Serous Cells in the Parotid Glands of Two Species of TamarinsPolarized Secretory Granules.код для вставкиСкачать
THE ANATOMICAL RECORD 291:1254–1261 (2008) Serous Cells in the Parotid Glands of Two Species of Tamarins: Polarized Secretory Granules BERNARD TANDLER* Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, Ohio ABSTRACT The parotid glands of two species of tamarins were examined by electron microscopy. Endpiece cells are typical in appearance, with an extensive rough endoplasmic reticulum, prominent Golgi apparatuses, and numerous serous granules. In the saddleback tamarin, the secretory granules contain a dense spherule pressed against the inner aspect of the limiting membrane, leading to a surface bulge. During the course of merocrine secretion (a form of exocytosis), such morphologically polarized granules approach the luminal plasma membranes with the bulge in the vanguard. It is these protuberances that fuse with the plasmalemma. In contrast, although serous granules in the cotton top tamarin contain a spherule, they lack surface bulges and their docking on luminal membranes seems to be a random event with respect to their surface morphology. Moreover, certain other types of cells in a taxonomically wide spectrum of species have granules with a less obvious structural polarity, as well as cells whose granules lack morphological polarity but have a functional polarity that comes into play during exocytosis of such secretory granules. Anat Rec, 291:1254–1261, 2008. Ó 2008 Wiley-Liss, Inc. Key words: merocrine secretion; exocytosis; salivary glands; secretion; secretory granules; tamarins; monkeys Since the original report by Palade (1959) on exocrine pancreas secretory phenomena, the basic morphological steps in the regulated liberation of exocrine protein products from serous cells are well recognized. When a secretory cell is signaled to secrete, mature storage granules move to and dock onto a luminal plasma membrane to which they fuse, forming a pentalaminar barrier between the contents of the secretory granule and the cell exterior (Palade, 1975). This barrier is reduced to a trilaminar one [in salivary gland mucous cells, it is reported that this reduction results from avulsion of the plasma membrane (Tandler and Poulsen, 1976)], which ultimately ruptures, allowing the outﬂow of the secretory product. There is little argument over the reality of these steps, but the mechanism of attachment of granule membrane to plasma membrane still is controversial. In the parotid gland of a particular species of tamarin, exocytosis of serous granules involves a morphological modiﬁcation of the granules that has implications for regulated exocytosis of secretory products in general. Ó 2008 WILEY-LISS, INC. MATERIALS AND METHODS This study is based on archival material. The animals were obtained from a breeding colony at the University of Texas Dental Science Institute at Houston, TX. Four adult tamarins were used in this study: one male and one female saddleback tamarin (Saguinus fuscicollis) and one male and one female cotton top tamarin (Saguinus oedipus). They had been housed in hanging wire mesh cages and food and water were available ad libitum. The two saddleback tamarins, which were being used for other experimental purposes, were sacriﬁced by injection of an overdose of *Correspondence to: Dr. Bernard Tandler, Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106-4905. E-mail: email@example.com Received 26 October 2007; Accepted 15 May 2008 DOI 10.1002/ar.20744 Published online in Wiley InterScience (www.interscience.wiley. com). AROTID GLANDS OF TAMARINS Fig. 1. Saddleback tamarin: Survey electron micrograph of several endpiece cells in the parotid gland. The cells include an extensive RER and numerous serous granules. The latter contain a dense spherule that often causes a surface bulge. Scale bar 5 2 mm. methoxyﬂuorane (‘‘Metofane,’’ Pitman-Moore, Indianapolis, IN). Their parotid glands were ﬁxed by immersion in phosphate-buffered half-strength Karnovsky’s (1965) ﬁxative. They were postﬁxed with 2% osmium tetroxide in the same buffer, and then, after rinsing in saline, they were soaked overnight in 0.5% uranyl acetate in saline. After another saline rinse, they were dehydrated in ascending concentrations of ethanol. In the case of the two cotton top tamarins, parotid glands were removed from monkeys anesthetized with Metofane; both animals survived salivary gland ablation. Gland specimens were ﬁxed by immersion in cacodylate-buffered 3% glutaraldehyde and then postﬁxed in 2% osmium. After a rinse in buffer, they were soaked overnight in acidiﬁed 0.25% uranyl acetate (Tandler, 1990), then rinsed and dehydrated in ethanol. Irrespective of the mode of ﬁxation, all dehydrated specimens were passed through propylene oxide and embedded in Maraglas:DER 732 (Erlandson, 1964). Thin sections were stained with acidiﬁed uranyl acetate (Tandler, 1990) followed by lead tartrate (Millonig, 1961) or by bismuth subnitrate (Riva, 1974) and then examined in a Siemens Elmiskop 1a electron microscope. RESULTS Histologically, parotid glands in both species of tamarin conform in structure to that exhibited by their 1255 Fig. 2. Saddleback tamarin: A localized specialization of the RER that to a degree resembles the ‘‘coat-of-mail’’ conﬁguration noted in other cell types. Scale bar 5 0.5 mm. namesakes in other primates. Their parenchyma consists of serous endpieces, short and inconspicuous intercalated ducts, prominent striated ducts, and occasional excretory ducts. In semithin sections, the endpiece serous granules are heavily stained with toluidine blue. In the saddleback tamarin, these granules tend to cluster near the luminal border of the serous cells and frequently outline intercellular canaliculi. At the ultrastructural level, the endpiece secretory cells in both species have all of the morphological hallmarks of serous cells, namely, an extensive, regular rough endoplasmic reticulum, a prominent supranuclear Golgi apparatus, and numerous dense secretory granules (Fig. 1) (Tandler and Phillips, 1993). In a few rare sites, the endoplasmic reticulum is locally modiﬁed (Fig. 2) to form a series of undulating, smooth-surfaced membranes corresponding in appearance to the ‘‘coat-of-mail’’ described in other cell types (Franke and Scheer, 1971). The Golgi apparatus consists of several slightly dilated, imbricated saccules that often show terminal expansions, a scattering of small vesicles, and some vacuoles. The vesicles and the expanded saccular termini often contain a dense spherule that is retained in the condensing vacuoles, ultimately in the mature granules. Fully mature serous granules in the saddleback tamarin parotid gland consist of a fairly dense matrix delimited by a single membrane. Many contain a dense spherule that usually is pressed against the granule mem- 1256 TANDLER Fig. 3. Saddleback tamarin: Higher magniﬁcation of a cluster of serous granules, each with a spherule-containing surface bulge. Scale bar 5 1 mm. brane, leading it to bulge outward to form a surface bump (Fig. 3). Those granules that appear to lack spherules might in fact possess them, but because these inclusions are small and acentrically positioned, they are often missed in thin sections. In a few granules, two spherules are present. The majority of granules lying near a lumen, whether endpiece or canalicular, are oriented in such manner that the surface bulge produced by the spherule points toward the lining plasmalemma (Figs. 4 and 5). In contrast to the saddleback tamarin, the serous granules of the cotton top tamarin are spherical in outline, without surface bulges, although they contain a lenticular dense inclusion that adheres to the inner aspect of the membrane (Fig. 6). Although the specimens of this species were ﬁxed in a slightly different manner from those originating from the saddleback tamarin, Tandler and Phillips (1993) found that other primate species similarly feature granules that always lack bulges, even when ﬁxed in precisely the same manner as was the saddleback tamarin—in other words, the difference in granule morphology between the two tamarin species in this study is not ascribable to a variation in ﬁxation. The granules in S. oedipus show no preferential orientation with respect to the plasma membranes. The lumina of the endpieces of the saddle-back tamarin, including those of the intercellular canaliculi, are scalloped as a result of extensive exocytosis. In addition to the expected X-shaped invaginations, there are deep, tortuous secretion channels resulting from compound (chain) exocytosis. A prominent feature of the lumina in these tamarins is the presence of assemblages of dense material, which consists in part of globules that resemble the spherules of the in situ serous granules in size, Figs. 4 and 5. Saddleback tamarin: Both ﬁgures show the relationship of serous granules to the lumina of intercellular canaliculi. Note that most of the granules are oriented with their surface bulges pointing toward the plasma membrane. Both illustrations show evidence of a recent exocytotic event. That the invaginations marked by an asterisk are of exocytotic origin rather than being intrinsic components of the luminal membrane is supported by their complete lack of microvilli, in contrast to the connected lumina. An aggregate of lipid-like bodies is present in the canaliculus shown in Fig. 5. Scale bars 5 1 mm. AROTID GLANDS OF TAMARINS 1257 Fig. 7. Saddleback tamarin: A large aggregate of lipid-like bodies in the lumen of an endpiece. The larger structures are similar to the serous granule spherules in size and density; the absence from the serous cell cytosol of obvious lipid droplets strongly bolsters the notion that the luminal structures originate in the secretory granules. Scale bar 5 1 mm. Fig. 6. Cotton top tamarin: In this species, the serous granules contain lenticular spherules, but lack surface bulges. The light granule in the center is not quite mature. Scale bar 5 1 mm. shape, and density (Figs. 4b, 7). The second component of the luminal material consists of substantially smaller and less dense particles. Both components frequently are present in the channels formed by compound exocytosis, where at ﬁrst glance they appear to be intracellular. In addition to particulate material, scattered oddments of membranes are present in the lumina. In the cotton-topped tamarin, the endpiece lumina contain only sparse spherule-sized dense globules. DISCUSSION The most interesting aspect of the tamarin parotid glands concerns their serous granules. Superﬁcially, these granules resemble their counterparts in the parotid glands of most of the few studied primate species: spider monkey (Leeson, 1969); squirrel monkey (Cowley and Shackleford, 1970); human being (Riva and RivaTesta, 1973); olive baboon (Tandler and Erlandson, 1976); green monkey (Messelt, 1981); and the tufted capuchin monkey (Watanabe, 1990)—also see Figs. 8–13 in Tandler and Phillips (1993) for a side-by-side comparison of primate serous granules in both the parotid and submandibular glands. Irrespective of matrix substructure, salivary serous granules in most primate species contain a dense inclusion, dubbed the ‘‘spherule’’ by Tandler and Erlandson (1972). Immunocytochemical tests of the serous granules in the submandibular glands of the longtailed macaque, Macaca fascicularis, that contain a prominent spherule have shown that these spherules consist at least in part of proline-rich proteins (PRP) (Kousvelari et al., 1982), suggesting that the spherules in the parotid granules in the two species of tamarins examined in this study also contain PRP. It should be noted, however, that in human parotid granules PRP is not conﬁned solely to the spherules, but is distributed throughout the granule content; instead, it appears that it is amylase that is localized predominantly in the spherules (Takano et al., 1993). Although PRP is present in parotid serous granules from other monkey species that have prominent spherules (Williams and Keller, 1973; Arneberg et al., 1976), the precise intragranular localization of these proteins has not been established. In the saddleback tamarin, the spherules are associated with surface bulges that confer a structural polarity on the granules. This bulge is intimately involved with granule exocytosis, because most granules near to a luminal membrane are oriented with the spherule-containing bulge toward the plasmalemma. A similar phenomenon occurs in the vervet monkey, Cercopithecus aethiops, where parotid serous granules approach luminal membranes with their spherule-containing pole leading the way (Messelt, 1981). Nevertheless, in this species there is no bulge at the point where the spherule 1258 TANDLER contacts the limiting membrane of the granule, thus no surface indication of polarity. It seems unlikely that PRP (or a related protein) in and of itself is responsible for the adherence of spherules to the inner side of the granule membrane because this happens only in the saddleback tamarin and not in the cotton-top tamarin nor in many other mammalian species that have salivary secretory granules that contain spherules, some of which have been determined to consist in part of PRP. It nonetheless is true that, in many species, spherules, sometimes lenticular in form, adhere to the bounding membrane of their encompassing granule (Tandler and Phillips, 1993). In the rat mammary gland, spherules—here called micelles—which consist of casein, are attached to the inner aspect of the granule membrane by ﬁne ﬁlaments of unknown nature (Franke et al., 1976). Several mechanisms for spherule position-determined exocytosis of secretory granules suggest themselves. One is that in these two species (S. fuscicollis and C. aethiops), the proximity of the spherule to the granule membrane produces a localized charge on that membrane opposite to that of the plasma membrane, resulting in an electrostatic attraction between the two. The molecular mechanisms involved in fusion of exocytotic structures with the plasma membrane have been reviewed by Watson (1999), Gerber and Sudhof (2002), Wasle and Edwardson (2002), and Burgoyne and Morgan (2003). The presence of the spherule adjacent to the granule membrane might result in the selective adherence of a protein from the cytosol [such as ADP-ribosylation factor (Arf) (Dohke et al., 1998), VAMP-2 (FujitaYoshigaki et al., 1998), rap1 (D’Silva et al., 1997), Rab3D (Ohnishi et al., 1996)] or Rab26 (Nashida et al., 2006), which ordinarily appear to be disposed over the entire granule surface and that could act as a docking protein. Another candidate is synexin or a synexin-like molecule, which has been proposed as a recognition protein that mediates fusion of chromafﬁn granules with the plasma membrane of cells in the adrenal medulla (Pollard et al., 1979). In sea urchin eggs, it has been found that the proteins involved in the fusion step of exocytosis are immobile within the granule membrane (Wong et al., 2007), suggesting that in the saddleback tamarin parotid gland the bulges are ﬁxed in position on individual granules. In a sense, the surface bulges on the tamarin granules might be analogous to the exocysts of yeast cells. Exocysts are complexes of proteins with some rod-like domains consisting of helical bundles (Munson and Novick, 2006) that are carried by secretory vesicles and target them to plasmalemmal exocytotic sites (Guo et al., 1999), which are marked by other units of the exocysts (Boyd et al., 2004). An additional possibility is that actin ﬁlaments, which are involved in the translocation of salivary serous granules to a secretory surface (Segawa and Riva, 1996; Jerdeva et al., 2005), are attached only to the spherulecontaining pole and pull this pole toward the cell surface so that it perforce becomes the forepart of the granule about to be exocytosed. The absence of obvious actin ﬁlaments in our preparations might be due to the wellknown propensity of osmium tetroxide to cause the depolymerization of this molecule (Boyles et al., 1985). The presence of an ostensibly polarized membrane on the saddleback tamarin serous granules raises some in- triguing questions. Freeze-fracture studies of rat parotid serous granules in the process of exocytosis reveal that at the zone of contact between granules and plasmalemma, there is a marked difference in the density of intramembrane particles, with the granule membranes having a low density and the plasma membrane having a high density (De Camilli et al., 1976; Tanaka, 1980). As a result, during release of the secretory product and brieﬂy thereafter, the luminal membrane has a mosaic arrangement. After (and even during) exocytosis, the excess luminal membranes are retrieved by endocytosis (Amsterdam et al., 1969) by a calcium-dependent mechanism (Koike and Meldolesi, 1981); this reuptake is highly speciﬁc for those patches originating from the exocytosed granules (Meldolesi and Ceccarelli, 1981). The fate of these recaptured membranes still is controversial. They clearly are recycled, but in what fashion? Are they used exclusively to delimit nascent granules? Do they become incorporated into the general membrane complement of the cell? Are they degraded and their components used in the de novo synthesis of the sundry cellular membranes? Because serous granules in the saddleback tamarin parotids were not subjected to freeze-fracture analysis, the status of intramembrane particles on the leader bulge versus the remainder of the granule membrane is unknown. Freeze-fracture studies of pancreatic islet cell granules, which have no obvious polarity in thin sections, reveal a highly circumscribed aggregate of particles on their P-face (Berger et al. 1975); in other words, the granule membranes in these cells do not have a homogeneous structure. If such aggregates overlie the bulges in the tamarin granules, are they leader domains and can they be distinguished from the plasmalemma once fusion has been achieved? Are such patches selectively removed from the cell surface during recovery from an exocytotic event? Once internalized, do they have the same or a different fate from indifferent exocytotic membranes? Comparative Considerations Compelling examples of structurally polarized secretory granules are found in certain ciliated protozoa, including Paramecium and Tetrahymena. In these protozoa, trichocysts, which are internally polarized elliptical secretory structures (Bannister, 1972), become joined to the plasmalemma only at their anterior, rounded end in a junction that involves a particulate annulus on their limiting membranes, and a little knob at the tip of the trichocyst (Beisson et al., 1980). In Tetrahymena, when an elliptical mucocyst approaches the plasma membrane, an annulus of particles forms at its leading end (Satir et al., 1973). A similar phenomenon occurs in the extrusosomes of the actinopod, Heterophrys marina (Davidson, 1976). In the lactating rat mammary gland, secretory granules are partially or completely coated with bristles (Franke et al., 1976). Most of the granules in early stages of exocytosis illustrated by these workers have a partial bristle coat and it is this membrane zone that is in contact with the plasmalemma, strongly suggesting that these granules are in fact polarized. In the same vein, a freeze-fracture study of bovine chromafﬁn granules revealed that in early stages of exocytosis there are AROTID GLANDS OF TAMARINS bridges, presumably proteinaceous, that connect the granule membrane to the plasmalemma (Aunis et al., 1979). An example of secretory granules that may have physiological polarity in the absence of structural indications in thin sections are pancreatic exocrine granules of the rat. Using Helix pomatia lectin-gold, Kan and Bendayan (1989) demonstrated immunocytochemically that the lectin-binding glycoconjugates are unevenly distributed on the granule surface and that the labeled surfaces are the ones that join the plasmalemma during exocytosis. Granule Pseudopodia In a number of cell types, including parotid serous cells of the rat (Schramm et al., 1972; Zellinger et al., 1974) and olive baboon (Tandler and Erlandson, 1976), secretory cells of the shell membrane-secreting gland of the duck (Sandborn et al., 1975), neurosecretory cells in the neurohypophysis of the mouse (Castel, 1977), and peripolar cells in the kidney of the sheep (Ryan et al., 1982), granules approaching luminal membranes often form pseudopodia that become attached to the plasmalemma. All of the foregoing examples of granule pseudopodia were observed by conventional transmission electron microscopy. Odajima and Nakane (1984) noted the same phenomenon in quick-frozen rat submandibular acinar cells, as did Tanaka (1980) in freeze-fractured pancreatic acinar cells. Granule contents are expelled via these extensions, leaving behind invaginations of the luminal membrane that are shaped like Florence ﬂasks. It is unclear as to whether these pseudopodia form only in relation to specialized zones of the granule membrane or can originate from any spot on the granule surface. If they are restricted as to point of origin, pseudopodia would be another indication of polarized secretory granules. An example of structurally polarized granules whose exocytosis remains conjectural is to be found in the so-called rodlet cells, a peculiar cell type occurring in relation to some, but not all, chemosensory cells of the zebraﬁsh (Dezfuli et al., 2007). The rodlet cells contain cytoplasmic ‘‘inclusions’’ (original authors’ term), which are implied to be secretory granules, albeit of unusual shape. These inclusions consist of a major portion that is ovate, and a single long, tubular projection that is pointed toward the cell apex. A dense core runs the length of the inclusion and may furnish the necessary stiffness to maintain its asymmetric form. The authors claim that the ends of these projections pierce the apical membrane, implying that the contents of the inclusions might eventually be exocytosed, but close inspection of their published micrographs suggests that the putative portions of the projections lying above the plasmalemma actually are microvilli. The precise excretory status of these polarized inclusions in the secretory cycle remains to be established, but whatever it may turn out to be, they are highly polarized and maintain a strict orientation vis-à-vis the apical plasma membrane. Compound Exocytosis In many species, e.g., the rat (Amsterdam et al., 1969), parotid endpiece cells engage in compound (chain) exocytosis where secretory granules fuse in turn to the 1259 granule membrane of their merocrine predecessor ultimately to form a channel of catenated empty granule membranes that debouches into a lumen. In the tamarin, the spherule-laden bulge is what attaches to the invaginated membranes. In this process, the membranes surrounding the now empty granule remnants must take on the characteristics of the plasmalemma so that further granules recognize them as potential docking sites. In the pancreatic acinar cell, it has been shown by confocal immunoﬂuorescence microscopy that protein attachment protein acceptors, such as syntaxin 2, extend from the plasmalemma into the membranes of fused granules (Pickett et al., 2005). In the rat parotid gland, secretory granules in unstimulated cells occasionally become closely apposed to form pentalaminar membranous barriers between their respective contents (Tanaka, 1982). In the saddleback tamarin, no examples of the granule bulge were encountered that formed such focal appositions with neighboring granules, indicating that the bulges are programmed to interact solely with surface membranes, whether of primary or secondary origin. Based on the many foregoing studies, it would appear that ultrastructural cyto- and immunocytochemical studies of molecules that have been implicated in secretory granule exocytosis where there is no obvious structural polarity might reveal an unsuspected differentiation of granule membranes. Luminal Contents Finally, the contents of the endpiece lumina in the saddleback tamarins deserve comment. This globular dense material is reminiscent of neutral lipids that have been blackened by osmium tetroxide. The larger globules precisely match in size and appearance the spherules of the serous granules. On the basis of the fact that the spherules in poorly ﬁxed serous granules in human submandibular glands sometimes form myelin ﬁgures, Tandler and Erlandson (1972) suggested that these structures consist in part of phospholipids. A histochemical study of the same gland failed to identify any lipid in the secretory granules (Sirigu et al., 1976), but similar studies of rat parotid gland revealed the presence of lipoidal material in serous granules (Simson et al., 1973). Biochemical studies of human saliva have established that lipids are present in small quantities in this ﬂuid (Mandel and Einstein, 1969; Rabinowitz and Shannon, 1975; Slomiany et al., 1985; Larsson et al., 1996). Small amounts of phospholipids conceivably could be added to saliva as the result of membrane shedding during exocytosis (Doljanski and Kapeller, 1976). In the saddleback tamarin, the dense luminal globules in the endpieces and proximal intercalated ducts are completely absent from the striated and excretory ducts, indicating that they have been disassembled. Saddleback tamarin saliva contains twice as much total lipid as does human saliva (Murty et al., 1984). 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