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Fine structure of the apex of absorptive cells from rat small intestine
1970 by Academic Press, Inc.
J. ULTRASTRUCTURERESEARCrI31, 291--311 (1970)
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Fine Structure of the Apex of Absorptive Cells from Rot Small Intestine s OSCAR BRUNSER2 and JOHN H. LUFT
Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington 98105 Received August 28, 1969 These cells were studied in rats, using buffered osmium or aldehyde fixatives followed by osmium. Three groups of filaments and the terminal web were visualized in the apex. One group, seen in aldehyde-fixed tissue formed the cores and rootlets of the microvilli; they showed no continuity with other filaments. Another group formed the desmosomal web, best seen in tissue stained en bloc with uranyl acetate of pbospbotungstic acid (PTA). The third group appeared as short, branched profiles between the rootlets after staining with PTA. The terminal web appeared as a dense layer with gaps, extending out to the zonula adhaerens. It seemed to embed the rootlets penetrating between their filaments. Although it appeared granular with the techniques used here, it is probable that at the molecular level the terminal web is formed by elongated branching elements. By supporting the rootlets and microvilli, the terminal web endows the apex with mechanical stability and anchors it to the body of the cell. The apices of the epithelial cells of the m a m m a l i a n small intestine are the structures t h r o u g h which most nutrients are obsorbed. They possess as conspicuous specializations of their free surfaces large numbers of vertically oriented, slender microvilli. These and the other components of the cell apex are so thoroughly interconnected that it is possible to isolate the whole cell apex as a discrete unit without serious disruption of its overall structure. Miller and Crane (19, 20) and others (4, 5, 7, 22), taking advantage of this characteristic, have been able to obtain purified preparations of apices with minimal amounts of the underlying cytoplasm by subjecting cells to osmotic shock and various other treatments. Similar results were obtained 1 Dr. Brunser was supported by a Fellowship from the John Simon Guggenheim Memorial Foundation. Supported by USPHS Grants GM-16598 and GM-00136 from the National Institutes of Health. Presented at the Eightieth Session of the American Association of Anatomists, Kansas City, Missouri, April 4-7, 1967. 2 Present address: Laboratorio de Investigaciones Pediatricas, Escuela de Medicina, Universidad de Chile, Casilla 5370, Santiago, Chile.
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by Harrison and Webster (9) using low frequency vibrations; this can be interpreted as indicating that the mechanical strength of this area is different from that of the rest of the cytoplasm. According to the early studies of Zetterqvist (36) and of Palay and Karlin (25), the microvillus has a filamentous axis that penetrates into the apical cytoplasm forming a rootlet that fuses with a network of horizontally oriented filaments called the "terminal web." This terminal web is described as being parallel to the luminal surface of the cell and inserting itself into the "terminal bar" at the periphery of the cellular apex. The maintenance of the orderly distribution of the microvilli and the rigidity and strength of the cellular apex have been attributed to the existence of this filamentous web and its attachment to other structures. Since these descriptive terms had originated from earlier light microscopic studies, a certain amount of confusion resulted when they were applied to electron microscopic observations (13, 14, 26). While studying our electron microscopic material, we were unable to identify a distinctly filamentous terminal web as had been previously described (1, 17, 18, 21, 24, 32). In order to find reasons for this descrepancy between our observations and those in previous publications, we studied the fine structure of the apex of the absorptive cell using different techniques of fixation and staining.
MATERIALS AND METHODS Samples of small intestine were obtained from an area about 1 cm beyond the pylorus from adult rats anesthetized with pentobarbital administered intraperitoneally. Rings of intestine, about 2 mm thick, were sectioned and immediately divided into four approximately equal quadrants. Contraction of the muscle coats transformed each quadrant into an everted hollow cylinder, with the outer surface covered by the mucosa with its villi slightly spread out radially and the inner surface covered by the serosa. These cylinders were fixed for 1 hour in bicarbonate- (34), phosphate- (34), or cacodylate- (34) buffered osmium tetroxide or in 2.4% glutaraldehyde in phosphate buffer (28) with and without 1% acrolein added (15), and postfixed for 1 hour in bicarbonate-buffered osmium. In one experiment, segments of intestine from two animals were isolated between ligatures without damaging the blood supply, and glutaraldehyde-acrolein mixture in phosphate buffer was injected under moderate pressure into the lumen in order to produce distention. After maintaining distention for 5 minutes, the loop was removed, divided in small pieces and further fixed by immersion in the glutaraldehyde-acrolein mixture and postfixed in osmium. All the tissue was dehydrated in an ethanol series. Some of the pieces of the bicarbonatebuffered osmium-fixed tissue were then transferred to absolute ethanol containing either 1% uranyl acetate or 1% phosphotungstic acid (PTA) for 1 hour (16). They were afterward washed in two changes of 10 minutes each of absolute ethanol. Of the PTA stained tissue, some pieces were subsequently transferred to 0.01% sodium hydroxide in absolute ethanol for 25 minutes, washed again in absolute ethanol, and transferred to propylene oxide previous to their infiltration and embedding in Epon 812 (16). The PTA-stained tissue that was
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FI~. 1. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. The filaments of the core (CO) and rootlet (R) of the microvilli (MV) are identifiable as well as some belonging to the desmosomal web (D W). There is no evidence of a filamentous terminal web at the level of the rootlets. L, intestinal lumen; ZO, zonula occludens; ZA, zonula adhaerens; D, desmosome, x 51,000.
not treated with alcoholic sodium hydroxide was embedded in Araldite (16). All the other pieces of tissue were embedded in E p o n 812 (16). Sections were cut with du Pont diamond knives on a Sorvall MT-2 ultramicrotome, collected on Parlodion and carbon-coated grids, and double stained with uranyl acetate and lead citrate (27). The observations were made with a modified R C A E M U - 2 C electron microscope using a 50-/~ objective aperture and an accelerating voltage of 50 kV. The observations were m a d e in cells of the upper third of each villus, except in the distended segments, where the boundary between the crypts of Lieberktihn and the base of the villi was also studied.
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OBSERVATIONS The apical area of the intestinal epithelial cell is characterized by the presence of uniformly shaped, regularly spaced microvilli (Figs. 1 and 2, MV) and by the absence of organelles that are scattered throughout the rest of the cytoplasm. Between and just beneath the bases of the microvilli and in the adjacent area there are pits (P), coated vesicles (V), and membrane-bounded canaliculi (CA) (Figs. 3 and 4). The microvilli are cylinders about 0.10-0.15 # in diameter, about 1.0-1.3 # long, and terminating in a hemispherical cap. In the tissue fixed in glutaraldehyde or the mixture of glutaraldehyde and acrolein, they have a core formed by a bundle of 40-50 straight, parallel nonbranching filaments separated from the lateral wall by finely granular material of low density (Fig. 2, CO). At their proximal end the filaments of the core penetrate about 0.5-0.7 # into the apical cytoplasm forming a rootlet (R) which terminates abruptly. There does not appear to be any continuity between the filaments of the rootlet and any other structures in the apical portion of the cell (Figs. 1 and 2). When compared to the core of the microvilli, the rootlet appears darker and this is due both to the filaments being more closely packed and to the existence of dense, apparently amorphous material between them. This material in which the rootlet filaments are embedded also extends between the rootlets and parallel to the cell surface in a location that corresponds to what has been called the terminal web (Fig. 2). This structure (Fig. 2, TW) appears as a band that is not continuous but presents gaps of variable size (Figs. 1 and 2, G). The terminal web is better appreciated in oblique sections as shown in Fig. 3 a, b; it extends laterally out to the zonula adhaerens (ZA). Sections taken parallel to the cell surface also show that the filament bundles become more compact as they penetrate the cytoplasm; the dense material between the filaments of the bundles appears to be continuous with the terminal web in the perpendicular sections (Figs. 4 and 5 a-d). As one examines progressively deeper sections of the cell apex, the terminal web appears first as isolated patches; in deeper sections it looks more like a perforated plate (Figs. 4 and 5 c, d, TW). In the spaces between the rootlets there are a few filaments that do not seem to make contact with the rootlets (Fig. 5 c, d, arrows). Examination of longitudinal and transverse sections does not reveal any evidence of microtubular structures within the core of the microvilli or in their rootlets. Some filaments can be seen to extend
Fie. 2. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. The filaments of the microvillous core (CO)and rootlet (R) are visible; they insert distally in an accumulation of dense material beneath the inner leaflet of the plasma membrane (arrow). The filaments of the rootlets of the microvilli (R) are more closely packed than those of the core and are embedded in a dense matrix that is continuous with the terminal web (TW); this structure shows visible gaps (G). × 124,000.
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from the desmosomes into the cytoplasm immediately below the level of the rootlets, but these give no evidence of continuity with the rootlets (Fig. 1, DW). In material fixed in bicarbonate-buffered osmium and stained during dehydration with uranyl acetate, the appearance is quite different (Fig. 6). The contents of the microvilli have a finely granular appearance. The rootlets (R), too, are finely granular, and they are slightly denser than the interior of the microvilli. The terminal web can again be seen as a finely granular discontinuous structure (Fig. 6, TW). It appears as patches whose density merges with that of the rootlets. An accumulation of material with the same textural and staining characteristics can be seen around the zonula adhaerens (Fig. 7, ZA). The filaments of the desmosomal web (D W) can be seen clearly extending from the desmosomes (D). Some of these filaments penetrate the terminal web and run between the rootlets but do not appear to merge with them (Figs. 6 and 7, arrow). When the tissue was stained with PTA during dehydration and embedded either in Epon or Araldite, the appearance (Fig. 8) is similar to what is observed when uranyl acetate was used as an en bloc stain (cf. Fig. 6). However, another component becomes evident: in the space between the rootlets there are short, branching structures that are quite different from the filaments of the desmosomal web (Fig. 9 a-c, arrows). These structures do not seem connected directly with either the terminal web or the rootlets. Fixation with phosphate- or cacodylate-buffered osmium tetroxide only, without aldehydes gives comparable results (Figs. 10 and 11). Even though the filaments of the core of the microvilli (CO) and rootlets (R) do not appear as clearly as in aldehyde-fixed tissue, they are somewhat more evident than in material fixed in bicarbonate-buffered osmium, whether the bicarbonate material was stained during dehydration or only after sectioning (Fig. 10). The terminal web (TW), too, is somewhat more prominent than with bicarbonate-buffered osmium, and gaps are evident (Fig. 10). A desmosomal web (DW) is clearly visible, and some filaments can again be traced penetrating the terminal web (Fig. 10, arrow). Those specimens that originated from the intestinal loops fixed while distended with the mixture of aldehydes have a distinctive appearance. By light microscopy the entire wall appears thin. The villi and crypts (Fig. 12, VI, CL) are short and are separated from each other by long stretches of thin mucosa covered with flattened
Fig. 3 a and b. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. (a) Oblique section through the apex. Sections of the microvilli (MV)and their rootlets (R) can be seen. The terminal web (TW) appears as a band extending laterally toward the zonula adhaerens (ZA). Some gaps (G) are very evident. (b) A gap as well as the nonfilamentous texture of the terminal web can be seen in this enlargement of the area enclosed in picture 3 a. P, pits; CA, canaliculi, a, × 26,500; b, × 50,000.
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epithelial cells (Fig. 12a, b, S). By electron microscopy, the villlous and crypt epithelial cells have a similar appearance to those in specimens fixed by immersion in the same fixative. However, those cells covering the areas between the base of the villi and the mouth of the crypts have relatively few microvilli, spaced irregularly and projecting at various angles of inclination, sometimes divergent (Fig. 13, MV). Their cores (CO) show the typical filaments seen with aldehyde fixation, but the rootlets (R) are short. Below the plasma membrane, mainly in areas where there are no microvilli, bundles of filaments can be seen that appear similar to rootlets sectioned at an angle (Fig. 14, R). The terminal web is sparse (Fig. 13, TW). At the cellular junction the desmosomes (D) nearest to the lumen are always stretched; the filaments of the desmosomal web emerge from them in thick bundles coursing parallel to the cell surface. These sometimes can be followed almost throughout the width of the cell (Fig. 13, DW). In contrast to these changes the zonula occludens remains closed as shown in Fig. 14 (ZO), and no changes are seen in the zonula adhaerens. The desmosomes located deeper in the cytoplasm have a normal appearance and the filaments connected to them radiate in all directions.
DISCUSSION Previous studies by light and electron microscopy have described the structure of the apical portion of the small intestinal epithelial cell. This paper is an attempt to clearly define the relationships between the various structures in the apex. The name "terminal web" was first used by Sauer (1935), who studied cells of the neural plate of vertebrate embryos, to describe a filamentous structure observed with the light microscope a n d which he considered as playing an important role in determining the shape of these cells (30, 31). In another light microscopic observation in 1958, Puchtler and Leblond described what they interpreted as a filamentous structure immediately below the brush border and running parallel to the luminal surface of the absorptive cells of the small intestine. They, too, called it the terminal web (26). According to their description, the terminal web inserts into terminal bars at the lateral cell wall. A similar arrangement was described later by the same authors and their coworkers in other cell types, both normal and abnormal (3, 13, 14). These findings were compatible with those of Chambers and de R6nyi; who demonstrated, with the use of the micromanipulaFI6. 4. Glutaraldehyde-acrolei:n, postfixed in bicarbonate-buffered osmium::~Section of the apex parallel to the liminal surface showing transverse sections of microvilli (~irV~'and their cores and rootlets (R). The terminal web (TW) appears as patches of nonfibrillar mater~al between the rootlets in the deeper parts of the~':hpex. Material of similar density surrounds: the zonula adhaerens (ZA). V, coated vesicles. × 34,000.
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tor, that the "striated cuticle" is a very stiff structure (2). Kallenbach showed that the terminal web has different staining properties from the brush border (lI). It is further of interest that this author described the appearance of the terminal web as "granular" in the light microscope. This appearance could be a reflection of the numerous gaps described in our studies. Palay and Karlin in their study of the ultrastructure of the intestinal epithelium in mice showed that the microvilli have a filamentous core that penetrates into the cytoplasm (25) forming a rootlet as previously described by Zetterqvist (36). They suggested that this rootlet merges with a dense "feltwork" of horizontal filaments corresponding to the "terminal web" previously described by light microscopy. They further suggested that these horizontal filaments could explain the negative birefringence observed by Schmidt in the apical area of tadpole intestine (32). Palay and Karlin postulated that the terminal web is a firm ectoplasmic gel "that stiffens and further stabilizes the apical surface by attaching to the terminal bars and thereby determines the regular arrangement of the microvilli" (25). Subsequent studies in other mammals, including man, produced descriptions similar to those of Palay and Karlin (1, 6, 8, 24, 33). M c N a b b and Sandborn, using material fixed in a mixture of glutaraldehyde and acrolein described "angular bends of the rootlet filaments" and suggested that there is direct continuity between the rootlets and the terminal web and that "the rootlet filaments represent an extension of filamentous elements from the terminal web." In their opinion, some of the rootlet filaments even merge with those of the desmosomal web to provide mechanical support for the microvillous border (17,
18, 29). The results of our study show that the apex of the absorptive cell in the rat contains several distinct groups of filaments as well as an apparently amorphous component. One group of filaments forms the core of the microvilli and the rootlets, which are best visualized in tissue fixed with either glutaraldehyde alone or glutaraldehydeacrolein, postfixed in osmium (Figs. 1-5, CO and R). Contrary to the observations of some authors (12, 21, 23), we saw no tubular components in the cores of the microvilli or the rootlets. The desmosomal web is another separate group of filaments; it shows no continuity with those of the rootlets (Figs. 1, 2, 6, and 8). A third group is formed by the short branching structures seen in the spaces between the rootlets but they are seen only in tissue fixed in bicarbonate-buffered osmium stained en bloc FI6. 5 a-d. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. Transverse sections of the apex of the same cell, where (a) is closest to the surface and (d) is deepest; (d) is an enlargement of a portion of Fig. 4. The terminal web (TW) can be seen in the deeper sections. In sections (c) and (d) some filaments course between the rootlets but are not continuous with them (arrows). The circles identify the same group of rootlets in the different sections, x 76,000.
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FIG. 6. Bicarbonate-buffered o s m i u m , u r a n y l acetate staining en bloc. F i l a m e n t s are n o t seen in t h e microvillous core or rootlets; t h e rootlets (R) still are denser t h a n the cores (CO). T h e terminal web (TW) is n o n f i l a m e n t o u s a n d h a s frequent gaps (G). Similar material s u r r o u n d s the z o n u l a adh a e r e n s (ZA). T h e d e s m o s o m a l web (DW)is p r o m i n e n t , a n d s o m e of its filaments course between t h e rootlets (arrow). × 64,0{)0.
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Fra. 7. Bicarbonate-buffered o s m i u m , u r a n y l acetate staining en bloc. S a m e structure as t h a t seen in Fig. 6, b u t in oblique section, x 17,700.
with PTA; they are separate and distinct from the two other groups described above (Fig. 9 a-c, arrows). Fixation with bicarbonate-buffered osmium tetroxide and staining during dehydration with uranyl acetate or PTA (Figs. 6--9) changes the appearance of the microvilli and their rootlets. The microvilli are filled with delicate, granular material at the expense of the core filaments. The contents of the rootlets appear to be similar though denser. This may be due to disorganization and partial extraction of the filaments of the cores and rootlets during fixation and dehydration, or to altered stainability. At the same time, however, the filaments of the desmosomal web are well visualized and can be followed individually as some of them course between the rootlets (Figs. 6 and 7, DW). The difference in staining characteristics between the filaments of the
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microvilli a n d those of the t e r m i n a l web, best seen in Figs. 6 a n d 7, suggest that these t w o structures are chemically different f r o m each other and, therefore, are unlikely to be continuous. A few filaments of the d e s m o s o m a l web traverse the terminal web into the space between the rootlets, b u t there are n o t e n o u g h t of t h e m to f o r m a weblike structure at this level (Figs. 6 a n d 7, arrows). The a p p a r e n t l y a m o r p h o u s c o m p o n e n t is the structure called the terminal web (1, 6, 8, 13, 14, 24-26, 33) a n d does n o t have an obvious filamentous texture in a n y of o u r pictures. I t a p p e a r s as an irregularly p e r f o r a t e d sheet in which the filaments of the rootlets are e m b e d d e d up to the bases of the microvilli (Fig. 2, TW). The presence of this a m o r p h o u s m a t e r i a l coupled with the closer p a c k i n g of the r o o t l e t filaments a p p e a r s to account for the increased density of the r o o t l e t bundles. The structural a n d staining characteristics of the terminal web clearly differentiate it f r o m the other c o m p o n e n t s of the cell apex. It persists as an a p p a r e n t l y g r a n u l a r layer u n d e r all fixation a n d staining conditions e m p l o y e d in this paper, whereas the filaments of t e e rootlets a n d cores, the d e s m o s o m a l filaments a n d the short b r a n c h i n g filaments are accentuated or obliterated with one p r o c e d u r e or another. F o r example, P T A p r o v i d e s the strongest c o n t r a s t for the short b r a n c h i n g filaments while this or u r a n y l acetate accentuate the d e s m o s o m a l filaments, b u t b o t h of these obliterate the core a n d r o o t l e t filaments. The latter filaments are best seen with a l d e h y d e fixation. By n o n e of these techniques is the terminal web a p p r e c i a b l y altered in its a p p e a r a n c e . T h u s it w o u l d seem to be different f r o m any of these c o m p o n e n t s , p r o b a b l y an entity u n t o itself. Since it c a n n o t be resolved as distinctly fibrillar by any of the fixation o r staining m e t h o d s e m p l o y e d here, the terminal web is described as being " g r a n u l a r " or " a m o r p h o u s . " In reality it m a y be c o m p o s e d of m a t t e d very fine irregular or b r a n c h i n g fibrils, b u t which c a n n o t be t r a c e d individually far enough in these pictures to give the a p p e a r a n c e of a thread. I n their p a p e r describing the a n a t o m i c relationships between the microvilli, the FIG. 8. Bicarbonate-buffered osmium, PTA staining en bloc. The overall image is very similar to that of the blocks stained with uranyl acetate (Fig. 6). x 61,009. Fig. 9 a-c. Bicarbonate-buffered osmium, PTA staining en bloc. X and Y identify the areas in Fig. 9 a from which enlargements 9 b and 9 c were made. Between the rootlets, some short, disconnected, branching profiles can be seen (arrows). These are quite distinct from the filaments of the desmosomal web (DW). a, ×73,500; b and c, x 143,000. Fro. 10. Cacodylate-buffered osmium. The contents of the microvillous core (CO) and rootlet (R) have a filamentous appearance (cf. Figs. 1 and 2). The terminal web (TW) is very prominent, extending laterally toward the zonula adhaerens (ZA) and containing gaps (G). Some filaments, similar to those of the desmosomal web, are seen between the rootlets (arrow). × 37,000. Fro. 11. Phosphate-buffered osmium. The terminal web (TW) is faintly outlined, at the level of the zonula adhaerens (ZA). Some filaments of the desmosomal web can be seen (DW). × 48,000. FIG. 12a and b. Effect of distension with glutaraldehyde-acrolein, postfixed in osmium. (a) Light micrograph of a 1 # Epon section of the base of a villus (VI) and its adjacent stretched segment (S). (b) Enlargement of the area enclosed in Fig. 12a. The epithelium of the stretched segment is very distorted and microvilli are sparse. Richardson's stain, a, × 500; b, × 2100.
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FIG. 15. Hypothetical reconstruction of the results obtained with the techniques described in this paper.
t e r m i n a l web a n d the terminal bar, Palay a n d K a r l i n h a d infused fixative into the l u m e n (25). We, therefore, employed a similar procedure except that we used a higher pressure a n d a different fixative, to see if, u n d e r conditions in which the cell apex is stretched, there would be orientation of the terminal web a n d filamentous structures would then become apparent. The aldehyde mixture was selected because of our observation that the rootlets a n d the terminal web are very p r o m i n e n t with this technique. We found, as J o h n s o n (10) did, that the distention of loops of small intestine with fixative caused extensive stretching a n d separation of villi a n d crypts (Fig. 12).
FIG. 13. Glutaraldehyde-acrolein, postfixed in osmium. Apex of a cell of the stretched segment. The microvilli (MV) are sparse and radiate from the cell surface at various angles. The terminal web (TW) is not prominent. The desmosomal plaques (D) seem to be pulled apart, and very thick bundles of filaments emerge from them (DW). CO, microvillous core; R, rootlet. Note: Figure is enlargement to show details of filaments. In the original picture, the filaments (D W) extended across the entire cell apex. × 47,000. Fio. 14a and b. Glutaraldehyde-acrolein, postfixed in osmium, both with bicarbonate buffer. (a) Junctional complex in cells of the stretched segment. The zonula occludens (ZO) remains tight while the subjacent desmosome (D) shows pulling apart of its plaques. The surface of the cells is undulating. Some rootlet filaments can be seen (R), but they do not seem to be connected to any microvilli. (b) A well preserved microtuble (MT) from a neighboring area suggesting that fixation has been adequate, a and b, × I 18,000.
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It is in the cells of the areas of thin mucosa between the base of the villi and the mouth of the crypts that changes in ultrastructure are evident because the stretching force has presumably acted with maximal intensity at this level. The terminal web is stretched and because the rootlets are embedded in it, they are pulled and the microvilli project from the cell surface at various angles of inclination. Even under these circumstances, no filamentous profiles become apparent in the terminal web. The thick horizontal bundles seen in the cell apex in this experiment are connected to the desmosomes (Figs. 13 and 14 a, D W). That the cell web is a highly resilient structure is further evidenced by the fact that the zonula occludens remains tight (Fig. 14 a, ZO).
The microvilli are thus cemented to and supported by this structure which inserts into the lateral wall of the cell at the level of the zonula adhaerens. This arrangement may well be responsible for the fact that the apex can be isolated as a unit by cell disruption and centrifugation. Figure 15 represents our own hypothetical reconstruction of the cell apex based on the results obtained with the different techniques described in this paper. The widely held concept of the structure of the intestinal epithelial cell apex is that the filaments of the core and rootlet of the microvilli are continuous with a horizontal, net-like, obviously filamentous terminal web that inserts at the lateral wall. F r o m the results of the present work we rather believe that the filaments of the rootlet are not in continuity with any other structure. As to the terminal web, it appears as a discontinuous, perforated, granular sheet in which the rootlets are embedded. In this way, the terminal web may provide considerable rigidity to the cell apex and at the same time permit exhange of nutrients. It is very probable that at the molecular level it is composed of elongated, multiple-branched asymmetrical elements, but this has not been resolved with the techniques used in this study. The authors acknowledge the review and helpful criticism of the manuscript by Drs. Daniel Szollosi, Shmuel Eidelman, and Douglas E. Kelly. Thanks are given to Miss Patricia C. Phelps and to Mr. T. G. Davies-Williams for their assistance. Mrs. Phyllis Wood made the drawing. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
CARDELL,R. R., JR., BADENHAUSEN,S. and PORTER, K. R., J. Cell Biol. 34, 123 (1967) CHAMBERS,R. and RI~NYI, G. S., Am. J. Anat. 35, 385 (1925). CLERMONT,Y. and PEREIRA,G., Anat. Record 156, 215 (1966). DONALDSON,R. M., MACKENZIE,I. L. and TRIER, J. S., d. Clin. Invest. 46, 1215 (1967). EICHHOLZ,A. and CRANE, R. K., J. Cell Biol. 26, 687 (1965). FARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). FAUST, R. G., Wu, S.=M. L. and FAGGARD, M. L., Science 155, 1261 (1967). FAWCETT,D. W., Circulation 26, 1105 (1962).
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9. HARRISON,D. D. and WEBSTER,H. L., Biochim. Biophys. Aeta 93, 662 (1964). 10. JOHNSON,F. P., Am. J. Anat. 14, 235 (1912-1913). 11. KALLENBACrhE., The cell web. A fibrillar component of the cytoplasm. Thesis, McGill University, Montreal, Canada (1963). 12. LAGUENS,R. and BRIONES, M., Lab. Invest. 14, 1616 (1965). 13. LEBLOND,C. P., PUCnTLER, H. and CLERMONT,Y., Nature 186, 784 (1960). 14. LEBLOND, C. P., SARKAR,K., KALLENBACH,E. and CLERMONT, Y. Cancer Res. 26, 2259 (1966). 15. LUFT, J. H., Anat. Record 133, 305 (1959). 16. - - - - J. Biophys. Biochem. Cytol. 9, 409 (196l). 17. Mc NABR, J. D., An electron microscope study of filaments and microtubules in the chief cells of the epithelium of the rat intestine. Thesis, McGill University, Montreal, Canada (1964). 18. Mc NABB, J. D. and SANDBORN,E., J. Cell Biol. 22, 701 (1964). 19. MILLER,D. and CRANE, R. K., Anal. Biochem. 2, 284 (1961). Biochim. Biophys. Acta 52, 281 (1962). 20. - 21. MILLINGTON,P. F. and F1NEAN, J. B., J. Cell Biol. 14, 125 (1962). 22. MILLINGTON,P. F., CRITCHLE¥,D. R. and TOVELL,P. W. A., J. CellSci. 1, 415 (1966). 23. MU~Z~ERJEE,T. M. and WILLIAMS,A. WYNN, J. Cell Biol. 34, 447 (1967). 24. PADYKULA,H., STRAUSS, E. W., LADMAN,A. J. and GARDNER, F. H., Gastroenterology 40, 735 (1961). 25. PALAY, S. and KARLIN, L., J. Biophys. Biochem. Cytol. 5, 363 (1959). 26. PUCHTLER,H. and LEBLOND,C. P., Am. J. Anat. 102, 1 (1958). 27. REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). 28. SABATINhD. D., BENSCrI, K. G. and BARRNETT,R. J., J. Cell Biol. 17, 19 (1963). 29. SANDBORN, E., KOEN, P. F., MCNABB, J. D. and MOORE, G., J. Ultrastruet. Res. 11, 123 (1964). 30. SAUZR,F. D., J. Comp. Neurol. 62, 377 (1935). J. Morphol. 61, 563 (1937). 31. - 32. SCHMIDT,W. J., Z. Zellforsch. Mikroskop. Anat. 33, 1 (1943). 33. TR~ER,J. S. and RUBIN, C. E., Gastroenterology 49, 676 (1965). 34. WooD, R. L. and LUET, J. H., J . Ultrastruct. Res. 12, 22 (1965). 35. ZAMBONI,L., Anat. Record 139, 290 (1961). 36. ZETTERQWST, H., The ultrastructural organization of the columnar absorbing cells of the mouse jejunum. Kar01inska Institutet, Aktiebolaget Godvil, Stockholm, Sweden, 1956.
J. ULTRASTRUCTURERESEARCrI31, 291--311 (1970)
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Fine Structure of the Apex of Absorptive Cells from Rot Small Intestine s OSCAR BRUNSER2 and JOHN H. LUFT
Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington 98105 Received August 28, 1969 These cells were studied in rats, using buffered osmium or aldehyde fixatives followed by osmium. Three groups of filaments and the terminal web were visualized in the apex. One group, seen in aldehyde-fixed tissue formed the cores and rootlets of the microvilli; they showed no continuity with other filaments. Another group formed the desmosomal web, best seen in tissue stained en bloc with uranyl acetate of pbospbotungstic acid (PTA). The third group appeared as short, branched profiles between the rootlets after staining with PTA. The terminal web appeared as a dense layer with gaps, extending out to the zonula adhaerens. It seemed to embed the rootlets penetrating between their filaments. Although it appeared granular with the techniques used here, it is probable that at the molecular level the terminal web is formed by elongated branching elements. By supporting the rootlets and microvilli, the terminal web endows the apex with mechanical stability and anchors it to the body of the cell. The apices of the epithelial cells of the m a m m a l i a n small intestine are the structures t h r o u g h which most nutrients are obsorbed. They possess as conspicuous specializations of their free surfaces large numbers of vertically oriented, slender microvilli. These and the other components of the cell apex are so thoroughly interconnected that it is possible to isolate the whole cell apex as a discrete unit without serious disruption of its overall structure. Miller and Crane (19, 20) and others (4, 5, 7, 22), taking advantage of this characteristic, have been able to obtain purified preparations of apices with minimal amounts of the underlying cytoplasm by subjecting cells to osmotic shock and various other treatments. Similar results were obtained 1 Dr. Brunser was supported by a Fellowship from the John Simon Guggenheim Memorial Foundation. Supported by USPHS Grants GM-16598 and GM-00136 from the National Institutes of Health. Presented at the Eightieth Session of the American Association of Anatomists, Kansas City, Missouri, April 4-7, 1967. 2 Present address: Laboratorio de Investigaciones Pediatricas, Escuela de Medicina, Universidad de Chile, Casilla 5370, Santiago, Chile.
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by Harrison and Webster (9) using low frequency vibrations; this can be interpreted as indicating that the mechanical strength of this area is different from that of the rest of the cytoplasm. According to the early studies of Zetterqvist (36) and of Palay and Karlin (25), the microvillus has a filamentous axis that penetrates into the apical cytoplasm forming a rootlet that fuses with a network of horizontally oriented filaments called the "terminal web." This terminal web is described as being parallel to the luminal surface of the cell and inserting itself into the "terminal bar" at the periphery of the cellular apex. The maintenance of the orderly distribution of the microvilli and the rigidity and strength of the cellular apex have been attributed to the existence of this filamentous web and its attachment to other structures. Since these descriptive terms had originated from earlier light microscopic studies, a certain amount of confusion resulted when they were applied to electron microscopic observations (13, 14, 26). While studying our electron microscopic material, we were unable to identify a distinctly filamentous terminal web as had been previously described (1, 17, 18, 21, 24, 32). In order to find reasons for this descrepancy between our observations and those in previous publications, we studied the fine structure of the apex of the absorptive cell using different techniques of fixation and staining.
MATERIALS AND METHODS Samples of small intestine were obtained from an area about 1 cm beyond the pylorus from adult rats anesthetized with pentobarbital administered intraperitoneally. Rings of intestine, about 2 mm thick, were sectioned and immediately divided into four approximately equal quadrants. Contraction of the muscle coats transformed each quadrant into an everted hollow cylinder, with the outer surface covered by the mucosa with its villi slightly spread out radially and the inner surface covered by the serosa. These cylinders were fixed for 1 hour in bicarbonate- (34), phosphate- (34), or cacodylate- (34) buffered osmium tetroxide or in 2.4% glutaraldehyde in phosphate buffer (28) with and without 1% acrolein added (15), and postfixed for 1 hour in bicarbonate-buffered osmium. In one experiment, segments of intestine from two animals were isolated between ligatures without damaging the blood supply, and glutaraldehyde-acrolein mixture in phosphate buffer was injected under moderate pressure into the lumen in order to produce distention. After maintaining distention for 5 minutes, the loop was removed, divided in small pieces and further fixed by immersion in the glutaraldehyde-acrolein mixture and postfixed in osmium. All the tissue was dehydrated in an ethanol series. Some of the pieces of the bicarbonatebuffered osmium-fixed tissue were then transferred to absolute ethanol containing either 1% uranyl acetate or 1% phosphotungstic acid (PTA) for 1 hour (16). They were afterward washed in two changes of 10 minutes each of absolute ethanol. Of the PTA stained tissue, some pieces were subsequently transferred to 0.01% sodium hydroxide in absolute ethanol for 25 minutes, washed again in absolute ethanol, and transferred to propylene oxide previous to their infiltration and embedding in Epon 812 (16). The PTA-stained tissue that was
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FI~. 1. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. The filaments of the core (CO) and rootlet (R) of the microvilli (MV) are identifiable as well as some belonging to the desmosomal web (D W). There is no evidence of a filamentous terminal web at the level of the rootlets. L, intestinal lumen; ZO, zonula occludens; ZA, zonula adhaerens; D, desmosome, x 51,000.
not treated with alcoholic sodium hydroxide was embedded in Araldite (16). All the other pieces of tissue were embedded in E p o n 812 (16). Sections were cut with du Pont diamond knives on a Sorvall MT-2 ultramicrotome, collected on Parlodion and carbon-coated grids, and double stained with uranyl acetate and lead citrate (27). The observations were made with a modified R C A E M U - 2 C electron microscope using a 50-/~ objective aperture and an accelerating voltage of 50 kV. The observations were m a d e in cells of the upper third of each villus, except in the distended segments, where the boundary between the crypts of Lieberktihn and the base of the villi was also studied.
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OBSERVATIONS The apical area of the intestinal epithelial cell is characterized by the presence of uniformly shaped, regularly spaced microvilli (Figs. 1 and 2, MV) and by the absence of organelles that are scattered throughout the rest of the cytoplasm. Between and just beneath the bases of the microvilli and in the adjacent area there are pits (P), coated vesicles (V), and membrane-bounded canaliculi (CA) (Figs. 3 and 4). The microvilli are cylinders about 0.10-0.15 # in diameter, about 1.0-1.3 # long, and terminating in a hemispherical cap. In the tissue fixed in glutaraldehyde or the mixture of glutaraldehyde and acrolein, they have a core formed by a bundle of 40-50 straight, parallel nonbranching filaments separated from the lateral wall by finely granular material of low density (Fig. 2, CO). At their proximal end the filaments of the core penetrate about 0.5-0.7 # into the apical cytoplasm forming a rootlet (R) which terminates abruptly. There does not appear to be any continuity between the filaments of the rootlet and any other structures in the apical portion of the cell (Figs. 1 and 2). When compared to the core of the microvilli, the rootlet appears darker and this is due both to the filaments being more closely packed and to the existence of dense, apparently amorphous material between them. This material in which the rootlet filaments are embedded also extends between the rootlets and parallel to the cell surface in a location that corresponds to what has been called the terminal web (Fig. 2). This structure (Fig. 2, TW) appears as a band that is not continuous but presents gaps of variable size (Figs. 1 and 2, G). The terminal web is better appreciated in oblique sections as shown in Fig. 3 a, b; it extends laterally out to the zonula adhaerens (ZA). Sections taken parallel to the cell surface also show that the filament bundles become more compact as they penetrate the cytoplasm; the dense material between the filaments of the bundles appears to be continuous with the terminal web in the perpendicular sections (Figs. 4 and 5 a-d). As one examines progressively deeper sections of the cell apex, the terminal web appears first as isolated patches; in deeper sections it looks more like a perforated plate (Figs. 4 and 5 c, d, TW). In the spaces between the rootlets there are a few filaments that do not seem to make contact with the rootlets (Fig. 5 c, d, arrows). Examination of longitudinal and transverse sections does not reveal any evidence of microtubular structures within the core of the microvilli or in their rootlets. Some filaments can be seen to extend
Fie. 2. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. The filaments of the microvillous core (CO)and rootlet (R) are visible; they insert distally in an accumulation of dense material beneath the inner leaflet of the plasma membrane (arrow). The filaments of the rootlets of the microvilli (R) are more closely packed than those of the core and are embedded in a dense matrix that is continuous with the terminal web (TW); this structure shows visible gaps (G). × 124,000.
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from the desmosomes into the cytoplasm immediately below the level of the rootlets, but these give no evidence of continuity with the rootlets (Fig. 1, DW). In material fixed in bicarbonate-buffered osmium and stained during dehydration with uranyl acetate, the appearance is quite different (Fig. 6). The contents of the microvilli have a finely granular appearance. The rootlets (R), too, are finely granular, and they are slightly denser than the interior of the microvilli. The terminal web can again be seen as a finely granular discontinuous structure (Fig. 6, TW). It appears as patches whose density merges with that of the rootlets. An accumulation of material with the same textural and staining characteristics can be seen around the zonula adhaerens (Fig. 7, ZA). The filaments of the desmosomal web (D W) can be seen clearly extending from the desmosomes (D). Some of these filaments penetrate the terminal web and run between the rootlets but do not appear to merge with them (Figs. 6 and 7, arrow). When the tissue was stained with PTA during dehydration and embedded either in Epon or Araldite, the appearance (Fig. 8) is similar to what is observed when uranyl acetate was used as an en bloc stain (cf. Fig. 6). However, another component becomes evident: in the space between the rootlets there are short, branching structures that are quite different from the filaments of the desmosomal web (Fig. 9 a-c, arrows). These structures do not seem connected directly with either the terminal web or the rootlets. Fixation with phosphate- or cacodylate-buffered osmium tetroxide only, without aldehydes gives comparable results (Figs. 10 and 11). Even though the filaments of the core of the microvilli (CO) and rootlets (R) do not appear as clearly as in aldehyde-fixed tissue, they are somewhat more evident than in material fixed in bicarbonate-buffered osmium, whether the bicarbonate material was stained during dehydration or only after sectioning (Fig. 10). The terminal web (TW), too, is somewhat more prominent than with bicarbonate-buffered osmium, and gaps are evident (Fig. 10). A desmosomal web (DW) is clearly visible, and some filaments can again be traced penetrating the terminal web (Fig. 10, arrow). Those specimens that originated from the intestinal loops fixed while distended with the mixture of aldehydes have a distinctive appearance. By light microscopy the entire wall appears thin. The villi and crypts (Fig. 12, VI, CL) are short and are separated from each other by long stretches of thin mucosa covered with flattened
Fig. 3 a and b. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. (a) Oblique section through the apex. Sections of the microvilli (MV)and their rootlets (R) can be seen. The terminal web (TW) appears as a band extending laterally toward the zonula adhaerens (ZA). Some gaps (G) are very evident. (b) A gap as well as the nonfilamentous texture of the terminal web can be seen in this enlargement of the area enclosed in picture 3 a. P, pits; CA, canaliculi, a, × 26,500; b, × 50,000.
3a~
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epithelial cells (Fig. 12a, b, S). By electron microscopy, the villlous and crypt epithelial cells have a similar appearance to those in specimens fixed by immersion in the same fixative. However, those cells covering the areas between the base of the villi and the mouth of the crypts have relatively few microvilli, spaced irregularly and projecting at various angles of inclination, sometimes divergent (Fig. 13, MV). Their cores (CO) show the typical filaments seen with aldehyde fixation, but the rootlets (R) are short. Below the plasma membrane, mainly in areas where there are no microvilli, bundles of filaments can be seen that appear similar to rootlets sectioned at an angle (Fig. 14, R). The terminal web is sparse (Fig. 13, TW). At the cellular junction the desmosomes (D) nearest to the lumen are always stretched; the filaments of the desmosomal web emerge from them in thick bundles coursing parallel to the cell surface. These sometimes can be followed almost throughout the width of the cell (Fig. 13, DW). In contrast to these changes the zonula occludens remains closed as shown in Fig. 14 (ZO), and no changes are seen in the zonula adhaerens. The desmosomes located deeper in the cytoplasm have a normal appearance and the filaments connected to them radiate in all directions.
DISCUSSION Previous studies by light and electron microscopy have described the structure of the apical portion of the small intestinal epithelial cell. This paper is an attempt to clearly define the relationships between the various structures in the apex. The name "terminal web" was first used by Sauer (1935), who studied cells of the neural plate of vertebrate embryos, to describe a filamentous structure observed with the light microscope a n d which he considered as playing an important role in determining the shape of these cells (30, 31). In another light microscopic observation in 1958, Puchtler and Leblond described what they interpreted as a filamentous structure immediately below the brush border and running parallel to the luminal surface of the absorptive cells of the small intestine. They, too, called it the terminal web (26). According to their description, the terminal web inserts into terminal bars at the lateral cell wall. A similar arrangement was described later by the same authors and their coworkers in other cell types, both normal and abnormal (3, 13, 14). These findings were compatible with those of Chambers and de R6nyi; who demonstrated, with the use of the micromanipulaFI6. 4. Glutaraldehyde-acrolei:n, postfixed in bicarbonate-buffered osmium::~Section of the apex parallel to the liminal surface showing transverse sections of microvilli (~irV~'and their cores and rootlets (R). The terminal web (TW) appears as patches of nonfibrillar mater~al between the rootlets in the deeper parts of the~':hpex. Material of similar density surrounds: the zonula adhaerens (ZA). V, coated vesicles. × 34,000.
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tor, that the "striated cuticle" is a very stiff structure (2). Kallenbach showed that the terminal web has different staining properties from the brush border (lI). It is further of interest that this author described the appearance of the terminal web as "granular" in the light microscope. This appearance could be a reflection of the numerous gaps described in our studies. Palay and Karlin in their study of the ultrastructure of the intestinal epithelium in mice showed that the microvilli have a filamentous core that penetrates into the cytoplasm (25) forming a rootlet as previously described by Zetterqvist (36). They suggested that this rootlet merges with a dense "feltwork" of horizontal filaments corresponding to the "terminal web" previously described by light microscopy. They further suggested that these horizontal filaments could explain the negative birefringence observed by Schmidt in the apical area of tadpole intestine (32). Palay and Karlin postulated that the terminal web is a firm ectoplasmic gel "that stiffens and further stabilizes the apical surface by attaching to the terminal bars and thereby determines the regular arrangement of the microvilli" (25). Subsequent studies in other mammals, including man, produced descriptions similar to those of Palay and Karlin (1, 6, 8, 24, 33). M c N a b b and Sandborn, using material fixed in a mixture of glutaraldehyde and acrolein described "angular bends of the rootlet filaments" and suggested that there is direct continuity between the rootlets and the terminal web and that "the rootlet filaments represent an extension of filamentous elements from the terminal web." In their opinion, some of the rootlet filaments even merge with those of the desmosomal web to provide mechanical support for the microvillous border (17,
18, 29). The results of our study show that the apex of the absorptive cell in the rat contains several distinct groups of filaments as well as an apparently amorphous component. One group of filaments forms the core of the microvilli and the rootlets, which are best visualized in tissue fixed with either glutaraldehyde alone or glutaraldehydeacrolein, postfixed in osmium (Figs. 1-5, CO and R). Contrary to the observations of some authors (12, 21, 23), we saw no tubular components in the cores of the microvilli or the rootlets. The desmosomal web is another separate group of filaments; it shows no continuity with those of the rootlets (Figs. 1, 2, 6, and 8). A third group is formed by the short branching structures seen in the spaces between the rootlets but they are seen only in tissue fixed in bicarbonate-buffered osmium stained en bloc FI6. 5 a-d. Glutaraldehyde-acrolein, postfixed in bicarbonate-buffered osmium. Transverse sections of the apex of the same cell, where (a) is closest to the surface and (d) is deepest; (d) is an enlargement of a portion of Fig. 4. The terminal web (TW) can be seen in the deeper sections. In sections (c) and (d) some filaments course between the rootlets but are not continuous with them (arrows). The circles identify the same group of rootlets in the different sections, x 76,000.
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FIG. 6. Bicarbonate-buffered o s m i u m , u r a n y l acetate staining en bloc. F i l a m e n t s are n o t seen in t h e microvillous core or rootlets; t h e rootlets (R) still are denser t h a n the cores (CO). T h e terminal web (TW) is n o n f i l a m e n t o u s a n d h a s frequent gaps (G). Similar material s u r r o u n d s the z o n u l a adh a e r e n s (ZA). T h e d e s m o s o m a l web (DW)is p r o m i n e n t , a n d s o m e of its filaments course between t h e rootlets (arrow). × 64,0{)0.
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Fra. 7. Bicarbonate-buffered o s m i u m , u r a n y l acetate staining en bloc. S a m e structure as t h a t seen in Fig. 6, b u t in oblique section, x 17,700.
with PTA; they are separate and distinct from the two other groups described above (Fig. 9 a-c, arrows). Fixation with bicarbonate-buffered osmium tetroxide and staining during dehydration with uranyl acetate or PTA (Figs. 6--9) changes the appearance of the microvilli and their rootlets. The microvilli are filled with delicate, granular material at the expense of the core filaments. The contents of the rootlets appear to be similar though denser. This may be due to disorganization and partial extraction of the filaments of the cores and rootlets during fixation and dehydration, or to altered stainability. At the same time, however, the filaments of the desmosomal web are well visualized and can be followed individually as some of them course between the rootlets (Figs. 6 and 7, DW). The difference in staining characteristics between the filaments of the
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microvilli a n d those of the t e r m i n a l web, best seen in Figs. 6 a n d 7, suggest that these t w o structures are chemically different f r o m each other and, therefore, are unlikely to be continuous. A few filaments of the d e s m o s o m a l web traverse the terminal web into the space between the rootlets, b u t there are n o t e n o u g h t of t h e m to f o r m a weblike structure at this level (Figs. 6 a n d 7, arrows). The a p p a r e n t l y a m o r p h o u s c o m p o n e n t is the structure called the terminal web (1, 6, 8, 13, 14, 24-26, 33) a n d does n o t have an obvious filamentous texture in a n y of o u r pictures. I t a p p e a r s as an irregularly p e r f o r a t e d sheet in which the filaments of the rootlets are e m b e d d e d up to the bases of the microvilli (Fig. 2, TW). The presence of this a m o r p h o u s m a t e r i a l coupled with the closer p a c k i n g of the r o o t l e t filaments a p p e a r s to account for the increased density of the r o o t l e t bundles. The structural a n d staining characteristics of the terminal web clearly differentiate it f r o m the other c o m p o n e n t s of the cell apex. It persists as an a p p a r e n t l y g r a n u l a r layer u n d e r all fixation a n d staining conditions e m p l o y e d in this paper, whereas the filaments of t e e rootlets a n d cores, the d e s m o s o m a l filaments a n d the short b r a n c h i n g filaments are accentuated or obliterated with one p r o c e d u r e or another. F o r example, P T A p r o v i d e s the strongest c o n t r a s t for the short b r a n c h i n g filaments while this or u r a n y l acetate accentuate the d e s m o s o m a l filaments, b u t b o t h of these obliterate the core a n d r o o t l e t filaments. The latter filaments are best seen with a l d e h y d e fixation. By n o n e of these techniques is the terminal web a p p r e c i a b l y altered in its a p p e a r a n c e . T h u s it w o u l d seem to be different f r o m any of these c o m p o n e n t s , p r o b a b l y an entity u n t o itself. Since it c a n n o t be resolved as distinctly fibrillar by any of the fixation o r staining m e t h o d s e m p l o y e d here, the terminal web is described as being " g r a n u l a r " or " a m o r p h o u s . " In reality it m a y be c o m p o s e d of m a t t e d very fine irregular or b r a n c h i n g fibrils, b u t which c a n n o t be t r a c e d individually far enough in these pictures to give the a p p e a r a n c e of a thread. I n their p a p e r describing the a n a t o m i c relationships between the microvilli, the FIG. 8. Bicarbonate-buffered osmium, PTA staining en bloc. The overall image is very similar to that of the blocks stained with uranyl acetate (Fig. 6). x 61,009. Fig. 9 a-c. Bicarbonate-buffered osmium, PTA staining en bloc. X and Y identify the areas in Fig. 9 a from which enlargements 9 b and 9 c were made. Between the rootlets, some short, disconnected, branching profiles can be seen (arrows). These are quite distinct from the filaments of the desmosomal web (DW). a, ×73,500; b and c, x 143,000. Fro. 10. Cacodylate-buffered osmium. The contents of the microvillous core (CO) and rootlet (R) have a filamentous appearance (cf. Figs. 1 and 2). The terminal web (TW) is very prominent, extending laterally toward the zonula adhaerens (ZA) and containing gaps (G). Some filaments, similar to those of the desmosomal web, are seen between the rootlets (arrow). × 37,000. Fro. 11. Phosphate-buffered osmium. The terminal web (TW) is faintly outlined, at the level of the zonula adhaerens (ZA). Some filaments of the desmosomal web can be seen (DW). × 48,000. FIG. 12a and b. Effect of distension with glutaraldehyde-acrolein, postfixed in osmium. (a) Light micrograph of a 1 # Epon section of the base of a villus (VI) and its adjacent stretched segment (S). (b) Enlargement of the area enclosed in Fig. 12a. The epithelium of the stretched segment is very distorted and microvilli are sparse. Richardson's stain, a, × 500; b, × 2100.
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FIG. 15. Hypothetical reconstruction of the results obtained with the techniques described in this paper.
t e r m i n a l web a n d the terminal bar, Palay a n d K a r l i n h a d infused fixative into the l u m e n (25). We, therefore, employed a similar procedure except that we used a higher pressure a n d a different fixative, to see if, u n d e r conditions in which the cell apex is stretched, there would be orientation of the terminal web a n d filamentous structures would then become apparent. The aldehyde mixture was selected because of our observation that the rootlets a n d the terminal web are very p r o m i n e n t with this technique. We found, as J o h n s o n (10) did, that the distention of loops of small intestine with fixative caused extensive stretching a n d separation of villi a n d crypts (Fig. 12).
FIG. 13. Glutaraldehyde-acrolein, postfixed in osmium. Apex of a cell of the stretched segment. The microvilli (MV) are sparse and radiate from the cell surface at various angles. The terminal web (TW) is not prominent. The desmosomal plaques (D) seem to be pulled apart, and very thick bundles of filaments emerge from them (DW). CO, microvillous core; R, rootlet. Note: Figure is enlargement to show details of filaments. In the original picture, the filaments (D W) extended across the entire cell apex. × 47,000. Fio. 14a and b. Glutaraldehyde-acrolein, postfixed in osmium, both with bicarbonate buffer. (a) Junctional complex in cells of the stretched segment. The zonula occludens (ZO) remains tight while the subjacent desmosome (D) shows pulling apart of its plaques. The surface of the cells is undulating. Some rootlet filaments can be seen (R), but they do not seem to be connected to any microvilli. (b) A well preserved microtuble (MT) from a neighboring area suggesting that fixation has been adequate, a and b, × I 18,000.
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It is in the cells of the areas of thin mucosa between the base of the villi and the mouth of the crypts that changes in ultrastructure are evident because the stretching force has presumably acted with maximal intensity at this level. The terminal web is stretched and because the rootlets are embedded in it, they are pulled and the microvilli project from the cell surface at various angles of inclination. Even under these circumstances, no filamentous profiles become apparent in the terminal web. The thick horizontal bundles seen in the cell apex in this experiment are connected to the desmosomes (Figs. 13 and 14 a, D W). That the cell web is a highly resilient structure is further evidenced by the fact that the zonula occludens remains tight (Fig. 14 a, ZO).
The microvilli are thus cemented to and supported by this structure which inserts into the lateral wall of the cell at the level of the zonula adhaerens. This arrangement may well be responsible for the fact that the apex can be isolated as a unit by cell disruption and centrifugation. Figure 15 represents our own hypothetical reconstruction of the cell apex based on the results obtained with the different techniques described in this paper. The widely held concept of the structure of the intestinal epithelial cell apex is that the filaments of the core and rootlet of the microvilli are continuous with a horizontal, net-like, obviously filamentous terminal web that inserts at the lateral wall. F r o m the results of the present work we rather believe that the filaments of the rootlet are not in continuity with any other structure. As to the terminal web, it appears as a discontinuous, perforated, granular sheet in which the rootlets are embedded. In this way, the terminal web may provide considerable rigidity to the cell apex and at the same time permit exhange of nutrients. It is very probable that at the molecular level it is composed of elongated, multiple-branched asymmetrical elements, but this has not been resolved with the techniques used in this study. The authors acknowledge the review and helpful criticism of the manuscript by Drs. Daniel Szollosi, Shmuel Eidelman, and Douglas E. Kelly. Thanks are given to Miss Patricia C. Phelps and to Mr. T. G. Davies-Williams for their assistance. Mrs. Phyllis Wood made the drawing. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
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