Striated anchoring fibrils-anchoring plaque complexes and their relation to hemidesmosomes of myoepithelial and secretory cells in mammary glands of lactating rats.код для вставкиСкачать
THE ANATOMICAL RECORD 237:318-325 (1993) Striated Anchoring Fibrils-Anchoring Plaque Complexes and Their Relation to Hemidesmosomes of Myoepithelial and Secretory Cells in Mammary Glands of Lactating Rats Y. CLERMONT, L. XIA, J.D. TURNER, AND L. HERMO Departments of Anatomy and Cell Biology (Y.C., L.H.) and Animal Sciences (L.X., J.D.T.,), McGill University, Montreal, Quebec, Canada ABSTRACT Striated anchoring fibrils (SAF) are associated with the basement membrane underlying myoepithelial and acinar cells of mammary glands. Their proximal extremities are inserted in electron-dense areas of the lamina densa, the anchoring plaques seen facing the hemidesmosomes of both myoepithelial and acinar cells. In the case of myoepithelial cells, the hemidesmosomes show a thick cytoplasmic plaque applied to the basal plasma membrane in which cytoplasmic filaments are inserted. Facing this plaque but on the extracellular aspect and at a short distance of 5-10 nm, there is a thin layer of electron-dense nodular material called the subcell membrane plate, which is connected to the plasma membrane by short filamentous bridges. Between this subcell membrane plate and the anchoring plaque, there is an abundance of fine anchoring filaments crossing the lamina lucida. Such anchoring filaments are less abundant in the lamina lucida outside the hemidesmosomal areas. In the case of acinar cells, the cytoplasmic plaques of the hemidesmosomes are thin and the associated cytoplasmic filaments less conspicuous. No distinct subcell membrane plate is seen on the extracellular aspect of the plasma membrane facing the cytoplasmic plaque of the hemidesmosomes. However, in this area numerous anchoring filaments cross the lamina lucida between the plasma membrane and the SAF-anchoringplaque complex. The abundance, in these cells, of hemidesmosomes and their association with SAFanchoring plaque complexes seen in the basement membrane must constitute a strong attachment for both myoepithelial and acinar cells and bind them to the underlying collagen fibrils, thus preventing their detachment from the connective tissue during the contractions of myoepithelial cells during milk ejection. o 1993 Wiley-Liss, Inc. Key words: Basement membrane, Striated anchoring fibrils, Anchoring filaments, Anchoring plaques, Hemidesmosomes, Myoepithelial cells, Acinar cells, Mammary glands Striated anchoring fibrils’ (SAF), first described as “special fibrils” in the amphibian skin by Palade and Farquhar (1965) and Bruns (1969), have since been described in association with the basement membrane underlying a wide variety of epithelia in mammalian tissues a s reviewed by Kawanami et al. (1978). More recently, SAF have been noted in other locations, such as the respiratory passages (Wasano and Yamamoto, 1985), cornea (Keene et al., 1987), and vas deferens (Clermont and Hermo, 1988). SAF have been shown by Burgeson and collaborators (1985) to be composed of type VII collagen (Bentz et al., 1983; Lundstrum et al., 1986; Morris et al., 1986; Sakai e t al., 1986; Keene et al., 1987). The tissue form of this collagen is a n antiparallel dimer with each monomer composed of 3 helical a chain subunits having a globular domain at the carboxy terminal. The two monomers overlap at their amino terminal ends where they are bridged by disulfide bonds (Morris et al., 1986). The carboxyl terminal globular domains of type VII ‘The term “striated anchoring fibrils,” or SAF, seen on the connective tissue aspect of the lamina densa is preferred to the more general term “anchoring fibril” to avoid confusion with the term “anchoring filaments” used to designate the fine threadlike structures that bridge the plasma membrane to the lamina densa across the lamina lucida. Received April 29, 1993; accepted May 24, 1993. Address reprint requests to Dr. Yves Clermont, Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montreal, Quebec, Canada H3A 2B2. 0 1993 WILEY-LISS, INC STRIATED ANCHORING FIBRILS-ANCHORING PLAQUE COMPLEXES Fig. 1. Electron microscope photograph of a small portion of a mammary gland acinus showing a myoepithelial cell (My) underlying an acinar cell (AC) containing secretory granules (Sg). Several hemidesmosomes (arrows) are seen along the plasma membrane of the myoepithelial cell. Even a t this low magnification, striated anchoring fibrils (arrowheads) can be seen associated with the underlying basement membrane (BM). N, nucleus; L, lipid. x 18,800. 319 320 Y. CLERMONT ET AL Figs. 2-4. 32 1 STRIATED ANCHORING FIBRILS-ANCHORING PLAQUE COMPLEXES in 3 consecutive baths of 1% tannic acid (3 x 20 min each, pH 7.0) at room temperature. The latter treatment was arrested in 1%buffered Na,SO, (20 min, pH 7.4) at room temperature. After postfixation, the tissue was dehydrated in ethanol and propylene oxide and embedded in Epon. Thick sections (0.5 pm) were cut and stained with toluidine blue to locate the acini and evaluate the quality of fixation of acinar cells. Thin sections of selected areas of the blocks were cut with a diamond knife and stained with both uranyl acetate (5 min) and lead citrate (2 min). Thin sections were examined with a Philips 400 and 400 T electron microscope. Stereoscopic images of striated anchoring fibrils were also obtained. In this case, sections of gold interference colour were prepared and placed on the goniometric stage of the electron microscope, and 2 photographs of the same field were taken at 12” from the MATERIALS AND METHODS original 0” position. A magnified 3-dimensional image Four lactating Sprague Dawley rats (350-450 g), of the SAF was obtained by looking a t stereopairs with whose pups were 8 days old, were utilized for the a standard stereoscopic binocular lens. present study. The animals were first anesthesized by RESULTS a n intraperitoneal injection of somnotol and then fixed The plasma membrane of myoepithelial cells facing by perfusion through the abdominal aorta in a n anterograde direction with 5% glutaraldehyde buffered in the basement membrane was slightly wavy and sodium cacodylate (0.1 M) containing 0.05% CaCl, showed many hemidesmosomes (Figs. 1-5). In conat pH 7.4. Ten min later, the mammary glands were trast, the basal cell membrane of acinar cells showed removed, cut into 1mm3 pieces, and placed in the same numerous large irregular folds or invaginations (see fixative for 1 h r at 4°C. The tissue was then washed in Figs. 63). Whereas the basement membrane followed several changes of 0.1 M sodium cacodylate buffer and closely the plasma membrane of myoepithelial cells, left overnight in the same buffer a t 4°C. On the follow- the basement membrane of acinar cells did not follow ing day, pieces of mammary gland tissue were either the sinuous contours of the cell membrane but estabpostfixed for 1 h r a t 4°C in reduced osmium (1:l mix- lished contact only with the tips of the basal folds. At ture of 2% aqueous osmium tetroxide and 3% aqueous these points of contact discreet hemidesmosomes were potassium ferrocyanide; Karnovsky 1971) or immersed visible (see Figs. 6,8). At the interface of myoepithelial cells and acinar cells, the plasma membranes of both cells were a t a short distance from each other except a t the level of desmosomes where they were closely apposed (Figs. 1, 3). Fig. 2. Electron microscope photograph showing the basal cell mem- collagen are inserted in plaques of electron-dense material that are usually associated with the lamina densa of the basement membrane. In a few locations such a s skin and cornea (Keene et al., 1987) and vas deferens (Clermont and Hermo, 19881, “anchoring plaques” associated with SAF are also separated from the basement membrane by a short distance. Whenever present, SAF encircle collagen fibrils and thus are considered as reinforced attachment devices that anchor the basement membrane and the overlying cells to the underlying connective tissue. It thus became of interest to examine more closely the structural features of the SAF-anchoring plaque complexes subjacent to myoepithelial and acinar cells of the lactating mammary gland. In addition, the relation of anchoring plaques and associated SAF with hemidesmosomes of myoepithelial and acinar cells is examined. brane of a myoepithelial cell facing the basement membrane (BM) of the acinus. Several hemidesmosomes are visible (open arrows). The basement membrane facing such hemidesmosomes shows areas of increased electron density which correspond to anchoring plaques (small white asterisks). Numerous striated anchoring fibrils (large straight arrows) are inserted in these anchoring plaques. Striated anchoring fibrils in cross section are also indicated (curved arrows) In the lamina lucida next to the cell membrane associated to the hemidesmosome, there are electron-dense lines (small arrows) referred to as a subcell membrane plates. f, cytoplasmic filaments; c, collagen fibrils. x 55,OO Fig. 3. Electron microscope photograph showing a small portion of a myoepithelial cell (My) underlying a n acinar cell (AC).Two hemidesmosomes of the myoepithelial cell are indicated (open arrows). Facing the cytoplasmic plaque of hemidesmosomes, there is a densification of the lamina densa, the anchoring plaques (asterisks) in which the extremities of a striated anchoring fibrils (SAF) are inserted. The fusiform segment of this SAF is indicated (curved arrows). In this section collagen fibrils are not stained. Several fine bridging filaments, i.e., the anchoring filaments are seen in the lamina lucida between the subcell membrane plate (straight arrows) and the anchoring plaque (asterisks). Such filaments are less abundant in the lamina lucida beyond the anchoring plaque areas. ER, endoplasmic reticulum with attached ribosomes of an acinar cell. x 55,000 Fig. 4. Electron microscope photograph of a hemidesmosome area of a myoepithelial cell (My). CM, cell membrane; CP, cytoplasmic plaque; SP, subcell membrane plate; AF, anchoring filaments between the subcell membrane plate (SP) and the anchoring plaque (AP); SAF, striated anchoring fibrils; c, cross section of collagen fibrils; LL, lamina lucida; LD, lamina densa. x 110,000 * Basement Membrane In our material prepared by a conventional method, the basement membrane showed a lamina densa, separated from the plasma membrane of both acinar cells and myoepithelial cells by a relatively wide lamina lucida (Figs. 2-4). Along the lamina densa there were thicker and denser areas facing hemidesmosomes. Such denser areas corresponded to anchoring plaques, which serve as sites of insertion of striated anchoring fibrils (Figs. 2-7). Areas of the lamina densa surrounding the anchoring plaques were composed of a relatively loose network of fine filaments referred to a s “cords” by Leblond and collaborators (Inoue and Leblond, 1988; Leblond and Inoue, 1989; Chan et al., 1993a,b) (Fig. 4). Some sparse fine filaments usually referred to as anchoring filaments bridged the basal plasma membrane of myoepithelial cells and acinar cells to the lamina densa (Figs. 3,4). Hemidesmosomes and Associated Basement Membrane of Myoepithelial Cells On the cytoplasmic aspect of the myoepithelial basal plasma membrane, there was a marked densification of the cytoplasmic matrix a t the level of the hemidesmosome. Into this cytoplasmic plaque, numerous cytoplas- 322 Y. CLERMONT ET AL. Figs. 5-8 STRIATED ANCHORING FIBRILS-ANCHORING PLAQUE COMPLEXES 323 mic filaments, presumably actin, were inserted (Figs. 2-4). Underlying the plasma membrane and at a very short distance (- 5-10 nm), there was a thin electrondense line 5-10 nm thick showing a nodular or punctate texture (Figs. 2-4,7). This particular layer, referred to as the subcell membrane plate, was only seen facing hemidesmosomes. Some very fine and short bridging filaments were seen between this plate and the plasma membrane (Figs. 43). Between the subcell membrane plate and the anchoring plaque lodged in the lamina densa, there was an abundance of fine filamentous threads crossing the lamina lucida (Figs. 3-51, Cross striated anchoring fibrils (SAF) were attached to the connective tissue face of the anchoring plaques (Figs. 2-5). These SAF presented various configurations. The longest ones arched with their widened extremities inserted into the anchoring plaques. They showed characteristic cross striations and a central fusiform segment (Figs. 3,7). Such SAF also branched (Fig. 3). Usually < 60 nm in diameter, they presented variable cross-sectional profiles (Fig. 2). Numerous cylindrical collagen fibrils were seen intermixed with SAF and within their loops or arches (Figs. 2,4). The cytoplasmic plaques of acinar cell hemidesmosomes were comparatively thinner than those of myoepithelial cells, and the cytoplasmic filaments associated with them were less conspicuous (Figs. 6,8). The subcell membrane plates were either absent or reduced to rare and barely visible punctate densities (Figs. 6,8). Numerous fine anchoring filaments were seen between the plasma membrane and the anchoring plaques fating these hemidesmosomes (Figs. 6,8). The anchoring plaques facing hemidesmosomes of acinar cells had a n electron density and increased thickness comparable to those facing hemidesmosomes of myoepithelial cells (Figs. 5,6). Whereas the anchoring plaques and associated SAF were abundant facing the folded basal plasma membrane of acinar cells, the reduced thickness of the cytoplasmic plaque and the relative absence of the subcell membrane plates made hemidesmosomes of acinar cells far less conspicuous than those of myoepithelial cells (compare Fig. 5 with Fig. 6 and Fig. 7 with Fig. 8). Fig. 5. Electron microscope photograph showing a hemidesmosome of a myoepithelial cell (open arrows). A characteristic subcell membrane plate (arrow) is seen a t a short distance of the cell membrane facing the cytoplasmic plaque, and the anchoring plaque located in the lamina densa is indicated with a white asterisk. A longitudinal view of a striated anchoring fibril is indicated (SAF). On the right (arrowhead) cross sections of SAF are visible. c, collagen fibrils. x 55,000 Radnor (1972) reported the presence of striated anchoring fibrils (SAF) in the basement membrane facing myoepithelial cells of mammary gland acini. However, the low power electron microscope photographs presented as supporting evidence did not permit their identification and distinction from collagen fibrils or their association with the hemidesmosomes of these contractile cells. The present report clearly demonstrates characteristic SAF in this particular basement membrane and defines their association with anchoring plaques and hemidesmosomes of both myoepithelial and acinar cells. Typical SAF have also been reported in the basement membrane facing myoepithelial cells and epithelial cells of ducts and glands of the dog trachea and bronchi (Kawanami et al., 1979). Fig. 6. Electron microscope photograph showing a hemidesmosome (arrow) along the basal plasma membrane of an acinar cell. Note that the basal plasma membrane shows numerous irregular folds (F). In this hemidesmosome the cytoplasmic plaque is thin. Facing it, in the lamina densa, there is a n electron-dense anchoring plaque (white asterisk) in which striated anchoring fibrils are inserted (SAF).The rest of the lamina densa is thin and less dense. Some small filaments are seen bridging the anchoring plaque and the cell membrane (curved arrow) but characteristically there is no distinct subcell membrane plate in the lamina lucida facing the hemidesmosome. ER, endoplasmic reticulum and associated ribosome; m, mitochondrion. x 55,OO Figs. 7 and 8. Electron microscope photographic stereopairs showing hemidesmosomes, anchoring plaques, and associated striated anchoring fibrils of a myoepithelial cell (Fig. 7) and of a n acinar cell (Fig. 8). A single magnified stereoscopic image may be obtained by utilizing a properly adjusted (at 65 mm) binocular lens. The magnification of the photographs of the two pairs is identical, i.e., x 100,000 Fig. 7. Striated anchoring fibrils (arrowheads) show their extremities inserted into a n electron dense anchoring plaque (white asterisk) lodged in the lamina densa facing a myoepithelial cell. The nodular texture of the subcell membrane plate is visible as well as bridges between this plate and the cell membrane (arrow). The cytoplasmic plaque of the hemidesmosome is indicated (white arrowhead). Fig. 8. Striated anchoring fibril (arrowhead) inserted in the anchoring plaque (asterisk) located in the lamina densa facing the hemidesmosome of an acinar cell. Whereas some bridging anchoring filaments are seen between the anchoring plaque and the plasma membrane, there is no well defined subcell membrane plate similar to the one seen in association with the hemidesmosome of myoepithelial cell (see Fig. 7). The cytoplasmic plaque of the hemidesmosome is indicated (white arrowhead). F, fold of the basal plasma membrane. Hemidesmosomes and Associated Basement Membrane of Acinar Cells DISCUSSION Association of SAF With the Basement Membrane of Mammary Gland Acini Relation of SAF With Hemidesmosomes of Myoepithelial and Acinar Cells SAF and anchoring plaques. It soon became evident in our material that SAF facing both myoepithelial and acinar cells were inserted in anchoring plaques, i.e., the thicker and denser areas of the lamina densa (Fig. 9). SAF-anchoring plaque complexes have been observed in the skin (Bruns, 1969; Keene et al., 1987; Rouselle et al., 1991) and vas deferens (Clermont and Hermo, 1988). In these locations, anchoring plaques were seen not only inserted in the basement membrane but also at a short distance from it (Keene e t al., 1987; Clermont and Hermo, 1988). Such distal anchoring plaques not only contained, as did the basement membrane associated ones, the carboxy terminal of type VII collagen (Sakai et al., 1986; Keene et al., 1987) but also type IV collagen, laminin, and heparan sulfate proteoglycan (Clermont and Hermo, 1988) a s did the basement membrane itself (Inoue, 1989; Leblond and Inoue, 1989). In the present study, distal anchoring plaques were not observed in the lamina propria of mammary gland acini, but those inserted in the basement mem- 324 Y. CLERMONT ET AL. MYOEPITHELIAL CELL HD ACINAR CELL HD W Fig. 9. Diagrams of hemidesmosomes and associated basement membrane components seen in association with myoepithelial cells and acinar cells of a mammary gland acinus. Lettering from top to bottom: HD: hemidesmosomes, f: cytoplasmic filaments, C P cytoplasmic plaque, CM: cell membrane with its two osmiophilic leaflets, S P subcell membrane plate, AF: anchoring filaments, L L lamina lucida, LD: lamina densa, A P anchoring plaque, SAF: striated anchoring fibrils, C: collagen fibrils. In both locations striated anchoring fibrils (SAF) are associated with anchoring plaques (AP), i.e., the electrondense areas of the lamina densa, which face hemidesmosomes. In the case of myoepithelial cells, an electron-dense subcell membrane plate (SP) is consistently seen next to the cell membrane underlying the hemidesmosome, whereas in the case of acinar cells, such a subcell membrane plate is not distinct. brane were well demarcated and always clearly related to SAF (Fig. 9). SAF-anchoring plaques and hemidesmosomes. The association of SAF and anchoring plaques with hemidesmosomes has been repeatedly observed in various epithelia (Susi, 1969; Susi et al., 1969; Briggaman and Wheeler, 1975; Kawanami et al., 1979; Ellison and Garrod, 1984; Keene et al., 1987). Although not reported in the vas deferens (Clermont and Hermo, 1988), a closer examination of our material indicated that there was a n association of SAF-anchoring plaque complexes with hemidesmosomes (Clermont and Hermo, unpublished). Such a feature may thus be of common occurrence in the SAF-anchoring plaques that were associated with basement membranes. However, within the same basement membrane a distinct difference was noted between the hemidesmosomes of myoepithelial and acinar cells. In the former the cytoplasmic plaques were thick and distinct subcell membrane plates were visible, whereas in the latter the cytoplasmic plaques were thin and subcell membrane plate was virtually absent (Fig. 9). In both locations, however, there was a n abundance of fine bridging anchoring filaments. An electron-dense subcell membrane plate was initially described in the skin (Briggaman and Wheeler, 1975) and was later observed in the respiratory passages (Kawanami et al., 1979; Wasano and Yamamoto, 1985). The composition of this subcell membrane plate remains to be determined. Several link proteins have been shown to be located in this subhemidesmosal location, e.g., a6P4 integrin; BPAG2180 protein, and kalinin (reviewed in Albeda and Buck, 1990; Luna and Hitt, 1992; Garrod, 1993). The nodular appearance of the plate may correspond to the extracellular ligand binding domains of the transmembrane protein integrin, i.e., the OL and P subunits, known to bind to laminin (Lee et al., 1992). Another possibility is that there is, at the level of this plate, the globular domain at kalinin, a 107 nmlong, rodlike molecular complex, composed of 3 nonidentical chains linked by disulfide bonds and found to be a component of anchoring filaments (Rousselle et al., 1991). In the present study a subcell membrane plate was well developed underlying the hemidesmosomes of myoepithelial cells, which showed a thick and dense cytoplasmic plaque. In contrast, no distinct subcell membrane plate was associated with hemidesmosomes of acinar cells in which the cytoplasmic plaque was poorly developed (Fig. 9). However, anchoring filaments that cross the lamina lucida to insert into anchoring plaques were equally abundant in both locations. This may indicate that the extracellular domains of the transmembranous link proteins either a6P4 integrin or BPAG2-180 proteins, are possibly more abundant in hemidesmosomes of myoepithelial than those of acinar cells, and may thus be responsible for the electron density of the subcell membrane plate. In view of recent evidence t h a t the lamina lucida is possibly a fixation artefact since it is not distinct from the lamina densa in tissues prepared by rapid freezing and freeze substitution (Goldberg and Escaig-Haye, 1986; Reale and Luciano, 1990; Chan et al., 1993b), one is led to consider the possible artefactual nature of this subcell membrane plate. On the one hand, in the skin STRIATED ANCHORING FIBRILS-ANCHORING PLAQUE COMPLEXES such a plate was observed to develop in synchrony with the formation of hemidesmosomes and thus appears structurally related to this junctional apparatus (Briggaman et al., 1971; Briggaman and Wheeler, 1975). On the other hand, in the present study these subcell membrane plates were consistently seen associated with the hemidesmosomes of myoepithelial cells but virtually absent facing the hemidesmosomes of acinar cells. Both cell types, adjacent to each other, are in close contact with the same basement membrane. These observations would indicate that such a plate is real and located in a layer, the lamina lucida, which is more or less apparent depending on the method of preparation utilized. Functional Significance of the Presence of SAF in the Basement Membrane of Mammary Gland Acini It is well known that with the sucking stimulus there is a n almost instantaneous release of oxytocin by the posterior pituitary, which provokes strong contractions of myoepithelial cells resulting in the rapid expulsion of milk from the lumen of the mammary gland acini into the collecting ducts and nipple. To maintain, during such contractions, the structural connection of myoepithelial and acinar cells to the underlying connective tissue, which includes the capillary networks, it is essential t h a t the cells of the acini remain tightly bound to the underlying type I or I11 collagen fibrils. This appears to be achieved by the system composed of hemidesmosomes, anchoring filaments, anchoring plaques, and SAF, which have so far been seen mainly in locations submitted to physical stress or tension such a s the skin, vas deferens, and now the acini of lactating mammary glands. ACKNOWLEDGMENTS This work was supported by grants of the MRC to Y.C. and L.H., and of the NSERC of Canada to J.D.T. The technical assistance of Ms. Jeannie Mui and Ms. Matilda Cheung is gratefully acknowledged. The secretarial assistance of Mrs. Ann Silkauskas is also appreciated. LITERATURE C [TED Albeda, S.M., and C.A. Buck (1990) Integrins and other cell adhesion molecules. FASEB J., 4t2868-2880. Bentz, H., N.P. 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