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Membrane labeling with cationized ferritin in isolated thyroid follicles
JUR-2520
JOURNAL OF ULTRASTRUCTURE RESEARCH
71, 203-221 (1980)
Membrane Labeling with Cationized Ferritin in Isolated Thyroid Follicles J.-F.
DENEF 1 AND
R.
EKHOLM 2
Department of Anatomy, University of G6teborg, S-400 33 G6teborg 33, Sweden Received February 27, 1980 Follicles, closed and open, isolated from rat, pig, and human thyroids, were incubated with polycationized ferritin (CF) in order to label the plasma membrane and follow the fate of internalized membrane. All parts of the plasma membrane, except the pseudopod membrane, were labeled. CF was rapidly (within 5 min) internalized from the apical surface by micropinocytosis. Macropinocytotic vacuoles, formed from pseudopods, were not primarily labeled but received CF by fusion with micropinocytotic vesicles. CF was early present in phagolysosomes and most of the CF was eventually accumulated in these structures. After 30 min CF was found in minute amounts in some Golgi saccules. In all labeled intracellular structures only some of the CF was clearly membrane associated. The labeling of Golgi saccules, although minimal, might indicate a recycling of membrane. However, since CF was not a pure marker of intracellular membranes in thyroid follicle cells, it could not be used to demonstrate the extent or route(s) of membrane recycling.
Endocytosis of thyroglobulin is a key step, controlled by TSH, in the hormone release from the thyroid follicle cell. Two different ways of colloid uptake have been described: macropinocytosis, involving the formation of colloid droplets from pseudopods (Nadler et al., 1962; Stein and Gross, 1964; Wollman et al., 1964) and micropinocytosis (Seljelid, 1967; Seljelid et al., 1970; Rocmans et al., 1978). The relative importance of these processes is not known. Previous studies from this laboratory (Ekholm et al., 1975; Ekholm, 1977; Ericson and Johansson, i977; Ericson and Engstr6m, 1978; Ericson et al., 1979) have shown that an endocytotic response to TSH requires addition of membrane to the apical cell surface by exocytosis. Furthermore, the amount of membrane appearing in endocytotic structures upon TSH stimulation corresponds to the amount of membrane added to the apical cell surface from exocytotic vesicles during the stimulation period. These observations indicate that exocytotic membrane, via the apical plasma membrane, is transferred into endocytotic membrane. Against this background it was i Present address: Laboratory of Histology, Louvain Medical School, U.C.L., B-1200 Brussels, Belgium. 2 To whom requests for reprints should be addressed.
deemed to be of interest to trace the fate of the membrane taken into the follicle cell by endocytosis and to explore whether such internalized membrane is recycled into exocytotic membrane, thus completing the membrane circuit. The present study was performed in an in vitro system, described in a preceding report (Denef et al., 1980) and consisting of isolated, closed and open, thyroid follicles. By incubation with polycationized ferritin, which binds to plasma membrane (Danon et al., 1972), we tried to track the m e m b r a n e taken into the follicle cells by endocytosis. MATERIALS AND METHODS Thyroid glands of about 50 rats and 6 pigs and thyroid biopsies from 6 humans were used in about 20 different labeling experiments. Closed and open follicles Were isolated as described in the preceding paper (Denef et al., 1980). The follicle preparations were preincubated for I hr at 37°C, under continuous gassing with 95% 02, 5% C02, in Tyrode solution supplemented with amino acids according to Eagle (1959), TAA, and containing 0.5% albumin and 2 f~g/ml DNAse. After preincubation, the samples were washed twice in TAA and then incubated in TAA with cationized ferritin (CF), prepared according to Danon et al. (1972) and obtained from Mfles-Yeda, Rehovot, Israel. Incubations with CF (0.1 or 0.5 mg/ml) were performed for 5-180 min at 37°C and under continuous Oe-CO2 gassing. When used, TSH (bovine TSH, a gift from NIH, Bethesda, Md.) was added to the incuba-
203 0022-5320/80/050203-19502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tion medium 10 min before CF at a concentration of 100 or 20 mIU/ml. After incubation, the samples were centrifuged (50g × 3 min), the supernatants were discarded and 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) was added to the pellets. After 60-90 min in glutaraldehyde the specimens were postfixed in osmium tetroxide and then prepared and examined as described in the preceding paper (Denef et al., 1980). RESULTS At all incubation times, most open follicles contained CF in their lumen. A p a r t from some differences in the labeling pattern of the apical plasma membrane, there were no qualitative differences in the labeling of open follicles in rat, pig, and h u m a n preparations. However, quantitatively the labeling of the apical plasma m e m b r a n e and intracellular structures in the pig follicles was greater t h a n the corresponding labeling in rat and h u m a n follicles. T h e pig follicles will therefore be used as basis for the description of the observations. OPEN PIG FOLLICLES Pig follicles showed endocytotic activity when incubated without T S H . Addition of T S H to the m e d i u m increased the n u m b e r of labeled endocytotic structures and the a m o u n t of internalized CF but did not induce any qualitative changes of the labeling of plasma m e m b r a n e or intracellular structures.
Labeling of Plasma Membrane Already at the shortest incubation times (5-15 min) all parts of the plasma membrane were labeled. At these times, the
apical plasma m e m b r a n e was generally more heavily labeled t h a n the other parts. As a rule, CF was accumulated in the pits at the base of the microvilli and to a varying extent in the spaces between the microvilli (Figs. 1, 5, and 6). After longer incubation times (30-60 min) the CF on the apical surface was redistributed into patches separated b y CF-free areas (Fig. 7). With increasing incubation time these patches became smaller and sparser. During the first hour of incubation the basal plasma m e m b r a n e was generally covered by a thin but continuous layer of CF of rather even thickness (Fig. 2). N o t until the second h o u r of incubation did the labeling became patchy, leaving some areas of the m e m b r a n e bare. After 3 hr of incubation some cells still had a basal plasma m e m b r a n e covered by CF while in other cells the basal plasma m e m b r a n e had no CF left. After 5 min of incubation some intercellular spaces were still devoid of CF but after 15 min the label was regularly found, alt h o u g h in varying amounts, along the lateral plasma membranes. M u c h CF was often remaining in the intercellular spaces after 3 hr incubation. In the apical and middle parts of the intercellular spaces, the CF was generally rather scarce and appeared as single particles or in small clusters (Fig. 3). T h e labeling stopped sharply at b o t h sides of the tight junctions (Fig. 3). T h e basal part of the intercellular spaces h a d a richer supply of CF which often filled narrow portions of the space (Fig. 4). T h e intercellular layer of CF was continuous
FIG. 1. Apical zone of a pig thyroid follicle cell. Incubation with CF for 5 rain; TSH. The CF is accumulated in the pits between the microvilli. × 35 000. FIG. 2. Basal zone of a pig follicle cell. Incubation with CF for 15 min. The CF layer is almost continuous but somewhat irregular in thickness. Clusters of CF are entrapped by membrane foldings. Note a labeled micropinocytotic vesicle (arrowhead). × 36 500. FIG. 3. Apical part of intercellular spaces between pig follicle cells. Incubation with CF for 1 hr. CF occurs in the intercellular spaces both in clusters and as single particles. No CF is present in the tight junction (arrowheads). Note the presence of some CF-loaded vesicles in the cytoplasm (arrows). × 39 300. FIG. 4. Basal part of an intercellular space between two pig follicle cells. Incubation with CF for 30 min. The CF forms clusters, sometimes only two molecules in thickness (arrowhead). Some endocytotic vesicles, loaded with CF, are located in the basal region of the cell (arrows). × 35 900.
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with the CF coating the basal plasma membrane (Figs. 2 and 4).
Labeling of Intracellular Structures Micropinocytotic vesicles. At the shortest incubation times (5-10 min) most of the intracellular CF was found in vesicular structures in the apical cell zone; these structures were interpreted to represent micropinocytotic vesicles. The labeled vesicles were smooth-surfaced or bristlecoated (Figs. 5 and 6). The smooth vesicles were round or irregular in shape and varied in diameter between 700 and 3000 A. Except for their content of CF, they appeared empty. The coated vesicles were always round and seemed to belong to two size classes with a diameter of about 700 and more than 1000/~, respectively. Many vesicles, both smooth and coated, were completely filled with CF. In the vesicles containing smaller amounts of label, some CF was concentrated along the membrane, but only exceptionally covering the entire membrane, and the remaining CF was found in the interior of the vesicle. In the smallest vesicles it was not possible to decide whether or not the CF particles were coating the membrane. With increasing incubation time the number of labeled micropinocytotic vesicles decreased and the CF particles showed a tendency to aggregate in small clusters in the interior of the vesicle or close to its membrane (Fig. 7). In addition to the described vesicles, another category of labeled vesicles was observed from 15 min and onward (Fig. 8). These vesicles were characterized by a
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moderately dense, homogeneous matrix and an extremely varying shape. The CF content was rather low and the particles were dispersed throughout the vesicles. At all incubation times, a few smooth and coated labeled vesicles were seen in the vicinity of the basal and lateral plasma membrane (Figs. 2-4).
Pseudopods and macropinocytotic vacuoles. At all incubation times the pseudopod membrane was much less labeled than the adjoining apical plasma membrane; as a rule the labeling was restricted to a few small patches of CF (Fig. 9). However, some small vesicles containing CF were usually present in the body and the base of the pseudopod. Large vacuoles, some of them more than 1 /tm in diameter, in the pseudopods and supranuclear parts of the cell body were interpreted as macropinocytotic vacuoles, corresponding to the colloid droplets in closed follicles (cf. Denef et al., 1980). Vacuoles located in pseudopods were devoid or almost devoid of CF, irrespective of the time of incubation (Fig. 9); only occasionally one or a few small clusters of CF could be seen close to the vacuole membrane. Vacuoles located in the cell body had a similar, very low labeling at short incubation times (Fig. 10). After longer incubations the vacuoles contained varying amounts of CF, generally in the form of confluent clusters of particles in the peripheral zone of the vacuole, close to its membrane (Fig. 12). These vacuoles were often surrounded by labeled micropinocytotic vesicles and pictures suggesting a fusion
FIG. 5. Apical zone of a pig follicle cell. Incubation with CF for 5 min; T S H . M o s t of t h e micropinocytotic vesicles are filled with CF (arrows) b u t in one relatively large vesicle, t h e CF s e e m s c o n c e n t r a t e d to t h e periphery (arrowhead). × 32 400. Fro. 6. Apical part of a pig follicle cell. Incubation with CF for 10 min; T S H . S o m e of t h e n u m e r o u s micropinocytotic vesicles h a v e a s m o o t h m e m b r a n e (arrowheads) while others have a bristle-coated m e m b r a n e . T h e latter vesicles s e e m to belong to two size classes (small arrows a n d big arrow). A macropinocytotic vacuole (D) with discontinuous peripheral labeling h a s close relation to a labeled micropinocytotic vesicle. × 46 300. FIG. 7. Apical part of a pig follicle cell. I n c u b a t i o n with CF for 1 hr; T S H . T h e apical m e m b r a n e is discontinuously labeled. S o m e labeled s t r u c t u r e s have a c o n t e n t of low density a n d are i n t e r p r e t e d as endocytotic vesicles (small arrows). O t h e r structures, having a denser m a t r i x a n d containing a few C F particles, are difficult to identify (secondary lysosome? (arrowhead) secondarily labeled colloid droplet? (large arrow)). × 38 700.
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Fro. 8. Apical part of a pig follicle cell. Incubation with CF for 15 min; TSH. One coated endocytotic vesicle is heavily labeled (small arrow). In addition several structures of varying size and shape and with a moderately dense matrix contain a few disseminated CF particles (arrowheads). Their nature is unclear; they could be endocytotic structures present in the cell before the opening of the follicle and labeled secondarily by fusion with newly labeled endocytotic vesicles; or they could be secondary lysosomes. The identity of the structures marked with asterisks is also unsettled; they might be invaginated macropinocytotic vacuoles. × 45 400.
between such vesicles and a vacuole were sometimes observed (Fig. 11). Typical colloid droplets, filled with a homogeneous substance, were observed in the supranuclear and paranuclear cell regions. These droplets, evidently formed before the
opening of the follicles, sometimes contained clusters of CF (Fig. 13). These clusters were always located in the matrix of the droplet without any obvious relation to the droplet membrane. Phagolysosomes. The labeled bodies in-
FIG. 9. Pseudopod and apical part of a pig follicle cell. Incubation with CF for 10 min; TSH. Although the apical plasma membrane is richly labeled, the pseudopod membrane is almost free of CF; the arrowheads mark small clusters of CF particles along the membrane. Some vesicles in the body and the base of the pseudopod also contain some CF (arrows). No CF is seen in the macropinocytotic vacuoles (D) of the pseudopod. × 44 200. FIG. 10. Macropinocytotic vacuole in the apical zone of a pig follicle cell. Incubation with CF for 15 min; TSH. The few CF particles (arrowheads) at the membrane are probably derived from the pseudopod membrane. × 65 100. FIG. 11. Macropinocytotic vacuole in the supranuclear region of a pig follicle cell. Incubation with CF for 30 min; TSH. The vacuole is surrounded by some labeled micropinocytotic vesicles (arrows) and has a CF-filled evagination which probably represents a labeled vesicle having fused with the vacuole. The small Clusters of CF (arrowheads) in another region of the vacuole could represent primary labeling achieved in the pseudopod. × 49 200. FIG. 12. Macropinocytotic vacuole in the supranuclear region of a pig follicle cell. Incubation with CF for 30 min. The label is concentrated to the periphery of the vacuole and much of the CF seems closely related to the membrane, L, phagolysosome (?). × 60 800. Fro. 13. Colloid droplet in the supranuclear part of a pig follicle cell. Incubation with CF for 30 min. The homogeneous content of the droplet suggests that it was formed before the opening of the follicle. Consequently, the CF in its matrix probably represents secondary labeling, obtained by fusion with a CF-labeled structure. A phagolysosome (L) has a rich, irregularly distributed supply of CF. × 51 800.
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terpreted as phagolysosomes constituted a heterogeneous population of structures. The most characteristic components of this population were multivesicular bodies (cf. Fig. 21, rat follicle cell). Other components were round or elongated bodies with a homogeneous matrix separated from the limiting membrane by an interspace of low density (Fig. 12). The most common components were polymorphous bodies containing a granular substance of moderate to high density and sometimes membrane remnants, particles, and small vesicles (Fig. 13; cf. also Figs. 20 and 23). CF was present in phagolysosomes already after 5 min incubation. The number of labeled lysosomes as well as the amount of CF per lysosome increased with increasing incubation time. The CF was rarely seen in close relation to the limiting membrane but was distributed in clusters of varying size and density in the interior of the lysosome. Golgi-related structures. Labeled Golgi saccules were observed after incubation for 30 min to 2 hr. However, labeling of Golgi saccules was rare; in only a few percent of the sections through cells with a rich intracellular labeling and including the Golgi region was a labeled saccule detected. Only exceptionally was CF seen in more than one saccule of a Golgi region. The number of CF particles seen in a saccule was generally small. The particles were sometimes closely related to the saccule membrane (Figs. 14 and 15) but often such a relation was not evident (Figs. 16, 17, and 24). Vesicles, both smooth and coated, present in the Golgi region were more often labeled than the saccules (Figs. 14 and 24).
Such labeled vesicles were occasionally seen after 15 min incubation but they were more frequent between 30 and 90 min. OPEN HUMAN FOLLICLES
All parts of the plasma membrane were labeled but the labeling pattern of the apical plasma membrane differed from that in the pig follicles: The CF was not concentrated to the pits between the microvilli but formed a continuous layer covering the microvilli: the latter seemed to adhere to each other (Fig. 18). In human follicles incubated without TSH little CF was found intracellularly. Also in the presence of TSH the number of micropinocytotic vesicles was much smaller than in TSH-stimulated pig follicles (Fig. 18). The labeling of the few pseudopods and macropinocytotic vacuoles was similar to that in the pig follicles. Labeled invaginations, some of them with a coated membrane, were rather common along the lateral and basal cell surfaces (Fig. 19). The labeled phagolysosomes did not differ from those in the pig follicles (Fig. 20). Labeled Golgi saccules were extremely rare (Fig. 17). OPEN
RAT FOLLICLES
As was the case in pig follicles, all parts of the plasma membrane were labeled with CF. However, the apical plasma membrane was much less labeled than in the pig follicles. The labeling was patchy at all incubation times; some CF was located in the pits between the microvilli but more CF was present in clusters along the microvilli (Fig. 21). After incubation without TSH very little
FIGs. 14-17. Golgi-related s t r u c t u r e s in pig follicle cells i n c u b a t e d with CF for 60 m i n (Fig. 14) or 30 m i n (Figs. 15 a n d 16) a n d in h u m a n follicle cells i n c u b a t e d with CF for 2 h r (Fig. 17); T S H in all incubations. FIG. 14. Two Golgi saccules contain CF (big arrows); in one saccule t h e CF is closely related to t h e m e m b r a n e . CF is also p r e s e n t in bristle-coated (arrowhead) a n d s m o o t h vesicles (small arrows). × 46 700. FIG. 15. CF particles are closely related to t h e m e m b r a n e in a Golgi saccule, partially coated (arrowheads). CF is also p r e s e n t in t h e o u t e r m o s t saccule in a Golgi stack (arrow). × 62 400. FIG. 16. CF particles lined up in a Golgi saccule. × 57 800. FIG. 17. In Golgi saccules, CF is often located in one e x t r e m i t y (arrow). × 45 300.
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CF was internalized. TSH stimulation increased the amount of CF taken up by the cells but it was far below that in the pig follicles. However, the intracellular labeling did not differ qualitatively from that in the pig follicles and the labeling of the various structures appeared in the same chronological order. Thus, labeled micropinocytotic vesicles appeared within 5 min and small amounts of CF were found in macropinocytotic vacuoles and colloid droplets. Labeled multivesicular bodies were observed already after 5 min incubation (Fig. 21) and after 60 min phagolysosomes were the dominating labeled structures (Fig. 23). Labeled Golgi saccules and Golgi-related vesicles were rarely seen (Fig. 24). CLOSEDFOLLICLES In closed follicles from all three species the labeling of the basal and lateral plasma membrane was similar to that in open follicles. The amount of intracellular CF was much less than in open follicles. After incubation for 30 min and more CF-labeled endocytotic structures were present in the vicinity of the basal and lateral cell surfaces (Figs. 25 and 27) and labeled phagolysosomes were observed in the supranuclear region (Figs. 25 and 26). In a few cases, round or oval vesicles with a rather dense content and containing a few scattered CF particles were found in the apical cell region, sometimes close to the apical plasma membrane (Fig. 26). Small clusters of CF were occasionally seen in follicle lumina (Figs. 26 and 27) and in intracellular lumina (Fig. 25), generally in the vicinity of the apical plasma membrane.
DISCUSSION
CF has been used as a plasma membrane marker in anterior pituitary cells to study membrane retrieval and reutilization (Farquhar, 1978). In this study it was shown that CF was taken up by endocytosis and was recovered in Golgi cisternae and in secretion granules. These observations were interpreted to show that membrane retrieved from the plasmalemma by endocytosis can be recycled to compensate for membrane added during exocytosis. In a similar study on thyroid follicles, published during the preparation of the present work, Herzog and Miller (1979) showed that CF was taken into the follicle cells by endocytosis and reached lysosomes and the Golgi apparatus. They concluded that CF-containing vesicles fuse with Golgi membranes. Most of the observations made by Herzog and Miller were corroborated in the present study but some new observations were made. Moreover, our interpretations are deviating in some respects.
Labeling of Plasma Membrane In open follicles all parts of the plasma membrane were labeled with CF but the apical cell surface was markedly more labeled in the pig and human follicles than in the rat follicles. A possible explanation of this difference might be that the pig and human follicles were more widely open than the rat follicles, making the apical surface more accessible to CF in the former. However, this seems not to be the case since the scanty apical labeling was also seen in rat follicles with plenty of CF in their lumen, and in rat thyroid cell strips which appar-
FIas. 18-20. Human follicle cells incubated with CF for 15 min (Fig. 18), 30 min (Fig. 19), or 3 hr (Fig. 20); TSH in all incubations. Fro. 18. The apical plasma membrane has a continuous layer of CF. The microvilli seem to stick together, separated only by the thin layer of CF. CF-labeled endocytotic vesicles are few and widely dispersed (arrows). ×41 000. Fro. 19. View of the lateral and basal plasma membrane. An active endocytosis is indicated by numerous CF-loaded vesicles (arrows). × 39 000. FIG. 20. After long-time incubation, the CF particles accumulate in phagolysosomes. Note the presence of a crescent-shaped labeled structure (arrowheads); it is probably the same structure as those marked with asterisks in Fig. 8, but sectioned in another plane. × 25 200.
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ently had free access to CF. Also, in rat follicles the CF on the apical cell surface had an irregular distribution, forming clusters both in the pits between the microvilli and along the microvilli, while in pig and human follicles the CF either was concentrated in the pits between the microvilli or formed a continuous layer along the apical surface. Consequently, the density and distribution of anionic sites accessible to CF binding on the apical plasma membrane seem to differ between the rat thyroid on one hand and the pig and human thyroid on the other. The CF labeling of the pseudopod surface was much less than the labeling of the other parts of the apical cell surface; generally only a few small patches of CF were found along the pseudopods. This indicates that a smaller number of negatively charged groups were exposed and available for CF binding on the pseudopod membrane than on the remaining parts of the apical plasma membrane. This in turn indicates that a rapid reorganization occurs of the apical plasma membrane in connection with its formation of the pseudopod. Such an inference is in accord with previous histochemical and autoradiographic observations (Tice and Wollman, 1974; Ekholm and Wollman, 1975) which have demonstrated peroxidase activity in the apical cell membrane but not in the pseudopod membrane. The possible significance of the low CF binding to the pseudopod membrane for exocytosis of thyroglobulin is not obvious. It could be speculated that a relatively low number of negative groups would facilitate
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the binding of the negatively charged thyroglobulin but, on the other hand, endocytosis of thyroglobulin by pseudopods-colloid droplets is certainly a bulk transport in which the proportion of thyroglobulin bound to the membrane should be of little importance. In contrast to the apical labeling, the labeling of the basal cell surface and the basal part of the lateral surfaces remained uninterrupted and rather even for at least 1 hr of incubation. It is likely that this difference in labeling patterns reflects differences in the properties between the apical and basal-lateral membranes. That such differences exist seems natural considering the special organization of the apical plasma membrane and the special functional demands on that membrane.
Internalization of CF The first intraceUular structures to be labeled were vesicular structures in the apical cell zone. These vesicles were consequently interpreted to be involved in the internalization of CF, hence representing endocytotic structures. The population of' labeled vesicles was polymorphous: Some of the vesicles were bristle-coated; others were smooth-surfaced, generally larger than the bristle-coated vesicles and of different shapes; the vesicles had a varying content, some appearing empty while others had a matrix of low to moderate density. The labeling pattern showed the following: characteristics: At short incubation times, the labeled vesicles were few but heavily labeled; with increasing time of incubation
FIGS. 21-24. Rat follicle cells incubated with CF for 5 min (Fig. 21), 30 min (Figs. 22 and 24), or 60 min (Fig. 23); TSH in all incubations. FIG. 21. The labeling of the apical surface is patchy; most of the CF is found in clusters along the microvilli. However, the presence of a few CF particles in a forming micropinocytotic vesicle (arrowheads in the inset), in an already formed pinocytotic vesicle (arrow), and in a multivesicular body (MV) indicates that endocytosis takes place. Fig. 21, × 25 000; inset, × 50 000. FIG. 22. The labeling of the basal plasma membrane is continuous. The arrow indicates a micropinocytotic vesicle. × 30 100. FIG. 23. After 60 min most of the CF is found in phagolysosomes. × 21 600. FIG. 24. A Golgi saccule is labeled with CF (arrowhead) and CF particles are also found dispersed in two vesicles (arrows). x 41 800.
FIGS. 25-27. CF labeling in closed follicles from rat (Figs. 25 and 26) and human (Fig. 27) thyroids after incubation with TSH. FIG. 25. Incubation for 3 hr. Low-magnification view of part of a closed follicle. (Compare the density of the lumen (upper right corner) and the extrafollicular space (lower left corner).) One cell contains an intracellular lumen with a content of the same density as that of the follicular lumen. CF is present in the intercellular space and is evidently endocytosed (arrowheads) from this. A labeled phagolysosome (asterisk) is located in the supranuclear region. A cluster of CF is seen in the intracellular lumen (arrow). × 14 000. FIG. 26. Incubation for 1 hr. CF particles are dispersed in some dense bodies, probably secondary lysosomes (small arrows). Smaller numbers of CF molecules are present in some less dense vesicles, maybe exocytotic vesicles (long arrows). Note also the presence of CF particles inside the filled lumen (arrowheads). × 46 600. 216
CATIONIZED FERRITIN IN THYROID FOLLICLES
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concentrated along the membrane; in these vesicles CF was probably a membrane marker. Other vesicles were completely filled with CF; a possible explanation of this could be that the portion of the apical plasma membrane from which the vesicle, was derived was coated with a thick layer of CF. It is evident that in these vesicles only the peripheral CF molecules could function as membrane markers. In still other vesicles the CF was dispersed in the, interior without any visible contact with the membrane; this CF could not be a membrane marker. It is noteworthy that the last distribution pattern was common in matrix-containing vesicles and it cannot be excluded that the matrix itself could modify the binding of CF. As suggested[ above, these structures might be of lysosomal nature. If not, this type of labeling would indicate that the internalized plasma membrane already in the endocytotic vesi.. cles undergoes modifications resulting in a loss of the ability to bind CF. FIG. 27. Incubationfor 3 hr. SomeCF particles are In tissue sections of the thyroid, bristlepresent in the closed lumen, in the vicinity of the coated micropinocytotic vesicles can easily apicalmembrane(arrowheads).The smallendocytotic vesicles (short arrows) might originate from the la- be recognized. However, this category of beled intercellular space (IS). The heavily labeled vesicles certainly represents only a portion body is probably a secondarylysosome(L). × 41 400. of the endocytotic vesicles in the follicle cells. The remaining micropinocytotic ves(but while there still was a rich labeling of icles have no very characteristic features the apical plasma membrane) the number and have been described (in the rat thyroid) of labeled vesicles increased but the label- as smooth-surfaced vesicles of somewhat ing per vesicle decreased. This pattern in- smaller size and lower density than the dicates that not all the labeled vesicles had exocytotic vesicles (Ericson et al., 1979). In obtained their CF by vesiculation of the the present study many bristle-coated veslabeled apical plasma membrane; some of icles contained CF but the majority of the them were probably formed before CF was labeled vesicles, considered to be endocyadded to the incubation medium and were totic, were smooth-surfaced and of rather labeled secondarily by--transient or per- varying size and shape. Most of these vesimanent--fusion with labeled vesicles. The cles had an empty appearance which should matrix-containing, generally sparsely la- be expected as they were formed in open beled, vesicles could not be definitely iden- follicles. However, since such empty-looktiffed; they could either represent endocy- ing vesicles are not seen in tissue sections totic vesicles present in the cell before the (or in isolated closed follicles, cf. Denef et opening of the follicle or they might be of al., 1980) one may ask if they represent lysosomal nature. artifacts, induced by the unphysiological The distribution of CF in the endocytotic environment at the apical cell surface, or if vesicles varied. In some vesicles CF was they correspond to the endocytotic vesicles
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described in tissue sections. The latter al- considered as membrane marker. In colloid ternative seems quite possible even if no droplets, CF was never found in close relastructural similarities could be demon- tion to the membrane but distributed in strated; the different content of the vesicles small aggregates throughout the matrix. It in vivo and in vitro--colloid material and seems possible that the content of the colincubation medium, respectively--could loid droplets was responsible for this labelprobably be responsible for differences in ing pattern. For example, the low pI of size and shape between these vesicles and thyroglobulin (Heidelberger and Pedersen, those observed in tissue sections. Two more 1935; Ui, 1971) should favor the binding of observations should be pointed out in this CF to thyroglobulin. connection: One is that the empty-looking vesicles were present after incubation with- CF in Phagolysosomes out CF (Denef et al., 1980) indicating that CF did not induce their formation, and the As soon as 5 rain after addition of CF to other is that the number of labeled empty the incubation medium, small numbers of looking vesicles increased after incubation CF molecules were observed in multivesicwith TSH, indicating that the vesicles were ular bodies. Since at this time endocytotic formed as a physiological process. vesicles were the only other intracellular In macropinocytotic vacuoles present component containing CF, it can be conafter short labeling times in pseudopods cluded that the multivesicular bodies had and the cell body no or very small numbers obtained their CF by fusion with endocyof CF particles were seen. This is in har- totic vesicles. With increasing incubation mony with the observation that the pseu- time the number of labeled phagolysosomes dopod membrane had a sparse CF labeling. as well as their content of CF increased With increasing incubation time the num- progressively. After the longest incubation ber of more heavily labeled vacuoles in- time (3 hr) phagolysosomes were almost creased. CF particles were also found in the only intracellular structure remaining well-filled colloid droplets, obviously labeled. This Shows that CF was accumuformed before the opening of the follicle. lated in phagolysosomes which hence seems Moreover, pictures indicating fusion be- • to be the terminal compartment for most of tween macropinocytotic vacuoles and la- the internalized CF. beled micropinocytotic vesicles were seen. In structures identified as phagolysoThese observations were interpreted to somes no preferential location of CF to the show that the CF present in macropinocy- membrane was seen. Abundantly labeled totic vacuoles was mainly derived from mi- phagolysosomes had CF also in their pecropinocytotic vesicles. Fusions between ripheral zone but it was not possible to micropinocytotic vesicles and colloid drop- decide ff some of this CF was bound to the lets have previously been suggested by Sel- membrane. In any case, the proportion of jelid et al. (1970). Whether the fusion be- CF possibly bound to the membrane was tween the structures responsible for the certainly very small. This could be due to observed transfer of CF was of transient or several factors. First, as pointed out above, already in the endocytotic structures a large permanent nature cannot be decided. In macropinocytotic vacuoles with rela- proportion of the CF was not membrane tively small amounts of CF, the label was bound. Second, CF bound to endocytotic generally concentrated in a peripheral membrane could be detached when endolayer, but with increasing labeling more and cytotic structures fuse with lysosomes. For more CF was found in the interior of the example, due to the protonation occurring vacuole. This indicates that only part of the in lysosomes (De Duve et al., 1974), conjuCF in macropinocytotic vacuoles can be gated basic groups could be transferred into
CATIONIZED FERRITIN IN THYROID FOLLICLES
219
cytotic structures could be responsible for the labeling of the Golgi saccules. The nature of the small labeled vesicles, sometimes bristle-coated, present in small numbers in the Golgi region after 15 min incubation and onward is not clear. However, since labeled vesicles of the same size, often coated, were observed in the apical cell region already after 5 min incubation, it seems possible that they were of endocytotic nature. Bristle-coated vesicles hawe been assumed to be involved in endocytosis and membrane retrieval in several cell types (for review see Silverstein et al., 1977). Although pictures indicating a fusion between labeled coated vesicles and Golgi saccules were never observed, partially coated labeled saccules were occasionally seen. In conclusion, these observations indiCF in Golgi-Related Structures cate that the direct transfer of membrane The rare and scanty labeling of the Golgi and content from the endocytotic compartsaccules could be due to a limited delivery ment to the Golgi compartment is very of CF to the Golgi or a rapid transfer of CF small. This is in agreement with the present through the Golgi to another compartment. knowledge about the physiology of the folIn the follicle cells, the quantitatively most licle cell which is incompatible with an eximportant receiving compartment should tensive direct transfer of thyroglobulin to be the exocytotic vesicles which transfer the Golgi. The possible communication bethyroglobulin into the follicular lumen. tween the apical surface and the Golgi via However, since no extensive labeling of typ- coated vesicles might serve a special purical exocytotic structures was observed, it pose, not related to thyroglobulin intake. Considering the possibility of a transfer seems likely that the sparse labeling of the Golgi was the result of a restricted supply of membrane from the phagolysosomes to the Golgi saccules it is interesting that in of CF. Since CF appeared in Golgi-related struc- pituitary mammotrophs there was a clear tures after the labeling of endocytotic struc- CF labeling in the Golgi (Farquhar, 19781) tures and phagolysosomes, CF in the Golgi whereas in macrophages, internalized CF was traced to lysosomes but could not be could be derived from these two sources. In endocytotic structures, as discussed detected in Golgi cisternae (Skutelsky and above, some of the CF was generally found Hardy, 1976). Thyroid follicle cells resemclose to the membrane, indicating that in ble macrophages in the respect that the these structures CF to a certain extent was traffic of endocytosed material is more or a membrane marker. At all incubation less completely directed toward the lysotimes the amount of CF (membrane-related somes. In this respect the thyroid follicle as well as free) in endocytotic structures cells differ from all other secretory cells. was very large compared to that in Golgi Therefore, the failure of demonstrating CF saccules. This means that even if all Golgi labeling in the Golgi of macrophages and CF was derived from endocytotic structures the poor labeling of the Golgi observed in only a very small proportion of the endo- the present study could indicate that CF, as
their acidic form, thus inducing a decrease of the net negative charge of the membrane. It also seems possible that intralysosomal hydrolysis of the protein part of the ferritin could occur, changing the physicochemical properties of the label. (This would imply that what is seen in the electron microscope is not CF but its iron core (Haggis, 1965).) Third, CF-labeled membrane portions originating from endocytotic structures could rapidly and selectively be pinched off and transferred to the Golgi. However, although this explanation would be in agreement with the observations by Schneider et al. (1979) indicating a selective recycling to the cell surface of plasma membrane constituents of secondary lysosomes, it does not fit with the rare occurrence of labeled Golgi membranes (cf. below).
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DENEF AND EKHOLM
soon as it has joined the lysosomal compartment, does not function as a membrane marker. This is in accord with the present observations that in phagolysosomes CF was rarely seen to be closely related to the membrane. It should also be pointed out that a loading of phagolysosomes with CF, a substance that evidently cannot be completely degraded by the lysosomal enzymes and is foreign to the follicle cell, might modify the normal behavior of the phagolysosomes, including their membrane. Consequently, the present observations could neither prove nor disprove that a transfer of membrane from the phagolysosomes to the Golgi occurs in vivo.
CF in Closed Follicles In closed follicles, the cells regularly contained CF but as a rule in much smaller amounts than cells in open follicles incubated under similar conditions. In closed follicles, the CF must have been taken into the cells via the basal and lateral cell surfaces which was also indicated by the presence of labeled micropinocytotic vesicles along these surfaces. Most of the internalized CF in closed follicles was present in structures belonging to the lysosomal family, which is to be expected considering the location of CF taken up via the apical surface. However, a more interesting observation was that small clusters of CF were occasionally present in the lumen of closed follicles and in closed intracellular lumina (Denef et al., 1980). The objection might be raised that CF had entered the follicle lumen during a transient leakage between follicle cells. However, it seems improbable that CF in the incubation medium could enter a colloid-filled lumen where the hydrostatic pressure should be higher than in the medium. Moreover, the presence of CF in intracellular lumina could not be explained by intercellular leakage. Hence, it is extremely likely that CF entered these lumina from the follicle cells. The mode of transcellular CF transport
cannot be definitely ascertained but two different routes seem possible. One route, indicated in Fig. 27, should be a direct migration of CF-containing micropinocytotic vesicles from the lateral cell surface to the apical surface where they should empty their content into the lumen. It is possible that this mode of transportation applies only to vesicles formed rather close to the apical surface (but below the cell junctions), thus having a rather short migration distance. The other mode of transport should involve exocytotic vesicles (as indicated in Fig. 26). However, this transportation includes several ambiguous steps. For example, the identification of exocytotic vesicles, a possible candidate for the last part of the intracellular route, is uncertain. Further, if the vesicles indicated in Fig. 26 are exocytotic we do not know where in the cell these vesicles were labeled with CF. Thus, the observations made clearly indicate that CF entering the follicle cell via the basal and lateral surfaces can be transferred into the follicle lumen, but how the transcellular transport occurs cannot be ascertained.
Concluding Remarks The present study shows that in the thyroid follicle cells, CF was internalized from the apical cell surface by micropinocytosis. By the same method some CF was also taken up from the basal and lateral surfaces. By fusion between vesicles, CF was redistributed among the micropinocytotic structures. Macropinocytosis also occurred at the apical cell surface; the macropinocytotic membrane, i.e., the pseudopod membrane, bound only minute amounts of CF but the macropinocytotic vacuoles were labeled secondarily by fusion with CF-containing micropinocytotic vesicles. In the endocytotic structures only part of the CF was clearly associated with the membrane and hence able to serve as a membrane marker. CF appeared early in phagolysosomes and most of the internalized CF was
CATIONIZED FERRITIN IN THYROID FOLLICLES
eventually accumulated in phagolysosomes. In these structures very little CF seemed to be associated with the membrane. In Golgi saccules the labeling was very scanty and the CF appeared only partially membrane-associated. Since the Golgi labeling occurred later than the labeling of endocytotic structures and phagolysosomes, it must be derived from one or both of these compartments. The fact that the amount of labeled Golgi membrane was minimal compared to the amount of labeled endocytotic membrane means that even if all labeled Golgi membrane was derived from endocytotic structures, this Golgi membrane could represent only an insignificant fraction of the labeled endocytotic membrane. Whether any labeled Golgi membrane emanated from phagolysosomes cannot be decided since very little CF was apparently associated with the phagolysosomal membrane. The fact that Golgi membranes were at all labeled suggests that a recycling of membrane occurs in the follicle cells but the extent of this recycling and the link between the endocytotic part of the loop and the Golgi can evidently not be elucidated by using CF as a marker. The occurrence of CF in the lumen of closed follicles showed that CF can be transferred through the follicle cell. However, the mode(s) of transport could not be ascertained. This work was supported by grants from the Swedish Medical Research Council (B80-12X-537) and the National Institutes of Health (AM-18842). We are grateful to Mrs. Gunnel Bokhede for excellent technical assistance and to Mrs. Maria Ekmark and Mrs. Karin Nllsson for generous assistance in preparing the manuscript. REFERENCES DANON, D., GOLDSTEIN, L., MARIKOVSKY,Y., AND SKUTELSKY, E. (1972) J. Ultrastruct. Res. 38, 500510. DE DUVE, C., DE BARSY,TH., POOLE, B., TROUET,A., TULKENS, P., AND VAN HOOF, f. (1974) Biochem. Pharmacol. 23, 2495-2531.
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DENEF, J.-F., BJORKMAN,U., AND EKHOLM,R. (1980) J. Ultrastruct. Res. 71, 185-202. EAGLE, H. (1959) Science 130, 432-437. EKHOLM, R. (1977) in DUMONT, J., AND NUNEZ, J. (Eds.), Hormones and Cell Regulation, Vol. 1, pp. 91-110, North-Holland, Amsterdam. EKHOLM, R. (1979) in DEGROOT,L. J. (Ed.), Endocrinology, Vol. 1, pp. 305-309, Grune & Stratton, New York. EKHOLM, R., ENGSTROM, G., ERICSON, L. E., AND MELANDER, A. (1975) Endocrinology 97, 337-346. EKHOLM, R., AND WOLLMAN, S. H. (1975) Endocrinology 97, 1432-1444. ERICSON, L. E., AND ENGSTROM, G. (1978) Endocrinology 103, 883-892. ERICSON, L. E,, ENGSTROM, G., AND EKHOLM, R. (1979) Endocrinology 104, 704-710. ERICSON, L. E., AND JOHANSSON, B. R. (1977) Acta Endocrinol. 86, 112-118. FARQUHAR,M. G. (1978) J. Cell Biol. 77, R35-R42. FRIEND, D. S., AND FARQUHAR,M. G. (1967) J. Cell Biol. 35, 357-376. HAGGIS, G. H. (1965) J. Mol. Biol. 14, 598-602. HEIDELBERGER, M., AND PEDERSEN, K. O. (1935) J. Gen. Physiol. 19, 95-108. HERZOG, V., AND FARQUHAR,M. G. (1977) Proc. Na~. Acad. Sci. USA 74, 5073-5078. HERZOG, V., AND MILLER, F. (1979) Eur. J. Cell Biol. 19, 203-215. HEUSER, I. E., AND REESE, T. S. (1973) J. Cell Biol. 57, 315-344. NADLER, N. J., SARKAR, S. K., AND LEBLOND, C. ]:'. (1962) Endocrinology 71, 120-129. NOVIKOFF, A. (1976) Proc. Nat. Acad. Sci. USA 7~;, 2781-2787. ROCMANS, P. A., KETELBANT-BALASSE,P., DUMONT, I. E., AND NEVE, P. (1978) Endocrinology 103, 1834-1848. SCHNEIDER, Y.-I., TULKENS, P., DE DUVE, C., AND TROUET, A. (1979) J. Cell Biol. 82, 466-474. SEI~ELID, R. (1967) J. Ultrastruct. Res. 18, 1-24. SELJELID, R., REITH, A., AND NAKKEN, K. F. (1970) Lab. Invest. 23, 595-605. SILVERSTEIN, S. C., STEINMANN~R. M., AND COHN, Z. A. (1977) Annu. Rev. Biochem. 46, 669-722. SKUTELSKY,E., AND HARDY, B. (1976) Exp. Cell Res. 101, 337-345. STEIN, 0., AND GROSS, J. (1964) Endocrinology 75, 787-798. TICE, L. W., AND WOLLMAN, S. H. (1974) Endocrinology 94, 1555-1567. UI, N. (1971) in FELLINGER,K., AND HOFER, R. (Eds.), Further Advances in Thyroid Research, Vol. 1, pp. 91-96, Verlag Wiener Medizin. Akad., Wien. WOLLMAN,S. H., SPICER, S. S., AND BURSTONE,M. S. (1964) J. Cell Biol. 21, 191-201.
JOURNAL OF ULTRASTRUCTURE RESEARCH
71, 203-221 (1980)
Membrane Labeling with Cationized Ferritin in Isolated Thyroid Follicles J.-F.
DENEF 1 AND
R.
EKHOLM 2
Department of Anatomy, University of G6teborg, S-400 33 G6teborg 33, Sweden Received February 27, 1980 Follicles, closed and open, isolated from rat, pig, and human thyroids, were incubated with polycationized ferritin (CF) in order to label the plasma membrane and follow the fate of internalized membrane. All parts of the plasma membrane, except the pseudopod membrane, were labeled. CF was rapidly (within 5 min) internalized from the apical surface by micropinocytosis. Macropinocytotic vacuoles, formed from pseudopods, were not primarily labeled but received CF by fusion with micropinocytotic vesicles. CF was early present in phagolysosomes and most of the CF was eventually accumulated in these structures. After 30 min CF was found in minute amounts in some Golgi saccules. In all labeled intracellular structures only some of the CF was clearly membrane associated. The labeling of Golgi saccules, although minimal, might indicate a recycling of membrane. However, since CF was not a pure marker of intracellular membranes in thyroid follicle cells, it could not be used to demonstrate the extent or route(s) of membrane recycling.
Endocytosis of thyroglobulin is a key step, controlled by TSH, in the hormone release from the thyroid follicle cell. Two different ways of colloid uptake have been described: macropinocytosis, involving the formation of colloid droplets from pseudopods (Nadler et al., 1962; Stein and Gross, 1964; Wollman et al., 1964) and micropinocytosis (Seljelid, 1967; Seljelid et al., 1970; Rocmans et al., 1978). The relative importance of these processes is not known. Previous studies from this laboratory (Ekholm et al., 1975; Ekholm, 1977; Ericson and Johansson, i977; Ericson and Engstr6m, 1978; Ericson et al., 1979) have shown that an endocytotic response to TSH requires addition of membrane to the apical cell surface by exocytosis. Furthermore, the amount of membrane appearing in endocytotic structures upon TSH stimulation corresponds to the amount of membrane added to the apical cell surface from exocytotic vesicles during the stimulation period. These observations indicate that exocytotic membrane, via the apical plasma membrane, is transferred into endocytotic membrane. Against this background it was i Present address: Laboratory of Histology, Louvain Medical School, U.C.L., B-1200 Brussels, Belgium. 2 To whom requests for reprints should be addressed.
deemed to be of interest to trace the fate of the membrane taken into the follicle cell by endocytosis and to explore whether such internalized membrane is recycled into exocytotic membrane, thus completing the membrane circuit. The present study was performed in an in vitro system, described in a preceding report (Denef et al., 1980) and consisting of isolated, closed and open, thyroid follicles. By incubation with polycationized ferritin, which binds to plasma membrane (Danon et al., 1972), we tried to track the m e m b r a n e taken into the follicle cells by endocytosis. MATERIALS AND METHODS Thyroid glands of about 50 rats and 6 pigs and thyroid biopsies from 6 humans were used in about 20 different labeling experiments. Closed and open follicles Were isolated as described in the preceding paper (Denef et al., 1980). The follicle preparations were preincubated for I hr at 37°C, under continuous gassing with 95% 02, 5% C02, in Tyrode solution supplemented with amino acids according to Eagle (1959), TAA, and containing 0.5% albumin and 2 f~g/ml DNAse. After preincubation, the samples were washed twice in TAA and then incubated in TAA with cationized ferritin (CF), prepared according to Danon et al. (1972) and obtained from Mfles-Yeda, Rehovot, Israel. Incubations with CF (0.1 or 0.5 mg/ml) were performed for 5-180 min at 37°C and under continuous Oe-CO2 gassing. When used, TSH (bovine TSH, a gift from NIH, Bethesda, Md.) was added to the incuba-
203 0022-5320/80/050203-19502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tion medium 10 min before CF at a concentration of 100 or 20 mIU/ml. After incubation, the samples were centrifuged (50g × 3 min), the supernatants were discarded and 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) was added to the pellets. After 60-90 min in glutaraldehyde the specimens were postfixed in osmium tetroxide and then prepared and examined as described in the preceding paper (Denef et al., 1980). RESULTS At all incubation times, most open follicles contained CF in their lumen. A p a r t from some differences in the labeling pattern of the apical plasma membrane, there were no qualitative differences in the labeling of open follicles in rat, pig, and h u m a n preparations. However, quantitatively the labeling of the apical plasma m e m b r a n e and intracellular structures in the pig follicles was greater t h a n the corresponding labeling in rat and h u m a n follicles. T h e pig follicles will therefore be used as basis for the description of the observations. OPEN PIG FOLLICLES Pig follicles showed endocytotic activity when incubated without T S H . Addition of T S H to the m e d i u m increased the n u m b e r of labeled endocytotic structures and the a m o u n t of internalized CF but did not induce any qualitative changes of the labeling of plasma m e m b r a n e or intracellular structures.
Labeling of Plasma Membrane Already at the shortest incubation times (5-15 min) all parts of the plasma membrane were labeled. At these times, the
apical plasma m e m b r a n e was generally more heavily labeled t h a n the other parts. As a rule, CF was accumulated in the pits at the base of the microvilli and to a varying extent in the spaces between the microvilli (Figs. 1, 5, and 6). After longer incubation times (30-60 min) the CF on the apical surface was redistributed into patches separated b y CF-free areas (Fig. 7). With increasing incubation time these patches became smaller and sparser. During the first hour of incubation the basal plasma m e m b r a n e was generally covered by a thin but continuous layer of CF of rather even thickness (Fig. 2). N o t until the second h o u r of incubation did the labeling became patchy, leaving some areas of the m e m b r a n e bare. After 3 hr of incubation some cells still had a basal plasma m e m b r a n e covered by CF while in other cells the basal plasma m e m b r a n e had no CF left. After 5 min of incubation some intercellular spaces were still devoid of CF but after 15 min the label was regularly found, alt h o u g h in varying amounts, along the lateral plasma membranes. M u c h CF was often remaining in the intercellular spaces after 3 hr incubation. In the apical and middle parts of the intercellular spaces, the CF was generally rather scarce and appeared as single particles or in small clusters (Fig. 3). T h e labeling stopped sharply at b o t h sides of the tight junctions (Fig. 3). T h e basal part of the intercellular spaces h a d a richer supply of CF which often filled narrow portions of the space (Fig. 4). T h e intercellular layer of CF was continuous
FIG. 1. Apical zone of a pig thyroid follicle cell. Incubation with CF for 5 rain; TSH. The CF is accumulated in the pits between the microvilli. × 35 000. FIG. 2. Basal zone of a pig follicle cell. Incubation with CF for 15 min. The CF layer is almost continuous but somewhat irregular in thickness. Clusters of CF are entrapped by membrane foldings. Note a labeled micropinocytotic vesicle (arrowhead). × 36 500. FIG. 3. Apical part of intercellular spaces between pig follicle cells. Incubation with CF for 1 hr. CF occurs in the intercellular spaces both in clusters and as single particles. No CF is present in the tight junction (arrowheads). Note the presence of some CF-loaded vesicles in the cytoplasm (arrows). × 39 300. FIG. 4. Basal part of an intercellular space between two pig follicle cells. Incubation with CF for 30 min. The CF forms clusters, sometimes only two molecules in thickness (arrowhead). Some endocytotic vesicles, loaded with CF, are located in the basal region of the cell (arrows). × 35 900.
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with the CF coating the basal plasma membrane (Figs. 2 and 4).
Labeling of Intracellular Structures Micropinocytotic vesicles. At the shortest incubation times (5-10 min) most of the intracellular CF was found in vesicular structures in the apical cell zone; these structures were interpreted to represent micropinocytotic vesicles. The labeled vesicles were smooth-surfaced or bristlecoated (Figs. 5 and 6). The smooth vesicles were round or irregular in shape and varied in diameter between 700 and 3000 A. Except for their content of CF, they appeared empty. The coated vesicles were always round and seemed to belong to two size classes with a diameter of about 700 and more than 1000/~, respectively. Many vesicles, both smooth and coated, were completely filled with CF. In the vesicles containing smaller amounts of label, some CF was concentrated along the membrane, but only exceptionally covering the entire membrane, and the remaining CF was found in the interior of the vesicle. In the smallest vesicles it was not possible to decide whether or not the CF particles were coating the membrane. With increasing incubation time the number of labeled micropinocytotic vesicles decreased and the CF particles showed a tendency to aggregate in small clusters in the interior of the vesicle or close to its membrane (Fig. 7). In addition to the described vesicles, another category of labeled vesicles was observed from 15 min and onward (Fig. 8). These vesicles were characterized by a
207
moderately dense, homogeneous matrix and an extremely varying shape. The CF content was rather low and the particles were dispersed throughout the vesicles. At all incubation times, a few smooth and coated labeled vesicles were seen in the vicinity of the basal and lateral plasma membrane (Figs. 2-4).
Pseudopods and macropinocytotic vacuoles. At all incubation times the pseudopod membrane was much less labeled than the adjoining apical plasma membrane; as a rule the labeling was restricted to a few small patches of CF (Fig. 9). However, some small vesicles containing CF were usually present in the body and the base of the pseudopod. Large vacuoles, some of them more than 1 /tm in diameter, in the pseudopods and supranuclear parts of the cell body were interpreted as macropinocytotic vacuoles, corresponding to the colloid droplets in closed follicles (cf. Denef et al., 1980). Vacuoles located in pseudopods were devoid or almost devoid of CF, irrespective of the time of incubation (Fig. 9); only occasionally one or a few small clusters of CF could be seen close to the vacuole membrane. Vacuoles located in the cell body had a similar, very low labeling at short incubation times (Fig. 10). After longer incubations the vacuoles contained varying amounts of CF, generally in the form of confluent clusters of particles in the peripheral zone of the vacuole, close to its membrane (Fig. 12). These vacuoles were often surrounded by labeled micropinocytotic vesicles and pictures suggesting a fusion
FIG. 5. Apical zone of a pig follicle cell. Incubation with CF for 5 min; T S H . M o s t of t h e micropinocytotic vesicles are filled with CF (arrows) b u t in one relatively large vesicle, t h e CF s e e m s c o n c e n t r a t e d to t h e periphery (arrowhead). × 32 400. Fro. 6. Apical part of a pig follicle cell. Incubation with CF for 10 min; T S H . S o m e of t h e n u m e r o u s micropinocytotic vesicles h a v e a s m o o t h m e m b r a n e (arrowheads) while others have a bristle-coated m e m b r a n e . T h e latter vesicles s e e m to belong to two size classes (small arrows a n d big arrow). A macropinocytotic vacuole (D) with discontinuous peripheral labeling h a s close relation to a labeled micropinocytotic vesicle. × 46 300. FIG. 7. Apical part of a pig follicle cell. I n c u b a t i o n with CF for 1 hr; T S H . T h e apical m e m b r a n e is discontinuously labeled. S o m e labeled s t r u c t u r e s have a c o n t e n t of low density a n d are i n t e r p r e t e d as endocytotic vesicles (small arrows). O t h e r structures, having a denser m a t r i x a n d containing a few C F particles, are difficult to identify (secondary lysosome? (arrowhead) secondarily labeled colloid droplet? (large arrow)). × 38 700.
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D E N E F AND EKHOLM
Fro. 8. Apical part of a pig follicle cell. Incubation with CF for 15 min; TSH. One coated endocytotic vesicle is heavily labeled (small arrow). In addition several structures of varying size and shape and with a moderately dense matrix contain a few disseminated CF particles (arrowheads). Their nature is unclear; they could be endocytotic structures present in the cell before the opening of the follicle and labeled secondarily by fusion with newly labeled endocytotic vesicles; or they could be secondary lysosomes. The identity of the structures marked with asterisks is also unsettled; they might be invaginated macropinocytotic vacuoles. × 45 400.
between such vesicles and a vacuole were sometimes observed (Fig. 11). Typical colloid droplets, filled with a homogeneous substance, were observed in the supranuclear and paranuclear cell regions. These droplets, evidently formed before the
opening of the follicles, sometimes contained clusters of CF (Fig. 13). These clusters were always located in the matrix of the droplet without any obvious relation to the droplet membrane. Phagolysosomes. The labeled bodies in-
FIG. 9. Pseudopod and apical part of a pig follicle cell. Incubation with CF for 10 min; TSH. Although the apical plasma membrane is richly labeled, the pseudopod membrane is almost free of CF; the arrowheads mark small clusters of CF particles along the membrane. Some vesicles in the body and the base of the pseudopod also contain some CF (arrows). No CF is seen in the macropinocytotic vacuoles (D) of the pseudopod. × 44 200. FIG. 10. Macropinocytotic vacuole in the apical zone of a pig follicle cell. Incubation with CF for 15 min; TSH. The few CF particles (arrowheads) at the membrane are probably derived from the pseudopod membrane. × 65 100. FIG. 11. Macropinocytotic vacuole in the supranuclear region of a pig follicle cell. Incubation with CF for 30 min; TSH. The vacuole is surrounded by some labeled micropinocytotic vesicles (arrows) and has a CF-filled evagination which probably represents a labeled vesicle having fused with the vacuole. The small Clusters of CF (arrowheads) in another region of the vacuole could represent primary labeling achieved in the pseudopod. × 49 200. FIG. 12. Macropinocytotic vacuole in the supranuclear region of a pig follicle cell. Incubation with CF for 30 min. The label is concentrated to the periphery of the vacuole and much of the CF seems closely related to the membrane, L, phagolysosome (?). × 60 800. Fro. 13. Colloid droplet in the supranuclear part of a pig follicle cell. Incubation with CF for 30 min. The homogeneous content of the droplet suggests that it was formed before the opening of the follicle. Consequently, the CF in its matrix probably represents secondary labeling, obtained by fusion with a CF-labeled structure. A phagolysosome (L) has a rich, irregularly distributed supply of CF. × 51 800.
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terpreted as phagolysosomes constituted a heterogeneous population of structures. The most characteristic components of this population were multivesicular bodies (cf. Fig. 21, rat follicle cell). Other components were round or elongated bodies with a homogeneous matrix separated from the limiting membrane by an interspace of low density (Fig. 12). The most common components were polymorphous bodies containing a granular substance of moderate to high density and sometimes membrane remnants, particles, and small vesicles (Fig. 13; cf. also Figs. 20 and 23). CF was present in phagolysosomes already after 5 min incubation. The number of labeled lysosomes as well as the amount of CF per lysosome increased with increasing incubation time. The CF was rarely seen in close relation to the limiting membrane but was distributed in clusters of varying size and density in the interior of the lysosome. Golgi-related structures. Labeled Golgi saccules were observed after incubation for 30 min to 2 hr. However, labeling of Golgi saccules was rare; in only a few percent of the sections through cells with a rich intracellular labeling and including the Golgi region was a labeled saccule detected. Only exceptionally was CF seen in more than one saccule of a Golgi region. The number of CF particles seen in a saccule was generally small. The particles were sometimes closely related to the saccule membrane (Figs. 14 and 15) but often such a relation was not evident (Figs. 16, 17, and 24). Vesicles, both smooth and coated, present in the Golgi region were more often labeled than the saccules (Figs. 14 and 24).
Such labeled vesicles were occasionally seen after 15 min incubation but they were more frequent between 30 and 90 min. OPEN HUMAN FOLLICLES
All parts of the plasma membrane were labeled but the labeling pattern of the apical plasma membrane differed from that in the pig follicles: The CF was not concentrated to the pits between the microvilli but formed a continuous layer covering the microvilli: the latter seemed to adhere to each other (Fig. 18). In human follicles incubated without TSH little CF was found intracellularly. Also in the presence of TSH the number of micropinocytotic vesicles was much smaller than in TSH-stimulated pig follicles (Fig. 18). The labeling of the few pseudopods and macropinocytotic vacuoles was similar to that in the pig follicles. Labeled invaginations, some of them with a coated membrane, were rather common along the lateral and basal cell surfaces (Fig. 19). The labeled phagolysosomes did not differ from those in the pig follicles (Fig. 20). Labeled Golgi saccules were extremely rare (Fig. 17). OPEN
RAT FOLLICLES
As was the case in pig follicles, all parts of the plasma membrane were labeled with CF. However, the apical plasma membrane was much less labeled than in the pig follicles. The labeling was patchy at all incubation times; some CF was located in the pits between the microvilli but more CF was present in clusters along the microvilli (Fig. 21). After incubation without TSH very little
FIGs. 14-17. Golgi-related s t r u c t u r e s in pig follicle cells i n c u b a t e d with CF for 60 m i n (Fig. 14) or 30 m i n (Figs. 15 a n d 16) a n d in h u m a n follicle cells i n c u b a t e d with CF for 2 h r (Fig. 17); T S H in all incubations. FIG. 14. Two Golgi saccules contain CF (big arrows); in one saccule t h e CF is closely related to t h e m e m b r a n e . CF is also p r e s e n t in bristle-coated (arrowhead) a n d s m o o t h vesicles (small arrows). × 46 700. FIG. 15. CF particles are closely related to t h e m e m b r a n e in a Golgi saccule, partially coated (arrowheads). CF is also p r e s e n t in t h e o u t e r m o s t saccule in a Golgi stack (arrow). × 62 400. FIG. 16. CF particles lined up in a Golgi saccule. × 57 800. FIG. 17. In Golgi saccules, CF is often located in one e x t r e m i t y (arrow). × 45 300.
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212
DENEF AND EKHOLM
CF was internalized. TSH stimulation increased the amount of CF taken up by the cells but it was far below that in the pig follicles. However, the intracellular labeling did not differ qualitatively from that in the pig follicles and the labeling of the various structures appeared in the same chronological order. Thus, labeled micropinocytotic vesicles appeared within 5 min and small amounts of CF were found in macropinocytotic vacuoles and colloid droplets. Labeled multivesicular bodies were observed already after 5 min incubation (Fig. 21) and after 60 min phagolysosomes were the dominating labeled structures (Fig. 23). Labeled Golgi saccules and Golgi-related vesicles were rarely seen (Fig. 24). CLOSEDFOLLICLES In closed follicles from all three species the labeling of the basal and lateral plasma membrane was similar to that in open follicles. The amount of intracellular CF was much less than in open follicles. After incubation for 30 min and more CF-labeled endocytotic structures were present in the vicinity of the basal and lateral cell surfaces (Figs. 25 and 27) and labeled phagolysosomes were observed in the supranuclear region (Figs. 25 and 26). In a few cases, round or oval vesicles with a rather dense content and containing a few scattered CF particles were found in the apical cell region, sometimes close to the apical plasma membrane (Fig. 26). Small clusters of CF were occasionally seen in follicle lumina (Figs. 26 and 27) and in intracellular lumina (Fig. 25), generally in the vicinity of the apical plasma membrane.
DISCUSSION
CF has been used as a plasma membrane marker in anterior pituitary cells to study membrane retrieval and reutilization (Farquhar, 1978). In this study it was shown that CF was taken up by endocytosis and was recovered in Golgi cisternae and in secretion granules. These observations were interpreted to show that membrane retrieved from the plasmalemma by endocytosis can be recycled to compensate for membrane added during exocytosis. In a similar study on thyroid follicles, published during the preparation of the present work, Herzog and Miller (1979) showed that CF was taken into the follicle cells by endocytosis and reached lysosomes and the Golgi apparatus. They concluded that CF-containing vesicles fuse with Golgi membranes. Most of the observations made by Herzog and Miller were corroborated in the present study but some new observations were made. Moreover, our interpretations are deviating in some respects.
Labeling of Plasma Membrane In open follicles all parts of the plasma membrane were labeled with CF but the apical cell surface was markedly more labeled in the pig and human follicles than in the rat follicles. A possible explanation of this difference might be that the pig and human follicles were more widely open than the rat follicles, making the apical surface more accessible to CF in the former. However, this seems not to be the case since the scanty apical labeling was also seen in rat follicles with plenty of CF in their lumen, and in rat thyroid cell strips which appar-
FIas. 18-20. Human follicle cells incubated with CF for 15 min (Fig. 18), 30 min (Fig. 19), or 3 hr (Fig. 20); TSH in all incubations. Fro. 18. The apical plasma membrane has a continuous layer of CF. The microvilli seem to stick together, separated only by the thin layer of CF. CF-labeled endocytotic vesicles are few and widely dispersed (arrows). ×41 000. Fro. 19. View of the lateral and basal plasma membrane. An active endocytosis is indicated by numerous CF-loaded vesicles (arrows). × 39 000. FIG. 20. After long-time incubation, the CF particles accumulate in phagolysosomes. Note the presence of a crescent-shaped labeled structure (arrowheads); it is probably the same structure as those marked with asterisks in Fig. 8, but sectioned in another plane. × 25 200.
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CATIONIZED FERRITIN IN THYROID FOLLICLES
ently had free access to CF. Also, in rat follicles the CF on the apical cell surface had an irregular distribution, forming clusters both in the pits between the microvilli and along the microvilli, while in pig and human follicles the CF either was concentrated in the pits between the microvilli or formed a continuous layer along the apical surface. Consequently, the density and distribution of anionic sites accessible to CF binding on the apical plasma membrane seem to differ between the rat thyroid on one hand and the pig and human thyroid on the other. The CF labeling of the pseudopod surface was much less than the labeling of the other parts of the apical cell surface; generally only a few small patches of CF were found along the pseudopods. This indicates that a smaller number of negatively charged groups were exposed and available for CF binding on the pseudopod membrane than on the remaining parts of the apical plasma membrane. This in turn indicates that a rapid reorganization occurs of the apical plasma membrane in connection with its formation of the pseudopod. Such an inference is in accord with previous histochemical and autoradiographic observations (Tice and Wollman, 1974; Ekholm and Wollman, 1975) which have demonstrated peroxidase activity in the apical cell membrane but not in the pseudopod membrane. The possible significance of the low CF binding to the pseudopod membrane for exocytosis of thyroglobulin is not obvious. It could be speculated that a relatively low number of negative groups would facilitate
215
the binding of the negatively charged thyroglobulin but, on the other hand, endocytosis of thyroglobulin by pseudopods-colloid droplets is certainly a bulk transport in which the proportion of thyroglobulin bound to the membrane should be of little importance. In contrast to the apical labeling, the labeling of the basal cell surface and the basal part of the lateral surfaces remained uninterrupted and rather even for at least 1 hr of incubation. It is likely that this difference in labeling patterns reflects differences in the properties between the apical and basal-lateral membranes. That such differences exist seems natural considering the special organization of the apical plasma membrane and the special functional demands on that membrane.
Internalization of CF The first intraceUular structures to be labeled were vesicular structures in the apical cell zone. These vesicles were consequently interpreted to be involved in the internalization of CF, hence representing endocytotic structures. The population of' labeled vesicles was polymorphous: Some of the vesicles were bristle-coated; others were smooth-surfaced, generally larger than the bristle-coated vesicles and of different shapes; the vesicles had a varying content, some appearing empty while others had a matrix of low to moderate density. The labeling pattern showed the following: characteristics: At short incubation times, the labeled vesicles were few but heavily labeled; with increasing time of incubation
FIGS. 21-24. Rat follicle cells incubated with CF for 5 min (Fig. 21), 30 min (Figs. 22 and 24), or 60 min (Fig. 23); TSH in all incubations. FIG. 21. The labeling of the apical surface is patchy; most of the CF is found in clusters along the microvilli. However, the presence of a few CF particles in a forming micropinocytotic vesicle (arrowheads in the inset), in an already formed pinocytotic vesicle (arrow), and in a multivesicular body (MV) indicates that endocytosis takes place. Fig. 21, × 25 000; inset, × 50 000. FIG. 22. The labeling of the basal plasma membrane is continuous. The arrow indicates a micropinocytotic vesicle. × 30 100. FIG. 23. After 60 min most of the CF is found in phagolysosomes. × 21 600. FIG. 24. A Golgi saccule is labeled with CF (arrowhead) and CF particles are also found dispersed in two vesicles (arrows). x 41 800.
FIGS. 25-27. CF labeling in closed follicles from rat (Figs. 25 and 26) and human (Fig. 27) thyroids after incubation with TSH. FIG. 25. Incubation for 3 hr. Low-magnification view of part of a closed follicle. (Compare the density of the lumen (upper right corner) and the extrafollicular space (lower left corner).) One cell contains an intracellular lumen with a content of the same density as that of the follicular lumen. CF is present in the intercellular space and is evidently endocytosed (arrowheads) from this. A labeled phagolysosome (asterisk) is located in the supranuclear region. A cluster of CF is seen in the intracellular lumen (arrow). × 14 000. FIG. 26. Incubation for 1 hr. CF particles are dispersed in some dense bodies, probably secondary lysosomes (small arrows). Smaller numbers of CF molecules are present in some less dense vesicles, maybe exocytotic vesicles (long arrows). Note also the presence of CF particles inside the filled lumen (arrowheads). × 46 600. 216
CATIONIZED FERRITIN IN THYROID FOLLICLES
217
concentrated along the membrane; in these vesicles CF was probably a membrane marker. Other vesicles were completely filled with CF; a possible explanation of this could be that the portion of the apical plasma membrane from which the vesicle, was derived was coated with a thick layer of CF. It is evident that in these vesicles only the peripheral CF molecules could function as membrane markers. In still other vesicles the CF was dispersed in the, interior without any visible contact with the membrane; this CF could not be a membrane marker. It is noteworthy that the last distribution pattern was common in matrix-containing vesicles and it cannot be excluded that the matrix itself could modify the binding of CF. As suggested[ above, these structures might be of lysosomal nature. If not, this type of labeling would indicate that the internalized plasma membrane already in the endocytotic vesi.. cles undergoes modifications resulting in a loss of the ability to bind CF. FIG. 27. Incubationfor 3 hr. SomeCF particles are In tissue sections of the thyroid, bristlepresent in the closed lumen, in the vicinity of the coated micropinocytotic vesicles can easily apicalmembrane(arrowheads).The smallendocytotic vesicles (short arrows) might originate from the la- be recognized. However, this category of beled intercellular space (IS). The heavily labeled vesicles certainly represents only a portion body is probably a secondarylysosome(L). × 41 400. of the endocytotic vesicles in the follicle cells. The remaining micropinocytotic ves(but while there still was a rich labeling of icles have no very characteristic features the apical plasma membrane) the number and have been described (in the rat thyroid) of labeled vesicles increased but the label- as smooth-surfaced vesicles of somewhat ing per vesicle decreased. This pattern in- smaller size and lower density than the dicates that not all the labeled vesicles had exocytotic vesicles (Ericson et al., 1979). In obtained their CF by vesiculation of the the present study many bristle-coated veslabeled apical plasma membrane; some of icles contained CF but the majority of the them were probably formed before CF was labeled vesicles, considered to be endocyadded to the incubation medium and were totic, were smooth-surfaced and of rather labeled secondarily by--transient or per- varying size and shape. Most of these vesimanent--fusion with labeled vesicles. The cles had an empty appearance which should matrix-containing, generally sparsely la- be expected as they were formed in open beled, vesicles could not be definitely iden- follicles. However, since such empty-looktiffed; they could either represent endocy- ing vesicles are not seen in tissue sections totic vesicles present in the cell before the (or in isolated closed follicles, cf. Denef et opening of the follicle or they might be of al., 1980) one may ask if they represent lysosomal nature. artifacts, induced by the unphysiological The distribution of CF in the endocytotic environment at the apical cell surface, or if vesicles varied. In some vesicles CF was they correspond to the endocytotic vesicles
218
DENEF AND EKHOLM
described in tissue sections. The latter al- considered as membrane marker. In colloid ternative seems quite possible even if no droplets, CF was never found in close relastructural similarities could be demon- tion to the membrane but distributed in strated; the different content of the vesicles small aggregates throughout the matrix. It in vivo and in vitro--colloid material and seems possible that the content of the colincubation medium, respectively--could loid droplets was responsible for this labelprobably be responsible for differences in ing pattern. For example, the low pI of size and shape between these vesicles and thyroglobulin (Heidelberger and Pedersen, those observed in tissue sections. Two more 1935; Ui, 1971) should favor the binding of observations should be pointed out in this CF to thyroglobulin. connection: One is that the empty-looking vesicles were present after incubation with- CF in Phagolysosomes out CF (Denef et al., 1980) indicating that CF did not induce their formation, and the As soon as 5 rain after addition of CF to other is that the number of labeled empty the incubation medium, small numbers of looking vesicles increased after incubation CF molecules were observed in multivesicwith TSH, indicating that the vesicles were ular bodies. Since at this time endocytotic formed as a physiological process. vesicles were the only other intracellular In macropinocytotic vacuoles present component containing CF, it can be conafter short labeling times in pseudopods cluded that the multivesicular bodies had and the cell body no or very small numbers obtained their CF by fusion with endocyof CF particles were seen. This is in har- totic vesicles. With increasing incubation mony with the observation that the pseu- time the number of labeled phagolysosomes dopod membrane had a sparse CF labeling. as well as their content of CF increased With increasing incubation time the num- progressively. After the longest incubation ber of more heavily labeled vacuoles in- time (3 hr) phagolysosomes were almost creased. CF particles were also found in the only intracellular structure remaining well-filled colloid droplets, obviously labeled. This Shows that CF was accumuformed before the opening of the follicle. lated in phagolysosomes which hence seems Moreover, pictures indicating fusion be- • to be the terminal compartment for most of tween macropinocytotic vacuoles and la- the internalized CF. beled micropinocytotic vesicles were seen. In structures identified as phagolysoThese observations were interpreted to somes no preferential location of CF to the show that the CF present in macropinocy- membrane was seen. Abundantly labeled totic vacuoles was mainly derived from mi- phagolysosomes had CF also in their pecropinocytotic vesicles. Fusions between ripheral zone but it was not possible to micropinocytotic vesicles and colloid drop- decide ff some of this CF was bound to the lets have previously been suggested by Sel- membrane. In any case, the proportion of jelid et al. (1970). Whether the fusion be- CF possibly bound to the membrane was tween the structures responsible for the certainly very small. This could be due to observed transfer of CF was of transient or several factors. First, as pointed out above, already in the endocytotic structures a large permanent nature cannot be decided. In macropinocytotic vacuoles with rela- proportion of the CF was not membrane tively small amounts of CF, the label was bound. Second, CF bound to endocytotic generally concentrated in a peripheral membrane could be detached when endolayer, but with increasing labeling more and cytotic structures fuse with lysosomes. For more CF was found in the interior of the example, due to the protonation occurring vacuole. This indicates that only part of the in lysosomes (De Duve et al., 1974), conjuCF in macropinocytotic vacuoles can be gated basic groups could be transferred into
CATIONIZED FERRITIN IN THYROID FOLLICLES
219
cytotic structures could be responsible for the labeling of the Golgi saccules. The nature of the small labeled vesicles, sometimes bristle-coated, present in small numbers in the Golgi region after 15 min incubation and onward is not clear. However, since labeled vesicles of the same size, often coated, were observed in the apical cell region already after 5 min incubation, it seems possible that they were of endocytotic nature. Bristle-coated vesicles hawe been assumed to be involved in endocytosis and membrane retrieval in several cell types (for review see Silverstein et al., 1977). Although pictures indicating a fusion between labeled coated vesicles and Golgi saccules were never observed, partially coated labeled saccules were occasionally seen. In conclusion, these observations indiCF in Golgi-Related Structures cate that the direct transfer of membrane The rare and scanty labeling of the Golgi and content from the endocytotic compartsaccules could be due to a limited delivery ment to the Golgi compartment is very of CF to the Golgi or a rapid transfer of CF small. This is in agreement with the present through the Golgi to another compartment. knowledge about the physiology of the folIn the follicle cells, the quantitatively most licle cell which is incompatible with an eximportant receiving compartment should tensive direct transfer of thyroglobulin to be the exocytotic vesicles which transfer the Golgi. The possible communication bethyroglobulin into the follicular lumen. tween the apical surface and the Golgi via However, since no extensive labeling of typ- coated vesicles might serve a special purical exocytotic structures was observed, it pose, not related to thyroglobulin intake. Considering the possibility of a transfer seems likely that the sparse labeling of the Golgi was the result of a restricted supply of membrane from the phagolysosomes to the Golgi saccules it is interesting that in of CF. Since CF appeared in Golgi-related struc- pituitary mammotrophs there was a clear tures after the labeling of endocytotic struc- CF labeling in the Golgi (Farquhar, 19781) tures and phagolysosomes, CF in the Golgi whereas in macrophages, internalized CF was traced to lysosomes but could not be could be derived from these two sources. In endocytotic structures, as discussed detected in Golgi cisternae (Skutelsky and above, some of the CF was generally found Hardy, 1976). Thyroid follicle cells resemclose to the membrane, indicating that in ble macrophages in the respect that the these structures CF to a certain extent was traffic of endocytosed material is more or a membrane marker. At all incubation less completely directed toward the lysotimes the amount of CF (membrane-related somes. In this respect the thyroid follicle as well as free) in endocytotic structures cells differ from all other secretory cells. was very large compared to that in Golgi Therefore, the failure of demonstrating CF saccules. This means that even if all Golgi labeling in the Golgi of macrophages and CF was derived from endocytotic structures the poor labeling of the Golgi observed in only a very small proportion of the endo- the present study could indicate that CF, as
their acidic form, thus inducing a decrease of the net negative charge of the membrane. It also seems possible that intralysosomal hydrolysis of the protein part of the ferritin could occur, changing the physicochemical properties of the label. (This would imply that what is seen in the electron microscope is not CF but its iron core (Haggis, 1965).) Third, CF-labeled membrane portions originating from endocytotic structures could rapidly and selectively be pinched off and transferred to the Golgi. However, although this explanation would be in agreement with the observations by Schneider et al. (1979) indicating a selective recycling to the cell surface of plasma membrane constituents of secondary lysosomes, it does not fit with the rare occurrence of labeled Golgi membranes (cf. below).
220
DENEF AND EKHOLM
soon as it has joined the lysosomal compartment, does not function as a membrane marker. This is in accord with the present observations that in phagolysosomes CF was rarely seen to be closely related to the membrane. It should also be pointed out that a loading of phagolysosomes with CF, a substance that evidently cannot be completely degraded by the lysosomal enzymes and is foreign to the follicle cell, might modify the normal behavior of the phagolysosomes, including their membrane. Consequently, the present observations could neither prove nor disprove that a transfer of membrane from the phagolysosomes to the Golgi occurs in vivo.
CF in Closed Follicles In closed follicles, the cells regularly contained CF but as a rule in much smaller amounts than cells in open follicles incubated under similar conditions. In closed follicles, the CF must have been taken into the cells via the basal and lateral cell surfaces which was also indicated by the presence of labeled micropinocytotic vesicles along these surfaces. Most of the internalized CF in closed follicles was present in structures belonging to the lysosomal family, which is to be expected considering the location of CF taken up via the apical surface. However, a more interesting observation was that small clusters of CF were occasionally present in the lumen of closed follicles and in closed intracellular lumina (Denef et al., 1980). The objection might be raised that CF had entered the follicle lumen during a transient leakage between follicle cells. However, it seems improbable that CF in the incubation medium could enter a colloid-filled lumen where the hydrostatic pressure should be higher than in the medium. Moreover, the presence of CF in intracellular lumina could not be explained by intercellular leakage. Hence, it is extremely likely that CF entered these lumina from the follicle cells. The mode of transcellular CF transport
cannot be definitely ascertained but two different routes seem possible. One route, indicated in Fig. 27, should be a direct migration of CF-containing micropinocytotic vesicles from the lateral cell surface to the apical surface where they should empty their content into the lumen. It is possible that this mode of transportation applies only to vesicles formed rather close to the apical surface (but below the cell junctions), thus having a rather short migration distance. The other mode of transport should involve exocytotic vesicles (as indicated in Fig. 26). However, this transportation includes several ambiguous steps. For example, the identification of exocytotic vesicles, a possible candidate for the last part of the intracellular route, is uncertain. Further, if the vesicles indicated in Fig. 26 are exocytotic we do not know where in the cell these vesicles were labeled with CF. Thus, the observations made clearly indicate that CF entering the follicle cell via the basal and lateral surfaces can be transferred into the follicle lumen, but how the transcellular transport occurs cannot be ascertained.
Concluding Remarks The present study shows that in the thyroid follicle cells, CF was internalized from the apical cell surface by micropinocytosis. By the same method some CF was also taken up from the basal and lateral surfaces. By fusion between vesicles, CF was redistributed among the micropinocytotic structures. Macropinocytosis also occurred at the apical cell surface; the macropinocytotic membrane, i.e., the pseudopod membrane, bound only minute amounts of CF but the macropinocytotic vacuoles were labeled secondarily by fusion with CF-containing micropinocytotic vesicles. In the endocytotic structures only part of the CF was clearly associated with the membrane and hence able to serve as a membrane marker. CF appeared early in phagolysosomes and most of the internalized CF was
CATIONIZED FERRITIN IN THYROID FOLLICLES
eventually accumulated in phagolysosomes. In these structures very little CF seemed to be associated with the membrane. In Golgi saccules the labeling was very scanty and the CF appeared only partially membrane-associated. Since the Golgi labeling occurred later than the labeling of endocytotic structures and phagolysosomes, it must be derived from one or both of these compartments. The fact that the amount of labeled Golgi membrane was minimal compared to the amount of labeled endocytotic membrane means that even if all labeled Golgi membrane was derived from endocytotic structures, this Golgi membrane could represent only an insignificant fraction of the labeled endocytotic membrane. Whether any labeled Golgi membrane emanated from phagolysosomes cannot be decided since very little CF was apparently associated with the phagolysosomal membrane. The fact that Golgi membranes were at all labeled suggests that a recycling of membrane occurs in the follicle cells but the extent of this recycling and the link between the endocytotic part of the loop and the Golgi can evidently not be elucidated by using CF as a marker. The occurrence of CF in the lumen of closed follicles showed that CF can be transferred through the follicle cell. However, the mode(s) of transport could not be ascertained. This work was supported by grants from the Swedish Medical Research Council (B80-12X-537) and the National Institutes of Health (AM-18842). We are grateful to Mrs. Gunnel Bokhede for excellent technical assistance and to Mrs. Maria Ekmark and Mrs. Karin Nllsson for generous assistance in preparing the manuscript. REFERENCES DANON, D., GOLDSTEIN, L., MARIKOVSKY,Y., AND SKUTELSKY, E. (1972) J. Ultrastruct. Res. 38, 500510. DE DUVE, C., DE BARSY,TH., POOLE, B., TROUET,A., TULKENS, P., AND VAN HOOF, f. (1974) Biochem. Pharmacol. 23, 2495-2531.
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DENEF, J.-F., BJORKMAN,U., AND EKHOLM,R. (1980) J. Ultrastruct. Res. 71, 185-202. EAGLE, H. (1959) Science 130, 432-437. EKHOLM, R. (1977) in DUMONT, J., AND NUNEZ, J. (Eds.), Hormones and Cell Regulation, Vol. 1, pp. 91-110, North-Holland, Amsterdam. EKHOLM, R. (1979) in DEGROOT,L. J. (Ed.), Endocrinology, Vol. 1, pp. 305-309, Grune & Stratton, New York. EKHOLM, R., ENGSTROM, G., ERICSON, L. E., AND MELANDER, A. (1975) Endocrinology 97, 337-346. EKHOLM, R., AND WOLLMAN, S. H. (1975) Endocrinology 97, 1432-1444. ERICSON, L. E., AND ENGSTROM, G. (1978) Endocrinology 103, 883-892. ERICSON, L. E,, ENGSTROM, G., AND EKHOLM, R. (1979) Endocrinology 104, 704-710. ERICSON, L. E., AND JOHANSSON, B. R. (1977) Acta Endocrinol. 86, 112-118. FARQUHAR,M. G. (1978) J. Cell Biol. 77, R35-R42. FRIEND, D. S., AND FARQUHAR,M. G. (1967) J. Cell Biol. 35, 357-376. HAGGIS, G. H. (1965) J. Mol. Biol. 14, 598-602. HEIDELBERGER, M., AND PEDERSEN, K. O. (1935) J. Gen. Physiol. 19, 95-108. HERZOG, V., AND FARQUHAR,M. G. (1977) Proc. Na~. Acad. Sci. USA 74, 5073-5078. HERZOG, V., AND MILLER, F. (1979) Eur. J. Cell Biol. 19, 203-215. HEUSER, I. E., AND REESE, T. S. (1973) J. Cell Biol. 57, 315-344. NADLER, N. J., SARKAR, S. K., AND LEBLOND, C. ]:'. (1962) Endocrinology 71, 120-129. NOVIKOFF, A. (1976) Proc. Nat. Acad. Sci. USA 7~;, 2781-2787. ROCMANS, P. A., KETELBANT-BALASSE,P., DUMONT, I. E., AND NEVE, P. (1978) Endocrinology 103, 1834-1848. SCHNEIDER, Y.-I., TULKENS, P., DE DUVE, C., AND TROUET, A. (1979) J. Cell Biol. 82, 466-474. SEI~ELID, R. (1967) J. Ultrastruct. Res. 18, 1-24. SELJELID, R., REITH, A., AND NAKKEN, K. F. (1970) Lab. Invest. 23, 595-605. SILVERSTEIN, S. C., STEINMANN~R. M., AND COHN, Z. A. (1977) Annu. Rev. Biochem. 46, 669-722. SKUTELSKY,E., AND HARDY, B. (1976) Exp. Cell Res. 101, 337-345. STEIN, 0., AND GROSS, J. (1964) Endocrinology 75, 787-798. TICE, L. W., AND WOLLMAN, S. H. (1974) Endocrinology 94, 1555-1567. UI, N. (1971) in FELLINGER,K., AND HOFER, R. (Eds.), Further Advances in Thyroid Research, Vol. 1, pp. 91-96, Verlag Wiener Medizin. Akad., Wien. WOLLMAN,S. H., SPICER, S. S., AND BURSTONE,M. S. (1964) J. Cell Biol. 21, 191-201.