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Vol. 40, No. 10, pp. 1501-1510, 1992 Printed in KKA.
The Joumal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.
Original Ar tide
I Endocytosis and Intracellular Transport of Transferrin Across the Lactating Rabbit Mammary Epithelial Cell TOURIA SEDDIKI, SERGE DELPAL, and MICHELE OLLIVIER-BOUSQUET' Laboratoire de Biologie Cellulaire et Molthhire, INRA (TS,MOB), and Laboratoire de Nutrition el SicuritZ Alimentaire, INRA, (SO) 78312 Jouy-enjosas, France. Received for publication November 13, 1991 and in revised form March 30, 1992; accepted May 4. 1992 (lA2504).
To study the transcytosis and segregation of ligand in the transit mammary epithelial cell, endocytosisand intracellula~ of human blood transferrin were followed in lactating rabbit mammary epithelial cells. Human transferrin labeled with biotin added to an incubation medium was bound to the basal membrane of mammary epithelial cells and carried aaoss the cell to the lumen of the acini within 5-60 min. At the same time, biotinylated human transferrin accumulated at the apex of the cell. After incubation with human transferrin labeled with colloidal gold, label was detected inside endosome-like structures, vesicles and saccules of the Golgi apparatus, and inside the lumen within 2-5 min. A
Introduction Endocytosis of iron-loaded transferrin (Tf) and of the transferrin receptor (TfR) occurs in many mammalian cells. After internalization, Tf releases its iron intracellularly at an acidic pH, and the Tf-TfR complex recycles to the plasma membrane where TfR and Tf dissociate (Dautry-Varsat et al., 1983; Klausner et al., 1983). The TfR has been well characterizedin the rat and mouse mammary epithelial cells. A significant increase in TfR occurs during mammary growth and the TfR level remains high during lactation in the rat (Grigor et al., 1988)and mouse (Schulman et al., 1989). The role of this high level of TfR in the lactating epithelial mammary cell remains unclear. The epithelial cell ferritin level decreases during lactation in the mouse, suggesting that there is no increased storage of iron (Schulman et al., 1989). Moreover, milk iron concentration decreases during lactation in the rat (Sigman and b n nerdal, 1990) and is very low in the rabbit (ref. in Tarvydas et al., 1968).The high level of TfR in the mammary epithelial cell could be associated not only with milk iron transport by Tf, but also to the transport of Tf itself. Tf has been implicated in differentiation and growth of various tissues (ref. in Lee et al., 1987). Correspondence to: M. Ollivier-Bousquet, Laboratoire de Biologie Cellulaire et Moliculaire, INRA, 78352 Jouy-en-Josas Cidex, France.
significant label accumulated at the apex of the cell after 30-60 min. Biotin labeling did not modify the time of transit of human transferrin, as attested by comparison with the time of transit of native transferrin. Human transferrin was never detected inside vesicles containing casein micelles. In contrast, rabbit milk transferrin was immunocytochemically detected inside vesicles containing casein micelles. These results indicate that transcytosis of human transferrin follows a pathway different from vesicles that carry casein micelles. (1Hisrochem C y r d e m 40:1501-1510, 1992) KEY WORDS:Transferrin; Mammary epithelial cells; Rabbit; Biotin;
Colloidal gold; Immunocytochemistry; Electron microscopy.
In the lactating rabbit mammary gland, in addition to a huge quantity of milk transferrin synthesized in the mammary epithelial cell (Jordan et al., 1967), a small amount of Tfis derived directly from the blood plasma (Jordan and Morgan, 1970). The mode of transport of this Tf across the mammary epithelial cell is not precisely known. It should occur through a receptor-mediated transcellular transport mechanism. A transepithelial mammary cell translocation has been described for polymeric immunoglobulins(Mostov et al., 1980). Many hormones are also transported across the mammary epithelial cell (Koldowsky, 1980). This is the case for prolactin (PIU),which is internalized in the lactating mammary epithelial cell and carried across the Golgi apparatus and the secretory vesicles to be released in the milk (Seddiki and Ollivier-Bousquet, 1991). The mechanisms involved in the endocytosis and segregation of ddferent molecules into specific carrier vesicles in the mammary epithelial cell remain unknown. To study this process, the endocytosis and intracellular transit of human blood Tf were studied in lactating rabbit mammary epithelial cells. Because human Tf (hTf) has been shown to bind to receptors in mammary epithelial cells of ddferent species (Schulman et al., 1989), mammary fragments of aggregates, were incubated with hTf labeled with biotin (biohTf) or colloidal gold (AuhTf). These localizationswere compared with those of endogenous rabbit milk transferrin. 1501
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Materials and Methods Animals. New Zealand female rabbits were used on Day 15 of lactation.
SEDDW, DELPAL, OLLIMER-BOUQUET
1:600 (30 min), and washed with 0.01 PBSIBSA 1%. The sections were mounted on a drop of Moviol and observed with a Polyvar Reichert microscope equipped with a filter set to fluorescein.Control sections were treated similarly with omission of the anti-hTf antibody. On the same section, the biohTf was localized by revealing biotin with streptavidin-Texas Red and by immunochemistry.To this aim, sections from tissue fragmentsincubated in the presence of biohTf were first treated for immunocytochemicaldetection of hTf with serum anti-hTf, then treated for cytochemical detection of biohTf with streptavidin-Texas Red before they were mounted. The sections were observed under a Polyvar Reichert microscope equipped with a filter set to FITC (which was used to photograph the sections immunostained with FITC) and a filter set to rhodamine (which was used to photograph the same section stained with Texas Red). To localize rabbit milk transferrin immunocytochemically,sectionsfrom tissue fragments incubated in the presence of biohTf were first treated for immunocytochemicaldetection of rabbit milk Tf using an anti-rabbit milk Tf antiserum (1:lOOO) and a second anti-sheep IgG-FITC antibody.
Materials. Hanks medium was obtained from Gibco (Paisley, UK), and collagenase from Seromed (Berlin, Germany). Biotinylated transferrin (bioTf) was prepared with a biotinylation kit (Amersham; Amersham, UK). One mg of iron-saturated hTf (Sigma; St Louis, MO) was labeled according to the instructions supplied by Amersham. Gold-labeled hTf (AuTf)was prepared with colloidal gold (5 nm and 15 nm) from Janssen (Beerse, Belgium) according to the instructions supplied by Janssen. The amount of hTf needed to stabilize the gold solution was determined by mixing 10 ~1 of gold solution with 10 pl of various amounts of hTf diluted in Tris buffer. After 5 min, 10 pI of 10% NaCl were added and the lowest hTf concentration that prevented color change from pink to blue was taken as the saturation point. Five ml of an hTf solution (70 pg/ml), which corresponds to a 10% excess of this saturation point, were added to 25 ml of gold solution. After 30 min at 4 ° C 3 ml of 0.1% polyethylene glycol, MW 20,000, were added. Then the AuhTf complex was harvested by centrifugation (40,000 x g160 min) and washed twice with 0.5% bovine serum albumin in phosphate-buffered saline (0.5% BSAIPBS). The final pellet was resuspended in 1 ml of 0.5% BSAIPBS, stored at 4 ° C and used within one week. Anti-hTf antiserum was obtained from Amersham. Anti-rabbit milk transferrin antibody prepared as described by Jahn et al. (1989) was a generous gift from these authors. Anti-goat IgG-fluorescein isothiocyanate (IgG-FITC) and anti-sheep IgG-FITC were obtained from Biosys (Compiegne, France), streptavidin-Texas Red from Amersham.
Detection of AuhTF. Aggregates of epithelial cells separated by collagenase wete incubated at 4'C in Hanks medium in the presence of 100 pl of PBS/BSA containing AuhTf for 30 min, then washed and incubated again at 37'C for 5, 15, or 30 min. To slow down the transport of AuhTf and to increase the label, some dissociated aggregateswete directly incubated at 25°C in the presence of AuhTf, in a continuous uptake experiment for 15 min. At the end of incubation, aggregateswere fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 1% os04 in the same buffer, then dehydrated and embedded in Epon.
Incubations. Each experiment was carried out with mammary fragments from one animal. The fragments were dissected to eliminate connective and adipose tissue and cut by hand with a razor blade (weight of each fragment 0.1-0.2 mg). Detection of bioTf was repeated with three animals. Detection of AuTf was repeated with five animals. Fragments were incubated in Hank's medium, pH 7.4, 37'C, atmosphere 95% 0 2 + 5% C02, containing Na bicarbonate (2.2 glliter). In some experiments epithelial cell aggregates were obtained by dissociation with collagenase (140UI/ml) at 37'C in Hanks medium for 90 min.
Immunoelectron Microscopy. Tissue fragments were fixed with 2% paraformaldehyde, dehydrated with ethanol, and embedded in Lowicryl G M at a low temperature (Armbruster et al., 1982). The sections were collected on gold grids, incubated with 50 mM NH&I and 0.2% gelatin, then immunolabeled (1 hr) with an anti-rabbitmilk Tantiserum (1:1000),washed with 0.01 M PBS containing 0.2%gelatin, incubated 1 hr with a biotinylated anti-sheep IgG (Amersham) 1:200in 0.01 M PBS containing 0.2%gelatin, washed in the same buffer, and incubated for 30 min with streptavidin-gold (15 nm) 1:200 (Amersham).
Cytochemical Detection of biox. After 15 min of pre-incubation in Hanks medium at 37'C, tissue fragments were incubated in the same medium at 4'C for 30 min in the presence of 100 pllml of PBS containing biohTf (corresponding to approximately 100 pglml of biohTf). After extensive washing, tissue fragments were either fixed with 2 % paraformaldehyde in 0.1 M cacodylate buffer or incubated for 5 , 15, 30, or 60 min at 37"C, then fixed with 2% paraformaldehyde(4 h) in the same buffer. Fixed tissues were infiltrated in 10% sucrose in PBS (17 h), frozen in liquid N2, and sectioned in 2-pm thick sections at -3S'C with a Reichert Cryocut (Leica; Rued Malmaison, France). The sections were collected on poly-^lysine-coated glass coverslips and sequentially incubated with SO mM NNCI (45 min), 1% BSA. streptavidin-Texas Red 1:300 in 0.01 M PBS containing 1% BSA (30 min), and washed with 0.01 M PBS, pH 8. The sections were mounted on a drop of glycerol and observed under a Polyvar Reichert microscopeequipped with a filter set to rhodamine. Control sections from fragments of tissue incubated in Hank's medium alone were similarly treated in parallel. ImmunofluorescentDetection of hTf and Rabbit Milk Transferrin. After 15 min of pre-incubation in Hanks medium at 37°C tissue fragments were incubated in the same medium at 4'C for 30 min in the presence of 2.5 mglml of iron-saturated hTf. After extensive washing, tissue fragments were either fixed with 2% paraformaldehyde in 0.1 M cacodylate buffer or incubated for 60 min at 37'C, then fixed in 2% paraformddehyde in the same buffer. Fixed tissue fragments were sectioned and treated as previously described until the 50 mM N&CI treatment, then incubated with horse serum in PBSIBSA 1% (13; vlv) (1 hr), anti-hTfantibody 1:lOOO in PBSlBSA 1% (30 min), then with a second antibody anti-goat IgG-FIK
Results Endocytosis of biohTf To examine the intracellular traffic of transferrin in the lactating mammary epithelial cell, mammary fragments were fixed after incubation with biohTf for various periods of time. Then biohTf was detected cytochemically. To demonstrate that biohTf conjugates used in our experiments bind to TfR, we carried out a series of 4% binding experiments in the presence or absence of native hTf. Fragments incubated at 4°C in Hank's medium for 30 min in the presence of 2 mg/ml of native hTf and of biohTf, fixed and treated for cytochemical detection of biohTf, did not show any labeling at the base or inside the cells (Figure la). In the absence of native hTf, label accumulated mainly at the base of the cell (Figure 1b). When mammary fragments were incubated at 4°C for 30 min, then at 37°C for 5 min, a fluorescent label was always seen on the basal membrane. Some fluorescence was also located throughout and at the apex of the cell, with slight labeling in the lumen (Figure IC).After 15 min at 37"C, bright fluorescence was seen throughout the whole cell, inside the cytoplasm, at the apex, and sometimes associated with secretory products within the lumen (Figure
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Figure 1. Cytochemical localization of biohTf in lactating rabbit mammary cells. Fragments were incubated with biohTf for 30 min at 4OC, washed, and incubated at 3PC for various periods of time before fixation. Cryosections were stained with streptavidin-Texas Red. L, lumen. (a) After 30 min at 4%. in the presence of 2' mglml of native hTf. no label is detectable. (b)After 30 min at 4OC. cells show marked labeling of the basal membrane (arrows). (c) After 30 min at 4OC and 5 min at 37%. label is detectable on the basal (large arrow) and apical membranes (arrowheads), in the cytoplasm, and on secretory products inside the lumen (small arrow). (d) After 30 min at 4% and 15 min at 3PC, cells show only faint labeling of the basal membranes (large arrows) but the cytoplasm is strongly labeled. Many fluorescent spots are located on the apical membranes (arrowheads) and on secretory products in the lumen (small arrow). (e) After 30 min at 4% and 60 min at 37% many fluorescent spots accumulate on the apical membrane (arrowheads) and on secretory products in the lumen (small arrow). Few fluorescent spots are located on the basal membrane (large arrow). Bar = 10 pm.
I
Id). After 60 min at 37°C. marked accumulation of fluorescent label was observed at the apex of the cell (Figure le). To ensure that the biotin remained associated with hTf during endocytosis, biotin was detected with streptavidin-Texas Red and. on the same section, hTf was detected by immunofluorescence with an antiserum to hTf and a second antibody anti-goat IgG-FIX. When mammary fragments were incubated at 4°C for 30 min. the label revealed with streptavidin-Texas Red was located at the base of the cell (Figure 2a). On the same section, the label revealed with F I X was also located on the basal region (Figure 2b). After incubation at 4°C for 30 min. then at 37°C for 30 min. labels revealed with streptavidin-Texas Red and FITC were located inside and at the apex of the cell (Figures 2c and 2d). The two labels did not give exactly the same picture but they shifted similarly between 4°C and 37°C to the cell apex. These results suggest that most of the detectable biotin remains associated with human Tf.
Endocytosis of Native
rf
To see if biotin modifies the time course of hTf transit, mammary
gland fragments were incubated with hTf at 4°C for 30 min and subsequently at 37'C for 60 min. hTfwas detected by immunofluorescence with an antiserum anti-hTf as before. When mammary fragments were incubated at 4'C for 30 min., a strong fluorescent label was detectable at the basis of the cell (Figure 3a). After washing and incubation at 37°C for 60 min. the base of the cell was no longer labeled. Fluorescent label was detectable inside the cell. at the apex, and accumulated in the secretory products (Figure 3b). This result shows that the intracellular transit of both native hTf and biohTf follows the same time course. Control sections from tissue fragment not previously incubated and similarly treated did not show any reaction. The anti-hTf antiserum did not recognize rabbit milk Tf. Results of both types of labeling experiment indicated that exogenous hTf is transported across the mammary cell and released in the lumen.
Localization of Rabbit Milh Transferrin Rabbit mammary epithelial cells synthesize a large quantity of Tf
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Figure 2. (a$) Cytochemical localization of biohTf and (b,d)immunocytochemical localization of hTf in lactating rabbit mammary cells. (a) After 30 min at 4OC. biohTf was detected on the basal membrane (arrows). (b)The same section stained with serum anti hTf shows a similar marked labeling of the basal membrane (arrows). (c) After 30 min at 4% then 30 min at 3PC. the biohTf was located throughout and at the apex of the cells (arrowheads) and on secretory products inside the lumen (arrow). (d) The same section stained with serum anti-hTf shows a more diffuse label. However, the label is located inside and at the apex Of the Cells (arrowheads) and on secretory products inside the lumen (arrow). Bar = 10 pm.
secreted in the milk (rabbit milk Tf) by a pathway that has not yet been well established. For this reason, we asked whether the last steps of transcytosis of exogenous hTf are common with the exocytosis of milk transferrin. Mammary fragments were incubated at 4°C for 30 min with biohTf, washed, and incubated at 37°C for 60 min. Cytochemical analysis of biohTf and rabbit milk transferrin was carried out on the same section. After incubation at 37°C for 5 min. biohTf was located mainly at the base of the cell as before (Figure 4a). Rabbit milk Tf was detected at the apex of the cell and associated with secretory products in the lumen (Figure 4b). After incubation at 37°C for 60 min, biohTfwas accumulated at the apex of the cell (Figure 4c). Rabbit milk Tf did not appear to co-localize with the apical accumulation of biohTf (Figure 4d), suggesting that the last steps of the exocytotic pathway of biohTf and milk rabbit Tf are not necessarily the same. Immunocytochemical localization of milk rabbit Tf on sections of lactating rabbit mammary gland embedded in Lowicryl revealed that milk rabbit
Tf was located inside secretory vesicles containing casein micelles (Figure 4e). This result precisely localizes the exocytotic pathway for milk rabbit Tf and shows that two milk proteins are carried by the same secretory vesicles.
Endocytosis of AubTJf The transit pathway of hTf across the cell was followed directly with aggregates exposed to AuhTf at 4°C for 30 min and warmed to 37°C for 5 and 30 min. At 4°C the periphery of the cell, coated pits, and endosome-like structures were labeled (Figures 5a-5c). After 5 min at 37"C, the basal membrane remained labeled. Gold particles were detectable in the endosome-like structures, microvesicles, and saccules of the Golgi region (Figure 5c). Moreover, AuhTf was located on the apical membrane and inside the lumen (Figure 5d). After 30 min at 37'C, very little AuhTfremained on the basal membrane, inside the endosomes, and in intracellular vesicles. AuhTf
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brane was labeled after 2 min at 25°C (Figure 6a). After 2 min. AuTf was located in small vesicles close the the apex, on the apical membrane, and in the lumen. Labeled vesicles close to the apex were coated (Figure 6b). In this case it ¬ possible to determine if AuhTf is in the process of exocytosis or is in the process of reendocytosis of material delivered to the cell surface which re-enters the cell. After 15 min, label accumulated in the lumen (Figure 6c). As at 37'C, AuTf was never localized inside the secretory vesicles containing casein micelles.
Discussion
Figure 3. Immunocytochemicallocalizationof hTf in lactating rabbit mammary cells. Fragments were incubated with native hTf. (a) After 30 min at 4% cells show marked labeling of the basal membrane (large arrows) and a few fluorescent spots inside the cytoplasm (small arrow). (b) After 30 min at 4OC, washing, and incubation for 60 min at 3pC.many fluorescent intracellular spots are detectable (small arrow). Apical membrane and secretory products in the lumen are strongly labeled. Very little label remains on the basal membrane (large arrow). Bar = 10 urn.
was never detected inside the vesicles containing casein micelles. The apical membrane and content of the lumen were strongly labeled. Under these experimental conditions, the hTf intracellular transit seemed to be very fast. Consequently, it is difficult to do quantitative analysis of the localization of the gold particles. With intent to slow down the endocytosis and to make the label more dense, aggregates of mammary epithelial cells were incubated at 25°C for 2 and 15 min in a continuous uptake experiment. The basal mem-
These results show, first, that hTfis carried across the lactating mammary cells by a transcytotic pathway and. second, that rabbit milk transferrin is secreted, mainly in the apical direction, via the secretory vesicles. The transcytosisof hTfwas c o n f i i e d by two different experhental modalities. BiohTf and native hTf, immunocytochemically detected, moved similarly from the basal to the apical pole of the cell. Differences concerning the aspect of the label (spots in the case of the biohTf and more diffuse fluorescence in the case of the native hTf) could result from the two systems of detection used. However, biohTf remains immunoreactive and appears a useful tool to study endocytosis. The transferrin biotin conjugate could be used for ultrastructural double label cytochemistry. The recycling of Tf during TfR-mediated endocytosis and iron delivery to the cells occurs within 1-8 min, depending on the cell type (Bali et al., 1991; Dautry-Varsat et al.. 1983). In the lactating mammary epithelial cell, Tf appears very quickly ( 5 min) in the lumen of the acini. However, with the approach used it is not possible to determine whether a part of biohTf if recycled to the basal membrane. The appearance of a strong label on the apical membrane and in the lumen suggests that a very fast polarized intracellular transport of hTf occurs. Receptor-mediated transcytosis of Tf has been described across hepatic endothelial cells (Kishimoto and Tavassoli, 1987). Transport of polymeric immunoglobulins across the mammary epithelium is mediated by a receptor component that serves as an Ig receptor at the basolateral surface of the cell and which is released in the lumen in association with Ig (Mostov et al., 1980). However, transcytosis of the poly Ig receptor from basolateral to apical membrane in the presence or absence of its ligand, described in transfected immortalized mammary cells, lasts half an hour (Schaerer et al., 1990). This difference in the velocity of transcytosis can be easily explained by the difference in the degree of polarization of these cells. This very quick transit of Tf across lactating mammary epithelial cells is different from the time course of newly synthesized caseins which are released in the lumen 15 to 25 min after a 3-min pulse (Ollivier-Bousquet and Denamur, 1975). These differences in the time of transit and the observation that hTf does not co-localize exactly with endogenous milk Tf and casein micelles contained in secretory vesicles suggest that the transcytotic pathway of exogenous hTf is different from the secretory pathway of milk proteins. Although it was not possible to describe precisely the organelles implicated in the hTf transport, the presence of AuhTf in vesicles and in the Golgi saccules suggests that hTf transits via the Golgi apparatus. In that case, this pathway would be in keeping with the
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Figure 4. Double localization of (a$) biohTf and (b,d,e) rabbit milk Tf on the same sections of lactating rabbit mammary tissue. Fragments were incubated with biohTf for 30 min at 4OC (a,b), washed, then incubated at 37% for 60 min (c,d). (a) After 30 min at 4OC, biohTf detected with streptavidin-Texas Red was mainly located on the basal membrane (large arrows). Apices of the cells are not labeled (arrowheads). (b) On the same section, rabbit milk Tf detected with ITCF was located at the apex of the cell (arrowheads) and on secretory products inside the lumen (small arrow). (c) After 30 min at 4OC, then 60 min at 37% biohTf was accumulated on the apical membrane (arrowheads). (d) On the same section, immunofluorescent localization of rabbit milk Tf revealed that endogenous Tf is not accumulated in the same apical region of the cell (compare apical membrane with arrowheads in c and d). (e) Electron microscopic demonstration of rabbit milk Tf in epithelial mammary cell. Thin sections of tissue embedded in Lowicryl were incubated with anti-rabbit milk Tf antiserum, biotinylated anti-sheep IgG, and streptavidin-gold (15 nm) as described in Materials and Methods. Rabbit milk Tf is detectable in Golgi vesicles and saccules (G) and in secretory vesicles (SV) containing casein micelles (c). Bars: a-d = 10 wm; e = 0.5 pm.
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Figure 5. Localization of AuhTf in dissociated lactating rabbit mammary epithelial cells, incubated at 4OC for 30 min in the presence of AuhTf, washed, then incubated at 37% for various times. After 30 min at 4OC. gold particles are located on the plasma membrane (a), inside a coated pit probably in the process of invaginating in a coated vesicle (b). and inside endosome-like structures (c). After 30 min at 4°C. then 5 min at 37% AuhTf is found inside Golgi saccules (d) and in the lumen (L) (e). Bars = 0.5 bm.
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SEDDIKI. DELPAL, OLLIVIER-BOUSQUET
1-7-
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.:
Cr.
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TRANSFERRIN TRANSPORT AND MAMMARY EPITHELIAL CELLS
-
casein micelle
e
Tf
*milk Tf
Most of the prolactin is not detected in the lumen of the acini before 15-25 min. Moreover, low temperature (25°C)slows down this process and does not allow prolactin to enter the lumen (Seddiki and Ollivier-Bousquet 1991), whereas under the same conditions Tf transcytosis is not inhibited. These results raise the question of the mechanism that controls the vesicular traffic along the exocytic and transcytotic pathways. Association of different proteins with each class of vesicles could be responsible for the sorting. Specific proteins characteristic of transcytotic vesicles have been described in hepatocytes (Sztul et al., 1991). The role of the transported molecules in the identification of the vesicular carrier remains unclear. Our results suggest that in mammary epithelial cells different ligands, such as hTf and PRL, could be carried by different pathways.These different ligands might be useful tools for the study of the complex intracellular transport across the mammary epithelial cell.
Acknowledgments
/ - r b
I
Tf-R
milk protein
We are very gratefulto Prcfissor M. C. Neville andto Professor H.Chuser for their helpfirladvice andcomments on the manuscript, and to J. Brug nolo, R. Scandolo, andE k r t for their contribution in preparing manuscn& and micrographs.
synthesis Literature Cited
Figure 7. Model for transcytosis of hTf across the mammary epithelial cells. We propose that after internalization Tf is carried to the Golgi complex and is transferred into transcytotic vesicles of the apical side in a few minutes. Fe3* could dissociate during this transport in endosomes at the requiredacidic environment and the TfR could recycle to the cell surface. In contrast, caseins and rabbit milk Tf, newly synthesized in the rough endoplasmic reticulum (RER) are transported across the Golgi apparatus (G) and sequestered in the same secretory vesicles (SV), then released into the lumen after 15-30 min.
accessibility of the trms-Golgi network by extracellular added Tf (Hedman et al., 1987). A connection between endocytosed Tf and secretory proteins in the trans-Golgi reticulum has been described in a human hepatoma cell line HepGz (Stoorvogel et al., 1988). However, we were not able to detect hTf in the secretory vesicles containing casein micelles. This result suggests that a putative pathway following the sorting in the trms-Golgi network could be distinct for hTf and secretory proteins. Since rabbit milk Tf, a protein synthesized inside the epithelial cells, co-localizes with casein micelles inside secretory vesicles, we can suggest the following working hypothesis, illustrated in figure 7. Caseins and rabbit milk Tf newly synthesized in the rough endoplasmic reticulum are transported across the Golgi apparatus and sequestered in the same secretory vesicles before being released into the lumen. Conversely, Tf after endocytosis could be carried in the Golgi apparatus where sorting to a transcytotic vesicular compartment occurs. We have shown previously that PRL is carried across the Golgi apparatus and within secretory vesicles containing casein micelles.
Armbruster BL, Carleman E, ChiovcttiR, Garavito RM, Hobot JA. Kellenberger E, Williger W (1982): Specimen preparation for electron microscopy using low temperature embedding resins. J Microsc 126:77 Bali PK, Zak 0, Aisen P (1991): A new role for the transferrin receptor in the release of iron from transferrin. Biochemistry 30:324 Dautry-Varsat A, Ciechanover A, Lodish HF (1983): pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci USA 80:2258 Grigor MR, Wilde CJ, Flint DJ (1988): Transferrin receptor activity in rat mammary epithelial cells. Biochem Int 17:747 Hedman K, Goldenthal KL, Rutherford, AV, Pastan I, Willingham MC (1987): Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry.J Histochem Cytochem 35:233
Jahn GA, Djiane J, Houdebine LM (1989): Inhibition of casein synthesis by progestins in vitro: modulation in relation to concentration of hormones that synergize with prolactin. J Steroid Biochem 32:373 Jordan SM,Kaldor I, Morgan EH (1967): Milk and serum iron and ironbinding capacity in the rabbit. Nature 215:76 Jordan SM,Morgan EH (1970): Plasma protein metabolism during lactadon in the rabbit. Am J Physiol 219:1549 Kishimoto T, Evassoli M (1987): Transendothelial transport (transcytosis) or iron-transferrin complex in the liver. Am J Anat 178:241 Klausner RD, Ashwell G, Van ReswoudeJ, HarfordJB, Bridges KR (1983): Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci USA 80:2263 Koldovsky MD (1980): Hormones in milk. Lifc Sci 26:1833 Lee EYH, Barcellos-Hoff MH. Chen LH,Parry G, Bise11MJ (1987): Trans-
Figure 6. Localization of AuhTf in dissociated lactating rabbit mammary epithelial cells incubated at 25OC. (a) After 2 min at 25OC AuhTf is found on the basal membrane (BM) of the mammary epithelial cell. A fibroblast (F) close to the epithelial cell is also labeled. (b) AuhTf is located in a transcytotic vesicle close to the apical membrane and also on the apical membrane (AM). (c) After 15 min at 25OC, AuhTf accumulates in the lumen. Secretory vesicles containing casein micelles are not labeled. Bars = 0.5 um.
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ferrin is a major mouse milk protein and is synthesized by mammary epithelial cells. In Vitro Cell Dev Biol 23:221 Mostov KE, Kraehenbuhl JP, Blobel G (1980): Receptor-mediated transcellular transport of immunoglobulin: synthesis of secretory component as multiple and large transmembraneforms. Proc Natl Acad Sci USA 77:7257 Ollivicr-Bousquct M, Denamur R (1975): Effct de Etat physiologique et du 3’5’ adenosine monophosphate cyclique sur le transit inuacellulairc et l’cxcrttion des proteines du lait. Etude autoradiographique en micronopie Slectronique. J Microsc Biol Cell 23:63 Schaerer E, Vcrrey F, Racine L, Tallichet C, Reinhardt M, Kraehenbuhl JP (1990: Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells. J Cell Biol 110:987 Schulman HM, Ponka P, Wilczynska A, Gauthier Y,Shyamala G (1989): Transferrinreceptor and ferritin levels during murine mammary gland development. Biochim Biophys Acta 101O:l
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Sed& T,Ollivier-Bousquet M (1991): Temperature dependenceof prolactin endocytosis and casein exocytosis in epithelial mammary cells. Eur J Cell Biol 55:60 Sigman M, L6nnerdal B (1990): Relationship of milk iron and the changing concentration of mammary tissue transferrin receptors during the c o w of lactation. J Nutr Biochcm 1:572 Stoorvogel W, Gcuze HJ, Griffith JM, Strous GJ (1988): The pathway of endocytosed transferrin and secretory protein are connected in the transGolgi reticulum. J Cell Biol 106:1821 Sztul E, Kaplin A, Saucan L, Palade G (1991): Protein traffic between distinct plasma membrane domains: isolation and characterizationof vesicular carriers involved in transcytosis. Cell 64:81 kvydas H, Jordan SM, Morgan EH (1968): Iron metabolism during lactation in the rabbit. Br J Nutr 22:565
Vol. 40, No. 10, pp. 1501-1510, 1992 Printed in KKA.
The Joumal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.
Original Ar tide
I Endocytosis and Intracellular Transport of Transferrin Across the Lactating Rabbit Mammary Epithelial Cell TOURIA SEDDIKI, SERGE DELPAL, and MICHELE OLLIVIER-BOUSQUET' Laboratoire de Biologie Cellulaire et Molthhire, INRA (TS,MOB), and Laboratoire de Nutrition el SicuritZ Alimentaire, INRA, (SO) 78312 Jouy-enjosas, France. Received for publication November 13, 1991 and in revised form March 30, 1992; accepted May 4. 1992 (lA2504).
To study the transcytosis and segregation of ligand in the transit mammary epithelial cell, endocytosisand intracellula~ of human blood transferrin were followed in lactating rabbit mammary epithelial cells. Human transferrin labeled with biotin added to an incubation medium was bound to the basal membrane of mammary epithelial cells and carried aaoss the cell to the lumen of the acini within 5-60 min. At the same time, biotinylated human transferrin accumulated at the apex of the cell. After incubation with human transferrin labeled with colloidal gold, label was detected inside endosome-like structures, vesicles and saccules of the Golgi apparatus, and inside the lumen within 2-5 min. A
Introduction Endocytosis of iron-loaded transferrin (Tf) and of the transferrin receptor (TfR) occurs in many mammalian cells. After internalization, Tf releases its iron intracellularly at an acidic pH, and the Tf-TfR complex recycles to the plasma membrane where TfR and Tf dissociate (Dautry-Varsat et al., 1983; Klausner et al., 1983). The TfR has been well characterizedin the rat and mouse mammary epithelial cells. A significant increase in TfR occurs during mammary growth and the TfR level remains high during lactation in the rat (Grigor et al., 1988)and mouse (Schulman et al., 1989). The role of this high level of TfR in the lactating epithelial mammary cell remains unclear. The epithelial cell ferritin level decreases during lactation in the mouse, suggesting that there is no increased storage of iron (Schulman et al., 1989). Moreover, milk iron concentration decreases during lactation in the rat (Sigman and b n nerdal, 1990) and is very low in the rabbit (ref. in Tarvydas et al., 1968).The high level of TfR in the mammary epithelial cell could be associated not only with milk iron transport by Tf, but also to the transport of Tf itself. Tf has been implicated in differentiation and growth of various tissues (ref. in Lee et al., 1987). Correspondence to: M. Ollivier-Bousquet, Laboratoire de Biologie Cellulaire et Moliculaire, INRA, 78352 Jouy-en-Josas Cidex, France.
significant label accumulated at the apex of the cell after 30-60 min. Biotin labeling did not modify the time of transit of human transferrin, as attested by comparison with the time of transit of native transferrin. Human transferrin was never detected inside vesicles containing casein micelles. In contrast, rabbit milk transferrin was immunocytochemically detected inside vesicles containing casein micelles. These results indicate that transcytosis of human transferrin follows a pathway different from vesicles that carry casein micelles. (1Hisrochem C y r d e m 40:1501-1510, 1992) KEY WORDS:Transferrin; Mammary epithelial cells; Rabbit; Biotin;
Colloidal gold; Immunocytochemistry; Electron microscopy.
In the lactating rabbit mammary gland, in addition to a huge quantity of milk transferrin synthesized in the mammary epithelial cell (Jordan et al., 1967), a small amount of Tfis derived directly from the blood plasma (Jordan and Morgan, 1970). The mode of transport of this Tf across the mammary epithelial cell is not precisely known. It should occur through a receptor-mediated transcellular transport mechanism. A transepithelial mammary cell translocation has been described for polymeric immunoglobulins(Mostov et al., 1980). Many hormones are also transported across the mammary epithelial cell (Koldowsky, 1980). This is the case for prolactin (PIU),which is internalized in the lactating mammary epithelial cell and carried across the Golgi apparatus and the secretory vesicles to be released in the milk (Seddiki and Ollivier-Bousquet, 1991). The mechanisms involved in the endocytosis and segregation of ddferent molecules into specific carrier vesicles in the mammary epithelial cell remain unknown. To study this process, the endocytosis and intracellular transit of human blood Tf were studied in lactating rabbit mammary epithelial cells. Because human Tf (hTf) has been shown to bind to receptors in mammary epithelial cells of ddferent species (Schulman et al., 1989), mammary fragments of aggregates, were incubated with hTf labeled with biotin (biohTf) or colloidal gold (AuhTf). These localizationswere compared with those of endogenous rabbit milk transferrin. 1501
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Materials and Methods Animals. New Zealand female rabbits were used on Day 15 of lactation.
SEDDW, DELPAL, OLLIMER-BOUQUET
1:600 (30 min), and washed with 0.01 PBSIBSA 1%. The sections were mounted on a drop of Moviol and observed with a Polyvar Reichert microscope equipped with a filter set to fluorescein.Control sections were treated similarly with omission of the anti-hTf antibody. On the same section, the biohTf was localized by revealing biotin with streptavidin-Texas Red and by immunochemistry.To this aim, sections from tissue fragmentsincubated in the presence of biohTf were first treated for immunocytochemicaldetection of hTf with serum anti-hTf, then treated for cytochemical detection of biohTf with streptavidin-Texas Red before they were mounted. The sections were observed under a Polyvar Reichert microscope equipped with a filter set to FITC (which was used to photograph the sections immunostained with FITC) and a filter set to rhodamine (which was used to photograph the same section stained with Texas Red). To localize rabbit milk transferrin immunocytochemically,sectionsfrom tissue fragments incubated in the presence of biohTf were first treated for immunocytochemicaldetection of rabbit milk Tf using an anti-rabbit milk Tf antiserum (1:lOOO) and a second anti-sheep IgG-FITC antibody.
Materials. Hanks medium was obtained from Gibco (Paisley, UK), and collagenase from Seromed (Berlin, Germany). Biotinylated transferrin (bioTf) was prepared with a biotinylation kit (Amersham; Amersham, UK). One mg of iron-saturated hTf (Sigma; St Louis, MO) was labeled according to the instructions supplied by Amersham. Gold-labeled hTf (AuTf)was prepared with colloidal gold (5 nm and 15 nm) from Janssen (Beerse, Belgium) according to the instructions supplied by Janssen. The amount of hTf needed to stabilize the gold solution was determined by mixing 10 ~1 of gold solution with 10 pl of various amounts of hTf diluted in Tris buffer. After 5 min, 10 pI of 10% NaCl were added and the lowest hTf concentration that prevented color change from pink to blue was taken as the saturation point. Five ml of an hTf solution (70 pg/ml), which corresponds to a 10% excess of this saturation point, were added to 25 ml of gold solution. After 30 min at 4 ° C 3 ml of 0.1% polyethylene glycol, MW 20,000, were added. Then the AuhTf complex was harvested by centrifugation (40,000 x g160 min) and washed twice with 0.5% bovine serum albumin in phosphate-buffered saline (0.5% BSAIPBS). The final pellet was resuspended in 1 ml of 0.5% BSAIPBS, stored at 4 ° C and used within one week. Anti-hTf antiserum was obtained from Amersham. Anti-rabbit milk transferrin antibody prepared as described by Jahn et al. (1989) was a generous gift from these authors. Anti-goat IgG-fluorescein isothiocyanate (IgG-FITC) and anti-sheep IgG-FITC were obtained from Biosys (Compiegne, France), streptavidin-Texas Red from Amersham.
Detection of AuhTF. Aggregates of epithelial cells separated by collagenase wete incubated at 4'C in Hanks medium in the presence of 100 pl of PBS/BSA containing AuhTf for 30 min, then washed and incubated again at 37'C for 5, 15, or 30 min. To slow down the transport of AuhTf and to increase the label, some dissociated aggregateswete directly incubated at 25°C in the presence of AuhTf, in a continuous uptake experiment for 15 min. At the end of incubation, aggregateswere fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 1% os04 in the same buffer, then dehydrated and embedded in Epon.
Incubations. Each experiment was carried out with mammary fragments from one animal. The fragments were dissected to eliminate connective and adipose tissue and cut by hand with a razor blade (weight of each fragment 0.1-0.2 mg). Detection of bioTf was repeated with three animals. Detection of AuTf was repeated with five animals. Fragments were incubated in Hank's medium, pH 7.4, 37'C, atmosphere 95% 0 2 + 5% C02, containing Na bicarbonate (2.2 glliter). In some experiments epithelial cell aggregates were obtained by dissociation with collagenase (140UI/ml) at 37'C in Hanks medium for 90 min.
Immunoelectron Microscopy. Tissue fragments were fixed with 2% paraformaldehyde, dehydrated with ethanol, and embedded in Lowicryl G M at a low temperature (Armbruster et al., 1982). The sections were collected on gold grids, incubated with 50 mM NH&I and 0.2% gelatin, then immunolabeled (1 hr) with an anti-rabbitmilk Tantiserum (1:1000),washed with 0.01 M PBS containing 0.2%gelatin, incubated 1 hr with a biotinylated anti-sheep IgG (Amersham) 1:200in 0.01 M PBS containing 0.2%gelatin, washed in the same buffer, and incubated for 30 min with streptavidin-gold (15 nm) 1:200 (Amersham).
Cytochemical Detection of biox. After 15 min of pre-incubation in Hanks medium at 37'C, tissue fragments were incubated in the same medium at 4'C for 30 min in the presence of 100 pllml of PBS containing biohTf (corresponding to approximately 100 pglml of biohTf). After extensive washing, tissue fragments were either fixed with 2 % paraformaldehyde in 0.1 M cacodylate buffer or incubated for 5 , 15, 30, or 60 min at 37"C, then fixed with 2% paraformaldehyde(4 h) in the same buffer. Fixed tissues were infiltrated in 10% sucrose in PBS (17 h), frozen in liquid N2, and sectioned in 2-pm thick sections at -3S'C with a Reichert Cryocut (Leica; Rued Malmaison, France). The sections were collected on poly-^lysine-coated glass coverslips and sequentially incubated with SO mM NNCI (45 min), 1% BSA. streptavidin-Texas Red 1:300 in 0.01 M PBS containing 1% BSA (30 min), and washed with 0.01 M PBS, pH 8. The sections were mounted on a drop of glycerol and observed under a Polyvar Reichert microscopeequipped with a filter set to rhodamine. Control sections from fragments of tissue incubated in Hank's medium alone were similarly treated in parallel. ImmunofluorescentDetection of hTf and Rabbit Milk Transferrin. After 15 min of pre-incubation in Hanks medium at 37°C tissue fragments were incubated in the same medium at 4'C for 30 min in the presence of 2.5 mglml of iron-saturated hTf. After extensive washing, tissue fragments were either fixed with 2% paraformaldehyde in 0.1 M cacodylate buffer or incubated for 60 min at 37'C, then fixed in 2% paraformddehyde in the same buffer. Fixed tissue fragments were sectioned and treated as previously described until the 50 mM N&CI treatment, then incubated with horse serum in PBSIBSA 1% (13; vlv) (1 hr), anti-hTfantibody 1:lOOO in PBSlBSA 1% (30 min), then with a second antibody anti-goat IgG-FIK
Results Endocytosis of biohTf To examine the intracellular traffic of transferrin in the lactating mammary epithelial cell, mammary fragments were fixed after incubation with biohTf for various periods of time. Then biohTf was detected cytochemically. To demonstrate that biohTf conjugates used in our experiments bind to TfR, we carried out a series of 4% binding experiments in the presence or absence of native hTf. Fragments incubated at 4°C in Hank's medium for 30 min in the presence of 2 mg/ml of native hTf and of biohTf, fixed and treated for cytochemical detection of biohTf, did not show any labeling at the base or inside the cells (Figure la). In the absence of native hTf, label accumulated mainly at the base of the cell (Figure 1b). When mammary fragments were incubated at 4°C for 30 min, then at 37°C for 5 min, a fluorescent label was always seen on the basal membrane. Some fluorescence was also located throughout and at the apex of the cell, with slight labeling in the lumen (Figure IC).After 15 min at 37"C, bright fluorescence was seen throughout the whole cell, inside the cytoplasm, at the apex, and sometimes associated with secretory products within the lumen (Figure
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TRANSFERRIN TRANSPORT AND MAMMARY EPITHELIAL CELLS
Figure 1. Cytochemical localization of biohTf in lactating rabbit mammary cells. Fragments were incubated with biohTf for 30 min at 4OC, washed, and incubated at 3PC for various periods of time before fixation. Cryosections were stained with streptavidin-Texas Red. L, lumen. (a) After 30 min at 4%. in the presence of 2' mglml of native hTf. no label is detectable. (b)After 30 min at 4OC. cells show marked labeling of the basal membrane (arrows). (c) After 30 min at 4OC and 5 min at 37%. label is detectable on the basal (large arrow) and apical membranes (arrowheads), in the cytoplasm, and on secretory products inside the lumen (small arrow). (d) After 30 min at 4% and 15 min at 3PC, cells show only faint labeling of the basal membranes (large arrows) but the cytoplasm is strongly labeled. Many fluorescent spots are located on the apical membranes (arrowheads) and on secretory products in the lumen (small arrow). (e) After 30 min at 4% and 60 min at 37% many fluorescent spots accumulate on the apical membrane (arrowheads) and on secretory products in the lumen (small arrow). Few fluorescent spots are located on the basal membrane (large arrow). Bar = 10 pm.
I
Id). After 60 min at 37°C. marked accumulation of fluorescent label was observed at the apex of the cell (Figure le). To ensure that the biotin remained associated with hTf during endocytosis, biotin was detected with streptavidin-Texas Red and. on the same section, hTf was detected by immunofluorescence with an antiserum to hTf and a second antibody anti-goat IgG-FIX. When mammary fragments were incubated at 4°C for 30 min. the label revealed with streptavidin-Texas Red was located at the base of the cell (Figure 2a). On the same section, the label revealed with F I X was also located on the basal region (Figure 2b). After incubation at 4°C for 30 min. then at 37°C for 30 min. labels revealed with streptavidin-Texas Red and FITC were located inside and at the apex of the cell (Figures 2c and 2d). The two labels did not give exactly the same picture but they shifted similarly between 4°C and 37°C to the cell apex. These results suggest that most of the detectable biotin remains associated with human Tf.
Endocytosis of Native
rf
To see if biotin modifies the time course of hTf transit, mammary
gland fragments were incubated with hTf at 4°C for 30 min and subsequently at 37'C for 60 min. hTfwas detected by immunofluorescence with an antiserum anti-hTf as before. When mammary fragments were incubated at 4'C for 30 min., a strong fluorescent label was detectable at the basis of the cell (Figure 3a). After washing and incubation at 37°C for 60 min. the base of the cell was no longer labeled. Fluorescent label was detectable inside the cell. at the apex, and accumulated in the secretory products (Figure 3b). This result shows that the intracellular transit of both native hTf and biohTf follows the same time course. Control sections from tissue fragment not previously incubated and similarly treated did not show any reaction. The anti-hTf antiserum did not recognize rabbit milk Tf. Results of both types of labeling experiment indicated that exogenous hTf is transported across the mammary cell and released in the lumen.
Localization of Rabbit Milh Transferrin Rabbit mammary epithelial cells synthesize a large quantity of Tf
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SEDDIKI, DELPAL, OLLMER-BOUSQUET
I
Figure 2. (a$) Cytochemical localization of biohTf and (b,d)immunocytochemical localization of hTf in lactating rabbit mammary cells. (a) After 30 min at 4OC. biohTf was detected on the basal membrane (arrows). (b)The same section stained with serum anti hTf shows a similar marked labeling of the basal membrane (arrows). (c) After 30 min at 4% then 30 min at 3PC. the biohTf was located throughout and at the apex of the cells (arrowheads) and on secretory products inside the lumen (arrow). (d) The same section stained with serum anti-hTf shows a more diffuse label. However, the label is located inside and at the apex Of the Cells (arrowheads) and on secretory products inside the lumen (arrow). Bar = 10 pm.
secreted in the milk (rabbit milk Tf) by a pathway that has not yet been well established. For this reason, we asked whether the last steps of transcytosis of exogenous hTf are common with the exocytosis of milk transferrin. Mammary fragments were incubated at 4°C for 30 min with biohTf, washed, and incubated at 37°C for 60 min. Cytochemical analysis of biohTf and rabbit milk transferrin was carried out on the same section. After incubation at 37°C for 5 min. biohTf was located mainly at the base of the cell as before (Figure 4a). Rabbit milk Tf was detected at the apex of the cell and associated with secretory products in the lumen (Figure 4b). After incubation at 37°C for 60 min, biohTfwas accumulated at the apex of the cell (Figure 4c). Rabbit milk Tf did not appear to co-localize with the apical accumulation of biohTf (Figure 4d), suggesting that the last steps of the exocytotic pathway of biohTf and milk rabbit Tf are not necessarily the same. Immunocytochemical localization of milk rabbit Tf on sections of lactating rabbit mammary gland embedded in Lowicryl revealed that milk rabbit
Tf was located inside secretory vesicles containing casein micelles (Figure 4e). This result precisely localizes the exocytotic pathway for milk rabbit Tf and shows that two milk proteins are carried by the same secretory vesicles.
Endocytosis of AubTJf The transit pathway of hTf across the cell was followed directly with aggregates exposed to AuhTf at 4°C for 30 min and warmed to 37°C for 5 and 30 min. At 4°C the periphery of the cell, coated pits, and endosome-like structures were labeled (Figures 5a-5c). After 5 min at 37"C, the basal membrane remained labeled. Gold particles were detectable in the endosome-like structures, microvesicles, and saccules of the Golgi region (Figure 5c). Moreover, AuhTf was located on the apical membrane and inside the lumen (Figure 5d). After 30 min at 37'C, very little AuhTfremained on the basal membrane, inside the endosomes, and in intracellular vesicles. AuhTf
TRANSFERRIN TRANSPORT AND MAMMARY EPITHELIAL CELLS
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brane was labeled after 2 min at 25°C (Figure 6a). After 2 min. AuTf was located in small vesicles close the the apex, on the apical membrane, and in the lumen. Labeled vesicles close to the apex were coated (Figure 6b). In this case it ¬ possible to determine if AuhTf is in the process of exocytosis or is in the process of reendocytosis of material delivered to the cell surface which re-enters the cell. After 15 min, label accumulated in the lumen (Figure 6c). As at 37'C, AuTf was never localized inside the secretory vesicles containing casein micelles.
Discussion
Figure 3. Immunocytochemicallocalizationof hTf in lactating rabbit mammary cells. Fragments were incubated with native hTf. (a) After 30 min at 4% cells show marked labeling of the basal membrane (large arrows) and a few fluorescent spots inside the cytoplasm (small arrow). (b) After 30 min at 4OC, washing, and incubation for 60 min at 3pC.many fluorescent intracellular spots are detectable (small arrow). Apical membrane and secretory products in the lumen are strongly labeled. Very little label remains on the basal membrane (large arrow). Bar = 10 urn.
was never detected inside the vesicles containing casein micelles. The apical membrane and content of the lumen were strongly labeled. Under these experimental conditions, the hTf intracellular transit seemed to be very fast. Consequently, it is difficult to do quantitative analysis of the localization of the gold particles. With intent to slow down the endocytosis and to make the label more dense, aggregates of mammary epithelial cells were incubated at 25°C for 2 and 15 min in a continuous uptake experiment. The basal mem-
These results show, first, that hTfis carried across the lactating mammary cells by a transcytotic pathway and. second, that rabbit milk transferrin is secreted, mainly in the apical direction, via the secretory vesicles. The transcytosisof hTfwas c o n f i i e d by two different experhental modalities. BiohTf and native hTf, immunocytochemically detected, moved similarly from the basal to the apical pole of the cell. Differences concerning the aspect of the label (spots in the case of the biohTf and more diffuse fluorescence in the case of the native hTf) could result from the two systems of detection used. However, biohTf remains immunoreactive and appears a useful tool to study endocytosis. The transferrin biotin conjugate could be used for ultrastructural double label cytochemistry. The recycling of Tf during TfR-mediated endocytosis and iron delivery to the cells occurs within 1-8 min, depending on the cell type (Bali et al., 1991; Dautry-Varsat et al.. 1983). In the lactating mammary epithelial cell, Tf appears very quickly ( 5 min) in the lumen of the acini. However, with the approach used it is not possible to determine whether a part of biohTf if recycled to the basal membrane. The appearance of a strong label on the apical membrane and in the lumen suggests that a very fast polarized intracellular transport of hTf occurs. Receptor-mediated transcytosis of Tf has been described across hepatic endothelial cells (Kishimoto and Tavassoli, 1987). Transport of polymeric immunoglobulins across the mammary epithelium is mediated by a receptor component that serves as an Ig receptor at the basolateral surface of the cell and which is released in the lumen in association with Ig (Mostov et al., 1980). However, transcytosis of the poly Ig receptor from basolateral to apical membrane in the presence or absence of its ligand, described in transfected immortalized mammary cells, lasts half an hour (Schaerer et al., 1990). This difference in the velocity of transcytosis can be easily explained by the difference in the degree of polarization of these cells. This very quick transit of Tf across lactating mammary epithelial cells is different from the time course of newly synthesized caseins which are released in the lumen 15 to 25 min after a 3-min pulse (Ollivier-Bousquet and Denamur, 1975). These differences in the time of transit and the observation that hTf does not co-localize exactly with endogenous milk Tf and casein micelles contained in secretory vesicles suggest that the transcytotic pathway of exogenous hTf is different from the secretory pathway of milk proteins. Although it was not possible to describe precisely the organelles implicated in the hTf transport, the presence of AuhTf in vesicles and in the Golgi saccules suggests that hTf transits via the Golgi apparatus. In that case, this pathway would be in keeping with the
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Figure 4. Double localization of (a$) biohTf and (b,d,e) rabbit milk Tf on the same sections of lactating rabbit mammary tissue. Fragments were incubated with biohTf for 30 min at 4OC (a,b), washed, then incubated at 37% for 60 min (c,d). (a) After 30 min at 4OC, biohTf detected with streptavidin-Texas Red was mainly located on the basal membrane (large arrows). Apices of the cells are not labeled (arrowheads). (b) On the same section, rabbit milk Tf detected with ITCF was located at the apex of the cell (arrowheads) and on secretory products inside the lumen (small arrow). (c) After 30 min at 4OC, then 60 min at 37% biohTf was accumulated on the apical membrane (arrowheads). (d) On the same section, immunofluorescent localization of rabbit milk Tf revealed that endogenous Tf is not accumulated in the same apical region of the cell (compare apical membrane with arrowheads in c and d). (e) Electron microscopic demonstration of rabbit milk Tf in epithelial mammary cell. Thin sections of tissue embedded in Lowicryl were incubated with anti-rabbit milk Tf antiserum, biotinylated anti-sheep IgG, and streptavidin-gold (15 nm) as described in Materials and Methods. Rabbit milk Tf is detectable in Golgi vesicles and saccules (G) and in secretory vesicles (SV) containing casein micelles (c). Bars: a-d = 10 wm; e = 0.5 pm.
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Figure 5. Localization of AuhTf in dissociated lactating rabbit mammary epithelial cells, incubated at 4OC for 30 min in the presence of AuhTf, washed, then incubated at 37% for various times. After 30 min at 4OC. gold particles are located on the plasma membrane (a), inside a coated pit probably in the process of invaginating in a coated vesicle (b). and inside endosome-like structures (c). After 30 min at 4°C. then 5 min at 37% AuhTf is found inside Golgi saccules (d) and in the lumen (L) (e). Bars = 0.5 bm.
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SEDDIKI. DELPAL, OLLIVIER-BOUSQUET
1-7-
'.-
--.
.:
Cr.
rr
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TRANSFERRIN TRANSPORT AND MAMMARY EPITHELIAL CELLS
-
casein micelle
e
Tf
*milk Tf
Most of the prolactin is not detected in the lumen of the acini before 15-25 min. Moreover, low temperature (25°C)slows down this process and does not allow prolactin to enter the lumen (Seddiki and Ollivier-Bousquet 1991), whereas under the same conditions Tf transcytosis is not inhibited. These results raise the question of the mechanism that controls the vesicular traffic along the exocytic and transcytotic pathways. Association of different proteins with each class of vesicles could be responsible for the sorting. Specific proteins characteristic of transcytotic vesicles have been described in hepatocytes (Sztul et al., 1991). The role of the transported molecules in the identification of the vesicular carrier remains unclear. Our results suggest that in mammary epithelial cells different ligands, such as hTf and PRL, could be carried by different pathways.These different ligands might be useful tools for the study of the complex intracellular transport across the mammary epithelial cell.
Acknowledgments
/ - r b
I
Tf-R
milk protein
We are very gratefulto Prcfissor M. C. Neville andto Professor H.Chuser for their helpfirladvice andcomments on the manuscript, and to J. Brug nolo, R. Scandolo, andE k r t for their contribution in preparing manuscn& and micrographs.
synthesis Literature Cited
Figure 7. Model for transcytosis of hTf across the mammary epithelial cells. We propose that after internalization Tf is carried to the Golgi complex and is transferred into transcytotic vesicles of the apical side in a few minutes. Fe3* could dissociate during this transport in endosomes at the requiredacidic environment and the TfR could recycle to the cell surface. In contrast, caseins and rabbit milk Tf, newly synthesized in the rough endoplasmic reticulum (RER) are transported across the Golgi apparatus (G) and sequestered in the same secretory vesicles (SV), then released into the lumen after 15-30 min.
accessibility of the trms-Golgi network by extracellular added Tf (Hedman et al., 1987). A connection between endocytosed Tf and secretory proteins in the trans-Golgi reticulum has been described in a human hepatoma cell line HepGz (Stoorvogel et al., 1988). However, we were not able to detect hTf in the secretory vesicles containing casein micelles. This result suggests that a putative pathway following the sorting in the trms-Golgi network could be distinct for hTf and secretory proteins. Since rabbit milk Tf, a protein synthesized inside the epithelial cells, co-localizes with casein micelles inside secretory vesicles, we can suggest the following working hypothesis, illustrated in figure 7. Caseins and rabbit milk Tf newly synthesized in the rough endoplasmic reticulum are transported across the Golgi apparatus and sequestered in the same secretory vesicles before being released into the lumen. Conversely, Tf after endocytosis could be carried in the Golgi apparatus where sorting to a transcytotic vesicular compartment occurs. We have shown previously that PRL is carried across the Golgi apparatus and within secretory vesicles containing casein micelles.
Armbruster BL, Carleman E, ChiovcttiR, Garavito RM, Hobot JA. Kellenberger E, Williger W (1982): Specimen preparation for electron microscopy using low temperature embedding resins. J Microsc 126:77 Bali PK, Zak 0, Aisen P (1991): A new role for the transferrin receptor in the release of iron from transferrin. Biochemistry 30:324 Dautry-Varsat A, Ciechanover A, Lodish HF (1983): pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci USA 80:2258 Grigor MR, Wilde CJ, Flint DJ (1988): Transferrin receptor activity in rat mammary epithelial cells. Biochem Int 17:747 Hedman K, Goldenthal KL, Rutherford, AV, Pastan I, Willingham MC (1987): Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry.J Histochem Cytochem 35:233
Jahn GA, Djiane J, Houdebine LM (1989): Inhibition of casein synthesis by progestins in vitro: modulation in relation to concentration of hormones that synergize with prolactin. J Steroid Biochem 32:373 Jordan SM,Kaldor I, Morgan EH (1967): Milk and serum iron and ironbinding capacity in the rabbit. Nature 215:76 Jordan SM,Morgan EH (1970): Plasma protein metabolism during lactadon in the rabbit. Am J Physiol 219:1549 Kishimoto T, Evassoli M (1987): Transendothelial transport (transcytosis) or iron-transferrin complex in the liver. Am J Anat 178:241 Klausner RD, Ashwell G, Van ReswoudeJ, HarfordJB, Bridges KR (1983): Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci USA 80:2263 Koldovsky MD (1980): Hormones in milk. Lifc Sci 26:1833 Lee EYH, Barcellos-Hoff MH. Chen LH,Parry G, Bise11MJ (1987): Trans-
Figure 6. Localization of AuhTf in dissociated lactating rabbit mammary epithelial cells incubated at 25OC. (a) After 2 min at 25OC AuhTf is found on the basal membrane (BM) of the mammary epithelial cell. A fibroblast (F) close to the epithelial cell is also labeled. (b) AuhTf is located in a transcytotic vesicle close to the apical membrane and also on the apical membrane (AM). (c) After 15 min at 25OC, AuhTf accumulates in the lumen. Secretory vesicles containing casein micelles are not labeled. Bars = 0.5 um.
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ferrin is a major mouse milk protein and is synthesized by mammary epithelial cells. In Vitro Cell Dev Biol 23:221 Mostov KE, Kraehenbuhl JP, Blobel G (1980): Receptor-mediated transcellular transport of immunoglobulin: synthesis of secretory component as multiple and large transmembraneforms. Proc Natl Acad Sci USA 77:7257 Ollivicr-Bousquct M, Denamur R (1975): Effct de Etat physiologique et du 3’5’ adenosine monophosphate cyclique sur le transit inuacellulairc et l’cxcrttion des proteines du lait. Etude autoradiographique en micronopie Slectronique. J Microsc Biol Cell 23:63 Schaerer E, Vcrrey F, Racine L, Tallichet C, Reinhardt M, Kraehenbuhl JP (1990: Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells. J Cell Biol 110:987 Schulman HM, Ponka P, Wilczynska A, Gauthier Y,Shyamala G (1989): Transferrinreceptor and ferritin levels during murine mammary gland development. Biochim Biophys Acta 101O:l
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