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Proc. Nati. Acad. Sci. USA Vol. 73, No. 8, pp.,2781-2787, August 1976 The : A cytochemist's view (A Review) * (electron microscopy/ localizations/GERL/) ALEX B. NOVIKOFF Department of Pathology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 Contributed by Alex B. Novikoff, March 25,1976

ABSTRACT Enzyme cytochemistry has been used, at the The contributions of AcPase cytochemistry to elucidating light and levels, to "mark" cytoplasmic the forms and functions of have been described by of mammalian cells. Catalase cytochemistry per- de Duve (19). Light microscopy of lysosomes with the AcPase mitted identification of microperoxisomes, apparently ubiq- uitous organelles that are attached by numerous slender cofl- procedure and of the Golgi apparatus with the TPPase proce- nections to the endoplasmic reticulum. Thiamine pyrophos- dure, in the small of rat dorsal root ganglia, initiated phatase and acid phosphatase cytochemistry can be used to a series of publications which led to appreciation of specialized distinguish between the Golgi apparatus and a specialized regions of ER. acid-phosphatase-rich region of smooth endoplasmic reticulum (ER) that appears to be involved in: (a) the formation of lyso- GERL and lysosomes somes and melanin granules: (b) the processing and ackaging of secretory materials in endocrine and exocrine cells; and (c) Light microscopic studies of a wide variety of cell types led us the of . The acronym GERL has been given to to identify a close relationship between the location of lysosomes this region of smooth ER because it is located at the inner or and the form and distribution of the apparatus (20, 2, 21). "trans" aspect of the Golgi apparatus and because it appears Golgi to produce various types of Lysosomes. In AcPase preparations of rat dorsal root ganglia not only the stained spherical lysosomes but also some larger stained areas Cytochemistry, "marker ," and specialized were encountered. From their distribution, size, and shape I endoplasmic reticulum regions considered that these areas could be fitted within the cres- By making possible the visualization of organelles in situ, cy- cent-shaped regions formed by portions of the Golgi apparatus tochemistry has aided interpretation of biochemical analyses of subcellular fractions isolated from homogenates. It has added Table 1. Cytochemical marker enzymes a new dimension to understanding electron microscope ob- servations. Cytoplasmic organelles not usually identifiable by Refer- light microscopy can be observed in normal and altered cell Enzyme ence states. In our laboratory we have chosen enzyme activities that Endoplasmic reticulum survive the sufficiently long aldehyde fixation required for in some cell types Nucleoside diphospha- 1, 2 adequate structural preservation of the organelles and that stain tase (NDPase) (EC ("mark") one or more cytoplasmic organelle (Table 1). The 3.6.1.6) manner of preparing the tissue sections and incubation media in a few cell types Peroxidase 3, 4 is described in the references listed in the table. (EC 1.11.1.7) During 1966-1970 a series of publications drew attention to GERL and Acid phosphatase 5 an effect of lead ions employed to trap the phosphate released lysosomes (AcPase) [orthophos- in the course of the phosphatase procedures: that the lead ions phoric-monoester could themselves induce hydrolysis of ATP or other phosphate phosphohydrolase esters. These papers (12-15) cast doubt upon the validity of the (acid optimum), cytochemical procedures. In reply I pointed to: (a) specificities EC 3.1.3.2] of the localizations obtained with specific substrates; (b) the Tyrosinase (mono- 6 similar localizations obtained under conditions where lead- phenol monoxy- induced hydrolysis does not occur; and (c) other observations genase) (EC 1.14.18.1) which made me confident that the observed localizations did (in melanocytes) indeed reveal the intracellular sites of the different phosphatases , Catalase 7, 8 (16-18). Thus, in 1963 we described the NDPase localization including (EC 1.11.1.6) in the endoplasmic reticulum (ER) of rat (Fig. 1) and we microperoxisomes confirmed it by assay of subcellular fractions (1). In addition, Golgi apparatus Thiamine pyrophospha- 9, 2 when purified from isolated fractions the enzyme tase (TPPase) the demonstrates same substrate specificity and optimal con- Mitochondria Enzymes, coenzymes 7, 10 ditions for activity as when studied by the cytochemical pro- that oxidize 3,3'-di- cedure (2). aminobenzidine Abbreviations: AcPase, acid phosphatase; ER, endoplasmic reticulum; NADH-tetrazolium re- 11 NDPase, nucleoside diphosphatase; TPPase, thiamine pyrophospha- ductase (for light tase. microscopy only) * By invitation. From time to time, reviews on scientific and techno- Plasma membrane Nucleoside phospha- 2 logical matters of broad interest are published in the PROCEED- tases INGS. 2781 Downloaded by guest on September 29, 2021 2782 Cell Biology: Novikoff Proc. Natl. Acad. Sci. USA 73 (1976)

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FIGS. 1-8. (Legend appears at bottom of the following page.) Downloaded by guest on September 29, 2021 Proc. Natl. Acad. Sci. USA 73 (1976) Endoplasmic reticulum (Review) 2783 (22). This was confirmed by incubating sections for AcPase contents of small ferritin-like grains, myelin-like figures, and activity and then for TPPase activity. With electron microscopy other structures (25). it was determined that the AcPase-rich region was smooth ER. I named it GERL (23) because this region of smooth ER is lo- GERL and processing and packaging cated at the inner or "trans" (24) aspect of the Golgi apparatus The cytochemical visualization of TPPase and AcPase activities and because it appears to produce various types of Lysosomes has shed new light on the packaging, and perhaps the final (25). processing, steps of secretory materials. With E. Essner, in the The structural relation of GERL and Golgi apparatus has early sixties, we had demonstrated the presence of AcPase ac- been studied best in the neurons of dorsal root ganglia of the rat tivity in nascent secretory granules of both endocrine and ex- (5) and mouse (26). In these cells the relationship appears to be ocrine glands (20). Recently we have returned to studies en- more complex than in other cell types studied in our laboratory gendered by these observations. In cells where TPPase activity (6, 4, 27). Figs. 5-7 illustrate some aspects of this relationship; is demonstrable in only one element of the Golgi apparatus it also see Fig. 40 in ref. 5. Smooth-surfaced , probably part is the trans element, adjacent to GERL, that shows the activity. of GERL, extend into each polygonal compartment of the trans Thus, by incubating sections for TPPase activity and parallel element of the Golgi apparatus. The polygonal arrangement ones for AcPase activity it can readily be determined whether provides a large surface area where molecular interchanges secretory granules arise from GERL or from the trans element between GERL and Golgi apparatus could occur. However, the of the Golgi apparatus. Thus far the findings are different from functional significance of the spatial relations of GERL and what is generally assumed (30, 31). Golgi apparatus remains to be elucidated. This is particularly In an -producing transplantable hamster tumor we important for understanding events in the secretory cells con- conclude (32) that most secretory granules arise from GERL sidered in the next section. and none from the Golgi apparatus, because the granules show Four types of lysosomes seem to arise from GERL: (a) re- AcPase activity and not TPPase activity (Fig. 9). sidual bodies (Fig. 8) in which presumably indigestible residues In the fi cells of the rat the granules also arise from of intracellular digestion are visible; (b) coated vesicles which GERL and not the Golgi apparatus as widely thought. Again may carry lysosomal hydrolases to other cell structures (if so the nascent granules show AcPase activity (Fig. 11) and no they would be primary lysosomes); (c) autophagic , TPPase activity. Unlike the insulinoma, however, the more type 1, in which portions of GERL envelop regions of mature secretory granules do not have demonstrable AcPase containing organelles like mitochondria, ER, or peroxisomes. activity. (When sealed off, lysosomal hydrolases are apparently released We (32) have indicated possible advantages of the trans- into the and the sequestered structures undergo deg- plantable insulinoma for biochemical studies required to test radation to become a .); and (d) autophagic vac- the assumption that when acid phosphatase activity is present uoles, type 2, in which a region of GERL enlarges and portions in granules it is likely that other lysosomal enzymes are there of its membrane are internalized, bringing in bits of cytoplasm as well, and to resolve the issues considered by Steiner et al. (31) like or glycogen. Degradation by lysosomal enzymes in their discussion of the possible involvement of lysosomes in leads to residual body formation, as in autophagic vacuoles, type converting to insulin. Unfortunately there is cur- 1. For a fuller description see another review (25). rently no valid cytochemical procedure generally accepted for An assumption made by cytochemists, apparently valid thus visualizing, at the electron microscope level, the sites of the far, is that when AcPase activity is found in a cell organelle other proteolytic enzymes involved in converting proinsulin to in- lysosomal hydrolases are probably there as well. Unfortunately, sulin. only two cytochemical procedures valid at the ultrastructural The presence of AcPase only in the nascent secretory granules level are currently available for other lysosomal enzymes, in is also seen in the exocrine cells of the pancreas. Fig. 10 shows most cells, and even these are not as convenient as the AcPase the absence of TTPase activity and Fig. 11, the presence of procedure. These enzymes are arylsulfatase and an esterase that AcPase in the nascent secretory granules in the pancreas of the hydrolyzes thiolacetate. Decker (28) has demonstrated both untreated guinea pig. The same is true in the fasted and refed enzyme activities in GERL of neurons. Bentfeld and Bainton guinea pig pancreas and in untreated hamster, rabbit, and rat (29) have shown arylsulfatase activity in GERL of mega- (33). The nascent granules are the "condensing vacuoles" karyocytes. considered by Palade and coworkers to be sites of processing In addition to the demonstrable acid hydrolase activities and packaging of secretory granules; for a review see Palade common to GERL and lysosomes, GERL often shows other (30). We view the condensing vacuole portions as expanded hallmarks of the residual body type of : a relatively cisternal portions of GERL; tubular ER elements attached to thick delimiting membrane, an electron-lucent "halo" between them often take the form of "rigid lamellae" described by membrane and residual body contents, and the presence in the Claude (34) (arrows in Figs. 10 and 12).

FIGS. 1-8 (on preceding page). Fig. 1. Section ofrat liver incubated for NDPase activity, with inosine diphosphate as substrate, from Novikoff and Heus (1). Reaction product is seen in the endoplasmic reticulum (ER) and (NM). The mitochondria (M) are unreactive; the is also labeled (Nu). X23,000. Fig. 2. Section of of C57 black mouse incubated for AcPase activity. The melanolysosomes of the pigment epithelial cell are ringed with reaction product, particularly evident at the arrows. X22,000. Fig. 3. Section ofmouse Harding- Passey melanoma incubated for tyrosinase activity, from Novikoff et al. (6). The Golgi apparatus (G) and rough endoplasmic reticulum (RER) are unreactive. Reaction product is present in GERL (GE) and in premelanosomes and (ME). X22,000. Fig. 4. Section of rat incubated for peroxidase activity, from Novikoff et al. (4). Reaction product is present in the endoplasmic reticulum (ER) and "A granules" (A), but is absent from the "B granules" and residual bodies (L). A portion of the nucleus is seen at N. X16,000. Figs. 5-7. Serial sections of a rat dorsal root ganglion incubated for TPPase activity, from P. M. Novikoff et al. (5). Reaction product is restricted to the trans element of the Golgi apparatus. GERL (GE) is unreactive. Long arrows indicate tubular structures, probably part of GERL. The short arrows indicate a small gap in the Golgi apparatus in Figs. 6 and 7 but this is closed over in Fig. 5, demonstrating that even at the electron microscope level the Golgi apparatus is a continuous organelle throughout the cytoplasm. X32,000. Fig. 8. A section, 0.5 ,um thick, ofa rat dorsal rat ganglion incubated for acid phosphatase activity, from P. M. Novikoff et al. (5). Reaction product is seen in GERL which shows a cisternal portion (C), tubular elements (T), and budding residual bodies (arrows). X40,000. Downloaded by guest on September 29, 2021 2784 Cell Biology: Novikoff Proc. Natl. Acad. Sci. USA 73 (1976)

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FIGS. 9-13. Fig. 9. Section of a transplantable hamster insulinoma, incubated for TPPase activity, from Novikoff et al. (32). Only the in- nermost element of the Golgi apparatus (G) shows reaction product. Most of the crescent-shaped area enclosed by the Golgi apparatus is occupied by GERL. It consists of tubular elements (arrowheads), with nascent secretory granules (S) attached to some, and coated vesicles (C), some still attached to tubular elements and others apparently free in the cytosol. Also labeled are two separated secretory granules (GR); note that they are unreactive. X36,000. Fig. 10. Section of guinea pig pancreas, incubated for TPPase activity, showing part of an exocrine cell. Reaction product is present only in the inner element of the Golgi apparatus (G). Condensing vacuoles are indicated by V and "rigid lamellae" by arrows; both are unreactive. X26,000. Fig. 11. Section of rat pancreas, incubated for AcPase activity, showing part of a j3 cell, from Novikoff et al. (32). The Golgi apparatus (G) is unreactive. Reaction product is seen in GERL (arrows) and nascent secretory granules (GR). Mature unreactive secretory granules are not included in this field. X37,000. Fig. 12. Section ofguinea pig pancreas, incubated for AcPase activity, showing part of an exocrine cell. The Golgi apparatus (G) is unreactive. Reaction product is seen in three condensing vacuoles (V), in "rigid lamellae" (arrows), and in a residual body (RB). X30,000. Fig. 13. Section ofliver from a male hamster fed a high- diet for 2 days, incubated for AcPase activity, from Nehemiah and Novikoff (27). The Golgi apparatus (G) is unreactive. Much reaction product is seen in GERL (GE) and lipolysosomes (LL), within which the lipid site is electron-lucent. Portions of GERL containing very low density lipoprotein-like particles show slight deposits of reaction product (arrows). X29,000. Strikingly similar results have been reported and conclusions direct access from ER to secretory granules for at least some drawn in a preliminary report by Hand and Oliver (35) for the secretory components. The functions of the Golgi apparatus in exorbital lacrimal gland. Earlier AcPase results on epineph- these particular instances remain to be elucidated. Again we rine-secreting cells of adrenal medulla were reported by are limited by the exceedingly few cytochemical procedures Holtzman and Dominitz (36). available Thus, it would be highly desirable to demonstrate Interesting questions raised by these findings are: (a) Do the cytochemically the glycosyl transferases known to be concen- elements of the Golgi apparatus contribute to the secretory trated in isolated subcellular fractions enriched in portions of material in GERL? (b) If so, what is the nature of this contri- the Golgi apparatus, but this is presently not possible. bution and by what mechanisms does transfer from Golgi to in GERL occur? (c) What roles do the coated vesicles derived from GERL, lysosomes, and lipid transformations GERL play? Despite such questions, it seems most likely to us that some We have studied two model situations which suggest an in- secretion processing and the secretion packaging in a variety volvement of GERL and lysosomes [identified cytochemically of secretory cells will prove to be a function of GERL, implying (AcPase) as well as by ultrastructural hallmarks] in lipid Downloaded by guest on September 29, 2021 Proc. Natl. Acad. Sci. USA 73 (1976) Endoplasmic reticulum (Review) 2785 transformations within hepatocytes. However, we will begin demonstrable in the premelanosomes and melanosomes (Fig. by referring to the studies of Essner and Oliver (37) on hepa- 3). These findings have been confirmed, in general, in other tocytes of the beige mouse, regarded as an analogue to the laboratories for other melanomas and for normal melanocytes human Chediak-Higashi disease. They wrote, "In addition to (4147). Since premelanosomes and melanosomes show AcPase marked enlargement and increased complexity, GERL (and (Fig. 2) as well as the typical tripartite delimiting membrane lysosomes) of beige hepatocytes was characterized by accu- of residual bodies, P. Leuenberger and I (48) proposed the term mulations of lipid-like inclusions, dense grains, and membra- melanolysosome. Maul (41) and Maul and Romsdahl (42) nous material...." Essner and Oliver suggest that the patho- suggest that AcPase may be involved in degrading enzymes genesis of Chediak-Higashi disease may involve a defect in lipid after melanization is completed or in degrading old melanin metabolism resulting in the accumulation of lipid within the granules but they present no evidence for the proposals. greatly enlarged GERL and the huge lysosomes (residual bodies) that arise from it. Microperoxisomes in mammalian cells J. Nehemiah and 1(27) have studied the hepatocytes of the The cytochemical 3,3'-diaminobenzidine procedure of Graham Syrian golden hamster, untreated and fed a high cholesterol and Karnovsky (49) has contributed to many areas of cell diet. Unlike any other mammalian hepatocytes we know of, in biology. In our laboratory we modified the procedure to stain the hamster hepatocytes lipid accumulates within lysosomes. peroxisomes and other organelles (3, 7, 10). The alkaline 3,3'- The lipid is stainable by Oil Red 0 and is birefringent; in diaminobenzidine procedure visualizing sites of electron micrographs its site is electron-lucent. Such lipid is catalase (7, 8) served to bridge morphology and biochemistry present in the periportal hepatocytes of untreated male ham- in studying peroxisomes (50). Unlike the -containing sters. On the high cholesterol diet all hepatocytes, in either male peroxisomes found among mammalian cells only in liver and or female hamsters, show enlargement of GERL and accu- kidney, anucleoid peroxisomes, or microperoxisomes, were first mulation of cholesterol-rich lipid in the residual bodies arising revealed by the diaminobenzidine procedure (Figs. 14-16). from GERL (also in adjacent areas of ER); cholesterol levels in Microperoxisomes are present in all cells save for mature the liver rise dramatically. Fig. 13 shows part of the Golgi zone erythrocytes and perhaps other specialized end-stage cells. They in a of a male hamster fed the cholesterol-rich diet differ from the nucleoid-containing peroxisomes in two other for 2 days. AcPase reaction product can be seen in GERL, respects. They are generally smaller and they possess numerous portions of which contain lipid-like (very low density lipopro- slender connections to the ER. So frequent are the continuities tein?) particles, and in lysosomes (residual bodies) filled with of their delimiting membranes with the ER membrane that large electron-lucent lipid. We have termed such lipid-filled microperoxisomes may well be considered as specialized regions lysosomes lipolysosomes. Recently, they have been described of ER in which catalase and other peroxisomal enzymes are in human hepatocytes of patients with Wilson's disease (38). compartmentalized. The roles of these enzymes in animal cells, judging from ultrastructure and from the early stages in their unlike those in plant cells (51), are still to be clarified. development as described by Wake (39), the vitamin-A-con- They taining "lipid droplets" in the "lipocytes" of rat liver are also appear in some manner to be involved in the metabolism, lipolysosomes. transport, and storage of lipid. The studies of P. M. Novikoff et al. (40) also suggest a role ER in thyroid epithelium of GERL in lipid alteration within hepatocytes. A fatty liver cells is induced in rats by feeding a semisynthetic diet rich in orotic If our interpretation (4) is correct there are two secretory acid. The hypolipidemic drug clofibrate (ethyl chlorophe- pathways that by-pass the Golgi apparatus in the thyroid epi- noxyisobutyrate) produces a mild fatty liver on this semisyn- thelial cells. One is like that in pancreas exocrine or endocrine thetic diet. If the two agents are fed together a fatty liver fails cells considered above. So-called "B" granules (Fig. 4) are to develop. In hepatocytes of the untreated rat GERL is not considered to arise from GERL and to carry noniodinated particularly conspicuous, but in rats fed clofibrate with the to the follicular where it is released by orotate GERL is very much enlarged and swollen with material . The second pathway involves the ER generally interpreted as lipid undergoing transformation. Stuffing of which in this cell type has endogenous peroxidase activity. GERL with lipid-like particles is even more prominent when Peroxidase-positive "A" granules (Fig. 4), smaller than the a fatty liver is first induced in the rat by orotate and then clo- peroxidase-negative "B" granules, appear to bud directly from fibrate is added to the diet, causing the fatty liver to disappear the apical ER and to bring the peroxidase to the lumen where and hepatocyte ultrastructure to revert to normal (P. M. No- it is secreted via exocytosis. vikoff and D. Edelstein, unpublished work). In the reversal studies GERL was identified not only by characteristic ultra- Conclusion structural features but also by AcPase cytochemistry (P. M. We have briefly reviewed how cytochemistry, by its demon- Novikoff, personal communication). stration of specific enzyme localizations in situ, can help solve Biochemical, autoradiographic, and morphological evidence problems in cell biology. We have emphasized a current focus (summarized in ref. 40) has established the ER as the organelle of our laboratory, namely, the complexity of the ER. In addition through which lipid is transported by the hepatocyte. In GERL, to its well-known roles in synthesis, the ER appears to and in lysosomes derived from it, the lipid acquires a different have regions with specialized functions. For example, in vir- appearance. The biochemical events associated with the tually all mammalian cells, microperoxisomes attached to the transformation cannot be revealed by cytochemistry. Isolation ER are sites where catalase and probably other peroxisomal procedures are required. enzymes are localized. In diverse cell types-including neurons, exocrine and endocrine cells, hepatocytes, and melanocytes- GERL in melanocytes the region of ER near the Golgi apparatus known as GERL In 1968 we reported that in the B-16 and Harding-Passey mouse possesses AcPase and morphological features in common with melanomas GERL possesses tyrosinase ("dopaoxidase") activity lysosomes (residual bodies) that appear to arise from it. We as well as AcPase activity (6). Both enzyme activities are also interpret our observations as revealing different transformations Downloaded by guest on September 29, 2021 2786 Cell Biology: Novikoff Proc. Natl. Acad. Sci. USA 73 (1976)

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A ( FIGS. 14-16. Fig. 14. Section of rat thyroid gland incubated in the alkaline 3,3'-diaminobenzidine for visualizing catalase sites (8), from Novikoff et al. (4). Only the microperoxisomes show reaction product. The arrows indicate two regions where microperoxisome membrane and membrane of the endoplasmic reticulum (ER) are probably continuous. X35,000. Fig. 15. Section of guinea pig pancreas incubated as in Fig. 14, from Novikoff et al. (52). Three reactive microperoxisomes are seen on two zymogen surfaces (Z). Note the proximity of endoplasmic reticulum (ER) to the upper left microperoxisome. X43,000. Fig. 16. Section of guinea pig ileum incubated as in Fig. 14, from Novikoff and Novikoff (53). Reactive microperoxisomes are numbered from 1 to 4. (R) are seen on some areas of ER. Arrowheads indicate continuities between microperoxisome membranes and membrane of the ER. X76,000.

within GERL-producing autophagic vacuoles and coated 8. Novikoff, A. B., Novikoff, P. M., Davis, C. & Quintana, N. (1972) vesicles as well as residual bodies, packaging secretory materials, J. Histochem. Cytochem. 20,1006-1023. changing the nature of lipid in transit within the ER, and 9. Novikoff, A. B. & Goldfischer, S. (1961) Proc. Nati. Acad. Sci. of USA 47,802-810. forming premelanosomes. Yet another type secretory 10. Novikoff, A. B. (1974) in The Proceedings of the Second Inter- mechanism is seen in thyroid epithelium cells, where some se- national Symposium on Electron Microscopy and Cytochem- cretory granules appear to bud from the apical ER. As in the istry, eds. Daems, W.Th., Molenaar, I. & Van Duijn, P. (North- mechanisms involving GERL, the Golgi apparatus would ap- Holland Publishing Co., Amsterdam), pp. 89-109. pear to be bypassed. The functional relations of the Golgi ap- 11. Novikoff, A. B., Shin, W.-Y. & Drucker, J. (1961) J. Biophys. paratus to GERL remain to be elucidated in all cell types we Biochem. Cytol. 9,47-61. have studied. 12. Rosenthal, A. S., Moses, H. L., Beaver, D. L. & Schuffman, S. S. (1966) J. Histochem. Cytochem. 14,698-701. 13. Moses, H. L., Rosenthal, A. S., Beaver, D. L. & Schuffman, S. S. I gratefully acknowledge the preparation of the final photographs (1966) J. Histochem. Cytochem. 14,702-710. by Mr. George Dominguez and the typing of successive versions by 14. Ganote, C. E., Rosenthal, A. S., Moses, H. L. & Tice, L. W. (1969) Ms. Fay Grad. This work was supported by U.S. Public Health Service J. Histochem. Cytochem. 17,641-650. Research Grant CA06576 and the John Polachek Foundation for 15. Rosenthal, A. S., Moses, H. L. & Ganote, C. E. (1970) J. Histo- Medical Research. The author is the recipient of the U.S. Public Health chem. Cytochem. 18,915. Service Research Career Award 5K6 CA14923 from the National 16. Novikoff, A. B. (1967) J. Histochem. Cytochem. 15,353-354. Cancer Institute. Needless to say, many colleagues have contributed 17. Novikoff, A. B. (1970) J. Histochem. Cytochem. 18,366. to the work and ideas expressed in this review, but any failings are mine 18. Novikoff, A. B. (1970) J. Histochem. Cytochem. 18,916. alone. 19. de Duve, C. (1969) in Lysosomes in Biology and Pathology, eds. Dingle, J. T. & Fell, H. B. (North-Holland Publishing Co., Am- 1. Novikoff, A. B. & Heus, M. (1963) J. Biol. Chem. 238, 710- sterdam), Vol. 1, pp. 3-40. 716. 20. Novikoff, A. B. & Essner, E. (1962) Fed. Proc. 21, 1130-1142. 2. Novikoff, A. B., Essner, E., Goldfischer, S. & Heus, M. (1962) in 21. Novikoff, A. B. (1963) in Ciba Foundation Symposium on Ly- The Interpretation of Ultrastructure, ed. Harris, R.J.C. (Aca- sosomes, eds. deReuck, A. V. S. & Cameron, M. P. (J. & A. demic Press, New York), Vol. 1, pp. 149-192. Churchill, Ltd.-Little, Brown, & Co., Boston), pp. 36-73. 3. Novikoff, A. B., Beard, M. E., Albala, A., Sheid, B., Quintana, N. 22. Novikoff, A. B. (1967) in The , ed. Hyden, H. (Elsevier & Biempica, L. (1971) J. Microsc. (Paris) 12,381-404. Publishing Co., Amsterdam), chap. 6, pp. 255-318. 4. Novikoff, A. B., Novikoff, P. M., Ma, M., Shin, W.-Y. & Quintana, 23. Novikoff, A. B. (1964) Biol. Bull. 127,358. N. (1974) in Advances in Cytopharmacology, eds. Ceccarelli, 24. Ehrenreich, J. H., Bergeron, J. J. M., Siekevitz, P. & Palade, G. B., Clementi, F. & Meldolesi, J. (Raven Press, New York), Vol. (1973) J. Cell Biol. 59, 45-72. 2, pp. 349-368. 25. Novikoff, A. B. (1973) in Lysosomes and Storage Diseases, eds. 5. Novikoff, P. M., Novikoff, A. B., Quintana, N. & Hauw, J.-J. Hers, G. & van Hoof, F. (Academic Press, New York), pp. (1971) J. Cell Biol. 50, 859-886. 1-41. 6. Novikoff, A. B., Albala, A. & Biempica, L. (1968) J. Histochem. 26. Boutry, J.-M. & Novikoff, A. B. (1975) Proc. Natl. Acad. Sci. USA Cytochem. 16, 299-319. 72,508-512. 7. Novikoff, A. B. & Goldfischer, S. (1969) J. Histochem. Cytochem. 27. Nehemiah, J. L. & Novikoff, A. B. (1974) Exp. Mol. Pathol. 21, 17,675-680. 398-423. Downloaded by guest on September 29, 2021 Proc. Nati. Acad. Sci. USA 73 (1976) Endoplasmic reticulum (Review) 2787

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