Ultrastructure of Basement Membranes

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 117

Ultrastructure of Basement Membranes SADAYUKI INOUE Department of Anatomy, McGill University, Montreal, Quebec, Canada, H3A 2B2

I. Introduction

The organism is composed of two main types of components: parenchyma and connective tissues. The parenchyma generally consists of tissues in which cells are closely adjacent to one another; this is the case of surface epithelia such as the epidermis and gastrointestinal lining, the epithelia composing endocrine and exocrine glands, the endothelia lining capillaries and other blood vessels, and the nervous system. Muscle fibers (striated, smooth, and cardiac) and fat cells are isolated individually, but are nevertheless considered to be part of the parenchyma. The Connective tissues are composed of cells scattered between fibers of various types; they include loose, dense, and other types of connective tissue; in addition, bone, cartilage, lymphatic, and myeloid tissues are generally included in a broad definition of connective tissues (Copenhaver et al., 1971). Between parenchyma and connective tissues are thin layers referred to as basement membranes. In simple epithelia and endothelia (Fig. 1, lower left) the cells are in contact with the basement membranes by their basal surface and with the lumen by their apical surface. In stratified epithelia, only a few of the cells contact the basement membrane (Fig. 1, upper left), as is also the case in the nervous system (Fig. 1, right). In the isolated muscle and fat cells, the surface is entirely covered with basement membrane. Finally, nerve fibers and the enclosing Schwann cells are also separated from connective tissue by a basement membrane. The basement membrane of the nervous system is continuous with that covering the Schwann cells of nerve fibers and, through it, establishes continuity with the basement membrane lining other parenchymal cells. In theory at least, the body may be considered as having a single, huge basement membrane separating all parenchyma from connective tissue. In practice, however, the basement membrane may be interrupted in places, for example, along the intestinal epithelium (McClugage e t al., 1986) or missing, as observed in liver (unpublished). The history of basement membrane (summarized from Berdal, 1906) begins with its discovery by Bowman, who also coined the name. It was 57 Copyright Q 1989 by Academic Preqs. Inc.

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FIG. 1. Schematic drawing of the location of basement membranes. They are thin layers (drawn as dark thick lines for emphasis) at the border between connective tissue and various parenchymas, such as epidermis, endothelia, muscle and fat cells, or the entire nervous system.

described as a thin, vitreous, barely distinguishable membrane underlying epithelia and generally not stained by routine stains. Several of its properties were recognized early. It was known to be crossed by nerve terminals but not by blood vessels and to swell when exposed to acetic acid-an early indication of the presence of collagen. The next important step followed from the discovery of the periodic acid-Schiff (PA-Schiff) as a tool for the detection of carbohydrates rich in 1,2-glycol groups (Hotchkiss, 1948). Even before the publication of this work, Lillie (1947) heard of the technique, used it, and noted the reactivity of basement membranes. A survey of many tissues showed that all investigated basement membranes were reactive, even after glycogen extraction, presumably as a result of mucoproteins or other carbohydrate-protein complexes, that is, substances referred to now as glycoproteins (Leblond, 1950). From a series of PA-Schiff-reactive sites, including two essentially composed of basement membranes (lens capsule, lung framework), extracts were obtained that contained protein as well as galactose, fucose, and other sugars, and were described as consisting of carbohydrate-protein complexes (Leblond et af., 1957). Hence glycoproteins were present in basement membrane, in addition to collagen.

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During the 1970s and, even more so, during the 1980s, work on basement membrane increased gradually and considerable progress took place in the biochemical knowledge of basement membrane composition. Recent developments were adequately summarized in several reviews (Kefalides, 1973; Kefalides et al., 1979; Heathcote and Grant, 1981; Martinez-Hernandez and Amenta, 1983; Timpl and Martin, 1982; Timpl and Dziadek, 1986; Abrahamson, 1986), in which biochemical developments were given particular emphasis. The present review, however, will mainly deal with morphological aspects. The first part will describe the ultrastructure and the second part the composition of basement membranes, but with emphasis being placed on the morphological role of its components. With regard to nomenclature, the term basement membrane is widely accepted by biochemists and morphologists, as is its division into three layers, for which the names lamina lucida, densa, and fibroreticularis are commonly used (Kefalides et al., 1979; Laurie and Leblond, 1985). Some authors considered the term basement membrane not altogether satisfactory and attempted to replace it by others, such as “boundary membrane” (Low, 1967) and “basal lamina” (Fawcett, 1962). The latter has met with some success but has also created much confusion, since basal lamina has not only been used to designate the whole basement membrane but has also been restricted by some authors to the association of lamina densa and lucida, and it is commonly used to refer to the lamina densa alone. Hence, the term “basal lamina” is avoided and “basement membrane” is used throughout this review. With regard to the names of the three layers of basement membrane, the term “lamina densa” has met with general acceptance. The term “lamina lucida” is occasionally replaced by “rara.” The third layer has been called-among other terms-pars reticularis, pars diffusa, and lamina fibroreticularis. The use of lamina densa, lucida, and fibroreticularis is recommended in the International Anatomical Nomenclature (Nomina Histologica, in “Nomina Anatomica,” Williams & Wilkins, Baltimore, 1983). We shall accordingly use the terms lamina densa and lamina lucida, but since the third layer does not form a continuous lamina as do the other two, but is composed of discrete elements, we shall replace the term “lamina fibroreticularis” by “pars fibroreticularis.” 11. Structure

Typical basement membranes are thin structures in which the main component is the lamina densa; they may be referred to as “simple” basement membranes (Fig. 2, left). Some basement membranes, such as

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mernbrone __v_

Connective

Capillary lumen

FIG 2 This drawing illustrates the two common types of basement membrane On the left, the most common type, referred to as rrniple basement membrane, is made up of three layers the lamina densa, a continuous layer composed of a dense network of fine “cords”, the kumrno Irrcida, a light layer between lamina densa and the plasmalemma of the associated cells, which is crossed by a few cords, and the parsfibroretrcularis, a poorly limited group of elements located next to the lamina densa and forming the transition with connective tissue. The pars fibroreticulans varies with the tissue and may include several structures. ( I ) collagen fibnls. presumed to correspond to the reticular fibers of histologists (as shown, for example. in Rambourg and Leblond, 1967), and usually embedded in extensions of the lamina densa, or (2) anchonng fibrils inserted at both ends into the lamina densa, or (3) microfibrils. one end of which may be inserted into the lamina densa. On the right IS a double batemenr membrane formed by the close association of an epithelial and an endothelial basement membrane As a result. the pars fibroreticulans disappears and the two laminae densae fuse into one The two laminae lucidae persist: the epithelial membrane is named lamina lucida externa and the endothelial. lamina lucida interna.

the glomerular basement membrane of kidney, arise from the fusion of two simple basement membranes and will be referred to as “double” (Fig. 2. right). Finally, a few basement membranes are quite thick, usually well over 200 nm. This is the case of the capsule of the lens and Reichert’s membrane; in the latter it is often possible to distinguish numerous layers similar to laminae densae. A. SIMPLE BASEMENT MEMBRANES As mentioned in the introduction, a typical basement membrane is a characteristic association of three layers (Figs. 2 and 3; Kefalides ef al., 1979; Vracko. 1974). In close apposition to the plasma membrane of the

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FIG. 3. Vas deferens of the rat. The opaque lamina densa (LD) of the basement membrane closely follows the contour of the base of the epithelial cell (Ep) and is separated from it by the lucent lamina lucida (LL). The latter is crossed by thin strands reaching the plasmalemma of the cell (white arrows). In the area of the lamina fibroreticularis (LFr) toward the connective tissue (CT), anchoring fibrils (AF) are present. Col, Collagin fibrils. ~66.400. Bar = 100 nm.

cells, the lamina lucida is a lucent zone that is rather thin (15-65 nm) and crossed by filamentous strands (Fig. 3). Next to it, the electron-dense lamina densa is usually uniform in thickness in a given location, but varies in thickness from 15 to 125 nm or more, according to the tissue and species. The third layer, pars fibroreticularis, is the transition zone between lamina densa and connective tissue, and consists of structures such as reticular fibers, microfibrils, and anchoring fibrils (Fig. 3) (Inoue and Leblond, 1988). Of the three layers of the basement membrane, the lamina densa is the most prominent (Fig. 3); in fact its absence implies a lack of basement membrane. The pars fibroreticularis i s missing in some basement membranes; this is frequently the case in developing tissues. The lamina lucida varies in thickness with the fixation; it is prominent after glutaraldehyde fixation, but it is much reduced after formaldehyde fixation (e.g. Fig. 5 in Grant and Leblond, 1988). Goldberg and Escaig-Haye (1986) claimed

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that, after rapid freezing and freeze-substitution fixation in an acetone solution of osmium tetroxide, a lamina lucida was not visible; they concluded that the lamina lucida might be a fixation artifact. However, it was possible that the procedure used by these authors could cause shrinking of the lamina lucida. In our experience, even though the size of the lamina lucida varies with the mode of fixation, we see at least a thin one. This thin lamina lucida may in fact correspond to the surface glycoproteins of the associated cell, that is, the glycoproteins composing the “cell coat” or glycocalyx. The fairly large lamina lucida currently observed may result from a widening of the space occupied by the cell coat during some types of fixation. The observation of the ultrastructure of the basement membrane has mainly been limited in the past to the glomerular basement membrane of the kidney and Reichert’s membrane of the parietal yolk sac (see later). In the few reports describing the structure of simple basement membranes, no detailed ultrastructure was given. In the capillary basement membrane of the lung, for example, the lamina densa was reported to be composed of thin fibrils, which may form a netlike organization (Vaccaro and Brody, 1981). Here the ultrastructure of simple basement membranes will be examined using the basement membrane of the epidermis of the rat footpad as a model (Fig. 4), since it is fairly typical and the layers composing it are readily distinguished (Inoue and Leblond, 1988). The electron-opaque lamina densa follows the contour of the base of the epithelial cells. The uniform, lucent lamina lucida, which is crossed by fine strands, separates the former from the basal plasmalemma of the cells. In the area of the pars fibroreticularis, processes extend from the lamina densa into spaces between collagen fibrils in the papillary layer of the dermis. Examination at high magnification (Fig. 5 ) shows that the lamina densa is composed of a network of irregular, fluffy linear elements. These anastomosing, poorly limited elements, referred to as “cords” (Inoue et al., 1983; Laurie et al., 1984; Inoue and Leblond, 1988), have highly variable thickness ranging from 1.8 to 5.3 nm, averaging 3.4 nm. Cords are often seen being continuous with a distinct fine filament. The openings of the network, o r spaces separating the cords-named “intercordal spaces’’-look empty or contain dustlike material. The size of these spaces on two-dimensional micrographs provides an index (“intercordal space diameter index”) of their diameter, which averages 12.8 nm in this particular basement membrane. Sections parellel to the surface of the epidermis and cut through the lamina densa show the cord network essentially identical to that in sections perpendicular to the epidermal

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FIG.4. Basement membrane of the epidermis of the rat footpad. The electron-opaque lamina densa (D) and intervening, lucent lamina lucida (L) follow the contour of the base of the epithelial cells (Ep). The lamina lucida is crossed by fine strands reaching the plasmalemma of the cell (arrows). Lamina densa-like extensions (Ext) extend toward the connective tissue of the papillary layer (Pa) of the dermis and fdl the space between collagen fibrils (Cc). ~ 5 0 , 1 0 2 .Bar = 500 nm. From Inoue and Leblond (1988).

surface, indicating that the network is, as expected, truly threedimensional. The lamina lucida is crossed by some cords that are more or less perpendicular to the plasmalemma (Fig. 5). A few, however, anastomose to form a loose network (Fig. 3). They are usually attached to the plasmalemma of the epidermal cells by their distal end and are in continuity with the cords of the lamina densa at their proximal end. Thus, the main ultrastructural component of the simple basement membrane consists of cords that are organized into a compact, threedimensional network in the lamina densa, and are loosely arranged in the lamina lucida. The extensions of the lamina densa into the subjacent connective tissue are also composed of a tridimensional network of cords (Inoue and Leblond, 1988). A structure frequently associated with the cords consists of 4.5- to 5-nm wide, ribbonlike entities composed of a set of two parallel lines separated by a light space. They are referred to as “double tracks” (Inoue and

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FIG. 5 . High-magnification view of the basement membrane of the epidermis of the rat footpad. The lamina densa (D) is composed of a network of irregular, anastomosinK ”cords.” which are separated by “intercordal spaces” (open arrows). Cords are composed of fuzzy material and in places associated with “double tracks” (paired arrows). Fine filaments are also present, either in continuity with a cord (arrows 1.2,4) or within cords (arrow 3). In between the plasmalemma of the epithelial cell (Ep) and the lamina densa, the lucent lamina lucida is crossed by cords (arrowhead). At the connective-tissue side of the lamina densa. inrercordal spaces are open toward the stroma (curved arrows). ~ 3 2 5 , 1 3 0 . Bar = SO nm. From lnoue and Leblond (1988).

Leblond, 1988; Inoue et al., 1989). Double tracks are distributed mainly along the cords in both lamina lucida and densa. As described later (see next section), they have been shown by immunohistochemical methods to be composed of heparan sulfate proteoglycan. Other simple basement membranes of diverse origins including trachea, jejunum, seminiferous tubuie, and vas deferens of the rat, seminiferous tubule of the monkey, and the mouse ciliary process, were observed to be composed of structures closely resembling those of the rat epidermis (Inoue and Leblond, 1988). The collagen fibrils identified by electron microscopy (EM) in the pars fibrorecticularis close to the basement membrane (Fig. 4) could be either the reticular fibers or the collagen fibers pf light microscopists. That they are usually reticular fibers has been shown in the basement membrane of the proximal convoluted tubule of the rat kidney (Rambourg and Leblond, 1967), where reticular fibers closely approximated to the basement

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membrane could be visualized by the PA-Schiff technique or more vividly by PA-silver methenamine staining. B, DOUBLEBASEMENT MEMBRANES These will be described in the glomerular basement membrane, but are also commonly encountered in association with the alveolar epithelium of the lung and around brain capillaries. They arise from the fusion of an epithelial basement membrane with the endothelial basement membrane of nearby capillaries. As a result the two laminae fibroreticulares disappear, the two laminae densae fuse into one, and the two laminae lucidae persist, one called “externa” (in contact with the epithelial cells) and the other “interna” (in contact with the endothelial cells) (Suzuki, 1959; Verrier and Birch-Andersen, 1962; Thorning and Vracko, 1977; Huang, 1979; Reeves et al., 1980). Similarly, in the lung the basement membrane of alveolar septa is made by fusion of the basement membrane of alveolar epithelium and that of capillary endothelium (Huang, 1978). In an early observation (Farquhar et al., 1961) the ultrastructure of the glomerular basement membrane was described as a network of poorly limited, 3- to 4-nm-thick fibrils. “Pores” were expected to be present in this basement membrane, since it functions as the main filtration barrier of the kidney glomerulus (Pappenheimer, 1953). However, pores could not be demonstrated by Farquhar et al. (1961). Hence the glomerular basement membrane was thought to be a gellike structure in which fine fibrils were embedded in an amorphous matrix. A fine fibrillar structure of the glomerular basement membrane was also reported by Latta (1970): the lamina densa was described as a dense feltlike layer composed of poorly stained fibrils 2-5 nm in thickness, while the laminae lucidae interna and externa were traversed by fine fibrils. After tracer experiments the lamina densa was said to be penetrated by fine “channels” of a size 510 nm (Latta, 1970). Rodewald and Karnovsky (1974) also observed fibrils of a diameter ranging from 3 to 10 nm in the laminae lucidae of the glomerular basement membrane, while the lamina densa was too compact to resolve its fine structure. Besides these fine fibrils, the presence of “large fibrils” was reported in the glomerular basement membrane (Farquhar el al., 1961; Latta, 1970; Farquhar, 1981). They were fairly straight, 10- to 11-nm-thick hollow structures and were localized mainly in the lamina lucida interna. Recent observations have shown that, in the glornerular basement rnernbrune of the rat kidney, cords with irregular diameter varying from 2 to 8 nm again anastomose into a three-dimensional network (Laurie et al., 1984). Cords are closely packed in the lamina densa, where the inter-

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cordal space diameter index averages only 8 nm, but they are loosely arranged in the laminae lucidae on both sides but without showing sharp boundaries between them. The average diameter of cords in both the lamina lucida interna and externa is 4 nm while their intercordal diameter index varies from 15 to 30 nm with an average of 20 nm. At areas near the slit between foot processes in the lamina lucida externa and also in front of the fenestrae of the endothelium in the lamina lucida interna, there are spaces largely devoid of cords (Laurie et al., 1984).

C. THICKBASEMENT MEMBRANES As a model, the basement membrane of the parietal wall of the yolk sac, known as Reichert’s membrane, will be used. Earlier reports indicated that, at high magnification, Reichert’s membrane was composed of fine fibrils (Clark et al., 1975; Jollie, 1968; Martinez-Hernandez et al., 1974; Wislocki and Dempsey, 1955; Hogan er al., 1980; Liotta et a!., 1981; Laurie and Leblond, 1982). Although no systematic observations or measurements on the ultrastructural features were made in these reports, such fine fibrils were described to be either arranged parallel to one another and to the surface of the membrane (Jollie, 1968), or organized into a fine feltwork, or meshwork (Clark et af., 1975; Wislocki and Dempsey, 1955). It has been observed in this laboratory that the membrane varies in thickness from 2 to 6 p m and is composed of many superimposed layers comparable to series of laminae densae (Inoue et a/., 1983).The layers are resolved into a network of interconnected “cords” (Fig. 6a, arrowheads). Cords also often cross between layers (Fig. 6b). Here again, the term cord has been used in preference to fibrils because it better reflects the unevenness and network arrangement of the structure. The cords occupy the bulk of the membrane, measuring 3-8 nm in thickness, with an average of 5 nm (Fig. 6a,b). The network arrangement of the cords is more easily seen in the specimen sectioned parallel to a layer (Fig. 6a). The meshes of the network vary in size, as the intercordal space diameter index measures from 7 to 60 nm with an average of 15 nm. Two other thick basement membranes have been examined. ( I ) The lens capsule, is comparable to Reichert’s membrane (Inoue and Leblond, 1988) and, like it, differs from simple basement membranes mainly by its thickness. The ultrastructure was initially thought to consist of filaments, or fibrils, arranged in orderly, parallel arrays (Jakus, 1964; Heathcote and Grant, 1981; Kefalides, 1971). Our observations show that the main structural component is a network of cords that, however, is somewhat stretched along the meridians of the eye. In specimens fixed in glutaral-

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FIG. 6. Reichert’s membrane of the parietal wall of the rat yolk sac. (a) Section parallel to the surface of the membrane. It is composed of a network of irregular, anastomosing cords of varying thickness (large and small arrowheads). A circle indicates a transversely sectioned basotubule. Occasionally, regular cross banding is seen on the surface of cords (arrow). (b) Section cut perpendicularly to the surface of the membrane. Successive layers (indicated by thick arrows at right) are composed of a network of cords (single thin arrows). Layers are closely interconnected with cords (paired thin arrows). Arrowheads indicate oblique-longitudinal section of basotubules. (a) ~180,000.(b) ~187,470. Bars = 100 nrn. From Inoue et al. (1983) by permission of the Rockefeller University Press (slightly modified).

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dehyde followed by postfixation in osmium tetroxide, the overall architecture is similar to that of specimens fixed in potassium permanganate (as seen, for example, in Fig. 5). Cords, however, are somewhat thicker and less clearly defined in the former, generally showing a hazier appearance as compared to the latter (Inoue and Leblond, 1988). (2) Descemet’s membrane of the cornea, in contrast, is very different from other basement membranes both in structure and in content (Jakus, 1956; Fitch et al., 1982; Sawada, 1982; Sawada et al., 1984; Grant and Leblond, 1988). Before concluding, it should be mentioned that at least Reichert’s membrane and the basement membranelike matrix of the EngelbrethHolm-Swarm (EHS) tumor (Inoue and Leblond, 1985a) contain two other structures in addition to the cord network, that is, basotubules and double pegs. Basotubules are unbranched, straight tubular structures, 7- 10 nm in diameter, running parallel to the surface of the Reichert’s membrane and to one another among the cord network (Fig. 7a,b); they are hollow structures (Fig. 7c; Inoue et al., 1983). The presence of basotubules in the tissue is more readily visualized after the specimen has been fixed with potassium permanganate (Inoue et al., 1983); they have been enumerated in cross sections and found to average 360 per square micron. At a higher magnification the cross section of the basotubule tends to appear pentagonal, with a light lumen containing a central dot from which spokelike lines are radiating. The tubule is surrounded by variable amounts of hazy material referred to as “peritubular feltwork” (see later). Individual basotubules are often fairly straight (Fig. 8) or gently curved, and their thickness can vary from 7 to 10 nm or larger. At high magnification and after the surrounding materials were eliminated by a brief treatment with the proteolytic enzyme, plasmin, the fine structure of basotubules was clearly seen. A basotubule is composed of a tubular core of superimposed pentagonal disks, which show a lumen containing a central dot and identified as molecules of amyloid P component (Inoue and Leblond, 1985b). Onto the surface of each column a ribbonlike helical wrapping is FIG. 7. Basotubules (i.e., basement membrane tubules) in the rat Reichert’s membrane. (a) Section approximately parallel to and cut through a level of the thickness of a layer in the membrane. Basotubules (arrowheads) are arranged approximately parallel to one another. Slightly off such level basotubules are no longer seen, and the cord network is the only structure present (asterisk). (b) Higher magnification view of basotubules running through a network of cords. (c) Section cut through a basotubule showing its tubular nature. (a) ~51,300. (b) ~118,700.(c) x288,200. Bars = 100 nm. From Inoue et al. (1983) by permission of the Rockefeller University Press.

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F I G . 8. Longitudinal section of basotubules in rat Reichert’s membrane treated with plasmin to eliminate neighboring material. (a) In several basotubules, two walls and a lumen as well as a peripheral dark material (“peritubular feltwork”) are visible. Arrow indicates basotubules that were sectioned only at their periphery. (b,c) Higher magnification view of areas indicated by asterisks in (a). (a) ~ 1 1 8 . 3 4 1 . Bar = 100 nm. (b,c) ~265,923.Bar = 10 nm. From lnoue e l al. (1983) by permission of the Rockefeller University Press.

tightly applied. Finally, a dense peripheral component, referred to as peritubular feltwork, associates with and surrounds the tubule proper. The presence of basotubules in basement membranes other than Reichert’s membrane has not been studied in a number of specimens. Two extreme examples have been described. First, the glomerular basement membrane of rat kidney shows the presence of only a limited number of basotubules, mainly in the lamina lucida interna and occasionally in the lamina densa (Laurie et ul., 1984). Basotubules had been described earlier in the glomerular basement membrane under the names “fibrils” and “large, straight fibrils,” respectively by Farquhar et al. (1961) and by Latta (1970). The second example is the basement membrane matrix of the EHS tumor of the mouse, in which basotubules are extraordinarily abundant (Inoue and Leblond, 1985a; see Section 11,D). The connective tissue contains a structure very similar to basotubules, the microfibrils. These are also hollow rods -8-10 nm wide and composed of superimposed disks made up of amyloid P component (Inoue rt ul., 1986a). Moreover, the microfibrils of connective tissue may join basement membranes as reported in the lung (Low, 1961, 1962). Since microfibrils may be present within the basement membrane(s) separating

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FIG. 9. High-magnification micrographs of rat Reichert’s membrane. (a) Minute particulate structures are seen scattered throughout the membrane and are particularly conspicuous at clear areas of intercordal spaces (arrowheads). (b) In places they are resolved as pairs of two parallel, 3.5-nm-long rodlets (circles) and are referred to as “double pegs,” because of their characteristic configuration. x428,500. Bar = 10 nm. From Inoue ef nl. (1983) by permission of the Rockefeller University Press.

capillaries from the alveolar epithelium and, in fact, the basement membrane may largely be made up of them-as was also described in human myocardium (Low, 1962), pig aortic media (Haust, 1965), cultured chick notochord (Carlson et a l . , 1974), the glomerular capillaries of normal (Farquhar et al., 1961; Farquhar, 1978; Hsu and Churg, 1979) and diseased kidney (Hsu and Churg, 1979; Hsu et al., 1980; Olsen, 1979),and ciliary epithelium (Inoue and Leblond, 1988)-the distinction between basotubule and microfibril is unclear. In the case of Reichert’s membrane where basotubules appear to be produced by the same cells that elaborate basement membranes, the endodermal cells, they should be given a name distinct from the microfibrils presumably produced by fibroblasts. Yet, at least in their amyloid P core, basotubules are identical to microfibrils. Whether the helical wrapping of basotubules (Inoue et al., 1983) is similar or not to the double-tracked “surface band” seen at the surface of microfibrils (Inoue and Leblond, 1986) has not been settled. If these peripheral components do indeed differ, they may result from their environment, since basotubules are embedded within basement membranes, whereas microfibrils are mainly loose in connective tissue. Double pegs are minute dotlike structures (Fig. 9) initially found

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FIG. 10. Double pegs released from fragmented extracellular matrix of the mouse EHS tumor before formaldehyde fixation and embedding in Lowicryl K4M. They are arranged in semicrystalline array. ~ 2 4 0 . 5 0 0 .Bar = I W nm.

scattered within the meshes of cord network in Reichert’s membrane (Inoue et al., 1983) and eventually observed within other basement membranes including rat glomerular basement membrane (Laurie et ul., 1984) and the EHS tumor matrix (see Fig. 12; Inoue and Leblond, 1985a). At high magnification when the orientation is favorable they tend to appear as sets of two parallel rodlets 3.5 nm in length and separated by -3.5 nm, a configuration from which the term “double pegs” has originally been derived. A large quantity of double pegs can be freed from the matrix of the EHS tumor. They have a tendency to arrange in three-dimensional semicrystalline array with a distance of -10 nm between neighboring ones (Fig. 10). At high magnification they are seen to be interconnected with one another by fine filaments, as reported previously in specimens from Reichert‘s membrane (Inoue et al., 1983). Double pegs are also observed, in addition to basement membranes, in the connective-tissue space, specifically within the vicinity of microfibrils (unpublished result). Close observation of double pegs at high magnification reveals that they may appear in configurations other than the parallel rodlets just de-

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scribed, including polygonal or circular ringlike structures or, as previously reported, sets of two tiny dots separated by 3.5 nm, depending on their orientation. As described later (Section III,B,2), amyloid P component is composed of 8.5- to 9.5-nm-wide pentagonal units with a small lumen. After high-resolution observation with negative staining these units were seen to be composed of an assembly of five subunits -3.5 nm in size. In a preparation of purified amyloid P component embedded in Epon and sectioned, units are seen, but in addition a significant number of freed individual subunits is also present. These subunits varied in configuration depending on their orientation. These configurations were identical to those observed on double pegs. Therefore, it is likely that double pegs are subunits of the amyloid P component (Inoue and Leblond, 1989). The observation that double pegs are abundant in areas rich in amyloid P component, that is, close to basotubules or microfibrils, supports this interpretation.

D. BASEMENTMEMBRANE MATRIX PRODUCED BY

THE

EHS TUMOR

The EHS tumor of the mouse is made up of clusters of cells surrounded by a large amount of extracellular matrix. It was initially shown by Orkin et al. (1977) that the tumor possessed biochemical properties of basement membrane and, eventually, various basement membrane components were identified and isolated from the tumor. The EHS tumor was first observed in the electron microscope by Merker and Barrach (1981)and its extracellular matrix was described as a series of successive layers, each of which resembled a common basement membrane. We have confirmed this finding (Inoue and Leblond, 1985a) and added that the layers are poorly defined in the area close to the cells (proximal region) but distinct at a distance from them (distal region). In the proximal region the bulk of the tissue is made up of a network of cords. Within the network, small numbers of 7- to I0-nm-thick, hollow rods typical of basotubules as well as double pegs are scattered. In addition to intact basotubules, many small structures with a configuration similar to that of the cross section of basotubules but thinner, are observed in the proximal region. The small structures are believed to be units of amyloid P component secreted in this form by the cells. In the distal region of the matrix, on the other hand, these units are believed, through self-association, to become basotubules. In this region basotubules are numerous and most prominent. They assume semicrystalline arrangements in picket-fence fashion along two parallel planes within each layer (Fig. 11). Cords are compacted between them. Double pegs are distributed throughout the matrix in this region and are readily seen at the clear interlayer spaces (Fig. 12).

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FIG. 11. Distal region of the basement membrane matrix of the mouse EHS tumor sectioned along one of the two planes of a layer. Rodlike structures in which a lumen is occasionally seen (circles) are identified as basotubules. They are arranged parallel to one another with a regular center-to-center distance of 40 nm to form the backbone of one of the two sheetlike planes in a layer of the matrix. x53.000. Bar = 100 nm. From Inoue and Leblond (1985a).

111. Composition

The basement membrane contains both collagenous and noncollagenous components (Timpl and Martin, 1982). Their insoluble nature and limited amount have been major obstacles to their biochemical characterization. Moreover, few tissues have been available from which basement membrane components could be isolated in substantial quantities. As a result, biochemical characterization did not progress until the extracellular matrix of the EHS tumor of the mouse was recognized as having the properties of a basement membrane (Orkin et a l . , 1977). This tumor became the most frequently used source for the extraction and purification of basement membrane components (Timpl and Martin, 1982). The basement membrane nature of the EHS tumor matrix was confirmed by structural study (Inoue and Leblond, 1985a) and by the immunogold detection of a series of recognized basement membrane components, particularly collagen IV, laminin, heparan sulfate proteoglycan, and entactin (Martin and Timpl, 1987). Recently, new ones were added, the amyioid P component (Inoue et al., 1986b3 and the protein BM-40, similar to osteonectin (Lankat-Buttgereit et al., 1988).

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FIG. 12. Semicrystalline array of double pegs seen in a widened, clear interlayer space in the distal region of the matrix of the mouse EHS tumor. A typical double-rodlets structure is indicated by arrow. X452,lOO. Bar = 10 nm. From Inoue and Leblond (1985a).

FIG. 13. Light micrograph of the rat kidney stained for type 1V collagen by an immunoperoxidase technique. Glomerular basement membrane (arrowheads), parietal basement membrane (Bowman’s capsule, arrow), and basement membranes of proximal (P) and distal (D) tubules are immunostained. x 197. Bar = 100 km. From Laurie et a/. (1983).

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A. TYPEIV COLLAGEN

Type IV collagen, also referred to as collagen IV, is a major component of basement membrane (Bornstein and Sage, 1980; Kefalides, 1973). It is composed of two types of (Y chains. two a l ( I V ) chains and one ar2(IV) chain wound into a helix (Gehron Robey and Martin, 1981; Kresina and Miller, 1979; Sage r t al., 1979: Temp1 et a/., 1982). At the level of light microscopy. collagen I V was located immunohistochemically within the basement membrane areas of several tissues (Timpl and Martin, 1982), including duodenum, trachea, kidney (Fig. 13), spinal cord, cerebrum, and incisor tooth (Laurie et af., 1983). In these specimens, collagen IV was colocalized with other basement membrane components, namely laminin, fibronectin, heparan sulfate proteoglycan, and entactin (Laurie et al., 1983). At the ultrastructural level, type IV collagen has been localized by immunostaining to the lamina densa of the basement membrane in a variety of tissues. including kidney glomeruli (Roll e t a / . , 1980;Courtoy et a/., 1982), epidermis (Yaoita et a]., 1978). lung alveolar epithelium (Sano et al.. 19811, capillary endothelium (Laurie et ul., 1980). and striated muscle (Sanes, 1982). Immunostaining of rat duodenum and incisor tooth (Laurie et al.. 1982a) for collagen IV using direct or indirect peroxidase methods, showed that the stain was present, not only along the thickness of the lamina densa, but also on narrow extensions of the former, stretching across the lamina lucida (Fig. 14). Similar observations were made on the glomerular (Fig. 15) and proximal tubule basement mem-

FIG. 14. Basement membrane of the outer-enamel epithelium of the rat incisor tooth immunostained for type IV collagen by an immunoperoxidase technique. The lamina densa (LD)and its narrow extensions (arrow) across the lamina lucida (LL) are intensely stained. Extensions on the connective-tissue side of the lamina densa are similarly stained. m, Mitochondrion of the epithelial cell. ~ 4 0 . 0 0 0Bar . = I00 nm. From Laurie e r n / . (1982b) by permission of the Rockefeller University Press.

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branes of rat kidney (Fig. 16), after treatment of frozen-thawed sections with sodium borohydride for “unmasking” of the antigenicity (Laurie et al., 1984). These authors further noted that the pattern of immunostaining, as described in the previous section, coincided with the distribution of the cords, since these were densely packed in the lamina densa and arranged as narrow extensions across the lamina lucida. It was suggested that collagen IV was localized in the cords (Laurie et al., 1984), a conclusion supported by observations on the EHS tumor matrix (Grant et al., 1985).

FIG. 15. Glomerular basement membrane of the rat kidney stained for type 1V collagen with PAP immunoperoxidase technique. (a) Control section showing no immunostaining. D, Lamina densa; LE and LI, lamina lucida externa and interna; Ep, epithelium; En, endothelium, (b) Section immunostained for type IV collagen. The stain can be seen throughout the lamina densa and in the form of fine cross bands (arrows) at the laminae lucidae. Details of such staining patterns are more easily seen in (c) at higher magnification. Narrow stained bands crossing the electron-lucent layers of laminae lucidae are either straight (vertical arrows) or irregular (horizontal arrow). The homogeneous darkening of the plasmalemmas and slit diaphragm is an artifact caused by diffusion of diaminobenzidine (see Laurie et a [ . , 1982b). (a, b) ~40,000.(c) X 130,000. Bars = 100 nm. From Laurie et al. (1984).

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FIG. 16. Tubule basement membrane of the rat kidney stained for type 1V collagen with PAP immunoperoxidase technique. (a) Control section of a distal convoluted tubule. The

lamina densa (upper D) of the epithelium (Ep), that (lower D) of the endothelium (En), or the laminae lucidae (L) are not stained. (b) Basement membrane of a proximal convoluted tubule immunostained for type IV collagen. Whole lamina densa (D) as well as bands (arrow) crossing the lamina lucida (L) are intensely stained. Plasmalemma of a cell at bottom presumed to be a fibroblast is artifactually stained. X40.000. Bar = 100 nm. From Laurie el a / . (1984).

In order to analyze further the localization of collagen IV at the ultrastructural level, use was made of Reichert’s membrane, which was known to show strong immunohistochemical staining for collagen 1V (Laurie et a / . , 1982b). After a 2-hour treatment with purified plasmin, a proteolytic enzyme believed to digest laminin and fibronectin effectively (Liotta et al., 1981), the cords showed different degrees of proteolytic FIG. 17. Rat Reichert’s membrane treated with plasmin for 2 hours. At the bottom, cords are nearly intact (arrow), whereas at the center the effect of plasmin is more advanced and the cord network has been replaced by a network of fine filaments. Dotlike thickenings irregularly distributed along the filaments may partly be true dots and others end-on views of filaments oriented perpendicularly. Inset shows an early stage of digestion at high magnification. Two partially digested cords (arrows) are made up of a filamentous core still associated with a sheath that appears to be composed of a few transverse threads. x 131,600. Bar = 100 nm. Inset, x380.500. Bar = 10 nm. From Inoue et a / . (1983) by permission of the Rockefeller University Press.

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FIG. 18. Higher magnification view of rat Reichert’s membrane treated with plasmin for 2 hours and stained for type IV collagen with direct irnmunoperoxidase technique. As the effect of plasmin i s more advanced in this particular area of the specimen, fewer filaments remain that are decorated with dark. tiny dots of immunoperoxidase reaction product

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digestion (Fig. 17). In what appears to be an early stage in digestion, the cord remnants are centered by a fine filament associated with various materials (Fig. 17, inset). At a more advanced stage these materials disappear, leaving only fine filaments as seen in the top center area of Figure 17. Each filament seems to run singly, but joins others to form a network. A similar network has also been observed in salt-extracted amniotic basement membrane (Yurchenco and Ruben, 1987). The interpretation of such observation is that the cords are composed of a thin “core filament” surrounded by a sheath in which transverse and other elements may be observed (Inoue et al., 1983). Immunoperoxidase staining for collagin 1V of the network of fine filaments produced after plasmin treatment of Reichert’s membrane showed a positive reaction (Inoue et a l . , 1983). At high magnification using preparations in which the effect of plasmin was more advanced, some filaments were lost, but the remaining ones were decorated by tiny dots of immunoperoxidase reaction product (Fig. 18a). In occasional sites, the junction between the filaments showed special patterns. Thus, a set of two joining filaments could be connected to another set through a 30-nm long vertical span, which appeared single (Fig. 18b, left) or double (Fig. 18b, right). In another pattern, filaments were interrupted by 3- to 4-nm dots that either were single (Fig. 17) or could be resolved into pairs (Fig. 18c). In two areas that had been lightly digested in plasmin, the mean distance between the dots was estimated at 819 and 859 nm (Inoue et al., 1983). However, it was later realized that the dots were more sensitive to plasmin than the filament and the longer the plasmin treatment, the fewer the dots were; therefore, we now conclude that these figures have little significance. Nevertheless, the junction patterns observed after a light plasmin digestion (Fig. 18b,cj were considered meaningful in the light of the demonstration by Timpl er al. (1981) and Bachinger et al. (1982) that collagen IV molecules were joined to one another by their extremities. Thus at the C-terminal end, each molecule carried a globule that could fuse with the globule of another molecule, whereas at the N-terminal end each molecule could join three (arrows). Occasional “dense islands” that remain after the plasmin treatment do not seem to be immunostained (asterisks). (b,c) Structural features of fine filaments exposed after a 2-hour plasmin digestion of rat Reichert’s membrane. (b) A short span connecting two pairs of filaments: left, upper and lower pairs ofjoining filaments are united by a thick, 30-nm-long span (between two horizontal lines); right, paired filaments (arrows) are united by a double span. (c) Dots observed along filaments (at the center of both micrographs) are resolved into pairs (each -3 nm in diameter) and are connected to a single filament. (a) X 166,700. Bar = 100 nm. (b,c) ~375,800.Bars = 10 nm. (b,c) From Inoue ef al. (1983) by permission of the Rockefeller University Press.

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other molecules and thus form what was called 7s collagen. It was therefore likely that the single (Fig. 17) or paired dots (Fig. 18c) corresponded to the complete o r partial fusion respectively of C-terminal globules. As for the patterns depicted in Fig. 18b, the 30-nm-long vertical span between two pairs of molecules was likely to correspond to 7s collagen. In a study of common, “thin” basement membranes (Inoue and Leblond, 1988) the basement membrane of rat seminiferous epithelium was treated with plasmin in a similar manner to that used for Reichert’s membrane. Cords were digested in various degrees, reduced in thickness, and finally became simple filaments of three different thicknesses: 1.5 nm, 2.0-2.5 nm, and 3 nm. High-magnification observation (Inoue and Leblond, 1988) of intact cord network of various thin basement membranes as well as lens capsule, a thick basement membrane, showed that the original network of cords, without plasmin treatment, also contained occasional areas where filaments of these three thicknesses could be identified. Such filaments were either free, continuing into a cord, or faintly recognizable at a center of a cord. These observations indicate that, in basement membrane in general, a cord is composed of a core of single, double, or triple filaments enclosed within an outer sheath that can occasionally be missing. The conclusions of this work are based on two key observations: (1) The main ultrastructural features of the filament network forming the skeleton of the cord network are the occasional finding of a 30-nm span connecting pairs of joining filaments as well as 3-4-nm dots and particularly the varying thicknesses of the axial filament; the latter observation was attributed to the lateral association of two or more molecules of type IV collagen, in accord with the demonstration by in v i m experiments of Yurchenco and Furthmayr (1984) that two or three molecules of type IV collagen could laterally aggregate. (2) A clear-cut result of immunostaining of filament network for type IV collagen was obtained, as described previously. It is concluded that the cords that constitute the bulk of basement membrane contain a core filament of collagen IV, which is enclosed within a plasmin-sensitive outer sheath (Fig. 19). The nature of the sheath associated with the collagen filament will be discussed presently.

B . NONCOLLAGENOUS COMPONENTS The presence of carbohydrate in basement membrane was proposed in the early 1950s as a result of staining by the PA-Schiff technique (Lillie, 1951; Leblond, 1950). It was later found that basement membranes contained carbohydrate-protein complexes of the type referred to now as

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Network of cords Network of filaments FIG. 19. Schematic representation of an interpretation of the cord network that constitutes the bulk of basement membrane. The diagram at left shows the network arrangement of cords; the diagram at right indicates the effect of a short plasmin treatment. Thus cords seem to be composed of plasmin-sensitive sheath containing laminin and other components that are gradually digested away, revealing the presence of a more plasmin-resistant axial filament consisting of type IV collagen. The prototype of this model was proposed by Inoue et a / . (1983).

glycoprotein (Leblond et al., 1957). During the past 25 years a variety of noncollagenous components has been identified in basement membrane, the glycoproteins laminin (Timpl et al., 1979a), fibronectin (Vaheri and Mosher, 1978), nidogen (Timpl et al., 1983), and amyloid P component (Inoue and Leblond, 1985b; Inoue et a/., 1986b; Dyck et al., 1980a), the sulfated glycoprotein entactin (Carlin et al., 1981), and heparan sulfate proteoglycan (Hassell et al., 1980, 1985). Immunohistochemical localization of these components in the basement membrane or, more specifically, their localization in two specific sites (i.e., cords and basotubules) will be discussed here. 1 . Cord Network

Evidence presented earlier indicates that the cords, which are organized into a network occupying the bulk of basement membrane, are made up of a core filament of type IV collagen, surrounded by a sheath of plasmin-digestible materials. The presence of various individual noncollagenous basement membrane components in the cord network will be described. a. Laminin. Laminin is a glycoprotein (Chung et al., 1979; Rohde et al., 1979; Timpl et al., 1979a) with MW 850,OOO-1,000,000 (Timpl et al., 1979b; Rohde et al., 1979; Engel et al., 1981). Laminin was initially assigned to the lamina lucida in the basement membrane of the epidermis (Foidart et al., 1980) and kidney glomeruli (Madri et al., 1980; Farquhar,

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FIG. 20. Immunoperoxidase localization of four components-laminin, entactin, heparan sulfate proteoglycan, and fibronectin-in tubule basement membrane of the rat kidney. D, Lamina densa; L , lamina lucida. (a) Left. basement membrane of a distal convoluted tubule imrnunostained for laminin. The stain is present in the lamina densa as well as wide extensions elongated toward connective-tissue spaces (dark arrow). Thin lamina lucida is

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1981; Courtoy et al., 1982; Martinez-Hernandez et al., 1982). However, other investigators observed it throughout the basement membrane, that is, in the lamina densa and certain structures within the lamina lucida, as seen in kidney glomeruli (Abrahamson and Caulfield, 1981, 1982; Abrahamson et al., 1983; Laurie et al., 1984) and tubules (Madri et al., 1980; Martinez-Hernandez et al., 1982; Laurie et al., 1984), striated muscle (Sanes, 1982), mammary gland (Monaghan et al., 1983), duodenum and incisor tooth (Laurie et al., 1982b), spleen, small intestine, and liver (Abrahamson and Caulfield, 1985). At the level of the light microscope, the reaction product of immunoperoxidase staining for laminin is usually observed throughout the entire basement membrane. In Reichert’s membrane, for example, the full width of the membrane is intensely stained, as previously reported (Leivo et al., 1980; Sakashita and Ruoslahti, 1980). At the ultrastructural level, as shown in the basement membrane of proximal tubules in the rat kidney (Fig. 20a, for example, the whole lamina densa is immunostained for laminin. The bulk of the lamina lucida is not stained, but it is crossed by numerous fine extensions from the lamina densa, which are also stained. The pars fibrorecticularis is generally clear, but in some tissues occasional wide strands extend from the lamina densa into connective tissue that is stained. In the case of the glomerular basement membrane, pretreatment of the specimens by borohydride and freezing was found helpful, as mentioned previously for type IV collagen immunostaining (Laurie et al., 1984). After such pretreatment, an intense stain was observed on the entire lamina densa and its narrow extensions traversing across laminae lucidae. With immunoferritin technique, the labeling was again localized at the same sites, as demonstrated by immunoperoxidase technique-that is, lamina densa and its narrow extensions. With this technique, no ferritin particles were localized along the plasmalemmas, suggesting that a homogeneous darkening observed at this site after immunoperoxidase staining is artifactual (presumably to diffusion of the diaminobenzidine reaction product; Seligman et al., 1973). Reichert’s membrane, immunostained for laminin by the peroxidase~~

~

~~

crossed by stained bands (white arrows). Right, at high magnification such bands appear straight (vertical arrow) or irregular (horizontal arrow). (b) Basement membrane of a distal convoluted tubule immunostained for entactin. The stain is present in the lamina densa, narrow bands (arrows) crossing the lamina lucida, and wide strands extending into the underlying connective tissue. (c) Basement membrane of a proximal convoluted tubule immunostained for heparan sulfate proteoglycan. The stain is present in the lamina densa as well as narrow bands (arrow) crossing the lamina lucida. (d) Tangentially sectioned basement membrane of a proximal convoluted tubule immunostained for fibronectin. The lamina densa is more reactive than the narrow lamina lucida. (a, left; b-d) X40,OOO. (a, right) ~130,000.Bars = 100 nm. From Laurie et al. (1984).

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antiperoxidase technique in sections parallel to its surface, was observed in the electron microscope at high magnification. Intense stain was specifically associated with variously oriented, elongated structures -10-13 nm in thickness, which were interpreted as cords somewhat thickened, probably as a result of stain accumulation at the surface (Inoue et ul., 1983). The extracellular matrix of the mouse EHS tumor, which has been shown to be suitable for a basement membrane model, has been immunostained for laminin with the protein A-gold technique (Grant et al., 1985). The whole matrix was labeled and gold particles were localized specifically on the cord network. In conclusion, laminin is present in the lamina densa and its extensions across the lamina lucida. In both cases, the fine localization at high magnification is restricted to the cords. b. Entuctin and Nidogen.

Entactin is a sulfated glycoprotein of M ,

158,000, originally isolated from the basement membranelike matrix of a mouse endodermal cell line (Carlin ef ul., 1981). Immunolocalization of

this component at the light microscope level has been reported in the basement membrane of placenta, smooth muscle, kidney, lung, and vascular tissues (Carlin e f al., 1981). The basement membranes of kidney glomerulus and tubules, as well as liver, spleen, uterus, and Reichert’s membrane, were also shown to be reactive (Bender et ul., 1981). In the rat mammary gland, entactin was localized to the basement membranes of secretory alveoli and blood vessels (Warburton et al., 1984). At the ultrastructural level, localization of entactin was done mainly on basement membranes of the kidney with immunoperoxidase technique. Carlin er al. (1981) and Bender et af. (1981) reported that the tubule basement membrane showed moderate homogeneous staining, but the glomerular basement membrane failed to show a consistency in the staining. Another report (Martinez-Hernandez and Chung, 1984) indicated that entactin was present throughout the glomerular basement membrane with strongest staining at the lamina lucida interna, while the full thickness of the tubule basement membrane was uniformly stained. In the basement membranes of other tissues, such as mammary glands (Warburton et al., 1984), duodenum, and smooth and skeletal muscle (Martinez-Hernandez and Chung, 19841, the lamina densa and some structures spanning between the former and the cell surface were reported to be immunostained for entactin. Localization of entactin was studied in this laboratory using the rat kidney and an immunoperoxidase technique as described earlier for collagen IV and laminin (Laurie et ul., 1984). The immunostaining pattern was similar t o that observed with this substance, that is, a reaction throughout the lamina densa and its narrow extensions across the laminae

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lucidae (Laurie el al., 1984). The somewhat different results observed by previous investigators (Bender et al., 1981 ; Martinez-Hernandez and Chung, 1984) might be caused by insufficient availability of antigenic sites. On the other hand, our result on tubule basement membrane (Fig. 20b), in which the lamina densa and its extensions across the lamina lucida were intensely stained, are similar to those of some previous authors (Carlin et al., 1981; Bender et al., 1981). Immunostain for entactin seems to localize on cords of the basement membrane, like laminin, since the lamina densa and its narrow extensions were stained and these two structures have been known to be mainly made up of a tight assembly and loose arrangement, respectively, of cords. In addition, an immunolabeling of basement membranelike matrix of the mouse EHS tumor for entactin with the protein A-gold technique showed that gold particles were seen preferentially attached to cords at the surface of the section (Grant et al., 1985). These results indicate that entactin is, like laminin, present within the cords, presumably as a component of the sheathlike material surrounding a core filament of type IV collagen. Nidogen is a recently reported glycoprotein, MW 150,000 (Dziadek and Timpl, 1985; Dziadek et a f . , 1985). It was initially isolated from the mouse EHS tumor as molecules of MW 80,000 (Timpl et a / . , 1983). With immunofluorescence, this material was localized at the basement membrane region of a variety of human and mouse tissues (Timpl et af., 1983). Close immunological and physiochemical similarities have been shown between entactin and nidogen (Paulsson et a f . , 1985). In situ formation of strong, stoichiometric complexes of nidogen with laminin was demonstrated (Dziadek et al., 1985; Dziadek and Timpl, 1985). Similar complexes were also shown to be formed between entactin and laminin (Carlin et al., 1983). Identity, or precise relationship, of these two components, entactin and nidogen, however, must await their detailed characterization. c. Heparan Sulfate Proteoglycan. A basement membrane-specific proteoglycan was purified from the mouse EHS tumor (Hassell et al., 1980). It was -750,000 in molecular weight and contained approximately equal amounts of protein (core protein) and covalently linked side chains of a glycosaminoglycan identified as heparan sulfate (70,000 MW). A heparan sulfate proteoglycan with a molecular weight varying from 130,000 to 185,000 has been isolated from the glomerular basement membrane (Kanwar et al., 1981, 1984; Kobayashi et al., 1983), and another of 400,000 from the basement membrane produced by the mouse PYS-2 cell line (Oohira et al., 1982), in contrast to 750,000 in

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BM-1. Two forms of heparan sulfate proteoglycans, low and high density, were reported in the EHS tumor (Fujiwara et ul., 1984). Hassell et af. ( 1989, who reexamined the EHS tumor proteoglycan by sequential extractions, confirmed the existence of two major forms of heparan sulfate proteoglycans-a large low-density form and a small high-density form-which also differ in the core protein size and the proteincarbohydrate ratio. Assuming a mixture of these two forms, the variation in the size of proteoglycans isolated from various basement membranes may be due to variation in the proportion of low- and high-density species. which may be regulated by still-unknown factors or, alternatively, determined by the procedures of isolation and purification (Hassell et ul., 1985). Some evidence suggests that the large proteoglycan is synthesized first and is then converted to the small proteoglycan as a result of physiological degradation with removal of part of the protein core (Ledbetter ef al., 1985). With immunohistochemical techniques at the light microscope level, heparan sulfate proteoglycan was localized in the basement membranes of various human and mouse tissues, such as epidermis, kidney glomeruli and tubules, Bowman’s and Descemet’s membrane of the cornea (Hassell et al., 1980) o r tooth (Thesleff et al., 1981). in the rat, it was localized in the basement membrane of various organs, including duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth (Laurie et al., 1983). At the ultrastructural level, rather than immunohistochemical techniques, indirect methods of applying cationic markers and dyes have commonly been used to detect the localization of the proteoglycan in the basement membranes. With such methods, it was localized at the lamina densa in embryonic salivary epithelium (Cohn et al., 1977; Gordon and Bernfield, 19801, on both sides of the lamina densa in embryonic corneal epithelium (Trelstad et al., 1974) and in embryonic lens (Hay and Meier, 1974), or at the lamina lucida in glomeruli (Kanwar and Farquhar, 1979a,b). in recent years. however, the results of ultrastructural immunostaining for heparan sulfate proteoglycan have been reported on basement membranes of a large variety of tissues such as rat duodenal epithelium, enamel-organ epithelium of incisor tooth with blood vessel endothelium (Laurie et al., 1982b), enamel epithelium (Laurie and Leblond, 1983), glomeruli and tubules of the rat kidney (Laurie et al., 1984), and rat ovarian follicles (Palotie et af., 1984). Using either immunoperoxidase or immunoferritin technique, this proteoglycan has been localized throughout the thickness of the lamina densa and also in its cordlike extensions traversing the lamina lucida (Fig. 20c). Immunostaining of the extracellular matrix of the EHS tumor for heparan sulfate proteoglycan with the protein A-gold technique showed gold particles localizedto cords. These

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observations indicate that heparan sulfate proteoglycan, like the other basement membrane components, is localized within basement membrane cords. To identify further the proteoglycan within cords at the ultrastructural level, immunogold labeling for this component was done using fine (5-nm) gold particles (Inoue et al., 1989). The result on the glomerular basement membrane of the mouse kidney showed that the only immunolabeled structure consisted of “double tracks,” a 4.5-nm-wide ribbonlike structure characterized by a set of two parallel lines separated by a light space. Double tracks in rat Reichert’s membrane were also immunostained for heparan sulfate proteoglycan with PAP immunoperoxidase method. Ultrastructural study of various thin and thick basement membranes showed that double tracks were mainly associated with cords (Inoue and Leblond, 1988). In the hope of obtaining an indirect confirmation, a preparation of basement membrane-specific heparan sulfate proteoglycan in Tris buffer was incubated at 35°C for 1 hour with or without other basement membrane components. Double tracks were produced as a result, probably by the polymerization of heparan sulfate proteoglycan molecules (Inoue et al., 1989). It was concluded that heparan sulfate proteoglycan exists in uiuo in the form of ribbonlike double tracks, which are closely associated with the cords and indeed are a constituent of the cords of basement membrane. d. Fibronectin. Fibronectin is a plasma component and also a major connective-tissue glycoprotein of MW -450,000 and composed of two similar subunits of MW 220,000 ? 20,000 joined together at their carboxy terminals by disulfide bonds (Vaheri and Mosher, 1978; Hynes and Yamada, 1982). Fibronectin is a multifunctional molecule, whose various domains interact with a variety of substances. At the light microscope level, localization of fibronectin was assigned by immunohistochernical methods to the basement membrane of duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth of the rat (Laurie et al., 1983). Although the immunoperoxidase reactions were generally weak, as compared to those given by other basement membrane components such as laminin, they were observed in all examined sites, a finding indicating that fibronectin may also be a component of basement membrane. Fibronectin was also identified in the basement membrane of intestine (Quaroni et al., 1978; Stenman and Vaheri, 1978), enamel (Lesot et al., 1981; Thesleff et al., 1981), epidermis, smooth and striated muscle, lung, thyroid, testis and epididymis (Stenman and Vaheri, 1978), and embryonic chick trunk (Mayer et al., 1981). At the ultrastructural level fibronectin was immunostained in the

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basement membrane of the rat duodenal epithelium, enamel-organ epithelium and blood vessel endothelium of incisor tooth (Laurie et al., 1982b). The immunostaining was observed along the lamina densa and narrow extensions across the lamina lucida. A generally similar localization was observed around rat skeletal muscle fibers (Sanes, 1982) and embryonic chick neural tube (Mayer et af., 1981). In the kidney, however, fibronectin was localized only to the laminae lucidae of the glomerular basement membrane (Courtoy et a f . , 1980), or it was not detected in either glomeruli or tubules (Martinez-Hernandez et ul., 1981). However, after using frozen sections subjected to sodium borohydride, immunostaining was detected in the basement membranes of both glomeruli and tubules (Laurie et a / . , 1984). The staining was fairly uniform in the lamina densa and cordlike extensions across the laminae lucidae in both sites (Fig. 2Od). Fibronectin, again, seems to localize in cords, since immunostain was observed on the lamina densa and its narrow extensions, which are known to be composed mainly of cords. In addition, one immunostaining of the EHS tumor matrix for fibronectin with the protein A-gold technique showed that gold particles were preferentially attached on cords (Grant et al., 1985). Before concluding, it may be mentioned that some investigators attribute the rather weak fibronectin immunostaining to diffusion of plasma and/or connective-tissue fibronectin into basement membrane (quoted by Abrahamson, 1986). An investigation has shown that, for reliable detection of fibronectin, the wash solutions must be free of serum. Under these conditions, they observed the presence of fibronectin in basement membrane. (CicadBo et al., 1988). e. Coexistence of Basement Membrane Components in an Integrated Complex That Constitutes the “Cords.” In the past, it was generally thought that the components of basement membrane were individually layered within the basement membrane region. Thus, the lamina densa was thought to be composed of type IV collagen (Yaoita et af., 1978), while the lamina lucida would be made up of laminin (Farquhar, 1981; Madriet a / . , 1980; Foidart et ul., 1980) and fibronectin (Madri et al., 1980; Courtoy et al., 1980). Heparan sulfate proteoglycan would exist at the interface of the layers (Trelstad et al., 1974; Hay and Meier, 1974). However, recent results on the immunohistochemical localization of various basement membrane components, as discussed earlier individually, suggest that these components are not layered within the basement membrane. Instead, the noncollagenous components-laminin, entactin,

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heparan sulfate proteoglycan, and fibronectin-appear to coexist in common sites, that is, the lamina densa and its narrow extensions across the lamina lucida. Ultrastructural studies indicate that cords are the major constituent of the lamina densa. As described in the previous section, basement membrane cords are compacted in the lamina densa and loosely arranged in the lamina lucida. The observed pattern of distribution of immunohistochemical staining at the ultrastructural level coincides with that of cords in all components described. Subsequent results of immunostaining of these components with the protein A-gold technique showed that gold particles were preferentially attached to cords (Grant et al., 1985). In conclusion, these substances, are therefore likely to be colocalized within cords. As shown before, the cords appear to be composed of a core filament of type IV collagen surrounded by a sheath, which would consist of the noncollagenous components. All of them, therefore, seem to be incorporated into an integrated complex, the cord. The formation of such a distinct complex is likely because of the well-known affinity of glycoproteins, such as laminin and fibronectin, to type IV collagen (Terranova et al., 1980; Kleinman et al., 1981; Ruoslahti et al., 1981) and to heparan sulfate (Ruoslahti et al., 1981; Sakashita et al., 1980). 2 . Basotubuies The presence of basotubules (abbreviation of “basement membrane tubules”), which are the second ultrastructural component of the basement membrane, was described at first in Reichert’s membrane, a thick basement membrane of the parietal wall of the rat yolk sac (Inoue et al., 1983); they were subsequently identified in the glomerular basement membrane (Laurie et al., 1984) and the matrix of the EHS tumor (Inoue and Leblond, 1985a). The nature of basotubules was partially clarified by the use of immunohistochemical methods. Staining with immunoperoxidase or the protein A-gold technique has shown the presence of the amyloid P component in basotubules of both Reichert’s membrane and mouse EHS tumor matrix (Inoue and Leblond, 1985b). The amyloid P component is a glycoprotein found in all types of amyloid deposits (Cathcart et af., 1971; Westermark et al., 1975; Shirahama et al., 1980; Breathnach et al., 1981), and is also a normal serum glycoprotein (Cathcart et al., 1967; Benson el al., 1976; Pepys et al., 1977, 1982; Le et al., 1981; Sipe et al., 1981). It has amolecular weight of 200,000-220,000 and is composed of subunits of M , 22,000 (human) and 23,000 (mouse) (Skinner et al., 1982; Anderson and Mole, 1982; Cathcart et al., 1967; Pepys et al., 1978, 1980, 1982; Skinner et al., 1974, 1980). In

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the electron microscope, the amyloid P component is seen as consisting of pentagonal units 8.5-9.5 nm wide and 2-3 nm in thickness, with a small central lumen. The units could be assembled into doublets or form elongated columnlike structures with a periodicity of -4 nm (Bladen et al., 1966; Glenner and Bladen. 1966; Cathcart e? af., 1967; Pinteric and Painter, 1979). Finally, as mentioned before, free subunits may be observed. Since the appearance and size of cross section of basotubules resembled those of a unit of amyloid P component, particularly in its roughly pentagonal outline with a lumen containing a central dot, it was likely that the amyloid P component was a component of basotubules. Indeed, it was mentioned earlier that immunostaining of EHS tumor matrix for the amyloid P component was restricted to traversely or tangentially sectioned basotubules (Inoue and Leblond, 1985b). An attempt was then made to isolate the amyloid P component from the EHS tumor. After a brief treatment of the homogenized tumor with collagenase, a component was isolated and purified by successive homogenization, calcium-dependent binding to agarose, followed by elution with ethylenediaminetetraacetic acid (EDTA). The purified material yielded a 23,000-Da band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed (1) immunochemical identity by immunodiffusion with the amyloid P component purified from amyloid deposits and (2) characteristic EM appearance as 8.5-nm pentagonal units often assembled into elongated columns, both being characteristic features of the amyloid P component (Inoue et al., 1986b). It was therefore likely that this substance was present within the numerous basotubules of the EHS tumor. As described earlier, the presence of basotubules has been shown in basement membranes of such diverse origins as the kidney glomerulus (Laurie et al., 1984), the parietal wall of the yolk sac (Inoue et al., 1983), and the EHS tumor matrix (Inoue and Leblond, 1985a). Therefore it seems likely that the amyloid P component previously immunohistochemically localized in other basement membranes such as those of kidney (Schneider and Loos, 1978: Dyck et al., 1980a,b), skin (Hamon and Walker, 1982), and retina (Inoue et ul., 1986a), or amyloid P componentlike protein isolated from the glomerular basement membrane of the human kidney (Dyck et al., 1980a) are also present as a component of basotubules. The amyloid P component has been shown to have affinity for hepardn sulfate proteoglycan (Pollak et al., 1982) and fibronectin (de Beer et al., 1981). Since these two components are known to be present in basement membrane (Laurie er a/., 1982b. 1984), the amyloid P component of

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basotubules may bind to these substances present in the cords, thus ensuring an intimate association of basotubule with the cord network.

IV. Conclusions While the biochemical characterization of basement membrane made considerable progress during the 1970s and 1980s, there were few systematic studies of the ultrastructure. Hence, our group undertook detailed studies of the fine structure of basement membranes in parallel with immunohistochemical investigations. This work, in relation with current progress in other laboratories, made it possible to reach the following conclusions. The main ultrastructural component of all basement membranes is a three-dimensional network of “cords. ” Cords are closely packed in the lamina densa of the basement membrane (average ‘‘intercordal spaces,” 8-14 nm) and loosely arranged in the lamina lucida. Cords are poorly defined irregular strands averaging 4-5 nm in thickness, which anastomose to form a network. The cords have a complex composition, since they seem to include a variety of substances. Thus, each cord has as its skeleton a core filament of type IV collagen. This is surrounded by a sheath containing the other components including laminin, entactin, and heparan sulfate proteoglycan. Like the cords, the collagen filament forms a tridimensional network resulting from the end-to-end as well as lateral association of type IV collagen molecules. Heparan sulfate proteoglycan is present along the periphery of the cord as a characteristic, 4.5-nm-wide ribbonlike structure with thickened edges and referred to as “double tracks. ” In addition to the cord network, there are minor ultrastructural components in basement membrane, namely “basotubules” and “double pegs.” The former are straight, 7- to 10-nm-thick tubular structures extending through the cord network. They contain the amyloid P component and resemble connective-tissue microfibrils. Their abundance varies from one basement membrane to another. Double pegs are tiny particulate structures, scattered among a network of cords. They are made up of a pair of 3.5-nm-long, parallel rodlets that resemble the subunits of the pentagonal units of the amyloid P component.

ACKNOWLEDGMENTS The author is deeply grateful to Dr. C. P. Leblond for his generous help and support for the completion of this review. Original works were done with the help of grants from the Medical Research Council of Canada and the National Institutes of Health (grant DE-05690).

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