HistochemicalJournal, 9 (1977), 525-551

Cytochemical contributions to differentiating GERL from the Golgi apparatus* A. B. N O V I K O F F a n d P. M. N O V I K O F F Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, USA

Received 11 April 1977

Synopsis. Recent studies from our laboratory are described which deal with endocrine cells (insulinoma, /3-cells of the pancreas, thyroid epithelial cells), pancreatic exocrine cells, and hepatocytes. These emphasize the importance of the hydrolase-rich specialized region of endoplasmic reticulum, known as GERL, in secretory cells. Also reviewed in this paper are the varied molecular transformations which apparently occur in GERL in different cell types, as reported from other laboratories as well as our own. Evidence of the continuity of GERL with rough endoplasmic reticulum is presented. Two hydrolytic enzyme activities in GERL, in addition to acid phosphatase activity, are recorded. Finally, the use of cytochemical staining procedures in the study of microperoxisomes is briefly described.

Introduction We have three purposes in this communication: (a) to indicate how phosphatase cytochemistry has led to formulation of new questions regarding secretory mechanisms in endocrine and exocrine cells; (b) to indicate the contribution made by cytochemistry [mostly phosphatase cytochemistry, and also 3,3'-diaminobenzidine (DAB) cytochemistry] in revealing specializations in the endoplasmic reticulum (ER) and organelles associated with ER, namely GERL and microperoxisomes; and (c) te record developments in acquiring knowledge of GERL, an organelle present in a variety of cell types in mammalian, invertebrate, and plant cells. In an abstract, one of us (A.B. Novikoff, 1964) summarized studies of small neurons of rat dorsal root ganglia and first usect tlie acronym, GERL. "Between the cell nucleus and the innermost of the three Golgi saccules there is a region of smooth ER with a complex, specialized structure in which localized accumulations of *The Histochemical Journal lecture 1976. Delivered to the Histochemistry and Cytochemistry Section of the Royal Microscopical Society on 14 September 1976.

9 1977 Chapman and HalILtd. Printed in GreatBritain.

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ferritin-like grains and continuities with 'coated vesicles' are common. The images suggest that both dense bodies (lysosomes) and 'coated vesicles' arise from it. Its continuity with ribosome-studded ER has been observed but not with Golgi saccules. 9 The specialized region of ER is referred to as GERL, to suggest that it is intimately related to the Golgi saccule (G), that it is part of the ER, and that it forms lysosomes (L)." Confidence in interpretation of the electron microscope images was based on the study by light and electron microscopy of frozen sections made from aldehyde-fixed tissue incubated for thiamine pyrophosphatase activity, to visualize the Golgi apparatus, and for acid phosphatase activity to visualize GERL and lysosomes (A.B. Novikoff, 1964; A.B. Novikoff et al., 1964). Continued study by electronmicroscopy strengthened the evidence that many cell types, but not necessarily all, possess GERL - a hydrolase-rich region of ER, spatially related to the Golgi apparatus (but not part of it) from which lysosomes appear to form. In early studies, the continuity of GERL with rough (i.e., ribosome-studded) ER was inferred from examination of serial sections. Direct continuities in the plane of a given thin section have now been observed and these will be reviewed here. Also recorded is evidence, from other laboratories, that not only acid phosphatase activity but aryl sulphatase activity and E-600 resistant esterase activity, both known to be localized in lysosomes, are also present in GERL. A description of the kinds of lysosomes apparently arising from GERL (coated vesicles, residual bodies and autophagic vacuoles) may be found in publications from our laboratory (P.M. Novikoff et al9 1971; A. B. Novikoff, 1973). Materials and m e t h o d s

The methods currently employed in our laboratory begin with aldehyde fixation, initially by perfusion, where possible. We use a formaldehyde-glutaraldehyde mixture of Karnovsky (1965). It consists of a final concentration of 2% formaldehyde (prepared from paraformaldehyde as he describes), 2.5% glutaraldehyde (purchased either from Ladd Research Industries, Burlington, Vermont, or TAAB Laboratories, Emmergren, Reading, England), and 0.025% CaC12 in 0.09 M cacodylate buffer, pH 7.4. Total fixation time of most tissues varies from 90 to 180 min (Figs. 6, 12, 13 & 1 7 - 1 9 are of unincubated tissue; the manner of fixation is indicated in the captions)9 Thiamine pyrophosphatase activity in the Golgi apparatus shows greater inhibition by glutaraldehyde than the acid phosphatase activity in GERL and lysosomes. Cells in monolayer or in suspension require much shorter fixation times (Lane & Novikoff, 1965). Localization of catalase is possible following even longer fixation in glutaraldehyde or in formaldehyde-glutaraldehyde mixtures. Following rinsing in 0.1 M cacodylate buffer, pH 7.4, with 5% sucrose, generally overnight, solid tissues are sectioned in either of two ways for light microscopy. Frozen sections are prepared with a routine freezing microtome or non-frozen sections are prepared with an Ivan Sorvall tissue sectioner (Smith & Farquhar, 1965). We now use only the latter for electron microsocopy. Incubations of freely-floating sections are carried out while being shaken at room temperatures, in the cold, or most frequently, at 37~ Incubation media consist of the following. (a) For thiamine pyrophosphatase activity, 25 mg thiamine pyrophosphate, sodium salt (Sigma Chemical Co, St. Louis,

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Mo.)*; 7 ml distilled water; 10 ml 0.2 M tris-maleate buffer, pH 7.2; 5 ml 0.025 M manganese chloride; and 3 ml 1% lead nitrate (A. B. Novikoff & Goldfischer, 1961). The medium is filtered before use and renewed after every 30 min of incubation. (b) For acid phosphatase activity, 25 mg cytidine-5'-monophosphate (CMP), sodium salt (Sigma Chemical Co); 12 ml distilled water; 10 ml 0.05 M acetate buffer, pH 5.0, and 3 ml 1% lead nitrate (A. B. Novikoff, 1963). The medium is filtered after a precipitate forms; and (c) for the localization of catalase, 20 mg DAB tetrahydrochloride (Sigma Chemical Co); 9.3 ml 0.05 M propanediol buffer, pH 9.7; adjuft pH to 9.7 with 1 N NaOH; 0.5 ml 0.1 M potassium cyanide (freshly prepared); 0.2 ml 2.5% hydrogen peroxide, freshly prepared from 30% H202 (Merck) (A. B. Novikoff et al., 1972). When sections are to be incubated for electron microscopy, the media contain 5% sucrose. Subsequent processing for electron microscopy is the same for unincubated and incubated tissue. The tissues are rinsed several times in cold 7.5% sucrose, treated with 0.5% uranyl acetate in veronal-acetate buffer, pH 5.0 for 60 min at room temperature in the dark (Kellenberger et al. 1958; Farquhar and Palade, 1965). After dehydration in ethanols, the tissues are treated with propylene oxide and embedded in Epon 812 according to Luft (1961). Thick sections (Figs. 1-3) and thin sections (Figs. 4 - 1 9 ) are cut on an LKB microtome with a DuPont diamond knife. They are observed. unstained (Figs. 1-3) or after staining in lead citrate (Reynolds, 1963), in either a Siemens Elmiskop I electron microscope or a Philips 300 electron microscope equipped with a goniometer stage. Results Neurons Figs. 20 and 21 show early light micrographs, taken in our laboratory in 1964, of small neurons in the rat dorsal root ganglia. Fig. 20 is of tissue incubated for thiamine pyrophosphatase activity which visualizes the Golgi apparatus. Fig. 21 is of tissue incubated for acid phosphatase activity which visualizes numerous dot-like lysosomes and occasional horseshoe-shaped areas of GERL. Focussing at the microscope suggested that the Golgi apparatus was a continuous structure. When sections were first incubated for acid phosphatase activity and then, following a rinse in water, for thiamine pyrophosphatase activity (prior to visualization of the reaction product by ammonium sulphide) the visualized areas of GERL appeared to be located within the visualized areas of the Golgi apparatus. Only electron microscopy could demonstrate unequivocally both this spatial relationship of GERL and Golgi apparatus and the continuity of the Golgi apparatus suggested by light microscopy (A. B. Novikoff, 1967; P. M. Novikoff et al., 1971). In the 1971 study, 0.5 gm as well as thin sections (including serial sections) were used. This established that: (a) GERL is situated close to the inner aspect, what we *Fig. 1 is from a section where IDP was the substrate rather than TPP.Since the work of Yamazaki & Hayaishi (1968) we have used thiamine pyrophosphatase and nucleoside diphosphatase interchangeably, recognizingthat their work was done only on a highly purified livermicrosomalnucleoside diphosphatase and extension to other cell types or other cell organdies may prove unwise.

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now refer to as the trans aspect (Ehrenreich et al., 1973) of the Golgi 'stack' (the portion of the Golgi apparatus as it appears in the thin sections used in electron microscopy); (b)the Golgi apparatus is a continuous network coursing through the cytoplasm, at the level of electron microscopy as well as light microscopy; (c)the thiamine pyrophosphatase procedure and the classical osmication procedures give the same images of the Golgi apparatus at the light microscope level. [In these neurons, as in many but not all cell types (Novikoff & Goldfischer, 1961; Novikoff & Essner, 1962; Novikoff et al., 1962) the osmium impregnates the outer, or cis element (Ehrenreich et al., 1973) of the Golgi apparatus and the thiamine pyrophosphatase procedure visualizes the trans element. All the Gotgi elements are parallel to each other and the distance between cis and trans elements is below the level of resolution of the light microscope.] ; and (d) in the neurons under study, the trans Golgi element is not a simple flat 'saccule' or 'cisterna' but, rather, it consists of membrane in the form of tubules which surround 'polygonal compartments'. Fig. 22 is a diagram attempting to show the three-dimensional relationships of these organelles to each other and to the ER. Figs. 1 - 4 are electron micrographs from the 1971 study. Others may be found in the original publication (P. M. Novikoff et al., 1971). Boutry & Novikoff (1975) reported observations on neurons in the mouse dorsal root ganglia. Two interesting differences between the ganglia of mouse and rat are that in the mouse: (a) in the small neurons, but not in the large neurons (Fig. 5), the Golgi elements, as well as GERL and the lysosomes apparently derived from GERL, show acid phosphatase activity; and (b) the residual bodies of the small neurons display

Figures 1 & 2. Portion of a small neuron from dorsal root ganglion of foetal rat, from P. M. Novikoff et al. (1971). Fixation: formaldehyde-glutaraldehyde, 4~ 60 min. Incubated in thiamine pyrophosphatase medium, 60 rain, 37~ 0.5 pm section not stained with lead. Fig. 1, photographed at a tilt of - 4 5 ~ and Fig. 2, at a tilt of +45 ~ from the initial 0 ~ position. Only the trans element of the Golgi apparatus shows reaction product. Its polygonal compartments (coloured orange in Fig. 22) show more clearly in Fig. 1 for the region marked 2, and in Fig. 2 for the region marked 1. In Fig. 2 the view of region 2 creates the impression of 2 or even 3 straight portions that most investigators would call 'saccules' or 'cisternae' as one of us (ABN) did earlier [Fig. 43 in A.B. Novikoff (1967)]; this impression is misleading, as Fig. 1 demonstrates, x 21 000 Figure 3. Portion of a small neuron from dorsal root ganglion ot fetal rat, from P. M. Novikoff et al. (1971). Fixation: as in Fig. 1. Incubated in acid phosphatase medium, 40 rain, 37~ 0.5 /am section not stained with lead, Reaction product is seen in GERL (see Fig. 22 coloured green). The portion of GERL seen in the figure includes a face view of a cisternal portion (C), connecting tubules (T) and residual bodies (RB) as if arising as swellings of GERL. x 17 000 Figure 4. Portion of a small neuron from dorsal root ganglion of foetal rat, from P. M. Novikoff et al. (1971). Fixation: as in Fig. 1. Incubated in acid phosphatase medium, 40 min, 37~ thin section stained with lead. No reaction product is present in the Golgi apparatus (G). In contrast, GERL shows reaction product. Included in the section is a portion of a cisterna (C) and portions of tubules (T). Also with reaction product is an autophagic vacuole, type 2 (see A. B. Novikoff, 1973). x 25 000

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Figure 5. A portion of a large neuron in the dorsal root ganglion of a C57 black mouse. Fixation: formaldehyde-glutaraldehyde, 4~ perfusion 10 rain, immersion 120 rain. Incubated in acid phosphatase medium, 18 rain, 37~ Reaction product is seen in GERL (arrows) and residual bodies (RB) apparently forming by swelling of GERL tubules. Another residual body is indicated by an arrowhead towards the left edge. Two coated areas, as if coated vesicles in formation, are seen at C. Reaction product is not present in the Golgi apparatus (G). x 21 000 'latency', i.e. they do not reveal their acid phosphatase activity unless they are damaged; for a listing of such latency in other cell types see Boutry & Novikoff (1975). Two other lysosomal enzymes have been described by Decker (1974) in GERL of neurons in the lateral motor columns of larval frogs. These are aryl sulphatase and thiolacetic esterase activities.

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Since our 1971 study, we have been struck by the large number of residual bodies (dense bodies) in neurons of foetal rat dorsal root ganglia (P. M. Novikoff et al., 1971). Their abundance may be explained in part by the observations of Gonatas etal. (1977). These authors demonstrated that internalized plasma membrane in neurons of cultured mouse embryo dorsal root ganglia (and also of cultured neuroblastoma cells) is transported to GERL. Under the conditions of their experiments, free horseradish peroxidase (HRP) was not endocytosed by these cells. However, when the HRP was conjugated to ricin or to phytohemagglutinin (by glutaraldehyde) it was internalized, presumably because the lectins bind to plasma membrane receptors and induce pinocytosis. The HRP was visualized by DAB cytochemistry. It was found, first, in pinocytic vesicles and tubules; later, it was found in GERL. In another study, Essner & Haimes (1977) followed an electron-opaque tracer, colloidal silver, in alveolar macrophages of beige mice to which the tracer had been administered by nasal instillation. The tracer, and presumably portions of the plasma membrane surrounding it, were transported to GERL. 15 rain after administration, the tracer was seen in numerous pinocytic vacuoles continuous with or adjacent to the plasma membrane. 15 rain later, the tracer was found in GERL and also in the residual bodies which have apparently derived from swollen areas of GERL. If plasma membrane translocation to GERL should prove to be a widespread pheonomenon, it would have significant functional implications. Rather than the recycling of more or less intact membrane implied by retrieval to the Golgi apparatus (Gonatas et al., 1975; see also Pelletier, 1973, and Farquhar et al., 1975), membrane transport to GERL would make it more likely that some membrane macromolecules are degraded by the action of lysosomal hydrolases. The products of such hydrolysis would be available for cell re-utilization or cell excretion.

Hepatocytes The first description from our laboratory of GERL in rat hepatocytes was tentative and rested heavily upon observations of changes during the reversal of fatty livers, induced in rats fed a semi-synthetic diet containing 1% orotic acid, by the addition of adenine to the diet (A. B. Novikoff etal., 1966; see particularly Figs. 27 and 28). These observations have been greatly strengthened by recent studies (P. M. Novikoff & Edelstein, 1977) on a similar reversal oforotic acid induced fatty livers by addition to the diet of the hypolipidoemic agent, ethyl chlorophenoxybutyrate (clofibrate or CPIB). Fig. 23 shows diagramatically the nature and sites of lipid deposits in the marked fatty liver produced by orotic acid and the mild fatty liver produced by CPIB. The enlargement of GERL when the orotic acid induced fatty liver is reversed by dietary CPIB is indicated. The enlargement is seen in electron micrographs (Figs. 6 & 7) to be due to the accumulation of lipid-like particles [probably VLDL (very low density lipoproteins) or other lipoproteins]. GERL, as in neurons, shows no thiamine pyrophosphatase activity (Fig. 6) and does show acid phosphatase activity (Fig. 7). Fig. 8 shows acid phosphatase reaction product inside an autophagic vacuole, type 1 (A. B. Novikoff, 1973), in which one or two mitochondria have been sequestered, presumably by GERL; the small acid phosphatase-positive structure above and to the left of the autophagic vacuole is interpreted as oart of GERL. The situation is enzymatically similar in hepatocytes of normal rats but GERL is

Figures 6 - 8 . Portions of hepatocytes from rats in which lipid accumulated within the ER as a result of feeding a semisynthetic, purine-free diet containing 1% orotic acid for 7 days, was made to disappear by addition of the hypolipidoemic drug, clofibrate, to this diet for 7 days. Fixation: f o r m a l d e h y d e - g l u t a r a l d e h y d e , 4~ 180rain. Incubations: Fig. 6, thiamine pyrophosphatase medium, 90 min, 37~ Figs. 7 and 8, acid phosphatase medium, 30 min, 37~ Fig. 6. Thiamine pyrophosphatase reaction product is seen in the trans element (T) of the Golgi apparatus (G). The arrowheads indicate swellings of the trans element in which VLDL-like particles are seen. Slight deposits of reaction product are seen in the endoplasmic reticulum (ER). GERL is free of reaction product; the portion seen includes a cisterna ['rigid lamella' (arrow); see Claude (1970)] and vacuole-like swellings containing VLDL-like particles (V). x 44 000 Fig. 7. The Golgi elements (G) are devoid of reaction product resulting from acid phosphatase activity. In contrast, GERL has much reaction product in its cisterna portion (arrow) and swollen vacuole-like areas containing VLDL-like particles (V); cf. Fig. 6. The electron-opaque b o d y at the lower right is probably a residual b o d y that had formed from a VLDL-containing vacuole; the more electron lucent area (L) is probably lipid, x 37 000 Fig. 8. Acid phosphatase reaction product is seen in an autophagic vacuole containing either one or two mitochondria. Reaction product is seen within the mitochondrial cristae. As in Fig. 7, the Golgi elements (G) show no reaction product, x 43 000

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less extensive than in the hepatocytes in the process of reversal of the fatty livers (P. M. Novikoff & Yam, 1977). The most conspicuous GERL found in hepatocytes thus far is that of the beige mouse, a mutant of the C57 black mouse and a homotogue of the human Ch~diak-Higashi syndrome. Essner & Oliver (1974) showed that the huge lysosomes ('anomalous granules') in such cells developed from enlargements of GERL. They wrote: 'The accumulation of lipid-like material in GERL and anomalous lysosomes suggests that a disturbance in lipid or lipoprotein synthesis or metabolism may be involved in the pathogenesis of Ch6diak-Higashi syndrome'. Fig. 9 confirms the observations of Essner & Oliver (1974). In addition, the micrograph shows transitions (small arrows) from rough (robosome-studded) ER to GERL. This was not as clear in their material, probably because of differences in processing the liver for electron microscopy. Another situation in which GERL appears to be involved in lipid transformations is that found in Syrian golden hamsters fed a high cholesterol diet (Nehemiah & A. B. Novikoff, 1974). In these hamsters, unlike other mammals we have studied, lipid accumulates within lysosomes, to form 'lipolysosomes'. GERL becomes enlarged and cholesterol.rich lipid accumulates in the lipolysosomes (residual bodies) apparently arising from GERL. In this study the differences between GERL and 'unspecialized ER' in regions near the bile canaliculi were emphasized. The unspecialized ER differed from GERL: (a) its delimiting membrane was more twisted and thinner; and (b) no acid phosphatase activity was demonstrable cytochemically. The transformation from small VLDL particles into large electron-lucent lipid drops seemed to occur in this unspecialized ER as well as in GERL. When we consider the pancreatic acinar cell in beige mice, we will suggest that ER removed from GERL can possess cytochemicallydemonstrable acid phosphatase and the other hallmarks of GERL and residual bodies. In this presentation we have emphasized lipid transformations, yet hepatocytes are also major secretory cells. An important area awaiting future study is the possible relation of events in GERL of hepatocytes to the secretion of lipoproteins. This will be briefly considered in the Discussion. Insulinoma and pancreatic ~-cells In the early sixties A. B. Novikoff & Essner (1962) and A. B. Novikoff et at. (1962) demonstrated the presence of acid phosphatase activity in immature secretory granules of both endocrine and exocrine cells. Holtzman & Dominitz (1968), in our laboratory, found acid phosphatase activity in many secretory granules of the epinephrinesecreting cells in the rat adrenal medulla. A. B. Novikoff et al. (1975) have reported the results of studies engendered by these earlier observations. As with the neurons and hepatocytes described above, thiamine pyrophosphatase activity was used to demonstrate the trans element of the Golgi apparatus (Fig. 10) and acid phosphatase to show GERL (Fig. 11). Thus it was possible to determine unequivocally that secretory granules arose from GERL and not from the Golgi apparatus in both a transplantable hamster insulinoma (one producing proinsulin and insulin) and in the normal g-cells of the rat pancreas. In the insulinoma, all secretory granules possessed cytochemically-demonstrable acid phosphatase activity but in the ~cells, Only the immature granules (in the Golgi zone) showed such activity.

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The A.B. Novikoff et al. (1975) paper may be consulted for further details, for discussion of findings by others on pancreatic ~-cells a n d , most importantly, on the possible relevance of the origin from GERL of secretory granules, possessing acid phosphatase activity, to the proteolytic cleavage of proinsulln to insulin (Steiner et al., 1972). Pancreatic exocrine cells The cell type is of central importance in modern research on secretion. In his Nobel prize lecture, George Palade assesses each step in the secretory process, pointing out the strengths as well as the weaknesses of current information (Palade, 1975). The least firmly established portion of the secretory pathway in the pancreatic acinar cell lies between the ER of the Golgi zone and the nascent secretory granules [the 'condensing vacuoles' of Caro & Palade (1964)]. Limitations in the cell fractionation procedures and in the interpretation of ultrastructural autoradiography for structures as small as the tubules and vesicles in the Golgi zone make firm conclusions difficult. In contrast, phosphatase cytochemistry permits such conclusions. It can yield direct and accurate localizations since adequate preservation of intracellular organelles can be maintained, and because reaction product remains at or very near the enzyme site. In pancreatic exocrine cells, as in cell types already discussed, the trans element of the Golgi apparatus shows thiamine pyrophosphatase activity and GERL and its derivatives show acid phosphatase activity. Figs. 12-16 show some of the results obtained. The results have been described m abstract form (A. B. Novikoff et al., 1976; A. B. Novikoff, 1976b; A. B. Novikoff & P. M. Novikoff, 1976); they are presented more fully elsewhere (A. B. Novikoff et al., 1977a, b.). In the pancreatic exocrine cells, GERL and derivatives consist largely of: (a) flattened areas of smooth ER for which we have adopted the term, 'rigid lameUae', applied by Claude (1970) to similar structures he described in hepatocytes; (b) dilated areas of smooth ER, the condensing vacuoles; and (c) numerous coated vesicles. Fig. 12 shows an unusually patent continuity of rough ER to a condensing vacuole;

Figure 9. Portion of a hepatocyte from a beige mouse (see Essner & Oliver, 1974). Fixation: cold 1% OsO4-0.1 M phosphate buffer, pH 7.4, 2 h. Unincubated tissue; thin section stained with lead. Portions of the Golgi apparatus (G) are seen. Parts of two residual bodies (RB) are included in the field. The upper one is large and contains more electron-lucent areas, probably lipid, and electron-opaque areas some of which may also be lipid. Two autophagic vacuoles, type 2 (see A. B. Novikoff, 1973) are seen at AV2. The three long arrows, in the lower left area of the figure, indicate some of the regions where GERL shows the hallmarks of residual bodies (see A. B. Novikoff, 1973): accumulation of electron opaque material, a relatively thick delimiting membrane and a clear area ('halo') beneath the membrane. See Figs. 11 and 15 of Essner & Oliver (1974) showing acid phosphatase activity. Three small arrows indicate regions of GERL that are continuous with rough ER; the ribosomes of the latter are labelled R. x 55 000

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Figure 10. Portion of a cell of a transplantable insulinoma of Syrian golden hamster, from A . B . Novikoff et al. (1975). Fixation: f o r m a l d e h y d e - g l u t a r a l d e h y d e , 4~ 180 min. Incubated in thiamine pyrophosphatase medium, 50 min, 37~ Reaction product is present only in the trans element (arrows) of the Golgi apparatus (G). Most of the Golgi apparatus is out of the field. The extensive region of GERL in the figure shows no reaction product; this includes cisternae (arrowheads), coated vesicles (C) and three areas swollen with accumulated secretory material (S). Apparently separated secretory granules, also without reaction product, are seen at GR. x 35 000 Figure 11. Portion of a c-cell from the rat pancreas, t'rom A. B. Novikoff et al. (1975). Fixation: as in Fig. 10. Incubated in acid phosphatase medium, 90 min, 37~C. Reaction product is not present in the Golgi apparatus (G). It is present in cisternae, probably 'rigid lamellae' (arrows), and in immature secretory granules (GR). x 38 000

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continuities,between the two are more common and are illustrated in A. B. Novikoffet al. (1977a). There are numerous continuities between condensing vacuoles and rigid lameltae [Fig. 13; also Novikoff et al. (1977a)] i Both condensing vacuoles and rigid lamellae may be quite far removed, in areas, from the Golgi stack (cf. Discussion in P.M. Novikoff etal., 1971, of similar findings on GERL in neurons). The rigid lamellae are regions of GERL in which the distance separating the two membrane surfaces (i.e., the cisternal space) is relatively uniform, 200-300 A. Images of coated vesicles arising from condensing vacuoles (Fig. 13) or from rigid lamellae [Fig. 12 in Novikoff et al. (1977a)] are also numerous. Fig. 14 shows that all portions of GERL - condensing vacuoles, rigid lameUae and coated vesicles- have no demonstrable thiamine pyrophosphatase activity. In contrast, all three structures show acid phosphatase activity (Fig. 15; Figs. 7, 9, 11-13, and 15 in A. B. Novikoff etal., 1977a). Mature zymogen granules, all arising from condensing vacuoles (Palade, 1975), show neither thiamine pyrophosphatase nor acid phosphatase activity (Figs. 14 & 15). It should be noted that identical results were obtained in the four mammalian species studied, guinea-pig, hamster, rat and rabbit. We were keenly interested in studying the pancreatic exocrine cells of the beige mouse, where the anomalous granules (lysosomes) are large (Oliver & Essner, 1973) and there was some suggestion that GERL, as in hepatocytes, was also enlarged [Fig. 14, Oliver & Essner (1973)]. We found (A.B. Novikoff et al., 1977b) that indeed GERL was much enlarged in these cells, that the enlarged regions showed the hallmarks of residuai bodies (A.B. Novikoff, 1973) as in beige mouse hepatocytes (Essner & Oliver, 1974) and in beige mouse macrophages (Essner & Haimes, 1977), and that the residual bodies apparently formed from the expanded regions of GERL (Fig. 16). Thus, an important question could be asked: would ER that is active in producing large acid phosphatase-positive lysosomes, at the same time send acid phosphatase to the condensing vacuoles? As seen in Fig. 16, it does so. As we noted above in the section on hepatocytes, in the pancreatic exocrine cells of the beige mouse, the acid phosphatase-positive areas of smooth ER, GERL, apparently extend farther from the Golgi apparatus than does the unspecialized 'neighbouring ER' in the hepatocytes of the Syrian golden hamster. However, unequivocal assertions regarding the distance from the Golgi zone requires serial sectioning, not performed for either the hamster hepatocytes or the beige mouse pancreatic exocrine cells. Strong support for our findings in the pancreas exocrine cells comes from the work of Hand & Oliver (1975, 1976a, b, 1977a, b) on the exocrine cells of five different salivary glands in three species. They considered 'immature secretory granules' (i.e., the condensing vacuoles) to be part of GERL. In their studies, condensing vacuoles and tubular elements displayed acid phosphatase but not thiamine pyrophosphatase activity; the trans Golgi element showed thiamine pyrophosphatase but not acid phosphatase activity, and mature zymogen granules showed neither phosphatase activity. Like us, Hand & Oliver also found no structural connections between Golgi elements and GERL. It should be noted that static electron micrographs cannot exclude the possibility that by some kind of 'membrane flow' the trans element of the Golgi apparatus can be converted into GERL with its condensing vacuoles and rigid lamellae (see Discussion in

Figure I2~ A portion of a pancreatic exocrine cell of the rabbit; from A. B. Novikoff et al. (1977a). Fixation: cold 1% O s O 4 - 0 . 1 M phosphate buffer, pH 7.4, 2 h. Rough endoplasmic reticulum (RER) is abundant at both cis and trans aspects of the Golgi

stack (G). Small vesicles (V) are seen at 'the lateral edges' of the stack (see A. B. Novikoff et al., 1977a) and at both its cis and trans aspects. Labelled components o f GERL are coated vesicles (C), rigid lamellae (L) and condensing vacuoles (CV). One vacuole (CV1) shows an unusually wide continuity with rough ER (RER). x 38 000 Figure 13. A portion of a pancreatic exocrine cell of the guinea-pig, fasted 43 h and then fed 95 min; from A. B. Novikoff et al. (1977a). Fixation: cold 1% OsO4-0.1 M phosphate buffer pH 7.4, 80 rain. Only a small portion of the Golgi stack is seen (G). Note that vesicles and larger membranous structures separate G E R L from the Golgi stack. Labelled portions of G E R L include coated vesicles (C), rigid lamellae (L) and condensing vacuoles (CV). In CV1 the upper arrow indicates the continuity o f a rigid lamella and vacuole; at the lower arrow the continuity is incomplete in the plane of section. The two arrowheads are only suggestive that such continuities might have been present in these areas outside the plane of section. Similarly, only suggestions of possible continuities are indicated at the two arrows in vacuole CV4. An arrowhead, about centre on the left border of the figure, indicates a rigid lamella sectioned so as to show its flattened nature. An arrow at the top centre of the figure indicates a small interruption in the Golgi stack in this plane of section. The coated vesicle (C) near the centre of the figure is attached to a tangentially-sectioned membranous structure, probably a condensing vacuole. Note that at the upper right the rough (ER (RER) is directed to the trans aspect of the Golgi stack, x 50 000 Figures 14 & 15. Portions of pancreatic exocrine cells of the rat; from A. B. Novikoff et al. (1977a). Fixation: perfusion ( 3 - 5 min) and immersion, formaldehyde-glutaraldehyde, for a total of 90 min. Incubations: Fig. 14. Thiamine pyrophosphatase medium, 90 min 37~ thin section stained with lead. Fig. 15. Acid phosphatase medium, 25 min 37~ thin section stained with lead. The Golgi stack (G) shows thiamine pyrophosphatase activity in its trans element and shows no acid phosphatase activity. In Fig. 14, the single trans element is so sectioned that its branches seem separated in areas (see P.M. Novikoff et al., 1971). In Fig. 15, acid phosphatase activity is shown by the two components of GERL seen in the field: condensing vacuoles (CV) and rigid lamellae (L). In Fig. 14, GERL structures, including coated vesicles (C), condensing vacuoles (CV) and rigid lamellae (L), do not show thiamine pyrophosphatase activity. Note, in Fig. 14, that rough ER (RER), portions of G E R L and a zymogen granule (Z) are seen in a 'passageway' (Novikoff et al., 1977a) across the Golgi stack; this is not evident in Fig. 15. The arrow in Fig. 14 indicates a dilated region in the trans element of the Golgi stack. In Fig. 15, LY indicates an autophagic vacuole; when a very light print was made from this negative, membranes (some resembling mitochondrial membranes) were evident within the vacuole (cf. Fig. 12 in A . B . Novikoff, 1976a and Fig. 9 in A. B. Novikoff et al., 1977a). The rough ER (RER) and mature zymogen granules show neither acid phosphatase nor thiamine pyrophosphatase activity. Fig. 14, x 26 000; Fig. 15, x 26 000 Figure 16. Portion of a pancreatic acinar cell from a beige mouse; from A. B. Novikoff et al. (1977b). Fixation: perfusion, 5 min, and immersion, formaldehyde-glutaraldehyde for a total of 35 rain. Incubated in acid phosphatase medium, 60 min, 37~ Heavy accumulations of reaction product mark extensive areas of ER, some at the trans aspect of the Golgi apparatus (G) and some apparently elsewhere in the cell (serial sections were not done). Expanded areas of this ER show 'membranous arrays' (M) and lipid-like areas (L). Some reaction product is seen at the periphery of condensing vacuoles (CV1 and CV2). The arrow indicates an area where a condensing vacuole and a cisternal portion of GERL were probably continuous. Zymogen granules (Z) show no reaction product, x 34 000

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A. B. Novikoff et aL, 1977a). On the other hand, such 'membrane flow' is unlikely for several reasons. The condensing vacuoles are often at a distance in the plane of section from the Golgi apparatus while still attached to the rigid lamellae, as in Fig. 13. The inner contents of the condensing vacuoles more frequently resemble those of the ER than those of the Golgi elements. Most importantly, as in the case of the neurons of dorsal root ganglia (P.M. Novikoff etaL, 1971) any such membrane flow would require the trans element of the Golgi apparatus to change its structure drastically, to lose its demonstrable thiamine pyrophosphatase activity and to gain demonstrable acid phosphatase activity. From the results herein presented it is easier to conceive that secretory material moves from the rough ER to the condensing vacuoles without passing through either the Golgi apparatus or some 'lock-gate' mechanism (Palade, I975) in the Golgi zone. It remains to be learned whether mechanisms exist, currently undetectable by electron microscopy, by which contributions may be made by the Golgi apparatus to this secretory material. In this context, it would be highly informative if a cytochemical procedure for the ultrastructural localizations of glycosyl transferases were to become available. Some of these transferases are known to be highly concentrated in subcelhilar fractions, isolated from different cell types, that are enriched in portions of the Golgi apparatus. This uncertainty regarding contributions to secretory product by the Golgi apparatus in cell types where GERL appears to concentrate and package secretory and other materials should not be extended to the many cell types where Golgi elements apparently do function in these capacities. These will be considered briefly in the Discussion.

Thyroid epithelial cells We balre suggested (A. B. Novikoff et aL, I974) that there are two secretory pathways that by-pass the Golgi apparatus in the thyroid epithelial cells. One is like that in pancreas exocrine or endocrine cells considered above. The 'B' granules (Fig. 24), are considered to arise from GERL and to carry uniodinated thyroglobulin to the

Figure 17. Autoradiograph of a portion of thyroid epithelial cell, from an 80 g rat on an iodine-deficient diet for 9 days, 45 rain following intravenous injection of 5.0 mC[3H]fucose, from A.B. Novikoff et al. (1974). Fixation by perfusion for 20 rain at room temperature followed by 80 rain immersion fixation at 4~ with 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 (Sabatini et al., 1963); post-fixation in 1% OsO4-0.1 M phosphate buffer at 4~ for 1 h. Two silver grains are seen over GERL (arrows). Coated vesicles (C), endoplasmic reticulum (ER), Golgi apparatus (G), residual body (RB) and mitochondrion (M) are seen. x 35 000 Figures 18 & 19. Portion of an absorptive cell of guinea-pig duodenum, from P. M. Novikoff and A.B. Novikoff (1972). Fixation: 2.5% glutaraldehyde-0.1 M cacodylate, pH 7.4, with 0.05% calcium chloride. Incubated in DAB, pH 9.7, medium 90 min, 37~ Fig. 18 is tilted at - 3 0 ~ and Fig. 19 at +30 ~ from the initial 0 ~ position. Three microperoxisome~, are seen, numbered 1, 2 and 3. Continuity (arrowhead) between the ER and the delimiting membrane of number 2 is seen in Fig. 18 but not in Fig. 19; conversely, the membrane of number 3 shows a continuity with ER (arrowhead) in Fig. 19 but not in Fig. 18. Note the dependence upon the degree of tilt of the visibility of the residual body (RB) membrane, x 36 000

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Figures 20 & 21. Light micrographs of frozen sections of rat dorsal root ganglia, fixed in 2.5% glutaraldehyde 0.1 Yl cacodylate buffer, pH 7.4 (Sabatini et al., 1963). Incubation: Fig. 20, in thiamine pyrophosphatase medium, with IDP as substrate, for 45 rain, 37~ Fig. 21, in acid phosphatase medium, with CMP as substrate, for 30 rain, 37~ See text for description of intracellular structures that are visualized by these procedures. The darkly stained extracellular structures in thiamine pyrophosphatase-incubated section are blood capillaries (see A. B. Novikoff, 1967). x 980 Figure 22. Diagram from P.M. Novikoff et al. (1971) 'depicting, tentatively, the relations of endoplasmic reticulum, GERL and Golgi apparatus in the small neurons of adult dorsal root ganglion and neurons of the 16-day foetal rat ganglion' (see Figs. 1 - 4 ) . ER from the Nissl bodies (NB, in red) carries materials to the Golgi stack via transitional vesicles (TV) and transitional sheets (TS) to the outer (or eis) element (OE) of the Golgi stack, in blue. This element is composed of irregularly anastomosing cisternae or tubules. Little is known about Golgi elements 2 and 3, except that they are fenestrated. The trans or innermost element (IE) of the stack, in orange, consists of a hexagonal array of tubules. A smooth tubule (T) at left, of smooth ER (coming from rough ER or from GERL) enters each polygonal compartment (PC). From a Nissl body (NB, at lower left) ER presumably transports material, including acid phosphatase, to GERL (GE, in green) which" consists of cisternal portions (C) and tubules (T). The apparent origin of residual bodies (DB) and of coated vesicles (CV) from GERL cisternae and tubules is indicated. The unlabelled vacuole, with an external coating represents an early autophagic vacuole, type 2 (A. B. Novikoff, 1973). Figure 23. Diagram, from P. M. Novikoff & Edelstein (1977), depicting some aspects of lipid distributions in rat hepatocytes. The lipid deposits are shown in red. In the normal hepatocyte, lipid which enters the cell at the space o f Disse (D) in the form of free fatty acids and glycerol traverse the plasma membrane and the ER membrane. Within the ER cisternae they are combined to form triacylgerycerols and other lipids. Most probably they travel via two pathways: (1) they are transported by the ER to the cytosol, where they take the form of lipid ('storage') spheres, smaller but otherwise as shown in the diagram; or (2) they may be transported, as very small 'liposomes,' via transitional sheets or vesicles (see Fig. 22), to the eis element of the Golgi apparatus where they are transformed to very low density lipoproteins (VLDL) that are packaged into vacuoles derived from Golgi elements (GV). These vaculoes move to the h e p a t o c y t e surface at the space of Disse, where, by exocytosis, the V L D L are released to the circulation. When rats are fed a purine-free semisynthetic diet rich in sucrose to which clofibrate (CPIB) is added, a mild fatty liver develops in which relatively small lipid spheres, concentrated at the sinusoidal surface, have a distinctive relation to the ER, as suggested in the diagram. In contrast, when orotic acid (OA) is added to this semisynthetic diet the ER vesiculates and within each of the vesicles lipid droplets are found. Lipid transport to the Golgi apparatus is interrupted; the Golgi elements are flat and lack V L D L and Golgi-derived vacuoles are not present. Addition of CPIB to the diet that still contains OA produces a dramatic reversal of the OA-induced fatty liver in which, at an early stage, large cytosol lipid spheres develop as shown in the diagram and, later, the normal lipoprotein secretory protein is re-established. During the reversal process GERL is enlarged (Figs. 6 and 7); coated vesicles (C) are shown arising from GERL. Figure 24. Diagram of rat thyroid epithelial cell, based upon A. B. Novikoff et al. (1974). This suggests that: ( 1 ) s m a l l peroxidase-positive ' A ' granules are derived directly from the apical endoplasmic reticulum (ER) (left panel); (2) larger peroxidasenegative 'B' granules (right panel) are derived from GERL (GE); these are thought to carry uniodinated thyroglobulin to the lumen of the follicle; see Fig. 17; and (3) GERL is the source of the residual bodies (DB) which fuse with the pinocytic vacuoles (colloid droplets) (CD) that transport iodinatecl thyroglobulin from the follicle lumen into the cell (middle panel). Following the fusion, the thyroglobulin is presumably partially hydr.olysed, releasing thyroid hormones to the circulation at the base of the epithelial cells.

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follicular lumen where it is released by exocytosis. The second pathway involves the ER which in this cell type has endogenous peroxidase activity. Peroxidase-positive 'A' granules (Fig. 24), smaller than the peroxidase-negative 'B' granules, appear to bud directly from the apical ER and to bring the peroxidase to the luminal border of the cell where it is secreted via exocytosis. DAB cytochemistry was employed to demonstrate the two kinds of apical vacuoles. Acid phosphatase cytochemistry was used to demonstrate the residual bodies, prior to and following fusion with the colloid droplets. The acid phosphatase work was done independently by Wetzel et al. (1965) and in our laboratory by A. B. Novikoff & Vorbrodt (t963) (see also A. B. Novikoff et aI., 1964). In addition, electron microscopic autoradiography with tritiated fucose (Fig. 17) was used with results that are consistent with the DAB cytochemistry, namely that uniodinated thyroglobulin is packaged by GERL into B granules. Other cell types This section is not intended to review the literature but rather to direct readers to a few relevant publications not referred to earlier. The wide range of cell types in which GERL has been described is illustrated by the work of Lane and her colleagues on invertebrates (Lane & Swales, 1976) and Dauwalder (Fig. 89 in Whaley, 1975) and Marty (1973, 1976) on plant cells. Marty's work demonstrates a dramatic role of GERL in the formation of the central vacuole in several plant cells. A. B. Novikoff (1976) has reviewed briefly GERL in melanocytes and its role in forming 'melanolysosomes'. Bentfeld & Bainton (1975) described the origin of primary lysosomes from GERL early in megakaryocyte differentiation. GERL is cytochemically demonstrable by its arylsulphatase activity as well as its acid phosphatase activity, as in neurons described by Decker (see above). Microperoxisomes Unlike the nucleoid-containing peroxisomes found among mammalian cells only in liver and kidney, anucleoid peroxisomes, or microperoxisomes (A. B. Novikoff & P. M. Novikoff, 1973), were first revealed by cytochemistry. These organdies are ubiquitous in mammalian cells. The cytochemical procedure employed (A. B. Novikoff et al., 1972) was a modification of the 3,3'-diaminobenzidine (DAB)procedure of Graham & Karnovsky (1966). Both peroxisomes and microperoxisomes seem to be involved in the metabolism, transport, or storage of lipid. Continuities of microperoxisomes with ER are so numerous (Figs. 18 & 19) that we believe it likely that they are at all times connected with ER, not necessarily by unchanging continuities. Therefore, we consider microperoxisomes as specialized regions of ER, more widely dispersed and with more delicate and attenuated attachments than GERL which is a more evident portion of ER. We chose to include Figs. 18 & 19 because a small lysosome (residual body) is attached to the ER close to where three microperoxisomes are present. We wrote of this (P. M. Novikoff & A. B. Novikoff, 1972): 'If, indeed peroxisomes are dilated regions of the ER, what kinds of controls operate to channel peroxisomal enzymes into the peroxisomes and to retain them there while excluding, e.g., lysosomal enzymes? As Figs. 31 and 32 indicate, a peroxisome and a lysosome may be attached to virtually the same ER region. In

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addition, particularly in the rat duodenum, there may be lipid droplets moving through neighbouring, perhaps the same, portions of ER (Fig. 36). In the guinea-pig duodenum, where we have observed GERL, an acid phosphatase-rich portion of ER at the inner aspect of the Golgi apparatus, large amounts of acid phosphatase and presumably other lysosomal hydrolases are transported to this region of ER. The extent to which ER channelling appears necessary staggers our present imagination.' Discussion

From our presentation (see also A. B. Novikoff, 1976) it is evident that, when present in a cell, GERL may be involved in a wide variety of molecular transformations. These transformations appear to include, in secretory cells, the final processing of secretory products as they are packaged into granules. On the other hand, there are a great many reports of cell types in which such processing and packaging occur in the Golgi apparatus. These have been reviewed by Beams & Kessel (1968) and Whaley (1975). Among the most strikin~ examples are odontoblasts (Weinstock & Leblond, 1974), neutrophils (Bainton & Farqulaar, 1968) and the marine alga Pleurochrysis (Brown er al., 1970). In the neutrophils, there is a very interesting shift ('modulation', as some investigators term it) in functional polarity of the Golgi stack. Early in development, when the lysosomal 'azurophflic granules' form, they appear in the trans Golgi elements. Later, when the 'specific granules' form, the eis Golgi elements are involved. Modulation in both Golgi apparatus and GERL has been described by Decker (1974), particularly in degenerating neurons. Paavola (1976) has reported that GERL is enzymatically different in corpus luteum cells of guinea-pigs at 26 days of pregnancy; it displays alkaline phosphatase activity rather than acid phosphatase activity. Variations in acid phosphatase localizations have been described in neurons following sciatic nerve crush (Holtzman & A.B. Novikoff, 1965) and in HeLa cells following either arginine deprivation or irradiation (Lane & Novikoff, 1965). Clearly, much remains to be learned about both function and structure of GERL. This brief review may help direct the attention of investigators to this region of ER, as distinct from the nearby Golgi 'cisternae'. GERL has often in the past not been recognized as distinct from trans Golgi elements, even when impressive acid phosphatase localizations were described [e.g., in interstitial cells of the guinea-pig testis (Frank & Christensen, 1968) and in exocrine cells of the rat parotid gland (Hand, 1971)]. The status of GERL as a distinct cellular organelle is firmer than at an earlier stage in its history (P. M. Novikoff etaL, 1971) for two reasons: (a) different laboratories have by now described it in a variety of cell types; and (b) the evidence for continuity with rough ER is considerably stronger. Figs. 9 and 12 show such continuities. Other recent reports may be seen in Fig. 1 of A. B. Novikoff etal. (1975) of an insulinoma cell, Figs. 19 and 20 of A. B. Novikoff etal. (1974) of a thyroid epithelial cell, Fig. 1 of P. M. Novikoff & Yam (submitted) of a rat hepatocyte, and Fig. 11 of Bentfeld & Bainton (1975) of a rat megakaryocyte. Although the status of GERL as a distinct organelle is now more firm, we wish to note that parts of ER other than GERL may show some of its properties. These properties include a delimiting membrane which is straighter and thicker than that of

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ER generally, an electron-lucent area ('halo') beneath the membrane, materials within the cisterna, sometimes consisting of electron-opaque grains, 'membranous arrays', lipid-like substances; and demonstrable acid phosphatase activity. In the beige mouse hepatocytes, this region of ER may or may not extend beyond the Golgi zone; from our observations (Fig. 9) most of GERL appears to be in the Golgi zone. In the pancreatic exocrine cells of the beige mouse ER with hallmarks of GERL probably extend considerably beyond the Golgi zone [A. B. Novikoff etal. (1977b); also Fig. 16]. However, the extent to which such ER is removed from the Golgi zone cannot be firmly established without serial sectioning. In concluding, we wish to indicate how cytochemical observations are currently helping guide our laboratory's study of lipoprotein metabolism in liver. As indicated in Figs. 6 and 7 and also in P.M. Novikoff & Yam (1977), two types of lipoproteincontaining vacuoles can be distinguished on the basis of demonstrable acid phosphatase activity. We are attempting to separate the two types of vacuoles. Would the acid phosphatase-positive vacuoles also contain hepatic lysosomal lipase activity (Guder et at., 1969; Hayase & Tappel, 1970; Assmann et al., 1973; Teng & Kaplan, 1974; Colbeau et al., 1974; Debeer, 1977)? If so, what role, if any, does lipase activity play in interconversions of VLDL and LDL (low density lipoproteins)? That such interconversions may occur is suggested by the data of Chapman et aL (1972, 1973). These investigators showed the presence of LDL, as well as VLDL, in a Golgi-enriched fraction isolated from homogenates of guinea-pig liver.

Acknowledgements We gratefully acknowledge the preparation of the final photographs by Mr George Dominguez; the typing of successive versions of the manuscript by Ms Fay Grad; Ms Jean-Marie Boutry for use of Fig. 5; Ms Aria Yam for assistance in the work illustrated by Figs. 6-8; Drs A. Hand and C. Oliver for sending us, prior to publication, two manuscripts listed in this paper (1977a, b); and the following colleagues for critical comments on the manuscript: Drs Paul H. Atkinson, Joseph C. Ehrlich, Herman W. Spater, Mr Howard Haimes and Ms Marianne Poruchynsky. This work was supported by the U.S. Public Health Service Research Grant CA06576. A. B. Novikoff is the recipient of the U.S. Public Health Service Research Career Award 5K6 CA14923 from the National Cancer Institute.

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azurophil and specific granules of polymorphonuclear leukocytes. II. Cytochemistry and electron microscopy of bone marrow cells. J. CellBiol. 39,299-317. BENTFELD, M.E. & BAINTON, D . F . (1975). Cytochemical localization of lyso-

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BOUTRY, J . - M . & NOVIKOFF, A . B . (1975). Cytochemical studies on Golgi apparatus, GERL, and lysosomes in neurons of dorsal root ganglia in mice. Proc. nat. Acad. ScL U.S.A. 7 2 , 5 0 8 - 1 2 . BROWN, R. M., JR., FRANKE, W. W., KLEINIG, H., FALK, H. & SITTE, P. (1970). Scale formation in chrysopllcean algae. I. Cellulosic and noncellulosic wall components made by the Golgi apparatus. J. Cell Biol. 4 5 , 2 4 6 - 7 1 . CARO, L. G . & PALADE, G . E . (1964). Protein synthesis, storage and discharge in the pancreatic exocrine cell. An autoradiographic study. J. Cell Biol. 2 0 , 4 7 3 - 9 5 . CHAPMAN, M.J., MILLS, G. L. & TAYLAUR, C . E . (1972). Lipoprotein particles from the Golgi apparatus of guinea-pig liver. Biochem. J. 1 2 8 , 7 7 9 - 8 7 . CHAPMAN, M. J., MILLS, G. L. & TAYLAUR, C. E. (1973). The effect of a lipid-rich diet on the properties and composition of lipoprotein particles from the Golgi apparatus of guinea-pig liver. Biochem. J. 131, 1 7 7 - 8 5 CLAUDE, A. (1970). Growth and differentiation of cytoplasmic membranes in the course of lipoprotein granule synthesis in the hepatic cell. I. Elaboration of elements of the Golgi complex. J. CetlBiot. 47, 7 4 5 - 6 6 . COLBEAU, A., CUAULT, F. & VIGNAIS, P.M. (1974). Characterization and subcellular localization of lipase activities in rat liver cell. Comparison with phospholipase A. Biochimie 5 6 , 2 7 5 - 8 8 . DEBEER, L . J . , THOMAS, J., MANNAERTS, G. & DESCHEPPER, P.J. (1977). Effect of sulfonylureas on trlglyceride metabolism in the rat liver. J. clin. Invest. 59, 1 8 5 - 9 2 . DECKER, R . S . (1974). Lysosomal packaging in differentiating and degenerating anuran lateral m o t o r column neurons. J. Cell Biol. 61, 5 9 9 - 6 1 2 . EHRENREICH, J.H., BERGERON, J o J . M . , S1EKEVITZ, P. & PALAI)E, G . E . (1973). Golgi fractions prepared from rat liver homogenates. J. Cell Biol. 59, 45-72 ESSNER, E. & HAIMES, H. (1977). Ultrastructural study of GERL in beige mouse alveolar macrophages. J. CellBiol. (in press). ESSNER, E. & OLIVER, C.(1974). Lysosome formation in hepatocytes of mice with Ch~diak-Higashi syndrome. Lab. Invest. 30, 5 9 6 - 6 0 7 . FARQUI-IAR, M. G. & PALADE, G. E. (1965). Cell junctions in amphibian skin. jr. CellBiol. 26. 2 6 3 - 9 1 . F A R Q U H A R , M. G., SKULTELSKY, E. H. & HOPKINS, C. R. (1975). Structure and function of the anterior pituitary and dispersed pituitary cells. In: The Anterior Pituitary (eds. A. Tixier-Vidal & M.G. Farquhar), pp. 8 4 - 1 2 8 . New York: Academic Press, Inc. FRANK, A. L. & cHRISTENSEN, A. K. (1968). Localization of acid phosphatase in lipofuscin granules and possible autophagic vacuoles in interstitial cells of the guinea pig testis. J. CellBioL 36, 1 - 1 3 . GONATAS, N. K., KIM, S.U., STIEBER, A. & AVRAMEAS, S. (1977). Internalization of lectins in neuronal GERL. J. Cell Biol. 73, 1 - 1 3 . GONATAS, N. K., STIEBER, A., KIM, S. U., GRAHAM, D. I. & AVRAMEAS, S. (1975). Internalization of neuronal plasma membrane ricin receptors into the Golgi apparatus. E x p I Cell Res. 94, 4 2 6 - 3 1 . GRAHAM, R. C., JR. & KARNOVSKY, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochern. Cytoehem. 14, 291-302. GUDER, W., WEISS, L. & WIELAND, O. (1969). Triglyceride breakdown in rat liver. The demonstration of three different lipases. Biochim. biophys. A cta 187, 1 7 3 - 8 5 .

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