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Endocytosis in thyroid follicle cells
© 1967 by Academic Press Inc.
J. ULTRASTRUCTURERESEARCH18, 479-488 (1967)
479
Endocytosis in Thyroid Follicle Cells V. On the Redistribution of Cytosomes following Stimulation with Thyrotropic Hormone ROLF SELJELID
The Department of Pathology, Sabbatsberg Hospital, Karolinska Institatet Medical School, Stockholm, Sweden Received June 23, 1966 In thyroid glands suppressed by the administration of thvroxine, the cytosomes were dispersed throughout the cytoplasm. After stimulation with TSH by the intravenous route, they tended to be located more apically. After stimulation by intrafollicular instillation of TSH, this tendency was even more pronounced; the cytosomes appeared to move toward the apex before colloid droplets were formed, they were often located inside pseudopods on the luminal cell surface, and were often abutting on the plasma membrane. When cysteine was injected into the follicle lumen, no endocytic activity was encountered. Simultaneous administration of TSH by the intravenous route led to an apical movement of cytosomes despite the absence of colloid droplets. The findings were interpreted as indicating that the apical movement of cytosomes is a direct effect of TSH, and not secondary to the occurrence of colloid droplets.
In their pioneering work on thyroid cytochemJstry, Dempsey and Singer (4) reported an apical localization of acid phosphatase-positive granules following stimulation of thyroid activity by exposure to cold. Wetzel et al. (16) and later Seljelid (11) described an apical movement of cytosomes ("lysosomes") after stimulation with thyrotropic hormone (TSH) simultaneously with the occurren~.e of endocytic vacuoles ("colloid droplets"). These findings appeared to be in keeping with observations in several other cell types, where cytosomes had been observed approaching and finally fusing with endocytic vacuoles (for a review, see 3). In fact, images suggesting such a coalescence have also been described in thyroid fddicl cells (13, 16). It appeared (11), however, that the redistribution of cytosomes following the administration of T S H could be observed before the occurrence of endocytic vacuoles. If this was true, it would imply that the apical movement of the cytosomes was a direct effect of T S H and not secondary to the formation of endocytic vacuoles. It was felt that if the endocytic activity of the follicle cells could be suppressed by the 31
671824 J . Ultrastructure Research
480
R. SELJELID TABLE I EXPERIMENTAL CONDITIONS Number of animals
3
Group
Ia
Thyroxine pretreatment
T S H treatment
+
Components of the microinjection fluid
m
1
II
+
m
2 3 4
II IIIa IV
+ + +
2
IV
-
6
V
+
-0.5 IU intravenously 0.01 IU/ml by microinjection 0.01 IU/ml by microinjection 0.5 IU intravenously
Ferritin, cysteine, trypan blue Ferritin, trypan blue Ferritin, TSH, trypan blue Ferritin, TSH, trypan blue Ferritin, cysteine, trypan blue
a A large number of animals treated in a similar way has been described in previous papers
(10, 12). intrafollicular injection of a n inhibitor, a n d the gland could be s i m u l t a n e o u s l y stimulated by the i n t r a v e n o u s a d m i n i s t r a t i o n of TSH, it w o u l d be possible to investigate whether the m o v e m e n t of the cytosomes was i n d e p e n d e n t of the endocytosis. MATERIALS A N D METHODS Young, male Sprague-Dawley rats were used throughout the investigation. A large number of preliminary experiments were carried out; the present report is based upon the results of experiments with 21 animals. In 19 of these, thyroid suppression was accomplished by the administration of 20/~g of sodium-L-thyroxine per animal and day for 5 days (10). Two rats received no thyroxine treatment. Microinjections were carried out in 15 animals. A summary of the materials and treatment of the animals is given in Table I. Some of the materials have been included in a previous paper (ll). In all cases, the microinjection fluid contained equine cadmium-free ferritin,1 20 mg/ml and 0.5 % trypan blue. In some experiments the injection fluid contained 0.01 I U TSH2/ml (group IV). Cysteine was used in concentrations of 10-a M or 5 × 10 -2 M (group V). The injections were performed as described previously (11). A total of 2000-4000/z 3 was injected into single follicles and the tissue fixed 20 minutes after the injection. The glass cannulae were kept in position until the fixation was finished. Six animals were not used for microinjection. All these were pretreated with thyroxine; three of them received 0.5 I U TSH intravenously 20 minutes 1 Pentex, Inc., Kankakee, Illinois, U.S.A. 2 TSH (NIH-TSH-B3) was supplied by the National Institutes of Health, Endocrinology Study Section, U.S.A. FIG. 1. Thyroxine-suppressed gland. The dark cytosomes (C) are dispersed throughout the follicle cell cytoplasm; most of them are located in the perinuclear area. The microvilli (MV) are short and straight. Holes in the section (x) represent artifacts caused by the fixation. N, nucleus; FL, follicle lumen; G, Golgi apparatus, x 18,000.
482
R. SELJELID
before fixation. All tissues were fixed by dripping 3.25 % glutaraldehyde in 0.1 M phosphate buffer (9) onto the gland for 10 minutes; the tissues were subsequently postfixed for 1 hour by immersion in 2 % osmium tetroxide in s-collidine buffer (1). The tissues were dehydrated and embedded in Epon (8). Thin sections were cut with a LKB Ultrotome microtome. They were stained with lead citrate (14) and uranyl acetate (15). Stained as well as unstained sections were examined in a Siemens Elmiskop I electron microscope.
OBSERVATIONS
General The general cytologic effects of thyroxine suppression as well as the effects of stimulation with T S H and the appearance of colloid droplets have been described in previous papers (10-13). The endocytosis following intrafollicular injection of ferritin and T S H has also been detailed elsewhere (11). In the present communication, only cytologic events directly related to the distribution of cytosomes will be described. As reported previously (10), the cytosomes had a dense homogeneous appearance following the fixation scheme here utilized. In sections stained with lead citrate and uranyl acetate, they appeared dark and contrasted strongly against the background cytoplasm, a feature that facilitated the observation of their overall distribution. They were only faintly visible in unstained sections.
Thyroxine-suppressed glands (group I) The cytosomes were dispersed throughout the cytoplasm (Fig. 1). Most of them were found in the perinuclear area; occasional cytosomes were observed in the apical cytoplasm. No pseudopods were seen. The microvilli appeared short and straight. Colloid droplets were only occasionally encountered.
Suppressed glands after intrafollicular administration offerritin and cysteine (group H) The distribution of cytosomes appeared to be the same as in the suppressed glands described above. No pseudopods were found. Colloid droplets were only occasionally observed; they were small and contained no ferritin particles.
Glands stimulated by intravenous administration of TSH (group III) After 20 minutes of T S H action there was a clear tendency toward a more apical localization of the cytosomes (Fig. 2), as compared to the suppressed glands. At the luminal cell surface, pseudopods were observed; the microvilli were long and tortuous. Colloid droplets were frequently encountered both in the pseudopods FIG. 2. Thyroxine-treated animal 20 minutes after the intravenous injection of 0.5 IU TSH. The cytosomes are located in the apical cytoplasm, some in close association (arrow) with a colloid droplet (CD). Note the pleomorphism of the microvilli (MV), as compared to Fig. 1. N, nucleus, x 18,500.
i
484
R. SELJELID
a n d in the apical cytoplasm proper. A very close spatial relationship between colloid droplets a n d cytosomes was frequently observed. Occasionally, cytosomes were present in the pseudopods.
Glands stimulated by intrafollicular administration of TSH (group IV) The findings were largely the same as in glands stimulated by the intravenous administration of TSH. The tendency toward an apical localization of the cytosomes was even more pronounced; cytosomes were frequently observed inside pseudopods and were often found in close association with the plasma membrane (Figs. 3 and 4). Pictures suggesting actual expulsion of cytosomes into the follicle lumen were not observed. Some of the pseudopods did not appear to contain endocytic vacuoles but seemed to be filled with cytosomes. A pronounced apical movement of cytosomes could be observed in many cells where colloid droplets had not occurred.
Suppressed glands after intrafollicular administration of ferritin and cysteine with simultaneous intravenous injection of TSH (group V) In initial experiments, an injection fluid containing 10-3 M cysteine was used. In these follicles there was no clear inhibition of the endocytosis although the impression was gained that the endocytosis was less pronounced than in follicles not treated with cysteine. After the administration of 5 × 10-2 M cysteine, the endocytosis appeared to be completely suppressed, however. No pseudopods were observed; the microvilli were short. Colloid droplets were only occasionally encountered. These were small and usually located basally in the cells. They contained no ferritin and seemed to have been present in the tissue before the microinjection. There was a clear tendency toward an apical distribution of the cytosomes (Fig. 5). DISCUSSION
General The literature o n the m o r p h o l o g y of endocytosis is vast. I n f o r m a t i o n o n the m e c h a n isms governing endocytosis is o n the other h a n d very limited a n d is with few exceptions derived from studies o n p r o t o z o a n s a n d phagocytes (2). Thus, the i n d u c t i o n or i n h i b i t i o n of endecytosis by chemical c o m p o u n d s has been extensively studied in FIG. 3. Follicle cell stimulated by the intrafollicular administration of TSH. A broad pseudopod is protruding into the follicle lumen (FL), where dense ferritin particles are seen. Microvilli (MV) are long and pleomorpbic. Note the close association between a cytosome and the plasma membrane indicated by arrow, x 28,000. FIG. 4. Same experimental conditions as in Fig. 3. Apparently free-floating cytoplasmic structure, assumed to represent pseudopod with the stalk out of the plane of the section. The pseudopod appears studded with cytosomes, some of which lies in close contact with the plasma membrane (arrows). FL, follicle lumen with ferritin particles; FC, follicle cell cytoplasm, x 32,000.
~L
FL
486
R. SELJELID
the amoebae (2). Corresponding studies in mammalian cells are rare (5), and provide little information concerning substances that might be expected to exert inhibitory effects on the type of endocytosis encountered in the thyroid. The engulfment of fluid by some protozoans is morphologically reminiscent of the colloid absorption in follicle cells (2, 7); it appears from studies on these cell types (2, 7) that cysteine in low concentration inhibits the endocytosis, an effect that probably involves the inhibition of an alkaline phosphatase activity on the cell surface, assumed to be essential for the endocytosis (6). Alkaline phosphatase activity has been described on the luminal surface of thyroid follicles (4). With this background, it seemed fruitful to investigate whether cysteine--in concentrations about 10 -~ M - - w o u l d inhibit the engulfment of colloid, Initial experiments were carried out with the above-mentioned concentration in the injection fluid. It appeared, however, that the amount injected, 2000-4000 #~, would be less than one-tenth of the volume of the follicle contents. To make up for the corresponding dilution, a concentration of 5 × 10 -2 M was adopted. The intrafollicular injection of cysteine appeared to inhibit the endocytosis normaUy evoked by T S H administration. Although the absence of signs of endocytosis might be incidental and related to the limited amount of material available, the appearance of the short microvilli (Fig. 6) appears to corroborate the assumption that endocytosis was inhibited. It is not clear whether the absence of endocytosis vacuoles depended solely upon a specific inhibition by cysteine or whether a more general, toxic effect, or the handling of the tissues, might have paralyzed the mechanisms involved in endocytosis under physiologic conditions. Yet the observation remains that a redistribution of cytosomes was encountered in the absence of endocytic vacuoles.
Interpretation of the results The finding that the cytosomes were dispersed throughout the cytoplasm in suppressed glands (10) seems to be at variance with previous communications (16) reporting a basal position of the cytosornes in these glands. It appears from some of the published pictures (Fig. 1 in 16), however, that the discrepancy may only reflect differences in nomenclature. There was a marked apical movement of cytosomes following the intravenous
Fro. 5. Follicle cell after the intrafollicular instillation of ferritin and cysteine with simultaneous administration of TSH by the intravenous route. The dense cytosomes are located in the apical cytoplasm. Note the absence of colloid droplets. The microvilli (MV) are only indistinctly resolved owing to the colloid-ferritin material in the follicle lumen. N, nucleus, x 25,000. FIG. 6. Same specimen as in Fig. 5. Note the short microvilli (arrows). Between these, the colloid substance with ferritin particles is seen. C, cytosomes, x 35,000.
488
R. SELJELID
administration of TSH. This finding is in agreement with previous communications (16). The cysteine experiments seem to indicate that the apical movement was a direct effect of TSH, and not secondary to the occurrence of colloid droplets. This interpretation is supported by the observation of an extreme apical distribution after intrafollicular T S H administration. In these experiments, pseudopods literally filled with cytosomes but devoid of endocytic vacuoles were frequently encountered. Cytosomes were often observed in close association with the plasma membrane. Pictures suggesting discharge of cytosomes through the plasma membrane were not encountered. The finding may indicate, however, that the mechanism governing the apical movement is similar to that responsible for the fusion between cytosomes and endocytic vacuoles (17). It appears that the thyroid follicle cells by virtue of their strict polarity of endocytosis, as well as the possibility of influencing them simultaneously from both sides with different agents, might serve as a convenient experimental system for further exploration of these mechanisms. The microinjections were performed at the Department of Cell Research, Karolinska Institutet; the facilities were made available through the courtesy of Professor Thorbj6rn Caspersson and Doctor Nils R. Ringertz. The skillful technical assistance of Miss Signe Fjeldsenden and Mrs. Sonja Larsson is gratefully acknowledged. The investigation was supported by grant K 66:848 from the Swedish Medical Research Council. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
12. 13.
14. 15. 16. 17.
BENNETT, H. S. and LUFT, J., J. Biophys. Biochem. Cytol. 6, 113 (1959). CHAPMAN-ANDRESEN,C., Compt. Rend. Tray. Lab. Carlsberg 33, 73 (1962). DE DUVE, C. and WATTIAUX,R., Ann. Rev. Physiol. 28, 435 (1966). DEMPSEY,E. W. and SINGER, M., Endocrinology 38, 270 (1946). KAYE, G. I. and DONN, A., Invest. Ophthalmol. 4, 844 (1965). LENTZ, T. L. and BARRNETT,R. J., J. Exptl. Zool. 147, 125 (1961). -J. Ultrastruct. Res. 13, 192 (1965). LUFT, J., J. Cell Biol. 9, 409 (1961). SABATINI,D. D., BENSCH,K. and BARRNETT,R. J., J. CellBiol. 17, 19 (1963). SELJELIO,R., .1". Ultrastruct. Res. In press. -ibid. In press, -ibid. In press. - - - - ibid. In press. VENABLE,J. H. and COGGESHALL,R., J. Cell Biol. 25, 407 (1965). WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). WETZEL,B. K., SPICER, S. S. and WOLLMAN, S. H., d. CelIBiol. 25, 593 (1965). ZUCKER-FRANKLIN,D. and HmSCH, J. G., ar. Exptl. Meat. 120, 569 (1964).
J. ULTRASTRUCTURERESEARCH18, 479-488 (1967)
479
Endocytosis in Thyroid Follicle Cells V. On the Redistribution of Cytosomes following Stimulation with Thyrotropic Hormone ROLF SELJELID
The Department of Pathology, Sabbatsberg Hospital, Karolinska Institatet Medical School, Stockholm, Sweden Received June 23, 1966 In thyroid glands suppressed by the administration of thvroxine, the cytosomes were dispersed throughout the cytoplasm. After stimulation with TSH by the intravenous route, they tended to be located more apically. After stimulation by intrafollicular instillation of TSH, this tendency was even more pronounced; the cytosomes appeared to move toward the apex before colloid droplets were formed, they were often located inside pseudopods on the luminal cell surface, and were often abutting on the plasma membrane. When cysteine was injected into the follicle lumen, no endocytic activity was encountered. Simultaneous administration of TSH by the intravenous route led to an apical movement of cytosomes despite the absence of colloid droplets. The findings were interpreted as indicating that the apical movement of cytosomes is a direct effect of TSH, and not secondary to the occurrence of colloid droplets.
In their pioneering work on thyroid cytochemJstry, Dempsey and Singer (4) reported an apical localization of acid phosphatase-positive granules following stimulation of thyroid activity by exposure to cold. Wetzel et al. (16) and later Seljelid (11) described an apical movement of cytosomes ("lysosomes") after stimulation with thyrotropic hormone (TSH) simultaneously with the occurren~.e of endocytic vacuoles ("colloid droplets"). These findings appeared to be in keeping with observations in several other cell types, where cytosomes had been observed approaching and finally fusing with endocytic vacuoles (for a review, see 3). In fact, images suggesting such a coalescence have also been described in thyroid fddicl cells (13, 16). It appeared (11), however, that the redistribution of cytosomes following the administration of T S H could be observed before the occurrence of endocytic vacuoles. If this was true, it would imply that the apical movement of the cytosomes was a direct effect of T S H and not secondary to the formation of endocytic vacuoles. It was felt that if the endocytic activity of the follicle cells could be suppressed by the 31
671824 J . Ultrastructure Research
480
R. SELJELID TABLE I EXPERIMENTAL CONDITIONS Number of animals
3
Group
Ia
Thyroxine pretreatment
T S H treatment
+
Components of the microinjection fluid
m
1
II
+
m
2 3 4
II IIIa IV
+ + +
2
IV
-
6
V
+
-0.5 IU intravenously 0.01 IU/ml by microinjection 0.01 IU/ml by microinjection 0.5 IU intravenously
Ferritin, cysteine, trypan blue Ferritin, trypan blue Ferritin, TSH, trypan blue Ferritin, TSH, trypan blue Ferritin, cysteine, trypan blue
a A large number of animals treated in a similar way has been described in previous papers
(10, 12). intrafollicular injection of a n inhibitor, a n d the gland could be s i m u l t a n e o u s l y stimulated by the i n t r a v e n o u s a d m i n i s t r a t i o n of TSH, it w o u l d be possible to investigate whether the m o v e m e n t of the cytosomes was i n d e p e n d e n t of the endocytosis. MATERIALS A N D METHODS Young, male Sprague-Dawley rats were used throughout the investigation. A large number of preliminary experiments were carried out; the present report is based upon the results of experiments with 21 animals. In 19 of these, thyroid suppression was accomplished by the administration of 20/~g of sodium-L-thyroxine per animal and day for 5 days (10). Two rats received no thyroxine treatment. Microinjections were carried out in 15 animals. A summary of the materials and treatment of the animals is given in Table I. Some of the materials have been included in a previous paper (ll). In all cases, the microinjection fluid contained equine cadmium-free ferritin,1 20 mg/ml and 0.5 % trypan blue. In some experiments the injection fluid contained 0.01 I U TSH2/ml (group IV). Cysteine was used in concentrations of 10-a M or 5 × 10 -2 M (group V). The injections were performed as described previously (11). A total of 2000-4000/z 3 was injected into single follicles and the tissue fixed 20 minutes after the injection. The glass cannulae were kept in position until the fixation was finished. Six animals were not used for microinjection. All these were pretreated with thyroxine; three of them received 0.5 I U TSH intravenously 20 minutes 1 Pentex, Inc., Kankakee, Illinois, U.S.A. 2 TSH (NIH-TSH-B3) was supplied by the National Institutes of Health, Endocrinology Study Section, U.S.A. FIG. 1. Thyroxine-suppressed gland. The dark cytosomes (C) are dispersed throughout the follicle cell cytoplasm; most of them are located in the perinuclear area. The microvilli (MV) are short and straight. Holes in the section (x) represent artifacts caused by the fixation. N, nucleus; FL, follicle lumen; G, Golgi apparatus, x 18,000.
482
R. SELJELID
before fixation. All tissues were fixed by dripping 3.25 % glutaraldehyde in 0.1 M phosphate buffer (9) onto the gland for 10 minutes; the tissues were subsequently postfixed for 1 hour by immersion in 2 % osmium tetroxide in s-collidine buffer (1). The tissues were dehydrated and embedded in Epon (8). Thin sections were cut with a LKB Ultrotome microtome. They were stained with lead citrate (14) and uranyl acetate (15). Stained as well as unstained sections were examined in a Siemens Elmiskop I electron microscope.
OBSERVATIONS
General The general cytologic effects of thyroxine suppression as well as the effects of stimulation with T S H and the appearance of colloid droplets have been described in previous papers (10-13). The endocytosis following intrafollicular injection of ferritin and T S H has also been detailed elsewhere (11). In the present communication, only cytologic events directly related to the distribution of cytosomes will be described. As reported previously (10), the cytosomes had a dense homogeneous appearance following the fixation scheme here utilized. In sections stained with lead citrate and uranyl acetate, they appeared dark and contrasted strongly against the background cytoplasm, a feature that facilitated the observation of their overall distribution. They were only faintly visible in unstained sections.
Thyroxine-suppressed glands (group I) The cytosomes were dispersed throughout the cytoplasm (Fig. 1). Most of them were found in the perinuclear area; occasional cytosomes were observed in the apical cytoplasm. No pseudopods were seen. The microvilli appeared short and straight. Colloid droplets were only occasionally encountered.
Suppressed glands after intrafollicular administration offerritin and cysteine (group H) The distribution of cytosomes appeared to be the same as in the suppressed glands described above. No pseudopods were found. Colloid droplets were only occasionally observed; they were small and contained no ferritin particles.
Glands stimulated by intravenous administration of TSH (group III) After 20 minutes of T S H action there was a clear tendency toward a more apical localization of the cytosomes (Fig. 2), as compared to the suppressed glands. At the luminal cell surface, pseudopods were observed; the microvilli were long and tortuous. Colloid droplets were frequently encountered both in the pseudopods FIG. 2. Thyroxine-treated animal 20 minutes after the intravenous injection of 0.5 IU TSH. The cytosomes are located in the apical cytoplasm, some in close association (arrow) with a colloid droplet (CD). Note the pleomorphism of the microvilli (MV), as compared to Fig. 1. N, nucleus, x 18,500.
i
484
R. SELJELID
a n d in the apical cytoplasm proper. A very close spatial relationship between colloid droplets a n d cytosomes was frequently observed. Occasionally, cytosomes were present in the pseudopods.
Glands stimulated by intrafollicular administration of TSH (group IV) The findings were largely the same as in glands stimulated by the intravenous administration of TSH. The tendency toward an apical localization of the cytosomes was even more pronounced; cytosomes were frequently observed inside pseudopods and were often found in close association with the plasma membrane (Figs. 3 and 4). Pictures suggesting actual expulsion of cytosomes into the follicle lumen were not observed. Some of the pseudopods did not appear to contain endocytic vacuoles but seemed to be filled with cytosomes. A pronounced apical movement of cytosomes could be observed in many cells where colloid droplets had not occurred.
Suppressed glands after intrafollicular administration of ferritin and cysteine with simultaneous intravenous injection of TSH (group V) In initial experiments, an injection fluid containing 10-3 M cysteine was used. In these follicles there was no clear inhibition of the endocytosis although the impression was gained that the endocytosis was less pronounced than in follicles not treated with cysteine. After the administration of 5 × 10-2 M cysteine, the endocytosis appeared to be completely suppressed, however. No pseudopods were observed; the microvilli were short. Colloid droplets were only occasionally encountered. These were small and usually located basally in the cells. They contained no ferritin and seemed to have been present in the tissue before the microinjection. There was a clear tendency toward an apical distribution of the cytosomes (Fig. 5). DISCUSSION
General The literature o n the m o r p h o l o g y of endocytosis is vast. I n f o r m a t i o n o n the m e c h a n isms governing endocytosis is o n the other h a n d very limited a n d is with few exceptions derived from studies o n p r o t o z o a n s a n d phagocytes (2). Thus, the i n d u c t i o n or i n h i b i t i o n of endecytosis by chemical c o m p o u n d s has been extensively studied in FIG. 3. Follicle cell stimulated by the intrafollicular administration of TSH. A broad pseudopod is protruding into the follicle lumen (FL), where dense ferritin particles are seen. Microvilli (MV) are long and pleomorpbic. Note the close association between a cytosome and the plasma membrane indicated by arrow, x 28,000. FIG. 4. Same experimental conditions as in Fig. 3. Apparently free-floating cytoplasmic structure, assumed to represent pseudopod with the stalk out of the plane of the section. The pseudopod appears studded with cytosomes, some of which lies in close contact with the plasma membrane (arrows). FL, follicle lumen with ferritin particles; FC, follicle cell cytoplasm, x 32,000.
~L
FL
486
R. SELJELID
the amoebae (2). Corresponding studies in mammalian cells are rare (5), and provide little information concerning substances that might be expected to exert inhibitory effects on the type of endocytosis encountered in the thyroid. The engulfment of fluid by some protozoans is morphologically reminiscent of the colloid absorption in follicle cells (2, 7); it appears from studies on these cell types (2, 7) that cysteine in low concentration inhibits the endocytosis, an effect that probably involves the inhibition of an alkaline phosphatase activity on the cell surface, assumed to be essential for the endocytosis (6). Alkaline phosphatase activity has been described on the luminal surface of thyroid follicles (4). With this background, it seemed fruitful to investigate whether cysteine--in concentrations about 10 -~ M - - w o u l d inhibit the engulfment of colloid, Initial experiments were carried out with the above-mentioned concentration in the injection fluid. It appeared, however, that the amount injected, 2000-4000 #~, would be less than one-tenth of the volume of the follicle contents. To make up for the corresponding dilution, a concentration of 5 × 10 -2 M was adopted. The intrafollicular injection of cysteine appeared to inhibit the endocytosis normaUy evoked by T S H administration. Although the absence of signs of endocytosis might be incidental and related to the limited amount of material available, the appearance of the short microvilli (Fig. 6) appears to corroborate the assumption that endocytosis was inhibited. It is not clear whether the absence of endocytosis vacuoles depended solely upon a specific inhibition by cysteine or whether a more general, toxic effect, or the handling of the tissues, might have paralyzed the mechanisms involved in endocytosis under physiologic conditions. Yet the observation remains that a redistribution of cytosomes was encountered in the absence of endocytic vacuoles.
Interpretation of the results The finding that the cytosomes were dispersed throughout the cytoplasm in suppressed glands (10) seems to be at variance with previous communications (16) reporting a basal position of the cytosornes in these glands. It appears from some of the published pictures (Fig. 1 in 16), however, that the discrepancy may only reflect differences in nomenclature. There was a marked apical movement of cytosomes following the intravenous
Fro. 5. Follicle cell after the intrafollicular instillation of ferritin and cysteine with simultaneous administration of TSH by the intravenous route. The dense cytosomes are located in the apical cytoplasm. Note the absence of colloid droplets. The microvilli (MV) are only indistinctly resolved owing to the colloid-ferritin material in the follicle lumen. N, nucleus, x 25,000. FIG. 6. Same specimen as in Fig. 5. Note the short microvilli (arrows). Between these, the colloid substance with ferritin particles is seen. C, cytosomes, x 35,000.
488
R. SELJELID
administration of TSH. This finding is in agreement with previous communications (16). The cysteine experiments seem to indicate that the apical movement was a direct effect of TSH, and not secondary to the occurrence of colloid droplets. This interpretation is supported by the observation of an extreme apical distribution after intrafollicular T S H administration. In these experiments, pseudopods literally filled with cytosomes but devoid of endocytic vacuoles were frequently encountered. Cytosomes were often observed in close association with the plasma membrane. Pictures suggesting discharge of cytosomes through the plasma membrane were not encountered. The finding may indicate, however, that the mechanism governing the apical movement is similar to that responsible for the fusion between cytosomes and endocytic vacuoles (17). It appears that the thyroid follicle cells by virtue of their strict polarity of endocytosis, as well as the possibility of influencing them simultaneously from both sides with different agents, might serve as a convenient experimental system for further exploration of these mechanisms. The microinjections were performed at the Department of Cell Research, Karolinska Institutet; the facilities were made available through the courtesy of Professor Thorbj6rn Caspersson and Doctor Nils R. Ringertz. The skillful technical assistance of Miss Signe Fjeldsenden and Mrs. Sonja Larsson is gratefully acknowledged. The investigation was supported by grant K 66:848 from the Swedish Medical Research Council. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
12. 13.
14. 15. 16. 17.
BENNETT, H. S. and LUFT, J., J. Biophys. Biochem. Cytol. 6, 113 (1959). CHAPMAN-ANDRESEN,C., Compt. Rend. Tray. Lab. Carlsberg 33, 73 (1962). DE DUVE, C. and WATTIAUX,R., Ann. Rev. Physiol. 28, 435 (1966). DEMPSEY,E. W. and SINGER, M., Endocrinology 38, 270 (1946). KAYE, G. I. and DONN, A., Invest. Ophthalmol. 4, 844 (1965). LENTZ, T. L. and BARRNETT,R. J., J. Exptl. Zool. 147, 125 (1961). -J. Ultrastruct. Res. 13, 192 (1965). LUFT, J., J. Cell Biol. 9, 409 (1961). SABATINI,D. D., BENSCH,K. and BARRNETT,R. J., J. CellBiol. 17, 19 (1963). SELJELIO,R., .1". Ultrastruct. Res. In press. -ibid. In press, -ibid. In press. - - - - ibid. In press. VENABLE,J. H. and COGGESHALL,R., J. Cell Biol. 25, 407 (1965). WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). WETZEL,B. K., SPICER, S. S. and WOLLMAN, S. H., d. CelIBiol. 25, 593 (1965). ZUCKER-FRANKLIN,D. and HmSCH, J. G., ar. Exptl. Meat. 120, 569 (1964).