- Email: [email protected]
Translocation of hepatocyte lysosomes following partial hepatectomy and its inhibition by colchicine
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/81/010025-06$02.00/0
Experimental
Cell Research 131 (1981) 25-30
TRANSLOCATION OF HEPATOCYTE LYSOSOMES FOLLOWING PARTIAL HEPATECTOMY AND ITS INHIBITION BY COLCHICINE MICHIO MORI, KATSUHIKO ENOMOTO, MASAAKI and TAMENORI ONOR Department
of Pathology,
Sapporo Medical
SATOH
College, S I, W17, Sapporo, Japan
SUMMARY Hepatocyte microtubules were studied during the translocation of lysosomes which follows partial hepatectomy. In hepatocytes of normal rat liver the lysosomes are seen mainly along the bile canaliculi. After partial hepatectomy, these lysosomes lose their pericanalicular arrangement and move towards large inclusions (‘protein droplets’) which result from the marked pinocytosis induced by the removal of about two-thirds of the liver mass. The lysosomes fuse with the inclusions, and by 4 h after partial hepatectomy most of the inclusions demonstrate acid phosphatase activity histochemically. Many microtubules are found in close proximity to the lysosomes. When the rats are treated with colchicine prior to the partial hepatectomy, events are markedly different. The lysosomes change their location but do not hit the cytoplasmic inclusions, and the inclusions remain negative for acid phosphatase activity even 4 h post-hepatectomy. Quantitative ultrastructural study shows that the volume density of microtubules in the hepatocytes decreases to one-third of that in control hepatocytes. This strongly suggests an involvement of microtubules in giving orientation to the translocation of hepatocyte lysosomes under these conditions.
In a previous study, it was found that partial hepatectomy induces a marked pinocytosis; as a consequence large cytoplasmic inclusions (protein droplets) are formed [ 11. The hepatocyte lysosomes move from their pericanalicular position to the vicinity of inclusions, with which they fuse; thus the inclusions acquire histochemically demonstrable acid phosphatase activity. By 4 h after partial hepatectomy essentially all inclusions show such activity. The role of microtubules in the translocation of hepatocyte lysosomes occurring in this situation was examined, since several lines of evidence have suggested that the microtubular system is engaged in the movement of organelles such as melano-
somes [2] and nuclei [3] in other cells. Morphometric analysis at the ultrastructural level was carried out and microtubule content in hepatocytes was compared in control and colchicine-treated rats. This was correlated with acid phosphatase histochemistry. MATERIALS
AND METHODS
Twenty-two male rats of Wistar strain, weighing 130200 I, were used in the present study. They were main&ned on a standard-solid diet (&iental Yeast Co., Tokyo, Japan) and given water freely. Three rats were partially hepatectomized without any prior treatment. Twelve rats were partially hepatectomized 1 h after intraperitoneal injection of colchicine (Nakarai Chemicals, Kyoto) at a concentration of 0.1 mg/loO g body weight. The surgical procedure was that of Higgins & AnExp Cell Res 131 (1981)
26
Mori et al.
demon [4], which involves removal of approximately two-thirds of the liver mass. Surgery was performed between 9 and 11 in the morning. The animals were fed until just before the operation. After periods varying between 30 min and 4 h following partial hepatectomy, the rats were lightly anesthetized with ether and the liver was perfused through the portal vein with 2.5% glutaraldehyde (Ladd Research Industries, Burlington, Vt) for a few minutes, and further fixed by immersion in Karnovsky’s fixative [5], as modified by Miller & Herzog [6]. Frozen sections approx. 10 pm thick were incubated for acid phosphatase activity by the lead procedure with cytidine monophosphate (Sigma Chemical Co., St Louis, MO) as substrate [7], for 30 min at 3PC, as done previously [ 11. For quantitative ultrastructural studies, livers of both colchicine-treated and untreated rats were fixed at 4 h after partial hepatectomy with 2.5% glutaraldehyde adjusted to pH 7.4 with 0.1 M cacodylate buffer, at 22°C. After perfusion fixation for a few minutes the liver was cut into small cubes and immersed in the same fixative at 22°C for 2 h. After rinsing in cacodylate buffer they were fixed in ice-cold 1% osmium tetroxide for 1 h. They were then immersed in 2.5% uranyl acetate in Verona]-acetate buffer for 1 h at room temperature, dehydrated in ethanols and embedded in Epon 812. Ultrathin sections were stained with lead citrate and examined with the Hitachi H-500 or JEOL 1OOCelectron microscope. Hepatocyte cytoplasm near the space of Disse and bile canaliculi were photographed at a direct magnitication of x 16000, at which magnification microtubules are not evident on the screen. Ten electron micrographs from two blocks of each rat were enlarged 2.2 times and the areas of ground substance of hepatocyte cytoplasm and microtubules were traced on transparent tracing papers. The areas were painted in black, leaving nuclei and cytoplasmic organelles blank, and the areas were measured using a photopattern analyzer (PR-350, Applied Electrics Industry, Tokyo). The volume density of microtubules in the hepatocyte cytoplasm was then calculated.
RESULTS Lysosomes in hepatocytes are readily visualized by acid phosphatase cytochemistry. In control rats, lysosomes visible by light microscopy are mainly found along the bile canaliculi (fig. 1). At 30 min after partial hepatectomy, the pericanalicular arrangement of lysosomes begins to be lost, as cytoplasmic inclusions begin to appear in the peripheral cytoplasm. These inclusions are acid phosphatasenegative. At 1 h after operation, some inclusions have become acid phosphatase-positive by fusion with lysosomes. At 4 h postExp Cell Res 131 (1981)
hepatectomy: (a) lysosomes are no longer demonstrable in the pericanalicular areas; (6) many are seen near the cytoplasmic inclusions; and (c) most inclusions are highly reactive for acid phosphatase (figs 2, 3). In contrast, in rats treated with colchitine prior to partial hepatectomy, most cytoplasmic inclusions are acid phosphatase-negative at 4 h after partial hepatectomy (fig. 4). Although their pericanalicular arrangement is not evident, most lysosomes appear to be scattered in the cytoplasm; they are not found near the inclusions. The number and size of inclusions appear essentially similar, 4 h after partial hepatectomy, whether or not the rats were previously treated with colchicine. Quantitative morphometric study showed that the volume density of microtubules in the hepatocyte cytoplasm is markedly lower if rats are treated with colchicine (table 1). Figs 5-8 show the appearance of the microtubules near the lysosomes in hepatocytes 4 h following partial hepatectomy. DISCUSSION It is well known that lysosomes are engaged in the digestion of various substances, derived from both outside and 1. Frozen section of the liver of control rat, incubated in CMP medium at 37°C for 30 min. The linear arrays of lysosomes in the hepatocytes reflect their pericanalicular arrangement. The Kupffer cells appear black, due to merging of reaction products in their large lysosomes. X200. Figs 2, 3. Frozen sections of the liver 4 h after partial hepatectomy, processed as in fig. 1. The pericanahcular lysosome distribution (fig. 1) is no longer evident. Lysosomes are now found near the surface of cytoplasmic inclusions. Many of the cytoplasmic inclusions (arrows) are already acid phosphatase-positive. (2) x300; (3) x600. Fig. 4. Frozen section of the liver 4 h after partial hepatectomy and 5 h after colchicine (0.1 m&JO g body weight) administration, processed as in fii. 1. Most cytoplasmic inclusions show no acid phosphatase activity. The lysosomes are not aggregated near the inclusions. x600. Fig.
Lysosome movement and microtubules
27
Exp Cell Res 131 (1981)
28
Mori et al.
Figs 5-8. Electron micrographs of portions of hepatocytes around Iysosomes in rats not treated with colchicine, 4 h after partial hepatectomy. Arrows indi-
cate some of the microtubules. (5) ~110000; (6) x60000; (7) x13000; (8) x60000.
inside of the cell. Lysosomes move toward the vacuoles containing these substances and fuse with them, thus bringing hydrolases which degrade the macromolecules.
Nevertheless, the mechanisms involved in this movement of lysosomes are pocrly understood. Although lysosomes might migrate con-
Exp Cell Res I31 (1981)
Lysosome movement and microtubules Table 1. Effect of colchicine on microtubule volume density in hepatocytes Microtubule volume density (X 10-S) Control 4 h after partial hepatectomy
3.979kO.678
Colchicine 0.1 mg/lOO g b.w. 1 h prior to partial hepatectomy; 4 h after partial hepatectomy
1.319kO.407”
a Significantly different from the control (P
tinuously throughout the cytoplasm, they are predominantly found along the bile canaliculi in normal rat hepatocytes, as shown in fig. 1. Soon after partial hepatectomy, they leave their pericanalicular sites and move toward forming cytoplasmic inclusions in order to fuse with them. Since hepatocyte lysosomes are clearly demonstrated by acid phosphatase histochemistry, their movement can be visualized. We used this system in the present study in an attempt to evaluate the state of the microtubules during the translocation of lysosomes by combining treatment of the rats with colchicine with histochemistry. It has been suggested that microtubules are involved in the release of lysosomal enzymes from polymorphonuclear leukocytes
Bl. When colchicine (0.1 mg/lOO g body weight) was administered, the number of microtubules in hepatocytes decreased significantly, by approximately one-third of that present in the controls. The point counting method employed by Reaven & Reaven [9] for measuring microtubules in hepatocytes was not used, since we are equipped with a system for automatic quantitative evaluation of images (photopattern
29
analyzer). With this system we can measure directly the areas of desired structures by scanning the electron micrographs with a narrow slit (0.5X0.04 mm). Following colchicine treatment lysosomes were not found near the cytoplasmic inclusions, and the number of acid phosphatase-positive inclusions at 4 h after partial hepatectomy decreased markedly. These results suggest that microtubules are involved in the translocation of lysosomes. Although the lysosomes changed their location following partial hepatectomy, they are not directed towards the inclusions. Consequently most inclusion bodies remained negative for acid phosphatase at 4 h after partial hepatectomy. Colchicine did not affect the induction of pinocytosis. Cytoplasmic inclusions (huge pinosomes) appeared at 30 min after operation and their average number at 4 h was similar to that in the control. This is in accordance with the data that colchicine has no effect on endocytosis in leukocytes
l-101. It seems unlikely that alterations in the property of the lysosomal membrane were induced by colchicine. Were this the case, the lysosomes should be found close to the inclusions. However, this is not the case. It would appear that the oriented movement of lysosomes to the inclusion bodies requires intact microtubules. This may account for the low bacteriocidal activity of leukocytes in Chediak-Higashi syndrome in man. In this rare genetic disease, a decrease in the microtubules has been noted [ll, 121 but the phagocytic activity and lysosomal enzyme content of the leukocytes are normal [ 133. The mechanism by which microtubules give orientation to lysosomes was not clarified from the present study. Our electron microscopic images strongly suggest that a Exp Cell Res 131 (1981)
30
Mori et al.
fixed array of microtubules may serve to The authors are grateful to Dr Alex B. Novikoff, Department of Pathology, Albert Einstein College of guide lysosomes towards these targets. Medicine, New York, for improving the language Although involvement of 10 nm filaments of the manuscript. This work was partially supported a grant-in-aid (357131) from the Ministry of Educaas well as microtubules in the movement of by tion, Science and Culture, Japan. nuclei has been reported recently [ 141,thus far 10 nm filaments have not been seen around lysosomes. Our conclusion is not completely in acREFERENCES cordance with recent studies of Marzella & 1. Mori, M & Novikoff, A B, J cell biol72 (1977)695. 2. Malawista, S E, J exp med 122 (1965) 361. Glaumann [15, 161, which indicated that 3. Holmes, K V & Choppin, P W, J cell biol39 (1968) vinblastine administration caused a re526. 4. Higgins, C M & Anderson, R M, Arch path 12 markable increase in fusion of lysosomes (1931) 186. with autophagic vacuoles. One of the likely 5. Kamovskv, M J. J cell bio127 (1965) 137a. 6 Miller, F & Herzog, V Z, Z Zellforsch 97 (1969) 84. explanations for the discrepancy is the difNovikoff, A B, Ciba found symp (1963) 36. ference in the type of lysosome studies. i. Zurier, R B, Hoffstein, S & Weissmann, G, J cell biol58 (1973) 27. Although they suggested that ferritin, which 9. Reaven, E P & Reaven, G M, J cell biol77 (1978) was used for labeling secondary lysosomes, 735. 10. Malawista, S E & Bode], P T, J clin invest 46 was frequently found in the autophagic (1967) 786. vacuoles, the idea that secondary lyso- 11. Hinds, K & Dames, B S, Lancet i (1976) 146. somes are involved in the delivery of hy- 12. Oliver, J M, Am j path 85 (1976) 395. 13. Root, R K, Rosenthal, A S & Balestra, D J, J clin drolases into newly formed autophagic invest 51 (1972) 649. 14. Wang, E, Cross, R K & Choppin, P W, J cell biol vacuoles is not generally accepted. Involve83 (1979) 320. ment of GERL is more likely [17]. On the 15. Marzella, L & Glaumann, H, Lab invest 42 (1980) other hand, lysosomes we studied are most16. 8- Ibid 42 (1980) 18. ly, if not exclusively, secondary lysosomes 17. Novikoff, A B, Proc natl acad sci US 73 (1976) 2781. as shown in figs 8 and 9 in ref. [I], and in figs 5-8 in the present paper. Sequential acid phosphatase cytochemistry after antiApril 2, 1980 microtubular reagent administration would Received Revised version received July 22, 1980 Accepted July 29, 1980 help elucidate this problem.
Exp Cell Res 131 (1981)
Printed
in Sweden
Experimental
Cell Research 131 (1981) 25-30
TRANSLOCATION OF HEPATOCYTE LYSOSOMES FOLLOWING PARTIAL HEPATECTOMY AND ITS INHIBITION BY COLCHICINE MICHIO MORI, KATSUHIKO ENOMOTO, MASAAKI and TAMENORI ONOR Department
of Pathology,
Sapporo Medical
SATOH
College, S I, W17, Sapporo, Japan
SUMMARY Hepatocyte microtubules were studied during the translocation of lysosomes which follows partial hepatectomy. In hepatocytes of normal rat liver the lysosomes are seen mainly along the bile canaliculi. After partial hepatectomy, these lysosomes lose their pericanalicular arrangement and move towards large inclusions (‘protein droplets’) which result from the marked pinocytosis induced by the removal of about two-thirds of the liver mass. The lysosomes fuse with the inclusions, and by 4 h after partial hepatectomy most of the inclusions demonstrate acid phosphatase activity histochemically. Many microtubules are found in close proximity to the lysosomes. When the rats are treated with colchicine prior to the partial hepatectomy, events are markedly different. The lysosomes change their location but do not hit the cytoplasmic inclusions, and the inclusions remain negative for acid phosphatase activity even 4 h post-hepatectomy. Quantitative ultrastructural study shows that the volume density of microtubules in the hepatocytes decreases to one-third of that in control hepatocytes. This strongly suggests an involvement of microtubules in giving orientation to the translocation of hepatocyte lysosomes under these conditions.
In a previous study, it was found that partial hepatectomy induces a marked pinocytosis; as a consequence large cytoplasmic inclusions (protein droplets) are formed [ 11. The hepatocyte lysosomes move from their pericanalicular position to the vicinity of inclusions, with which they fuse; thus the inclusions acquire histochemically demonstrable acid phosphatase activity. By 4 h after partial hepatectomy essentially all inclusions show such activity. The role of microtubules in the translocation of hepatocyte lysosomes occurring in this situation was examined, since several lines of evidence have suggested that the microtubular system is engaged in the movement of organelles such as melano-
somes [2] and nuclei [3] in other cells. Morphometric analysis at the ultrastructural level was carried out and microtubule content in hepatocytes was compared in control and colchicine-treated rats. This was correlated with acid phosphatase histochemistry. MATERIALS
AND METHODS
Twenty-two male rats of Wistar strain, weighing 130200 I, were used in the present study. They were main&ned on a standard-solid diet (&iental Yeast Co., Tokyo, Japan) and given water freely. Three rats were partially hepatectomized without any prior treatment. Twelve rats were partially hepatectomized 1 h after intraperitoneal injection of colchicine (Nakarai Chemicals, Kyoto) at a concentration of 0.1 mg/loO g body weight. The surgical procedure was that of Higgins & AnExp Cell Res 131 (1981)
26
Mori et al.
demon [4], which involves removal of approximately two-thirds of the liver mass. Surgery was performed between 9 and 11 in the morning. The animals were fed until just before the operation. After periods varying between 30 min and 4 h following partial hepatectomy, the rats were lightly anesthetized with ether and the liver was perfused through the portal vein with 2.5% glutaraldehyde (Ladd Research Industries, Burlington, Vt) for a few minutes, and further fixed by immersion in Karnovsky’s fixative [5], as modified by Miller & Herzog [6]. Frozen sections approx. 10 pm thick were incubated for acid phosphatase activity by the lead procedure with cytidine monophosphate (Sigma Chemical Co., St Louis, MO) as substrate [7], for 30 min at 3PC, as done previously [ 11. For quantitative ultrastructural studies, livers of both colchicine-treated and untreated rats were fixed at 4 h after partial hepatectomy with 2.5% glutaraldehyde adjusted to pH 7.4 with 0.1 M cacodylate buffer, at 22°C. After perfusion fixation for a few minutes the liver was cut into small cubes and immersed in the same fixative at 22°C for 2 h. After rinsing in cacodylate buffer they were fixed in ice-cold 1% osmium tetroxide for 1 h. They were then immersed in 2.5% uranyl acetate in Verona]-acetate buffer for 1 h at room temperature, dehydrated in ethanols and embedded in Epon 812. Ultrathin sections were stained with lead citrate and examined with the Hitachi H-500 or JEOL 1OOCelectron microscope. Hepatocyte cytoplasm near the space of Disse and bile canaliculi were photographed at a direct magnitication of x 16000, at which magnification microtubules are not evident on the screen. Ten electron micrographs from two blocks of each rat were enlarged 2.2 times and the areas of ground substance of hepatocyte cytoplasm and microtubules were traced on transparent tracing papers. The areas were painted in black, leaving nuclei and cytoplasmic organelles blank, and the areas were measured using a photopattern analyzer (PR-350, Applied Electrics Industry, Tokyo). The volume density of microtubules in the hepatocyte cytoplasm was then calculated.
RESULTS Lysosomes in hepatocytes are readily visualized by acid phosphatase cytochemistry. In control rats, lysosomes visible by light microscopy are mainly found along the bile canaliculi (fig. 1). At 30 min after partial hepatectomy, the pericanalicular arrangement of lysosomes begins to be lost, as cytoplasmic inclusions begin to appear in the peripheral cytoplasm. These inclusions are acid phosphatasenegative. At 1 h after operation, some inclusions have become acid phosphatase-positive by fusion with lysosomes. At 4 h postExp Cell Res 131 (1981)
hepatectomy: (a) lysosomes are no longer demonstrable in the pericanalicular areas; (6) many are seen near the cytoplasmic inclusions; and (c) most inclusions are highly reactive for acid phosphatase (figs 2, 3). In contrast, in rats treated with colchitine prior to partial hepatectomy, most cytoplasmic inclusions are acid phosphatase-negative at 4 h after partial hepatectomy (fig. 4). Although their pericanalicular arrangement is not evident, most lysosomes appear to be scattered in the cytoplasm; they are not found near the inclusions. The number and size of inclusions appear essentially similar, 4 h after partial hepatectomy, whether or not the rats were previously treated with colchicine. Quantitative morphometric study showed that the volume density of microtubules in the hepatocyte cytoplasm is markedly lower if rats are treated with colchicine (table 1). Figs 5-8 show the appearance of the microtubules near the lysosomes in hepatocytes 4 h following partial hepatectomy. DISCUSSION It is well known that lysosomes are engaged in the digestion of various substances, derived from both outside and 1. Frozen section of the liver of control rat, incubated in CMP medium at 37°C for 30 min. The linear arrays of lysosomes in the hepatocytes reflect their pericanalicular arrangement. The Kupffer cells appear black, due to merging of reaction products in their large lysosomes. X200. Figs 2, 3. Frozen sections of the liver 4 h after partial hepatectomy, processed as in fig. 1. The pericanahcular lysosome distribution (fig. 1) is no longer evident. Lysosomes are now found near the surface of cytoplasmic inclusions. Many of the cytoplasmic inclusions (arrows) are already acid phosphatase-positive. (2) x300; (3) x600. Fig. 4. Frozen section of the liver 4 h after partial hepatectomy and 5 h after colchicine (0.1 m&JO g body weight) administration, processed as in fii. 1. Most cytoplasmic inclusions show no acid phosphatase activity. The lysosomes are not aggregated near the inclusions. x600. Fig.
Lysosome movement and microtubules
27
Exp Cell Res 131 (1981)
28
Mori et al.
Figs 5-8. Electron micrographs of portions of hepatocytes around Iysosomes in rats not treated with colchicine, 4 h after partial hepatectomy. Arrows indi-
cate some of the microtubules. (5) ~110000; (6) x60000; (7) x13000; (8) x60000.
inside of the cell. Lysosomes move toward the vacuoles containing these substances and fuse with them, thus bringing hydrolases which degrade the macromolecules.
Nevertheless, the mechanisms involved in this movement of lysosomes are pocrly understood. Although lysosomes might migrate con-
Exp Cell Res I31 (1981)
Lysosome movement and microtubules Table 1. Effect of colchicine on microtubule volume density in hepatocytes Microtubule volume density (X 10-S) Control 4 h after partial hepatectomy
3.979kO.678
Colchicine 0.1 mg/lOO g b.w. 1 h prior to partial hepatectomy; 4 h after partial hepatectomy
1.319kO.407”
a Significantly different from the control (P
tinuously throughout the cytoplasm, they are predominantly found along the bile canaliculi in normal rat hepatocytes, as shown in fig. 1. Soon after partial hepatectomy, they leave their pericanalicular sites and move toward forming cytoplasmic inclusions in order to fuse with them. Since hepatocyte lysosomes are clearly demonstrated by acid phosphatase histochemistry, their movement can be visualized. We used this system in the present study in an attempt to evaluate the state of the microtubules during the translocation of lysosomes by combining treatment of the rats with colchicine with histochemistry. It has been suggested that microtubules are involved in the release of lysosomal enzymes from polymorphonuclear leukocytes
Bl. When colchicine (0.1 mg/lOO g body weight) was administered, the number of microtubules in hepatocytes decreased significantly, by approximately one-third of that present in the controls. The point counting method employed by Reaven & Reaven [9] for measuring microtubules in hepatocytes was not used, since we are equipped with a system for automatic quantitative evaluation of images (photopattern
29
analyzer). With this system we can measure directly the areas of desired structures by scanning the electron micrographs with a narrow slit (0.5X0.04 mm). Following colchicine treatment lysosomes were not found near the cytoplasmic inclusions, and the number of acid phosphatase-positive inclusions at 4 h after partial hepatectomy decreased markedly. These results suggest that microtubules are involved in the translocation of lysosomes. Although the lysosomes changed their location following partial hepatectomy, they are not directed towards the inclusions. Consequently most inclusion bodies remained negative for acid phosphatase at 4 h after partial hepatectomy. Colchicine did not affect the induction of pinocytosis. Cytoplasmic inclusions (huge pinosomes) appeared at 30 min after operation and their average number at 4 h was similar to that in the control. This is in accordance with the data that colchicine has no effect on endocytosis in leukocytes
l-101. It seems unlikely that alterations in the property of the lysosomal membrane were induced by colchicine. Were this the case, the lysosomes should be found close to the inclusions. However, this is not the case. It would appear that the oriented movement of lysosomes to the inclusion bodies requires intact microtubules. This may account for the low bacteriocidal activity of leukocytes in Chediak-Higashi syndrome in man. In this rare genetic disease, a decrease in the microtubules has been noted [ll, 121 but the phagocytic activity and lysosomal enzyme content of the leukocytes are normal [ 133. The mechanism by which microtubules give orientation to lysosomes was not clarified from the present study. Our electron microscopic images strongly suggest that a Exp Cell Res 131 (1981)
30
Mori et al.
fixed array of microtubules may serve to The authors are grateful to Dr Alex B. Novikoff, Department of Pathology, Albert Einstein College of guide lysosomes towards these targets. Medicine, New York, for improving the language Although involvement of 10 nm filaments of the manuscript. This work was partially supported a grant-in-aid (357131) from the Ministry of Educaas well as microtubules in the movement of by tion, Science and Culture, Japan. nuclei has been reported recently [ 141,thus far 10 nm filaments have not been seen around lysosomes. Our conclusion is not completely in acREFERENCES cordance with recent studies of Marzella & 1. Mori, M & Novikoff, A B, J cell biol72 (1977)695. 2. Malawista, S E, J exp med 122 (1965) 361. Glaumann [15, 161, which indicated that 3. Holmes, K V & Choppin, P W, J cell biol39 (1968) vinblastine administration caused a re526. 4. Higgins, C M & Anderson, R M, Arch path 12 markable increase in fusion of lysosomes (1931) 186. with autophagic vacuoles. One of the likely 5. Kamovskv, M J. J cell bio127 (1965) 137a. 6 Miller, F & Herzog, V Z, Z Zellforsch 97 (1969) 84. explanations for the discrepancy is the difNovikoff, A B, Ciba found symp (1963) 36. ference in the type of lysosome studies. i. Zurier, R B, Hoffstein, S & Weissmann, G, J cell biol58 (1973) 27. Although they suggested that ferritin, which 9. Reaven, E P & Reaven, G M, J cell biol77 (1978) was used for labeling secondary lysosomes, 735. 10. Malawista, S E & Bode], P T, J clin invest 46 was frequently found in the autophagic (1967) 786. vacuoles, the idea that secondary lyso- 11. Hinds, K & Dames, B S, Lancet i (1976) 146. somes are involved in the delivery of hy- 12. Oliver, J M, Am j path 85 (1976) 395. 13. Root, R K, Rosenthal, A S & Balestra, D J, J clin drolases into newly formed autophagic invest 51 (1972) 649. 14. Wang, E, Cross, R K & Choppin, P W, J cell biol vacuoles is not generally accepted. Involve83 (1979) 320. ment of GERL is more likely [17]. On the 15. Marzella, L & Glaumann, H, Lab invest 42 (1980) other hand, lysosomes we studied are most16. 8- Ibid 42 (1980) 18. ly, if not exclusively, secondary lysosomes 17. Novikoff, A B, Proc natl acad sci US 73 (1976) 2781. as shown in figs 8 and 9 in ref. [I], and in figs 5-8 in the present paper. Sequential acid phosphatase cytochemistry after antiApril 2, 1980 microtubular reagent administration would Received Revised version received July 22, 1980 Accepted July 29, 1980 help elucidate this problem.
Exp Cell Res 131 (1981)
Printed
in Sweden