A three-dimensional reconstruction study of the [PDF]

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Journal of Cell Science 107, 517-528 (1994) Printed in Great Britain © The Company of Biologists Limited 1994

A three-dimensional reconstruction study of the rough ER-Golgi interface in serial thin sections of the pancreatic acinar cell of the rat Antonio Sesso*, Flávio Paulo de Faria, Edna Sadayo Miazato Iwamura and Hélio Corrêa† Laboratory of Molecular Pathology, Department of Pathology, Faculty of Medicine, University of São Paulo, CP 8100, Av. Dr Arnaldo, 455 - CEP 01246-903, São Paulo, SP, Brazil *Author for correspondence †Technical assistant

SUMMARY Distinctive views of the tubulo-vesicular elements interposed between the endoplasmic reticulum (ER) and the Golgi apparatus were obtained in thin sections. The tubules that protrude from the transitional rough ER (tRER) are of dissimilar length. The numbers of tubules and of the nearby omega- and pear-shaped profiles decrease after fasting and are partially restored by refeeding. This formation is designated herein as the budding chamber of the tRER. Close to the budding chamber, clusters of 56 nm diameter vesicles are consistently observed. In some of the cells, convoluted tubules appear enmeshed with the presumptive transport vesicles of 56 nm diameter and with irregular, vesicular formations. Apparently structureless, electron-lucent ellipsoidal areas are found adjacent to these membranous elements. Serial and semi-serial sections show that the budding chamber, the sinuous tubules, the irregular vesicles, the structureless regions and the 56 nm vesicles fill tunnel-like spaces limited by the outermost

Golgi cisterna (OGC) on one side and by the tRER on the other. Curved tubules appear to link the lumen of the OGC with that of smooth membranous occupants of these tunnel-like spaces. A presumptive luminal connection between these membranous occupants and the tubules of the budding chamber can also be seen. The predominant configuration of the OGC is that of a perforated, flat saccule. However, OGC regions exhibiting progressively lower densities of fenestrae, including smooth surfaced sectors eventually accumulating an intraluminal content are seen. Two such dilated, saccular portions of the OGC were analyzed through reconstruction of serial sections. Bundles of microtubules run closely apposed to the cis side of the OGC.

INTRODUCTION

issues. We decided to re-examine the rough ER-Golgi interface in the pancreatic acinar cell. This cell type has been a model system for a number of studies on the various steps in the secretory process (Palade, 1975). In freeze-fracture replicas, the configurations of the P and E fracture faces of the cis-most Golgi cisterna of the rat pancreatic acinar cell are those of a perforated cisterna with E-face pits of 50-60 nm diameter, which correspond to fenestrae (Sesso et al., 1983). Rambourg et al. (1988) have described the cis-most Golgi element from the same pancreatic cell as a network of anastomosing tubules. These authors schematically represented the network as wide-meshed and identical to that previously described in other cell types (Rambourg et al., 1981, 1984, 1987). While some authors observed saccular portions in the stacked, cis-most Golgi element (Lindsey and Ellisman, 1985), others did not (Rambourg and Clermont, 1990). In this report, we show in thin sections that sectors of the outermost Golgi cisterna (OGC) occasionally appear expanded. Such dilated profiles represent either a real accumulation of cisternal contents or a face view of a twisted, flat portion of the cisterna, sectioned through the lumen. To

Immunohistochemical (Saraste and Kuismanen, 1984; Saraste et al., 1987; Pelham, 1989; Schweizer et al., 1990; Saraste and Svensson, 1991; Lotti et al., 1992; Hobman et al., 1992) and biochemical (Tooze et al., 1988; Beckers and Balch, 1989; Pelham, 1989; Schweizer et al., 1991) studies have revealed the existence of a tubulo-vesicular, post-ER, pre-Golgi compartment (see Saraste and Kuismanen, 1992; Hauri and Schweizer, 1992; Lippincott-Schwartz, 1993). How this intermediate compartment is related to the transitional ER and the cis-Golgi region has not yet been accurately defined (Kao and Draper, 1992). Neither has it been unequivocally established to what extent the cis-most, stacked Golgi cisterna is part of the intermediate compartment. Also undetermined is whether the boundaries of this compartment vary among different cell types. The first, stacked Golgi component itself has been considered to be the intermediate compartment (Duden et al., 1991). Detailed knowledge of the morphology of the ER-Golgi interface as well as of the cis-most, stacked Golgi structure is a basic step in the process of obtaining answers to the above

Key words: transport vesicles, intermediate compartment, rough ERGolgi interface, outermost Golgi cisterna, transitional rough ER, cisternal maturation model

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address this issue, a reconstruction study in serial thin sections was undertaken. The study revealed that these dilated profiles represent a hitherto undescribed, accumulation of intraluminal material in saccular portions of the OGC. Further, bundles of microtubules are frequently noted running in close apposition to the cis side of the OGC. In thick sections of tissues impregnated with heavy metals, the OGC may appear fused on its cis side with a network of tortuous tubules. Seen in thin section, these tubules exhibit circular profiles similar to those of the nearby transport vesicles (Geuze et al., 1977). We have obtained sharper images from silver-gold sections of pancreases fixed with aldehydes and subsequently with reduced osmium than those previously published of the above cited tubular web. Some of these convoluted tubules lie close to the tRER while others are situated near the Golgi cisternae. Tubular elements appear to establish continuity between the lumen of the OGC and the nearby sinuous tubules, which were observed in only some of the cell profiles examined. Continuity between these sinuous tubules and those that protrude from the tRER is possible but as yet unsubstantiated.

MATERIALS AND METHODS Processing of the glands and groups of fed, fasted and refed animals Pancreas fragments from four fed, adult rats, weighing some 250 g, were rapidly removed under ether anesthesia and fixed. Fixation was initially performed in 1.5% glutaraldehyde and 1% paraformaldehyde in 0.08 M cacodylate buffer, pH 7.3, for 2 hours at about 4°C and subsequently in a 1:1 (v/v) mixture of 3% potassium ferrocyanide and of 2% osmium tetroxide, either at approximately 20°C or in a water bath at 37°C for 2 hours. The fragments were contrasted overnight in an aqueous, saturated, uranyl acetate solution, dehydrated in a graded ethanol series, and embedded in Spurr’s resin. Serial or semi-serial, silver-gold sections were picked up on Formvar-coated copper grids with a central orifice measuring 600 µm diameter. To evaluate the effects of fasting on the morphology of the rough ER-Golgi interface, glands from at least two adult rats subjected to the following treatments were examined: (a) fasting for 4, 10 and 20 hours; and (b) 20 hours of fasting followed by refeeding for 1, 3 and 5 hours. All sections were stained with uranyl acetate and subsequently with lead citrate. Micrographs were obtained in either a Philips 301 or a Zeiss EM 9S2 electron microscope. To estimate the mean diameter of the presumptive transport vesicles found between the transitional rough ER (tRER) and the outermost Golgi cisternae (OGC), slides were made from negatives photographed at ×9100 and projected to a final magnification of about ×500,000. The outlines of vesicles clearly exhibiting a limiting membrane were traced on a sheet of paper and two orthogonal diameters were measured. Data are given as the mean value ± the standard error of the mean (s.e.m. = s.d./√n). Thiamine pyrophosphatase and β-glycerophosphatase or cytidine monophosphatase activities were determined as indicated by Carneiro and Sesso (1987). Three-dimensional reconstruction From three series of 20, 28 and 48 consecutive sections, we obtained 16, 21 and 40 semi-serial sections, respectively. The two sequences of consecutive sections presented here were obtained from the latter two semi-series. Serial prints magnified to ×38,000 were used. Data acquisition was performed by tracing the contours of the desired structure on the micrographs on a digitalizing tablet. Before tracing

the contours, reference points were marked at the geometric center of the profiles of at least three zymogen granules that appeared in at least two successive sections. The program uses these points of reference to align the sequence of the serially sectioned material. From each closed profile traced, the area and perimeter are obtained. Thus, the profile volume in the section is the product of the section thickness times the area. By summing up the sectional volumes the aggregate volume can be obtained. Reconstruction was done with a package of programs known as the IBM PC-based three-dimensional reconstruction system (HVEM-3D) developed in the Laboratory for High Voltage Electron Microscopy, University of Colorado, Boulder (USA). This system uses a monochrome video monitor, and an enhanced graphics adapter (EGA) and color monitor, to follow the program and the reconstruction, respectively.

RESULTS Thin sections After reduced osmium treatment, the intensity of osmiophilia of the Golgi cisternae varies both within and between organ fragments. Although membranes are consistently clearly perceptible, in some fragments all the cisternae exhibit little or no blackening, owing to the luminal deposition of electron-opaque material. In other fragments, either all cisternae or the two or three outermost cisternae from peripheral cells are strongly electron-dense. These samples frequently exhibit a peripherocentral gradient of decreasing electron opacity of the cisternae. The analysis of semi-serial sections showed that the Golgi cisternae, as demonstrated previously for this (Noda and Ogawa, 1984) and other cell types (Rambourg et al., 1979), appear in the cytoplasm as a long, tortuous pile of curved, tightly connected ribbons. In our preparations, each stacked cisterna could occasionally be recognized by its peculiar electron density (e.g. see Figs 5a-d and 7a-d). In these serial and semi-serial sections, a given cisterna was never seen to change position or to be replaced by an adjacent cisternal element. Thus in the figures, the OGC and the successive, subjacent cisternae are indicated by the numerals 1, 2, etc. In pancreatic acinar cells from adult rats, transverse sections of these stacked ribbons at any given level always show at least four saccules or cisternae (Fig. 1). The presence of a 5th element, which is frequently a forming condensing vacuole, is the prevailing finding. A sixth piled profile, which may be either a rigid lamella or more rarely an immature, condensing vacuole, may also be seen. In complete agreement with previously published results (Novikoff et al., 1977; Rambourg et al., 1988) we observed that the 4th stacked component is thiamine pyrophosphatase positive. Occasionally this cisterna reveals a weak reaction for acid phosphatase. The 5th and 6th stacked components and the periphery of some of the condensing vacuoles may show β-glycero- or cytidine monophosphatase activities at pH 5.2. These results indicate that the 5th and, when present, the 6th stacked elements belong to the transGolgi network (TGN) (Griffiths and Simons, 1986; Mellman and Simons, 1992). Thus, when the stack appears to be formed of only four piled cisternae (e.g. as in Figs 1 and 2), the TGN is possibly missing at that level. The commonly encountered face view of the OGC showing fenestral size and density, appears in Fig. 1. The fenestrae are roughly circular, of 50.5+1.20 nm (n=150) diameter, measured frontally. The vesicles found between the tRER and the nearby

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Abbreviations used in legends. In the thin sections, successive Golgi cisternae from the cis to the trans regions are indicated by numbers. The OGC is indicated by 1 and the successively stacked elements by 2, 3, 4, 5 and 6 or C; C, condensing vacuole. In Figs 6 and 8 the first to 12th or 13th serial sections are referred to as I to XII or XIII, respectively. tRER, transitional rough ER; star, structureless region; t, sinuous tubules; i, large or small, irregular, vesicular profiles found close to the tRER; tu, tubular protrusion; tc, tubules connecting the OGC with nearby sinuous tubules. Fig. 11 is from an animal fasted for 20 hours and subsequently having free access to food for 5 hours. Fig. 22 is from a rat fasted for 20 hours. All other figures are from normally fed, adult rats. Figs 1-2. The frontal view of the OGC (1) in Fig. 1, shows commonly found patterns of fenestral size and density per area. The OGC face view (1) in Fig. 2 exhibits a lower density of fenestrae than that inf Fig. 1. In both figures arrowheads point to omega profiles on the smooth side of the tRER. In Fig. 2, the horizontal thin arrow indicates an exposed pore region connecting the OGC to an elongated, membranous profile. The latter has a coated extremity (thick arrow). It is uncertain whether the structure indicated by the small 4 is an incipient condensing vacuole.This formation exhibits omega profiles (O) on its surface, as condensing vacuoles often do. Fig. 1, ×52,000; Fig. 2, ×47,500.

Figs 3 and 4. In Fig. 3, stars mark dilated profiles of the OGC while asterisks indicate conglomerates of 56 nm diameter vesicles, other irregular vesicles and short tubules interposed between the tRER and the OGC. A growing condensing vacuole of notable size (C) is seen as a swollen portion of the 5th (5) stacked element. In both figures arrows indicate microtubules. Fig. 3, ×22,800; Fig. 4, ×45,000.

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Fig. 5. Sections XII(a), IX(b), VI(c), and I(d) from a sequence of 10 consecutive sections, plus the 12th, are shown. These sections are part of a more extensive collection of semi-serial sections. An ellipsoidal region (star in a) of low electron density and devoid of structural elements and the adjacent 56 nm vesicles, could be followed from section I to section XXI. a to d, ×24,500. Fig. 6. A computer-generated, three-dimensional reconstruction of the OGC (Fig. 5a and d) presented under a chosen observation angle of the coordinate (x, y and z) system. Given that the origin of these axes is the point at the lower left corner of a square paper plane, if x varies positively, the paper rotates around the x axis, which lies close and parallel to the frontal plane of the observer; the distal part of the square elevates to an angle n (x=n, y=0 and z=0). When y varies, e.g. positively, the square dislocates to an angle n from the right to the left around the y axis, which is situated orthogonally to the observer’s frontal plane (x=0, y=n and z=0). When z varies positively, the plane rotates clock-wise around the z axis. This axis passes perpendicularly through the lower left corner of the square (x=0, y=0 and z=n). Fig. 6, x=240, y=0 and z=180. Owing to the depth of focus of the electron microscope, all the structures contained in the section thickness are represented in the micrographs as lying within a single plane. The distance between two such planes is the thickness of the section, assumed to be 100 nm, since, in the silver-gold sections, the gold shade was predominant.

OGC are of 56.2+0.63 nm (n=121) diameter. Most of these putative, transport vesicles are either larger than, or appear to fit tightly into, the OGC pores (Fig. 1). Occasionally, OGC sectors either lack, or possess a low density of, fenestrae (Fig. 2). These latter regions may appear in longitudinal sections as uninterrupted or poorly interrupted OGC profiles (see Fig. 10). Bundles of microtubules are seen closely apposed to the cis side of the OGC (arrows in Figs 3, 4). To determine the significance of the dilated profiles occasionally exhibited by the OGC (stars in Fig. 3) successive sections of two cis-most cisternae with swollen saccular portions (Figs 5a-d and 7a-d) were used to provide computer aided, three-dimensional reconstruction images. These images are presented under chosen angles of observation (Figs 6 and 8). The aggregate volumes of the dilated OGC regions, marked by asterisks in Fig. 5a and d and Fig. 7a and d, are 0.87 and 0.41 µm3, respectively. Cisternal profiles with localized dilations are also observable

Fig. 7. Sections II(a), VI(b), VIII(c) and X(d) from 13 serial sections through a stack of Golgi saccules are shown. A roughly circular zone (star) identical in texture and electron density to that marked by the star in Fig. 5a was present in all sections. In cross-section, the fenestrae appear as gaps in the cisternal profile (arrowheads in, e.g., Fig. 7a). In these serial sections, such breaches represent a relatively small fraction of the area of the OGC, as shown in Figs 8 and 9. ×38,000. Fig. 8. Three-dimensional reconstruction of the OGC denoted as 1 in a and d and followed in 13 consecutive sections. x=60, y=0 and z=0. Most of the gaps along the OGC profile, are considerably less in width than the section thickness. Thus, each gap probably represents an individual fenestra contained totally or mainly within the section. ×38,000. Fig. 9. Although this drawing represents the OGC seen under the observation angle of Fig. 8, various other observation angles were also used to elaborate the final scheme. To represent fairly the diameters of the fenestrae in the scheme, we used the coordinates x=90° y=0° and z=0° that show the 13 sectioned profiles as interrupted (each interruption representing a fenestral gap) straight, parallel lines. ×38,000.

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Figs 10 and 11, respectively. From a normally fed rat (Fig. 10) and from an animal fasted for 20 hours subsequently having free access to food for 5 hours (Fig. 11). In Fig. 10, many 56 nm vesicles overlie the middle and upper parts of the longitudinally cross-sectioned OGC (1). These OGC parts reveal very few breaches. The stack of cisternae also longitudinally sectioned seen in Fig. 11 exhibits relatively few 56 nm vesicles on its cis side. In other sectors of this gland the density of 56 nm diameter vesicles is similar to that seen in Fig. 10. This stack of cisternae appears as two subgroups. The smaller group is at the right center position. Both groups are formed by four cisternae and share the fourth cisterna in common. Two forming condensing vacuoles (C) are seen as swollen portions of the fourth cisterna. Figs 10, 11, ×34,000.

either in the second or in any of the four stacked elements subjacent to the OGC. In fed animals, forming condensing vacuoles are consistently found as swollen portions of the 5th stacked element of the pile that pertains to the TGN. Unpublished observations from this laboratory derived from the analysis of semi-serial sections in fed animals reveal that forming condensing vacuoles grow markedly only when connected to the 5th cisterna (e.g. C in Figs 3 and 10) by a tubular connection. However, in these glands, the fourth cisterna may exhibit profiles of incipient forming condensing vacuoles. In fasted rats subjected to refeeding, possibly because of the different secretory status of the cells, growing condensing vacuoles are commonly noted as appendages of the fourth cisterna (Fig. 11). This result is compatible with the view that incipient pro-secretory granules may be seen, in normally fed, adult rats, as relatively small, swollen portions of the fourth cisterna as indicated by the small 4 in Fig. 3. Jamieson and Palade (1968) consider the peripheral region of the Golgi complex as comprising both the tRER and the adjacent vesicular elements, thought to transport proteins from the rough ER to the Golgi zone. These vesicles and the nearby tubules have been designated as ‘peripheral elements’ (Slot and Geuze, 1976) while the space they occupy has recently been referred to as a ‘transitional area’ (Oprins et al., 1993). In many cells (Figs 12-14), omega-shaped and tubule-like

profiles protrude from the smooth side of the tRER. Some of the protruding tubules attain 200 nm in length (arrowhead in Fig. 12) and are of a diameter comparable to that of the nearby putative, transport vesicles of 56 nm diameter. Some of the omega- and tubule-like profiles with a bulging extremity exhibit a thin coat of dense, fibrillar material (e.g. arrow in Figs 15 and 16). In freeze-fracture replicas, these protrusions exhibit P and E fracture faces with textures that differ markedly from those of the corresponding faces of the rough ER and of the non-budding regions of the tRER (unpublished results from this laboratory). We refer to these formations as the budding chamber of the tRER. Adjacent to the budding chamber clusters of 56 nm diameter vesicles occasionally appear enmeshed by tortuous, tubular profiles of variable length. The sinuous tubules exhibit omegaand pear-shaped, terminal portions (Figs 13 and 17). Adjacent to these membranous structures, an area devoid of structural elements is occasionaly seen. Two such apparently structureless regions, the nearby 56 nm vesicles and variable amounts of tubular elements extend over appreciable distances (Figs 5ad and 7a-d). The fact that the budding chamber, the 56 nm and irregular vesicles (Fig. 18) and the sinuous tubules cover considerable segments of the OGC is also noted in favourable single sections (e.g. Figs 3 and 10). These elements occupy a region limited by the tRER on one side and by the OGC on the

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decrease, not only in the number of profiles that bud off from the smooth side of the tRER, but also of the membranous elements normally found close to the tRER. This effect was maximal after 20 hours (Fig. 22) of fasting. Although refeeding for up to 5 hours after the 20 hour fast was insufficient to completely restore the density of these tubules and the 56 nm vesicles to the levels observed in fed rats in all cells, many cells exhibited clusters of tubulo-vesicular elements close to the tRER, similar to those seen in fed control rats. DISCUSSION The predominant architecture of the OGC is that of a flat, perforated saccule. However, poorly fenestrated and dilated, unperforated areas are also seen. The volumes of the swollen portions observed in OGC sections (of at least 0.87 and 0.41 µm3) are in the range of the volumes of the largest condensing vacuoles found in fed adult rats (Ermak and Rothman, 1981). As discussed below, these various phenotypes of the OGC may bear some relation with an ongoing process of cisternal maturation. Fig. 12. The tRER exhibits three tubular profiles. The tubule indicated by the arrowhead is some 200 nm in length. ×60,000.

other. Such a space can be grossly compared to a tunnel of variable height. The density of the tortuous, tubular profiles varies in the different tunnel-like regions overlying the OGC in different cells. Fig. 15 shows the initial section of a semi-series of sections containing sections I, III, IV, VI, VII, X, XII, XIII, XIV and XVII. The region neighboring the tRER indicated by a single asterisk could be followed in all sections while that indicated by the double asterisks was absent from sections X to XVII. Both regions consistently exhibited many sinuous, tubular profiles. Fig. 16 shows the region labelled by a single asterisk, in section X. Other similar regions, followed in two sets of 21 and 13 semi-serial and serial sections, respectively (Figs 5a-d and 7a-d), showed a lower density of tortuous tubules. It is not clear whether all the tubular profiles seen in successive sections are part of a single, intercommunicating, tubular web. However, in some single sections, the threedimensional, convoluted disposition of the tubules can be easily perceived (e.g. t in Figs 13 and 17). Occasionally, in cross-sectioned, stacked Golgi cisternae, a restricted region of the OGC lacks the bidimensional, stacked condition and seems to connect the OGC to elements situated in the central portion of the region interposed between the tRER and the OGC (tc in Figs 19-21). We have obtained some micrographs suggesting continuity between the lumen of the tRER and that of the nearby tortuously tubules (e.g. vertical arrows in Figs 14, 17 and 18). Unequivocal demonstration of such a continuity is missing. Pear-shaped profiles are visible at the extremities of the short and longer tubules of the budding chamber (arrowheads in Figs 13 and 20). Effects of fasting and refeeding on the morphology of the region close to the smooth side of the tRER The deprivation of food for 4 to 20 hours causes a progressive

The sinuous tubules in the vicinity of the tRER A high density of sinuous tubules is observed close to the tRER of some but not all cells. In the acinar cells of low secretory rate from suckling rats, particularly in the early post-natal days, tortuous tubules near the cis-Golgi are found much less frequently than in adults (unpublished results from this laboratory). It is not yet clear whether the presence of many sinuous tubules is related to the level of secretory activity and thereby to the prevalence of antero- or eventually retro-grade membrane traffic (Lippincott-Schwartz, 1993) across the rough ER-Golgi interface. It is also possible that the presence of these tubules derives from an undetermined cause. The immunochemical reaction used to reveal the 58 kDa marker protein of the intermediate compartment (p58) showed that in pancreatic acinar cells, the clusters of smooth, membranous elements found close to the rough ER are composed of both p58-positive and p58-negative tubulo-vesicular structures (Saraste and Kuismanen, 1992). The electron-lucent areas indicated by stars in Figs 5a, 7a-d, and 14 appear structureless in our preparations. These regions are of a shape and localization comparable to those described by Merisko et al. (1986) as fibrillar aggregates in anoxic, pancreatic cells. Although the precise meaning of such domains devoid of membranous elements is undetermined these may correspond to the aggregates of β-COP positive material described by Oprins et al. (1993) in the cis-Golgi region. A cis tubule network that runs parallel to the OGC has been described in neurons (Lindsey and Ellisman, 1985). Further studies are needed to establish whether, and to what extent, this network in neurons, and the tortuous tubules demonstrated here in pancreatic cells, are equivalent structures. Continuity between the tRER and the cis-Golgi The possibility of such luminal continuity between the tubules protruding from the tRER and the adjacent sinuous tubules is apparent in some micrographs. However, given the dimensions of the apparently continuous elements, one cannot exclude the possibility that these structures, which appear in the same plane

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Fig. 13. to and tu, respectively, indicate omega, and short tubular profiles. The arrowhead and curved arrow point to coated, pearshaped profiles in the tRER and in a sinuous tubule, respectively. 1, the OGC. ×57,000. Fig. 14. tu1, tu2 and tu3 show tubular profiles of various lengths. The vertical arrow indicates a tubular protrusion possibly fusing with vesicles. ×62,000. Figs 15 and 16. Sections I and X, of a semi-series of sections. The single and double asterisks in Fig. 15 show two conglomerates of smooth membranous tubules and vesicles. The white arrow points to the coated extremity of a relatively long, tubular protrusion. In Fig. 16, the white arrow and the arrowhead point to the dense coat and to

a pear-shaped profile, respectively, found at the extremities of tubular protrusions from the tRER. Fig. 15, ×28,500; Fig. 16, ×51,000. Fig. 17. The vertical arrow indicates the close proximity between the tRER and a tubule that appears to enmesh with the tortuous tubules nearby. The curved arrow points to a coated, pear-shaped part of a convoluted tubule. The thick arrow shows a fusion pinching-off profile found in the sinuous tubules. This profile indicates either fusion or fission of vesicles. ×80,000. Fig. 18. Close to the tRER are several large irregular vesicular profiles of 100-200 nm in their largest dimension (i). ×57,000.

of focus in the electron micrographs, in fact lie at different levels in the section. Should such images correspond to true continuity between compartments, these together with the results showing connecting tubules linking the OGC to membranous elements interposed between the tRER and the cis-

Golgi indicate direct continuity between the tRER and the OGC. Only rarely have we obtained images suggesting continuity between the tRER and the nearby convoluted tubules. This is compatible with the existence of a non-permanent link between the two tubular systems. Geuze et al. (1977) have

Figs 19-21. tc indicates tubular elements that appear to connect the OGC with sinuous tubules found in the region interposed between the tRER and the first cisterna. Arrowheads in Fig. 20 point to the extremity of pear-shaped profiles. Fig. 19, ×54,000; Fig. 20, ×60,000; Fig. 21, ×56,000. Fig. 22. Animal fasted for 20 hours. The star indicates an area limited by both the tRER and the cis-Golgi cisternae, possessing visibly less membranous structures than in fed rats. A few vesicles occupy an otherwise structureless sector. ×30,400.

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Fig. 23. The tRER is represented as spheroidal regions (1) of the rough ER (2) cisternae. The tubules and vesicles budding from (1) and the sinuous tubules supposedly formed by microvesicular fusion are indicated by 3, 4 and 5, respectively. The elements connecting the OGC with the sinuous tubules are indicated by 6. The possible continuity between (1) and (6) is not represented in this scheme.

suggested a transport mechanism by which membrane and intraluminal material could flow through tubules that intermittently fuse, forming a sinuous pathway between the ER and the cis-Golgi. One of the models of membrane traffic between the Golgi apparatus and the rough ER discussed by Saraste and Kuismanen (1992) suggests that the tRER-Golgi interface is a cytoplasmic region where the sorting and recycling of membrane from the cis-Golgi back to the tRER occur on a prominent scale. Tubulo-vesicular elements or even direct, tubular continuity between the tRER and the cis-Golgi may mediate this backward flow of membrane (Saraste and Kuismanen, 1992). The omega profiles frequently seen in the sinuous tubules may indicate vesicular fusion or pinching-off. Many of these profiles appear coated in our sections. To the extent that the coat is required to provide the mechanochemical force necessary to promote budding followed later by fission (Klausner et al., 1992; Peter et al., 1993), the coated, pearshaped parts that we have observed in the sinuous tubules may be revealing vesicular pinching-off (Figs 13 and 17) from these tubules. Should this be the case, then at least some of the sinuous tubules may be in the process of dismantling, generating vesicles that, considering the size of the pear-shaped segment, would be indistinguishable from the 56 nm diameter vesicles. The disassembling of this part of the complex formed by the OGC and the nearby, continuous, sinuous tubule may be related to sorting mechanisms occurring in this region with possible membrane retrieval to the rough ER. Fig. 23 schematically represents the lumen to lumen communication between the OGC and the convoluted tubules located nearby. In this scheme, the boundary of the area occupied by these tubules and the 56 nm vesicles is represented

as tunnel-like. This particular configuration was inferred from observations of serial and semi-serial, silver-gold sections. Tubular protrusions of variable length are noted in the tRER. Several micrographs of thin sections of this region published by various authors show these tubular profiles with dissimilar lengths. This fact, plus the observation that the number of omega and tubular protrusions from the tRER declines in the cells of rats fasted for 4, 10 or 20 hours and is restored to normal by refeeding, support the notion that the tubules of the budding chamber are dynamic structures that grow and retract. These tubules may initially protrude from the tRER as coated omega profiles. After the growing membranous cylinder attains a certain length in conditions of forward traffic, a 56 nm vesicle would pinch off from the free end of the tubule. The remaining tubular stump would recede until the stage of an uncoated, omega profile. Subsequently, a flat, smooth, cisternal membrane surface would be reconstituted. Coated and uncoated omega profiles are normally encountered on the smooth side of the tRER. Mechanisms promoting a reduction in the number of fenestrae in the OGC ought to be postulated in case the theory proposing that the Golgi cisternae migrate from the cis to the trans zone is correct. This imposition is derived from the fact that the second cisterna has a visibly lower pore density than the OGC as stated by Rambourg et al. (1988) and that is apparent in our data (e.g. Figs 1, 5a-d, 7a-d). Thus, if a segment (e.g. of the size necessary to contain sufficient intraluminal material to form at least one condensing vacuole, when the maturing cisterna arrives at the trans-most position) of the beltlike first cisterna, migrates to the trans-Golgi zone, it must, on occupying the position of the second cisterna, exhibit a lower density of pores than present when it constituted the outermost cis cisterna. The number of fenestrae per unit surface area must

Morphology of post-ER membranous elements be reduced either before or during migration to the position of second cisterna. Two concepts, appearing in succession, have been proposed to explain how products, transferred from the tRER to the OGC, ultimately arrive at the trans-most portion of the Golgi zone: the cisternal progression (Morré et al., 1979; Lindsay and Ellisman, 1985) and the stationary cisternae models (Dunphy and Rothman, 1985; Farquhar, 1985). Although the latter concept appears to be currently more widely accepted (Farquhar, 1985), both theories are under review (Lippincott-Schwartz, 1993). An argument illustrating that the subject is not yet settled can be made for each one of the two theses: (a) cisternal migration has been demonstrated to occur in a combined light and electron microscopic study of the alga Pleurochrysis scherfelli. Cisternal vesicles containing scales are visible under the light microscope and the kinetics of their formation and ejection has been recorded (Brown, 1969; Brown et al., 1970). Additional observations on flagellate algae confirm that the transport of scales through the Golgi apparatus occurs by cisternal progression (Melkonian et al., 1991). The contention that, in these algae, the cisternae on the cis side are stationary and that only the trans-most cisternae are migratory, is as yet unwarranted. The occurrence of vesicular transport between cisternae is not supported, since the electron-dense scaly material found in the cisternal lumen has never been observed in any of the 350 putative transport vesicles examined (Melkonian et al. 1991); (b) studies carried out in hybrid cells formed by the fusion of wild-type with mutant cells devoid of specific glycosyl transferases revealed the transfer of a glycoprotein from the more proximal cisternae of the defective cells to the more distal (medial or trans) cisternae of the wild-type cell (Rothman et al., 1984a,b). The glycoprotein undergoing terminal processing would be progressively transported from the cis to the trans direction by vesicles transiting between adjacent stationary cisternae. Assays on cell-free systems support the notion that transport of proteins between Golgi subcompartments demands the movement of vesicles between cisternae (Orci et al., 1989; Rothman and Orci, 1992; Söllner et al., 1993). The scheme for the formation of the condensing vacuole proposed by Rambourg et al. (1988) reveals the occurrence of cisternal migration. The present article contains data that may support the cisternal maturation model. If the structure denoted by the small 4 (indicating that it pertains to the 4th saccule) in Fig. 2 is a forming condensing vacuole, then the simplest and indeed only means by which this formation could be transposed to C at position 5 (indicating that it is in the 5th stacked element) in Fig. 10, to undergo further growth, would be by cisternal migration. Given the cisternal migration hypothesis, our present results support the conjecture that, prior to migration towards the position of second cisterna, OGC regions with a reduced number of fenestrae and occasional dilations become structurally organized like second cisternae. We thank Maria Margarida Teixeira Monteiro for preparation of the plates. The drawings were made by Raul Cecílio Menezes Júnior. The English manuscript was revised by Dr John C. McNamara. This work was supported by grants from FAPESP (89/3805, 90/1778-1 and 92/3170-6), CNPq (40.005/89-8) and FINEP (PSME).

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(Received 23 August 1993 - Accepted 30 November 1993)