial tracer uptake; terminals exposed to HRP and Mg++ for up to 16 h show little tracer uptake into vesicles. Nor is the decrease likely to the due to loss of HRP enzyme activity; HRP is very stable in solution. During the chases there is a striking accumulation of HRP in perikarya that is far more extensive in cultures initially exposed to tracer with strychnine than 10 mM Mg++ regardless of chase conditions. Much of the tracer ends up in large dense bodies. These findings suggest that synaptic vesicle membrane turnover involves retrograde axonal transport of membrane to neuronal perikarya for further processing, including lysosomal degradation. The more rapid (4-8 h) loss of tracer-containing vesicles with strychnine may reflect vesicle membrane reutilization for exocytosis.