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Cytoskeleton of intestinal goblet cells in rabbit and monkey
GASTROENTEROLOGY
1984:87:1313-25
Cytoskeleton of Intestinal Goblet Cells in Rabbit and Monkey The Theta ROBERT D. SPECIAN and MARIAN
R. NEUTRA
Department of Anatomy, Louisiana State University School of Medicine, Shreveport, and Department of Anatomy, Harvard Medical School, Boston. Massachusetts
The ultrastructure and function of the cytoskeleton in intestinal goblet cells was investigated in colonic mucosa from rabbits and monkeys. This exocrine cell is unusual in that its secretory granules are stored as a compact apical mass limited by a dense, cup-shaped layer of cytoplasm called the “theta.” Ultrastructural analysis of this cytoplasmic layer in rabbit goblet cells permeabilized with Triton X-100, treated with S4 fragments of heavy meromyosin, and fixed in the presense of tannic acid revealed that it contains an orderly arrangement of microtubules and intermediate filaments, but no detectable actin filaments. Microtubules are arranged vertically, like barrel staves, along the inner aspect of the theta. Intermediate filaments are arranged in two contiguous layers: an inner, basketlike network and an outer series of circumferential bundles resembling the hoops of a barrel. Autoradiography of [“Hlglucosamine-labeled human and rabbit cells maintained in organ culture without secretagogues had provided preIimi.nary data suggesting that labeled secretory granules migrate preferentially along the periphery of the apical granule mass, adjacent to the theta, toward the Iuminal cell surface. In this study, we confirm this observation and show that colchicine inhibits this movement. Cholinergic secretagogues induce rapid release of mucin by compound exocy-
Received November 8. 1983. Accepted June 27, 1984. Address requests for reprints to: Marian R. Neutra, Ph.D., Department of Anatomy, Harvard Medical School, 25 #Shattuck Street, Boston, Massachusetts 02115. This work was supported by National Institutes of Health Research Grant AM-21505, Research Career Development Award AM-00407. and a Research Fellowship (to R.D.S.) from the Cystic Fibrosis Foundation. The authors thank Dr. David Begg for the generous gift of S, myosin fragments, and Dr. Jerry S. Trier for collaboration in the studies using human mucosal tissue. 0 1984 by the American Gastroenterological Association 0016-5085/84/$3.00
Louisiana
tosis of the granules stored in the theta. This secretory activity is not inhibited by colchicine, presumably because it does not require granule movement. The cuplike shape of the theta is unaltered during rapid release of stored granules. Because the shape is unaltered after 6 h of colchicine treatment, it appears to be maintained by the intermediate filament layers. The process of secretion in exocrine cells requires directed movement of secretory granules from the region of the Golgi complex to the plasma membrane. Experimental disruption of the microtubular system in a wide variety of secretory cell types has been shown to inhibit the release of secretory products not by preventing exocytosis, but primarily by preventing the movement of granules through the cell (l-6). Inhibition of secretion by drugs such as colchicine has been measured either as a decrease in the normal baseline secretory rate or a decrease in the rapid secretory response evoked by secretagogues, or both. In some cell types, positioning of granules for compound exocytosis occurs only after exposure to a secretagogue. In these cells a functional microtubular system is presumably required for the movement of granules into close apposition with each other before tandem fusion of granule membranes. Intestinal goblet cells are unlike most other secretory cells in that their mucin-containing secretory granules are always positioned in close contact with one another, priming the cell for compound exocytosis (7,8). In this highly polarized epithelial cell, mature secretory granules are stored in a compact apical mass, delimited by a cup-shaped, filamentrich layer of cytoplasm, the “theta.” Within the Abbreviations ethane sulfonic
used in this paper: acid].
PIPES, piperazine-N.N’-bis]2-
1314 SPECIAN AND NEUTRA
apical mass, adjacent granule membranes are in close contact with little or no intervening cytoplasmic matrix, and the uppermost granules are generally separated from the apical plasma membrane only by a thin layer of cytoplasm. Exposure of rat and rabbit intestinal mucosa to cholinergic agonists induces the rapid release of mucin from crypt goblet cells by compound exocytosis (8,9). Release of the contents of most or all of the granules in the theta seems to occur without significant movement of granules and without alteration in cell shape. Slow, baseline secretion, in contrast, is accomplished by the intermittent exocytosis of a single secretory granule, usually located at the lateral edge of the granule mass and adjacent to the upper edge of the theta (8,10,11). In autoradiographic studies of the intracellular transport of [“Hlglucosaminelabeled secretory granules in human goblet cells maintained in mucosal organ culture without secretagogues (12), it was noted that under baseline conditions, granules tend to preferentially follow a peripheral path from the Golgi region to the apical cell surface, moving slowly up the inner surface of the theta. Although the cytoskeleton is generally thought to play an important role in the organization and intracellular movement of membrane-limited organelles, it is not known whether the organization of cytoskeletal elements in the filament-rich theta of the goblet cell is an important determinant of this distinctive pattern of granule transport. Nor is it known whether cytoskeletal elements play any role during accelerated secretion of intestinal mucin. Indeed, the exact arrangement of cytoskeletal elements in the intestinal goblet cell has not been carefully explored. The purpose of this study is to examine in detail the substructure and function of the goblet cell theta. We have found that the theta consists of an elaborate, orderly arrangement of intermediate filaments and microtubules. The importance of these elements for the maintenance of the distinctive shape of the goblet cell, for the slow transport of secretory granules under baseline conditions, and for the rapid secretion in response to cholinergic agents is the subject of this report.
Materials
and Methods
Tissues Mucosal samples were obtained from the descending colon of seven 2-3 kg, nonfasted female New Zealand white rabbits under urethane anesthesia. A segment of sigmoid colon was exposed at laparotomy and opened along the antimesenteric surface without interrupting
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blood flow. The mucosal surface was gently rinsed clean with oxygenated culture medium consisting of 90% chloromycetin-free Trowel1 T8 medium and 10% fetal calf serum (both from Gibco Laboratories, Grand Island, N.Y.), and containing 100 pg/mI gentamicin and 60 U/ml Mycostatin (Squibb and Sons, Inc., Princeton, N.J.). Up to 40 mucosal explants from each rabbit, each 2-4 mm in diameter, were excised with fine scissors and immediately immersed in the appropriate solution. Multiple samples of colonic mucosa were also obtained from a single adult rhesus monkey (Macaca mulatta) under sodium pentobarbital anesthesia. Explants of human rectal mucosa, 2-3 mm in diameter, were obtained after written informed consent from healthy adult volunteers as part of a separate published study (12).
Organ
Culture
and
Autoradiography
To follow the movements of secretory granules in rabbit and human goblet cells, glycoproteins were pulselabeled by immersing mucosal samples for 20-30 min at 37°C in 1 ml of oxygenated culture medium (as described above but prepared without glucose] containing 200 &i/ml of [3H]o-gIucosamine hydrochloride (sp act 18.8 Ciimmol; New England Nuclear, Boston, Mass.). The samples were then rinsed and mounted mucosal side up on stainless steel wire screens, with two or three samples in each plastic organ culture dish as previously described (13,14). The screens were then floated on nonradioactive chase medium and maintained at 37°C in an atmosphere of 95% O,-5% C02. Rabbit mucosal explants were harvested and fixed after l-8 h of culture, and human biopsy specimens were harvested after 2-24 h. Two mucosal samples from each time point were prepared for lightmicroscopic autoradiography by fixation in potassium dichromate-buffered 1% Os04, postfixation in sodium phosphate-buffered 4% formaldehyde, and embedment in Epon. One-micrometer sections were stained with periodic acid-Schiff and iron hematoxylin (15), coated for autoradiography by dipping in Ilford K5 photographic emulsion (Ilford Ltd., Basildon, Essex, England) diluted l:l, exposed at 4°C for 4-16 wk, and developed in Kodak D-19 developer [Eastman Kodak Co., Rochester, N.Y.) for 4 min. For each time point, 25-50 cells were examined and the positions of labeled mucin granules were recorded.
Electron
Microscopy
To examine the ultrastructure of the theta by transmission electron microscopy, rabbit and monkey mucosal slices were fixed for 3 h at 23°C in a solution consisting of 2% formaldehyde, 2.5% glutaraldehyde, and 0.4% CaCl, in 0.1 M sodium cacodylate buffer pH 7.4, postfixed for 2 h at 4°C with 1% 0~0~ in 0.1 M sodium cacodylate buffer pH 7.4, stained en bloc for 4 h at 4°C in 1% uranyl acetate in sodium acetate buffer pH 6.0, and embedded in Epon-Araldite. Thin sections were stained with uranyl acetate and lead citrate, and were examined and photographed with JEOL lOOS, lOOB, and 1OOCX electron microscopes.
December 1984
Colchicine
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Experiments
To inhibit the polymerization of microtubules and to examine the effects of this inhibition on the movement and release of goblet cell secretory granules under baseline conditions, rabbit mucosal explants were pulse-labeled for 30 min as described above and maintained in organ culture for 2, 4, or 6 h on oxygenated medium containing 10 PM colchicine (Sigma Chemical Co., St. Louis, MO.). Other explants were first maintained on medium containing 10 PM colchicine for 3 h, then were pulse-labeled for 30 min in colchicine-free medium to minimize possible inhibition of glycosyltransferase activity [IS], and finally were maintained for another 2, 4, or 6 h on colchicinecontaining medium. At each time interval, two explants from each treatment group were fixed and processed for light-microscopic autoradiography. At least 10 sections from each tissue sample were autoradiographed, and 2550 goblet cells from each time point and each treatment group were examined. To determine the effect of colchicine on accelerated secretion, eight mucosal samples were maintained for 3 h on medium containing 10 PM colchicine and were then transferred to medium containing 1 PM acetylcholine chloride and 3 mM eserine sulfate (Sigma) for 30 min before fixation. To examine the effects of colchicine on the structure of the cytoskeleton of the goblet cell theta, eight additional mucosal samples were maintained for 3 or 6 h on organ culture medium containing 10 PM colchicine, and then were either fixed directly, or were treated with Triton X-100 and further processed as described below.
Triton Extraction
and SI Labeling
Colchicine-treated and untreated rabbit mucosal samples were incubated for 5, 10, 15, or 20 min, with agitation, in a solution consisting of 0.1% Triton X-100, 2 mM MgC12, and 2 mM ethylenediaminetetraacetic acid in 0.1 M piperazine-N,N’-bis[Z-ethane sulfonic acid] (PIPES) buffer pH 6.9, at 37°C. In some samples, actin microfilaments were decorated by subsequent incubation for 10,15, or 20 min. in the same solution to which had been added 8 mg/ml of S1 subfragments of heavy meromyosin (kindly provided by Dr. David Begg). All Triton-extracted samples were fixed for 3 h at room temperature in a solution consisting of 2% glutaraldehyde and O.ZO~tannic acid in 0.1 M phosphate buffer pH 7.2. Samples were rinsed with buffer and postfixed with 1% 0s04 in 0.1 M phosphate buffer, and further processed for electron microscopy as already described. For each treatment, at least 30 different cells were examined in multiple (but not serial] thin sections.
Results Secretory
Granule
Storage and Transport
Goblet cells in the surface epithelium of rabbit and monkey intestine display the major structural features typical of this cell type (Figure 1). The nucleus occupies the basal cytoplasm, the extensive
Figure 1. The surface epithelium of rabbit colon, illustrating the dramatic polarization of goblet cells. Above the dense basal nucleus is an extended supranuclear region containing the Golgi complex (Go) and newly formed mucin granules. Mature secretory granules are stored as a compact apical mass limited by a cup-shaped layer of dense cytoplasm, the “theta.” (Periodic acid-schiffiron hematoxylin x1210.)
Golgi complex and newly-formed secretory granules occupy the “stem” of the goblet, and a dense cytoplasmic layer defines the apical cup within which mucin granules are stored. This filament-rich layer, the “theta” (Latin: sheath), separates mature secretory granules from the Golgi region below and from the lateral plasma membrane, but not from the luminal cell surface. The membranes of the uppermost granules are separated from the luminal plasma membrane by a variable, often very thin layer of cytoplasm [Figure 2). Within the granule mass, adjacent granule membranes are closely apposed (Figures 2 and 3). At its base, the theta appears ultrastructurally as a dense, discontinuous network of filament bundles [Figure 3), the relative concentration of which explains the dense layer by nonspecific stains (Figure
staining 1).
of this
Small groups of newly formed secretory granules are associated with the Golgi complex in the supranuclear region [Figure 3). When nascent glycoprotein was pulse-labeled with [3H]glucosamine and followed by autoradiography, groups of labeled granules formed during the pulse period were seen to remain in the supranuclear region for 1-2 h. and then to move up through the base of the theta into the mass of stored granules. Their subsequent movement was visualized most readily in human goblet cells, having relatively large apical mass of granules (12).
Labeled
granules
were
consistently
located
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Figure
2. The apical surface of a monkey colonk goblet cell. Secretory granule membranes are separated from the plasma membrane bl a thin layer of cytoplasm (cyto). Membranes of adiacent granules are closely apposed (clrroL\rheuds). rnt~: microvillus. .X68.395.
among the peripheral the theta (Figure 4).
Substructure
layer
of granules
adjacent
to
of the Theta
The density of the cytoplasmic matrix of goblet cells after conventional fixation tends to obscure the substructure of the filament-rich theta. Brief treatment of the mucosa with Triton X-100 in PIPES buffer rendered the membranes of surface epithelial cells permeable and resulted in loss of cytoplasmic matrix. Although secretory granule membranes were ruptured, plasma membranes remained morphologically recognizable and cell shape was maintained. In Triton models treated with myosin S, subfragments and fixed in the presence of tannic acid (Figure 5). actin filaments were recognized by their characteristic decoration with S1 “arrowheads”, intermediate filaments were identified by their lack of decoration and their IO-nm diameter, and microtubules were readily visualized as hollow tubes approximately 25 mm in diameter, both in cross-section (Figure 6a) and longitudinal section [Figure 6b). &-decorated actin filaments were present in the microvillar cores and “rootlets,” and also in the region of the zonula adherens of goblet cells, as in adjacent absorptive cells (Figure 5b).Along the later-
al cell margins, decorated thin filaments were present in the interdigitations of both goblet and absorptive cells. Decorated actin filaments were not detected in the theta (Figure 5~). A system of intermediate filaments, associated with desmosomes and resembling the cytokeratin networks previously described in intestinal columnar cells (17,18), coursed along the lateral cell membranes and did not appear to contribute to the substructure of the theta (Figure 5a). The upper border of the theta was visualized as a thin layer of intermediate filaments beginning just below the dense filamentous mat associated with the adhering junction: this layer became increasingly prominent at lower levels of the theta (Figure 5a). In longitudinal sections through the lateral wall of the theta (Figure 5c), intermediate filaments were arranged in two contiguous layers: the medial layer was oriented vertically and the lateral layer, consisting of thick bundles, was arranged circumferentially. Intermediate filaments of the theta were of the same lo-nm diameter as those associated with desmosomes. In cells sectioned perpendicular to the long axis of the cell [Figure 6a), microtubules were invariably cut in cross-section and were seen as dense rings positioned at regular intervals along the inner aspect of the intermediate filament layers. In the repre-
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Figure
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3. Central region of a monkey colonic goblet cell. The supranuclear cytoplasm, containing multiple Golgi stacks and newly formed secretory granules, is separated from the apical mass of mucin granules (mg) by the theta. This filament-rich cytoplasmic layer forms an apical cup that segregates mature secretory granules from other cell organelles. x 18.800
sentative surface goblet cell shown in Figure 6a, a series of eight microtubules were counted along a span of the thecal wall that accounted for 60' or 17% of the total circumference of the theta. By extrapolation to the entire circumference, we estimate that
-45-50 such microtubules are present around the periphery of a single theta. A tangential section grazing the wall of the theta [Figure 6b) illustrated the architecture of these layers relative to the inner mass of secretory granules. The inner, vertical layer
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4a of goblet cells in a huFigure 4. Autoradiographs man rectal biopsy specimen pulse-labeled with [3H]glucosamine and maintained in organ culture for 12 h (a) or 24 h (b) before fixation. Groups of labeled secretory granules have migrated various distances toward the luminal cell surface, but they are consistently located peripherally (arrows) adjacent to the theta. x1060.
of intermediate filaments actually consisted of interwoven flattened bundles that generally followed an filament network upward course. This vertical seemed to be continuous with a basketlike layer of intermediate filaments that formed the base of the theta (Figure 7a). In Triton models of this region (Figure i’b) microtubules were occasionally seen crossing the filament layer, implying that the vertical microtubules originated in more basal regions of the cell. The upper and lower ends of the vertical microtubules were not identified. The data obtained from sections of Triton-extracted and conventionally prepared cells are consistent with the three-dimensional model shown in Figure 8. Briefly, the goblet cell theta is composed of three major layers: an inner array of vertical microtubules (Figure 8a), an interwoven basketlike network of intermediate filaments (Figure 8b) and an outer series of intermediate filament bundles that encircle the theta like the hoops of a barrel (Figure 8~).
Effects of Colchicine After culture of rabbit mucosal explants on medium containing 10 PM colchicine for 3 h, some microtubules were still identified along the inner aspect of the theta. After 6 h on colchicine-containing medium, microtubules were rarely detected in the theta but they were still present in the Golgi region below. Autoradiographs of goblet cells in control rabbit colonic explants, pulse-labeled with [“Hlglucosamine and maintained for up to 6 h on nonradioactive medium before fixation, showed radioactive secre-
tory product in the supranuclear region and, less frequently, in the basal region of the theta at 2 h (Figure 9a). By 6 h after pulse-labeling there was considerable variability in the intracellular position of labeled granules; nevertheless, all surface goblet cells in control explants showed some labeled granules near or at the luminal surface. Labeled granules at 6 h were generally restricted to the lateral borders of the apical granule mass (Figure 9b). In explants pulse-labeled and maintained for up to 6 h on colchicine-containing medium, in contrast, labeled secretory product never reached the luminal surfaces of goblet cells. In the majority of goblet cells, labeled granules remained in the supranuclear region and at the base of the theta (Figure 9c). Similar results were obtained when explants were preincubated in colchicine-containing medium for 3 h, pulse-labeled, and then maintained for an additional 6 h before fixation and autoradiography. Exposure of rabbit mucosal explants to the secretagogue acetylcholine, in the presense of the cholinesterase inhibitor eserine, has been shown to result in rapid secretion of mucin granules from crypt goblet cells by compound exocytosis, resulting in deep cavitation of the apical cell surface (8). When explants were maintained for 3 or 6 h on colchicinecontaining medium and then exposed to acetylcholine and eserine, crypt goblet cells showed a secretory response comparable to that observed in control explants (Figure 10). Thus, although exposure to 10 PM colchicine for 3 or 6 h inhibits the slow granule movement that accompanies baseline secretion, it does not inhibit rapid secretion by compound exocytosis; this activity seems to be independent of granule movement in this cell type.
December
Figure
5
1984
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a. Portions of a goblet Rabbit colonic epithelial cells permeabilized with Triton X-100 and treated with myosin S, fragments. cell (left) and a columnar cell (right] are joined by an apical junctional region (above) and by a series of desmosomes. At left. mucin from disrupted secretory granules fills the goblet cell apex. In both cells, lateral intermediate filament networks are associated with desmosomes (desm IF). The theta consists primarily of intermediate filaments; medially, they are oriented vertically (vert IF) and laterally, they are cross-sectioned, indicating a circumferential orientation (circ IF). X61.400. b. Apical surface of a goblet cell (gc, Jeff) and a columnar cell (cc, right] including the junctional region and the terminal webs. Actin filaments in the microvilli (mv) of both cells are decorated with S, fragments. ~68.000. (c.)Intermediate filaments of the theta, sectioned as in 5n. The inner, vertical filaments pass obliquely through the section. The circumferential filaments are arranged in IRWP hllnrlles. ~118.800.
1320
Figure
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6. Goblet cells of rabbit colon, prepared as in Figure 5. o. The lateral border long axis of the cell. Cross-sectioned microtubules (arrows) are spaced disrupted secretory granules, cc, columnar cell. X100.000. b. A tangential intermediate filament (IF) bundles, next through crisscrossing bands microtubules (arrows), and finally through the interior mass of disrupted
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of a goblet cell (gc) sectioned perpendicular to the at intervals around the periphery of the mass of section passes first through the circumferential of vertical IF, then through a series of vertical mucin granules. ~56,000.
December
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7. Vertical sections (parallel to the long axis of preparation; a higher magnification of a portion form a barrier between the secretory granules goblet cell, Triton model. Although secretory intact. A microtubule (mt) passes through the
CYTOSKELETON
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the cell], showing the base of the theta. a. Monkey goblet cell. conventional of the section shown in Figure 3. Intermediate filament bundles of the theta above, and the Golgi complex and other organelles below. * 51.500. b. Rabbit granule membranes are disrupted. the filament bundles dehning the theta are base of the theta. *56,800.
Discussion The theta of the intestinal goblet cell is a specialized cytoskeletal structure consisting of intermediate filaments and microtubules, but not of actin. The distribution of actin in goblet cells is comparable to that of neighboring absorptive cells (19-23) in that bundles of actin filaments form the cores and rootlets of goblet cell microvilli and a filamentous accumulation containing actin is associated with the zonula adherens. Because of the crowd of mucin granules at the apical pole of the goblet cell, however, the terminal web is discontinuous and does not form a uniform plate at the level of the zonula adherens, as in absorptive cells (17). Immunocytochemical staining of absorptive cells has revealed relatively little actin below the brush border: an actin network is present along basal cell surfaces (22)
and actin has been detected in lateral interdigitations (Tokuyasu KT, personal communication) but no actin has been detected in more central regions. Similarly, we detected no actin in the theta of goblet cells. The presence of actin cannot be ruled out by lack of S1 labeling, however, because short actin assemblies might not be recognized by this method (24).
We have shown that the cuplike theta of goblet cells consists primarily of a well-organized system of intermediate filaments. The arrangement and composition of the IO-nm filaments in intestinal absorptive cells have been studied in detail (17,18,25). like those of many other These “tonofilaments,” vertebrate epithelial cells, consist of cytokeratins, a class of proteins related to epidermal prekeratin (18.25-28). Although structurally identical lo-nm filaments can be formed in different types of cells
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8. Schematic cut-away Vertical microtubules oriented intermediate
view of the architecture of the goblet cell theta: a model based on our ultrastructural observations. o. lie closely apposed to the central mass of stored secretory granules. b. A basketlike network of vertically filaments (IF) defines the cup shape of the theta. c. Circumferential bundles of IF encircle the basket,
from different cytokeratins (25,283, we consider it likely that the intermediate filaments of goblet cells are biochemically comparable to those of absorptive cells as the two cell types arise by differentiation from the same epithelial stem cell population (29). In absorptive cells, most lo-nm filaments are arranged as a peripheral network of bundles that often attach to the lateral cell membrane by looping through the dense plaques of material associated with the cytoplasmic face of spot desmosomes (17). At the apical poles of absorptive cells, lo-nm filaments emanating from the apical ring of spot desmosomes interweave to form a dense “subapical disk” immediately beneath the actinand myosin-rich terminal web (17,181. Goblet cells also have a lateral, desmosomeassociated system of tonofilament bundles, but this system seems to be separate from those that form the theta. From our thin sections we were not able to follow the entire three-dimensional course of the circumferential and interwoven filament bundles of the theta so we cannot rule out attachments of this system to lateral desmosomes. Evidence from many cell types indicates that intermediate filaments provide an intracellular scaffold that influences the arrangement of organelles but that does not directly participate in cellular or organellar motility (27,28). These concepts are consistent with our data showing that the goblet cell theta seems to be structurally stable during accelerated secretion. Mucin is apparently expelled by the rapid expansion that accompanies hydration and decondensation of the mucin granule contents, and
not by collapse or contraction of the theta. A recently emptied goblet cell resembles an empty cup. The cup shape of the theta is maintained, even after 6 h of colchicine treatment when the theta is largely depleted of microtubules. The elaborate architecture of the intermediate filament networks, resembling a reinforced basket, suggests that these elements play
.. I._
Figure
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9. Autoradiographs of goblet cells in the surface epitheliurn of rabbit colonic mucosa, maintained in organ culture and pulse-labeled with [“Hlglucosamine. o. Control mucosa, pulse-labeled for 30 min and fixed 2 h later. Labeled secretory granules (arrow!) are present in the supranuclear region and at the base of the theta. x1020. b. Control mucosa, pulse-labeled for 30 min and fixed 6 h later. Most labeled granules have moved out of the supranuclear region and are located along the periphery of the apical granule mass. x 1020. c. Colchtine-treated mucosa. fixed 6 h after pulse-labeling. Labeled granules remain in the supranuclear region and at the base of the theta. x1020.
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10. Apical portion of a rabbit colonic goblet cell, maintained for 3 h on colchicine-containing medium and then exposed to acetylcholine and eserine in the presence of colchicine. The apical cell surface is deeply cavitated by compound exocytosis of many mucin granules. Some intact granules (mg) remain in the base of the theta. This secretory response is identical to that seen in control cells that were not exposed to colchicine. x 15,330.
the major role in segregating the apical granule mass and in maintaining the distinctive shape of the cell apex. The intestinal goblet cell theta thus provides a dramatic example of the importance of intermediate filaments in the organization of intracellular space (271.
An intact, functional system of microtubules is generally believed to be necessary for the directed movement of secretion granules toward the cell surface. Although the precise modes of interaction between secretory granule membranes and polymerized tubulin are not yet clear, there is evidence to suggest that microtubuleand perhaps membraneassociated proteins play roles in the movement of granules along microtubule paths (30). In any case, inhibition of tubulin polymerization by drugs such as colchicine is now well documented and the dependence of normal microtubule functions on tubulin polymerization/depolymerization, or tread-
CYTOSKELETON OF INTESTINAL GOBLET CELLS
1323
milling, is supported by compelling evidence (31,321. Colchicine has been shown to inhibit secretion by halting granule movement in many secretory cell types, including endocrine cells, connective tissue cells, and a variety of exocrine epithelial cells (l-6). Transport of membrane-associated glycoprotein to the luminal (but not basal-lateral) cell surfaces of intestinal columnar cells was also blocked by colchicine (33-37). Inhibition of mucin glycoprotein secretion by colchicine treatment of the respiratory mucosa was detected biochemically by some authors (38) although not by others (39). It is thus not surprising that inhibition of secretory granule movement was observed in the colchicine-treated goblet cells in this study. The observation is underscored in this cell, however, by the unusual barrel-stave arrangement of the microtubules in the theta and the preferential upward migration of peripheral secretory granules, apparently along these orderly microtubular pathways. Microtubules were generally not observed within the mass of stored granules, and central granules did not seem to participate in baseline secretion. In a preliminary autoradiographic study of goblet cells continuously labeled with [3H]glucosamine for 12 h (40), we observed that central granules remained unlabeled despite heavy labeling of the peripheral layer of granules. This suggests that central granules are synthesized early in the life of the goblet cell and that in the absense of secretagogues, they remain unmoved and unsecreted through most of the cell’s 4-6-day lifetime. In acetylcholine-stimulated cells, in contrast, compound exocytosis begins with the uppermost central granules, spreads sequentially through granules at the center of the apical granule mass, and finally includes peripheral granules (8). Studies on mucin secretion from the respiratory epithelium in explants of tracheal mucosa reported that colchicine inhibited the accelerated release of glycoconjugates normally observed in response to methacholine (39) and cholera toxin (41). Tracheal goblet cells are ultrastructurally very similar to their intestinal counterparts (42). These findings were thus inconsistent with our studies of accelerated intestinal goblet cell secretion, in which compound exocytosis in response to acetylcholine did not seem to require granule movement (8). Unlike most other secretory cells, in which rapid exocytosis of stored secretory product requires movement of many granules toward the cell surface and toward each other, the goblet cell need only close the narrow gap between the plasma membrane and a single apical granule membrane to initiate a long series of exocytotic events. This cytoplasmic gap contains no microtubules; it is comparable in width and in compo-
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sition to that described between peripheral secretory granules and plasmalemma in Limulus amebocytes (43). Through the fusion of multiple granule membranes, the luminal plasma membrane in effect moves down to progressively deeper granules until the theta is empty. The present study confirms that functional microtubules are not needed for this process to occur.
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AM, Muller P, Rossingnol B. Microtubules and protein secretion in rat lacrimal glands. Relationship between colchicine binding and its inhibitory effect on the intracellular transport of proteins. J Biol Chem 1978: 253:3870-6. Dustin P. Microtubules. Berlin: Springer-Verlag, 1978:1-452. Lacy PE. Endocrine secretory mechanisms. Am J Path01 1975: 79:170-87. Malaisse-Lagae F, Amherdt M, Ravazzola M. et al. Role of microtubules in the synthesis, conversion, and release of (pro)insulin. A biochemical and radioautographic study in rat islets. J Clin Invest 1979:63:1284-96. Redman CM, Banerjee D, Yu S. The effect of colchicine on the synthesis and secretion of rat serum albumin. In: Hand AR, Oliver C, eds. Basic mechanisms of cellular secretion. Methods in cell biology. New York: Academic, 1981;23:231-46. Williams JA. Effects of antimitotic agents on ultrastructure and intracellular transport of protein in pancreatic acini. In: Hand AR, Oliver C, eds. Basic mechanisms of cellular secretion. Methods in cell biology. New York: Academic, 1981: 23:247-58. Neutra MR, Schaeffer SF. Membrane interactions between adjacent mucous secretion granules. J Cell Biol 1977:74:98391. Specian RD, Neutra MR. Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine. J Cell Biol 1980; 85:626-40. Specian RD. Neutra MR. Regulation of goblet cell secretion. I. Role of parasympathetic stimulation. Am J Physiol 1982; 242:G370-9. Palade G. Intracellular aspects of protein synthesis. Science 1975;183:347-58. Trier JS. Madara JL. Functional morphology of the mucosa of the small intestine. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York:Raven. 1981:925-62. Neutra MR, Grand RJ, Trier JS. Glycoprotein synthesis, transport and secretion by epithelial cells of human rectal mucosa: normal and cystic fibrosis. Lab Invest 1977;36:535-47. Eastwood GL. Trier JS. Organ culture of human rectal mucosa. Gastroenterology 1973;64:375-82. Neutra MR. The use of human intestinal mucosa in organ culture for the study of cystic fibrosis. Birth Defects 1980: 16:261-73. Shantz A, Schecter A. Iron hematoxylin and safranin 0 as a polychrome stain for Epon sections. Stain Technol 1965: 40:279-82.
16. Mitranic MM, Boggs JM. Moscarello MA. An effect of colchitine on galactosyland sialyltransferase of rat liver Golgi membranes. Decrease in glycosyltransferase activity produced by colchicine. Biochim Biophys Acta 1981;672:57-62. 17. Hull BE, Staehelin LA. The terminal web. A reevaluation of its structure and function. J Cell Biol 1979:81:67-82. 18. Franke WW, Appelhans B, Schmidt E. Freudenstein C. Os-
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born M, Weber K. The organization of cytokeratin filaments in the intestinal epithelium. Eur J Cell Biol 1979;19:255-68. 19. Mooseker MS, Tilney LG. Organization of an actin filamentmembrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol 1975;67:725-43. 20. Bretscher A, Weber K. Localization of actin and microfilament-associated proteins in the microvilli and terminal web of intestinal brush border by immunofluorescence microscopy. J Cell Biol 1978;79:839-45. 21. Mooseker MS. Pollard TP, Fujiwara K. Characterization and localization of myosin in the brush border of intestinal epithelial cells. J Cell Biol 1978:79:444-53. 22. Drenckhahn D, Groschel-Stewart U. Localization of myosin. actin and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels. J Cell Biol 1980;86:475-82. 23. Mooseker MS. Howe CL. The brush border of intestinal epithelium: a model system for analysis of cell-surface architecture and motility. Methods Cell Biol 1982;25:143-74. 24. Forer A. Actin localization within cells by electron microscopy. Methods Cell Biol 1982;25:131-42. 25. Franke WW, Winter S, Grund C, Schmid E, Schiller 13, Jarasch ED. Isolation and characterization of desmosome-associated tonofilments from rat intestinal brush border. J Cell Biol 1981;90:116-27. C 26. Franke WW, Weber K. Osborn M, Schmid E. Freudenstein Antibody to prekeratin decoration of tonofilament-like arrays in various cells of epithelial character. Exp Cell Res 1978: 116:429-45. 27. Lazarides E. Intermediate filaments as mechanical integrators of intracellular space. Nature 1980;283:249-56. 28. Lazarides E. Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem 1982:51:219-50. 29. Cheng H, Leblond CP. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am J Anat 1974:141:537-61. KA. Dentler J, Association between endocrine 30. Suprenant pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins. J Cell Biol 1982;93:164-74. 31. Kirschner MW. Implications of treadmilling for the stability and polarity of actin and tubulin polymers in viva. J Cell Biol 1980;86:330-4. 32. Margolis RL, Wilson L. Treadmill model of MT-directed movements. Proc Nat1 Acad Sci USA 1977:74:3466-70. 33. Bennett GC, Carlet E. Wild G, Parsons S. Influence of colchitine and vinblastine on the intracellular migration of secretory and membrane glycoproteins. III. Inhibition of intracellular migration of membrane glycoproteins in intestinal columnar cells and hepatocytes as visualized by light and electron microscope radioautography after ,‘H-fucose injection. Am J Anat 1984 (in press). 34. Bennett G. Hugon J. Effect of colchicine on migration of ‘Hfucose labeled glycoproteins in mouse jejunal absorptive cells in organ culture: an electron microscope radioautographic study (abstr). J Cell Biol 1982:95:401a. 35. Blok J. Ginsel LA, Mulder-Stapel AA, Onderwater JJ, Daems W. The effect of colchicine on the intracellular transport of “H-fucose labeled glycoproteins in the absorptive cells of cultured human small intestinal tissue. An autoradiographical and biochemical study. Cell Tissue Res 1981;215:1-12. 36. Michaels J. Effects of colchicine on the location of vesicles containing cell coat glycoproteins in murine colonic epithelial cells (abstr). J Cell Biol 1981;91:400a.
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37. Quaroni
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A, Kirsch K, Weiser MM. Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of L-(1,5,6-"H)fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes. Biochem J 1979;182:213-21. 38. Adler KB, Brody AR, Craighead JE. Studies on the mechanism of mucin secretion by cells of the porcine tracheal epithelium. Proc Sot Exp Biol Med 1981;166:96-106. 39. Coles SJ, Reid L. Inhibition of glycoconjugate secretion by colchicine and cytochalasin p. An in vitro study of human airwav. Cell Tissue Res 1981;214:107-18.
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40. Specian RD, Neutra MR. Mucous granule subpopulations in intestinal goblet cells (abstr). J Cell Biol 1982;95:403a. 41. Adler KB. Hardwick DH. Craighead JE. Effect of cholera toxin on secretion of mucin by explants of guinea pig trachea. Lab Invest 1981;45:372-7. 42. Adler KB. Hardwick DH, Craighead JE. Porcine tracheal goblet cell ultrastructure: a three-dimensional reconstruction. Exp Lung Res 1982:3:69-80. 43. Ornberg RL, Reese TS. Beginnings of exocytosis captured by rapid-freezing of Limulus amebocptes. J Cell Biol1981;90:4054.
1984:87:1313-25
Cytoskeleton of Intestinal Goblet Cells in Rabbit and Monkey The Theta ROBERT D. SPECIAN and MARIAN
R. NEUTRA
Department of Anatomy, Louisiana State University School of Medicine, Shreveport, and Department of Anatomy, Harvard Medical School, Boston. Massachusetts
The ultrastructure and function of the cytoskeleton in intestinal goblet cells was investigated in colonic mucosa from rabbits and monkeys. This exocrine cell is unusual in that its secretory granules are stored as a compact apical mass limited by a dense, cup-shaped layer of cytoplasm called the “theta.” Ultrastructural analysis of this cytoplasmic layer in rabbit goblet cells permeabilized with Triton X-100, treated with S4 fragments of heavy meromyosin, and fixed in the presense of tannic acid revealed that it contains an orderly arrangement of microtubules and intermediate filaments, but no detectable actin filaments. Microtubules are arranged vertically, like barrel staves, along the inner aspect of the theta. Intermediate filaments are arranged in two contiguous layers: an inner, basketlike network and an outer series of circumferential bundles resembling the hoops of a barrel. Autoradiography of [“Hlglucosamine-labeled human and rabbit cells maintained in organ culture without secretagogues had provided preIimi.nary data suggesting that labeled secretory granules migrate preferentially along the periphery of the apical granule mass, adjacent to the theta, toward the Iuminal cell surface. In this study, we confirm this observation and show that colchicine inhibits this movement. Cholinergic secretagogues induce rapid release of mucin by compound exocy-
Received November 8. 1983. Accepted June 27, 1984. Address requests for reprints to: Marian R. Neutra, Ph.D., Department of Anatomy, Harvard Medical School, 25 #Shattuck Street, Boston, Massachusetts 02115. This work was supported by National Institutes of Health Research Grant AM-21505, Research Career Development Award AM-00407. and a Research Fellowship (to R.D.S.) from the Cystic Fibrosis Foundation. The authors thank Dr. David Begg for the generous gift of S, myosin fragments, and Dr. Jerry S. Trier for collaboration in the studies using human mucosal tissue. 0 1984 by the American Gastroenterological Association 0016-5085/84/$3.00
Louisiana
tosis of the granules stored in the theta. This secretory activity is not inhibited by colchicine, presumably because it does not require granule movement. The cuplike shape of the theta is unaltered during rapid release of stored granules. Because the shape is unaltered after 6 h of colchicine treatment, it appears to be maintained by the intermediate filament layers. The process of secretion in exocrine cells requires directed movement of secretory granules from the region of the Golgi complex to the plasma membrane. Experimental disruption of the microtubular system in a wide variety of secretory cell types has been shown to inhibit the release of secretory products not by preventing exocytosis, but primarily by preventing the movement of granules through the cell (l-6). Inhibition of secretion by drugs such as colchicine has been measured either as a decrease in the normal baseline secretory rate or a decrease in the rapid secretory response evoked by secretagogues, or both. In some cell types, positioning of granules for compound exocytosis occurs only after exposure to a secretagogue. In these cells a functional microtubular system is presumably required for the movement of granules into close apposition with each other before tandem fusion of granule membranes. Intestinal goblet cells are unlike most other secretory cells in that their mucin-containing secretory granules are always positioned in close contact with one another, priming the cell for compound exocytosis (7,8). In this highly polarized epithelial cell, mature secretory granules are stored in a compact apical mass, delimited by a cup-shaped, filamentrich layer of cytoplasm, the “theta.” Within the Abbreviations ethane sulfonic
used in this paper: acid].
PIPES, piperazine-N.N’-bis]2-
1314 SPECIAN AND NEUTRA
apical mass, adjacent granule membranes are in close contact with little or no intervening cytoplasmic matrix, and the uppermost granules are generally separated from the apical plasma membrane only by a thin layer of cytoplasm. Exposure of rat and rabbit intestinal mucosa to cholinergic agonists induces the rapid release of mucin from crypt goblet cells by compound exocytosis (8,9). Release of the contents of most or all of the granules in the theta seems to occur without significant movement of granules and without alteration in cell shape. Slow, baseline secretion, in contrast, is accomplished by the intermittent exocytosis of a single secretory granule, usually located at the lateral edge of the granule mass and adjacent to the upper edge of the theta (8,10,11). In autoradiographic studies of the intracellular transport of [“Hlglucosaminelabeled secretory granules in human goblet cells maintained in mucosal organ culture without secretagogues (12), it was noted that under baseline conditions, granules tend to preferentially follow a peripheral path from the Golgi region to the apical cell surface, moving slowly up the inner surface of the theta. Although the cytoskeleton is generally thought to play an important role in the organization and intracellular movement of membrane-limited organelles, it is not known whether the organization of cytoskeletal elements in the filament-rich theta of the goblet cell is an important determinant of this distinctive pattern of granule transport. Nor is it known whether cytoskeletal elements play any role during accelerated secretion of intestinal mucin. Indeed, the exact arrangement of cytoskeletal elements in the intestinal goblet cell has not been carefully explored. The purpose of this study is to examine in detail the substructure and function of the goblet cell theta. We have found that the theta consists of an elaborate, orderly arrangement of intermediate filaments and microtubules. The importance of these elements for the maintenance of the distinctive shape of the goblet cell, for the slow transport of secretory granules under baseline conditions, and for the rapid secretion in response to cholinergic agents is the subject of this report.
Materials
and Methods
Tissues Mucosal samples were obtained from the descending colon of seven 2-3 kg, nonfasted female New Zealand white rabbits under urethane anesthesia. A segment of sigmoid colon was exposed at laparotomy and opened along the antimesenteric surface without interrupting
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blood flow. The mucosal surface was gently rinsed clean with oxygenated culture medium consisting of 90% chloromycetin-free Trowel1 T8 medium and 10% fetal calf serum (both from Gibco Laboratories, Grand Island, N.Y.), and containing 100 pg/mI gentamicin and 60 U/ml Mycostatin (Squibb and Sons, Inc., Princeton, N.J.). Up to 40 mucosal explants from each rabbit, each 2-4 mm in diameter, were excised with fine scissors and immediately immersed in the appropriate solution. Multiple samples of colonic mucosa were also obtained from a single adult rhesus monkey (Macaca mulatta) under sodium pentobarbital anesthesia. Explants of human rectal mucosa, 2-3 mm in diameter, were obtained after written informed consent from healthy adult volunteers as part of a separate published study (12).
Organ
Culture
and
Autoradiography
To follow the movements of secretory granules in rabbit and human goblet cells, glycoproteins were pulselabeled by immersing mucosal samples for 20-30 min at 37°C in 1 ml of oxygenated culture medium (as described above but prepared without glucose] containing 200 &i/ml of [3H]o-gIucosamine hydrochloride (sp act 18.8 Ciimmol; New England Nuclear, Boston, Mass.). The samples were then rinsed and mounted mucosal side up on stainless steel wire screens, with two or three samples in each plastic organ culture dish as previously described (13,14). The screens were then floated on nonradioactive chase medium and maintained at 37°C in an atmosphere of 95% O,-5% C02. Rabbit mucosal explants were harvested and fixed after l-8 h of culture, and human biopsy specimens were harvested after 2-24 h. Two mucosal samples from each time point were prepared for lightmicroscopic autoradiography by fixation in potassium dichromate-buffered 1% Os04, postfixation in sodium phosphate-buffered 4% formaldehyde, and embedment in Epon. One-micrometer sections were stained with periodic acid-Schiff and iron hematoxylin (15), coated for autoradiography by dipping in Ilford K5 photographic emulsion (Ilford Ltd., Basildon, Essex, England) diluted l:l, exposed at 4°C for 4-16 wk, and developed in Kodak D-19 developer [Eastman Kodak Co., Rochester, N.Y.) for 4 min. For each time point, 25-50 cells were examined and the positions of labeled mucin granules were recorded.
Electron
Microscopy
To examine the ultrastructure of the theta by transmission electron microscopy, rabbit and monkey mucosal slices were fixed for 3 h at 23°C in a solution consisting of 2% formaldehyde, 2.5% glutaraldehyde, and 0.4% CaCl, in 0.1 M sodium cacodylate buffer pH 7.4, postfixed for 2 h at 4°C with 1% 0~0~ in 0.1 M sodium cacodylate buffer pH 7.4, stained en bloc for 4 h at 4°C in 1% uranyl acetate in sodium acetate buffer pH 6.0, and embedded in Epon-Araldite. Thin sections were stained with uranyl acetate and lead citrate, and were examined and photographed with JEOL lOOS, lOOB, and 1OOCX electron microscopes.
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Colchicine
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Experiments
To inhibit the polymerization of microtubules and to examine the effects of this inhibition on the movement and release of goblet cell secretory granules under baseline conditions, rabbit mucosal explants were pulse-labeled for 30 min as described above and maintained in organ culture for 2, 4, or 6 h on oxygenated medium containing 10 PM colchicine (Sigma Chemical Co., St. Louis, MO.). Other explants were first maintained on medium containing 10 PM colchicine for 3 h, then were pulse-labeled for 30 min in colchicine-free medium to minimize possible inhibition of glycosyltransferase activity [IS], and finally were maintained for another 2, 4, or 6 h on colchicinecontaining medium. At each time interval, two explants from each treatment group were fixed and processed for light-microscopic autoradiography. At least 10 sections from each tissue sample were autoradiographed, and 2550 goblet cells from each time point and each treatment group were examined. To determine the effect of colchicine on accelerated secretion, eight mucosal samples were maintained for 3 h on medium containing 10 PM colchicine and were then transferred to medium containing 1 PM acetylcholine chloride and 3 mM eserine sulfate (Sigma) for 30 min before fixation. To examine the effects of colchicine on the structure of the cytoskeleton of the goblet cell theta, eight additional mucosal samples were maintained for 3 or 6 h on organ culture medium containing 10 PM colchicine, and then were either fixed directly, or were treated with Triton X-100 and further processed as described below.
Triton Extraction
and SI Labeling
Colchicine-treated and untreated rabbit mucosal samples were incubated for 5, 10, 15, or 20 min, with agitation, in a solution consisting of 0.1% Triton X-100, 2 mM MgC12, and 2 mM ethylenediaminetetraacetic acid in 0.1 M piperazine-N,N’-bis[Z-ethane sulfonic acid] (PIPES) buffer pH 6.9, at 37°C. In some samples, actin microfilaments were decorated by subsequent incubation for 10,15, or 20 min. in the same solution to which had been added 8 mg/ml of S1 subfragments of heavy meromyosin (kindly provided by Dr. David Begg). All Triton-extracted samples were fixed for 3 h at room temperature in a solution consisting of 2% glutaraldehyde and O.ZO~tannic acid in 0.1 M phosphate buffer pH 7.2. Samples were rinsed with buffer and postfixed with 1% 0s04 in 0.1 M phosphate buffer, and further processed for electron microscopy as already described. For each treatment, at least 30 different cells were examined in multiple (but not serial] thin sections.
Results Secretory
Granule
Storage and Transport
Goblet cells in the surface epithelium of rabbit and monkey intestine display the major structural features typical of this cell type (Figure 1). The nucleus occupies the basal cytoplasm, the extensive
Figure 1. The surface epithelium of rabbit colon, illustrating the dramatic polarization of goblet cells. Above the dense basal nucleus is an extended supranuclear region containing the Golgi complex (Go) and newly formed mucin granules. Mature secretory granules are stored as a compact apical mass limited by a cup-shaped layer of dense cytoplasm, the “theta.” (Periodic acid-schiffiron hematoxylin x1210.)
Golgi complex and newly-formed secretory granules occupy the “stem” of the goblet, and a dense cytoplasmic layer defines the apical cup within which mucin granules are stored. This filament-rich layer, the “theta” (Latin: sheath), separates mature secretory granules from the Golgi region below and from the lateral plasma membrane, but not from the luminal cell surface. The membranes of the uppermost granules are separated from the luminal plasma membrane by a variable, often very thin layer of cytoplasm [Figure 2). Within the granule mass, adjacent granule membranes are closely apposed (Figures 2 and 3). At its base, the theta appears ultrastructurally as a dense, discontinuous network of filament bundles [Figure 3), the relative concentration of which explains the dense layer by nonspecific stains (Figure
staining 1).
of this
Small groups of newly formed secretory granules are associated with the Golgi complex in the supranuclear region [Figure 3). When nascent glycoprotein was pulse-labeled with [3H]glucosamine and followed by autoradiography, groups of labeled granules formed during the pulse period were seen to remain in the supranuclear region for 1-2 h. and then to move up through the base of the theta into the mass of stored granules. Their subsequent movement was visualized most readily in human goblet cells, having relatively large apical mass of granules (12).
Labeled
granules
were
consistently
located
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Figure
2. The apical surface of a monkey colonk goblet cell. Secretory granule membranes are separated from the plasma membrane bl a thin layer of cytoplasm (cyto). Membranes of adiacent granules are closely apposed (clrroL\rheuds). rnt~: microvillus. .X68.395.
among the peripheral the theta (Figure 4).
Substructure
layer
of granules
adjacent
to
of the Theta
The density of the cytoplasmic matrix of goblet cells after conventional fixation tends to obscure the substructure of the filament-rich theta. Brief treatment of the mucosa with Triton X-100 in PIPES buffer rendered the membranes of surface epithelial cells permeable and resulted in loss of cytoplasmic matrix. Although secretory granule membranes were ruptured, plasma membranes remained morphologically recognizable and cell shape was maintained. In Triton models treated with myosin S, subfragments and fixed in the presence of tannic acid (Figure 5). actin filaments were recognized by their characteristic decoration with S1 “arrowheads”, intermediate filaments were identified by their lack of decoration and their IO-nm diameter, and microtubules were readily visualized as hollow tubes approximately 25 mm in diameter, both in cross-section (Figure 6a) and longitudinal section [Figure 6b). &-decorated actin filaments were present in the microvillar cores and “rootlets,” and also in the region of the zonula adherens of goblet cells, as in adjacent absorptive cells (Figure 5b).Along the later-
al cell margins, decorated thin filaments were present in the interdigitations of both goblet and absorptive cells. Decorated actin filaments were not detected in the theta (Figure 5~). A system of intermediate filaments, associated with desmosomes and resembling the cytokeratin networks previously described in intestinal columnar cells (17,18), coursed along the lateral cell membranes and did not appear to contribute to the substructure of the theta (Figure 5a). The upper border of the theta was visualized as a thin layer of intermediate filaments beginning just below the dense filamentous mat associated with the adhering junction: this layer became increasingly prominent at lower levels of the theta (Figure 5a). In longitudinal sections through the lateral wall of the theta (Figure 5c), intermediate filaments were arranged in two contiguous layers: the medial layer was oriented vertically and the lateral layer, consisting of thick bundles, was arranged circumferentially. Intermediate filaments of the theta were of the same lo-nm diameter as those associated with desmosomes. In cells sectioned perpendicular to the long axis of the cell [Figure 6a), microtubules were invariably cut in cross-section and were seen as dense rings positioned at regular intervals along the inner aspect of the intermediate filament layers. In the repre-
6
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Figure
1984
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3. Central region of a monkey colonic goblet cell. The supranuclear cytoplasm, containing multiple Golgi stacks and newly formed secretory granules, is separated from the apical mass of mucin granules (mg) by the theta. This filament-rich cytoplasmic layer forms an apical cup that segregates mature secretory granules from other cell organelles. x 18.800
sentative surface goblet cell shown in Figure 6a, a series of eight microtubules were counted along a span of the thecal wall that accounted for 60' or 17% of the total circumference of the theta. By extrapolation to the entire circumference, we estimate that
-45-50 such microtubules are present around the periphery of a single theta. A tangential section grazing the wall of the theta [Figure 6b) illustrated the architecture of these layers relative to the inner mass of secretory granules. The inner, vertical layer
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4a of goblet cells in a huFigure 4. Autoradiographs man rectal biopsy specimen pulse-labeled with [3H]glucosamine and maintained in organ culture for 12 h (a) or 24 h (b) before fixation. Groups of labeled secretory granules have migrated various distances toward the luminal cell surface, but they are consistently located peripherally (arrows) adjacent to the theta. x1060.
of intermediate filaments actually consisted of interwoven flattened bundles that generally followed an filament network upward course. This vertical seemed to be continuous with a basketlike layer of intermediate filaments that formed the base of the theta (Figure 7a). In Triton models of this region (Figure i’b) microtubules were occasionally seen crossing the filament layer, implying that the vertical microtubules originated in more basal regions of the cell. The upper and lower ends of the vertical microtubules were not identified. The data obtained from sections of Triton-extracted and conventionally prepared cells are consistent with the three-dimensional model shown in Figure 8. Briefly, the goblet cell theta is composed of three major layers: an inner array of vertical microtubules (Figure 8a), an interwoven basketlike network of intermediate filaments (Figure 8b) and an outer series of intermediate filament bundles that encircle the theta like the hoops of a barrel (Figure 8~).
Effects of Colchicine After culture of rabbit mucosal explants on medium containing 10 PM colchicine for 3 h, some microtubules were still identified along the inner aspect of the theta. After 6 h on colchicine-containing medium, microtubules were rarely detected in the theta but they were still present in the Golgi region below. Autoradiographs of goblet cells in control rabbit colonic explants, pulse-labeled with [“Hlglucosamine and maintained for up to 6 h on nonradioactive medium before fixation, showed radioactive secre-
tory product in the supranuclear region and, less frequently, in the basal region of the theta at 2 h (Figure 9a). By 6 h after pulse-labeling there was considerable variability in the intracellular position of labeled granules; nevertheless, all surface goblet cells in control explants showed some labeled granules near or at the luminal surface. Labeled granules at 6 h were generally restricted to the lateral borders of the apical granule mass (Figure 9b). In explants pulse-labeled and maintained for up to 6 h on colchicine-containing medium, in contrast, labeled secretory product never reached the luminal surfaces of goblet cells. In the majority of goblet cells, labeled granules remained in the supranuclear region and at the base of the theta (Figure 9c). Similar results were obtained when explants were preincubated in colchicine-containing medium for 3 h, pulse-labeled, and then maintained for an additional 6 h before fixation and autoradiography. Exposure of rabbit mucosal explants to the secretagogue acetylcholine, in the presense of the cholinesterase inhibitor eserine, has been shown to result in rapid secretion of mucin granules from crypt goblet cells by compound exocytosis, resulting in deep cavitation of the apical cell surface (8). When explants were maintained for 3 or 6 h on colchicinecontaining medium and then exposed to acetylcholine and eserine, crypt goblet cells showed a secretory response comparable to that observed in control explants (Figure 10). Thus, although exposure to 10 PM colchicine for 3 or 6 h inhibits the slow granule movement that accompanies baseline secretion, it does not inhibit rapid secretion by compound exocytosis; this activity seems to be independent of granule movement in this cell type.
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Figure
5
1984
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a. Portions of a goblet Rabbit colonic epithelial cells permeabilized with Triton X-100 and treated with myosin S, fragments. cell (left) and a columnar cell (right] are joined by an apical junctional region (above) and by a series of desmosomes. At left. mucin from disrupted secretory granules fills the goblet cell apex. In both cells, lateral intermediate filament networks are associated with desmosomes (desm IF). The theta consists primarily of intermediate filaments; medially, they are oriented vertically (vert IF) and laterally, they are cross-sectioned, indicating a circumferential orientation (circ IF). X61.400. b. Apical surface of a goblet cell (gc, Jeff) and a columnar cell (cc, right] including the junctional region and the terminal webs. Actin filaments in the microvilli (mv) of both cells are decorated with S, fragments. ~68.000. (c.)Intermediate filaments of the theta, sectioned as in 5n. The inner, vertical filaments pass obliquely through the section. The circumferential filaments are arranged in IRWP hllnrlles. ~118.800.
1320
Figure
SPECIAN
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6. Goblet cells of rabbit colon, prepared as in Figure 5. o. The lateral border long axis of the cell. Cross-sectioned microtubules (arrows) are spaced disrupted secretory granules, cc, columnar cell. X100.000. b. A tangential intermediate filament (IF) bundles, next through crisscrossing bands microtubules (arrows), and finally through the interior mass of disrupted
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of a goblet cell (gc) sectioned perpendicular to the at intervals around the periphery of the mass of section passes first through the circumferential of vertical IF, then through a series of vertical mucin granules. ~56,000.
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Figure
1984
7. Vertical sections (parallel to the long axis of preparation; a higher magnification of a portion form a barrier between the secretory granules goblet cell, Triton model. Although secretory intact. A microtubule (mt) passes through the
CYTOSKELETON
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GOHLET
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the cell], showing the base of the theta. a. Monkey goblet cell. conventional of the section shown in Figure 3. Intermediate filament bundles of the theta above, and the Golgi complex and other organelles below. * 51.500. b. Rabbit granule membranes are disrupted. the filament bundles dehning the theta are base of the theta. *56,800.
Discussion The theta of the intestinal goblet cell is a specialized cytoskeletal structure consisting of intermediate filaments and microtubules, but not of actin. The distribution of actin in goblet cells is comparable to that of neighboring absorptive cells (19-23) in that bundles of actin filaments form the cores and rootlets of goblet cell microvilli and a filamentous accumulation containing actin is associated with the zonula adherens. Because of the crowd of mucin granules at the apical pole of the goblet cell, however, the terminal web is discontinuous and does not form a uniform plate at the level of the zonula adherens, as in absorptive cells (17). Immunocytochemical staining of absorptive cells has revealed relatively little actin below the brush border: an actin network is present along basal cell surfaces (22)
and actin has been detected in lateral interdigitations (Tokuyasu KT, personal communication) but no actin has been detected in more central regions. Similarly, we detected no actin in the theta of goblet cells. The presence of actin cannot be ruled out by lack of S1 labeling, however, because short actin assemblies might not be recognized by this method (24).
We have shown that the cuplike theta of goblet cells consists primarily of a well-organized system of intermediate filaments. The arrangement and composition of the IO-nm filaments in intestinal absorptive cells have been studied in detail (17,18,25). like those of many other These “tonofilaments,” vertebrate epithelial cells, consist of cytokeratins, a class of proteins related to epidermal prekeratin (18.25-28). Although structurally identical lo-nm filaments can be formed in different types of cells
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_
8. Schematic cut-away Vertical microtubules oriented intermediate
view of the architecture of the goblet cell theta: a model based on our ultrastructural observations. o. lie closely apposed to the central mass of stored secretory granules. b. A basketlike network of vertically filaments (IF) defines the cup shape of the theta. c. Circumferential bundles of IF encircle the basket,
from different cytokeratins (25,283, we consider it likely that the intermediate filaments of goblet cells are biochemically comparable to those of absorptive cells as the two cell types arise by differentiation from the same epithelial stem cell population (29). In absorptive cells, most lo-nm filaments are arranged as a peripheral network of bundles that often attach to the lateral cell membrane by looping through the dense plaques of material associated with the cytoplasmic face of spot desmosomes (17). At the apical poles of absorptive cells, lo-nm filaments emanating from the apical ring of spot desmosomes interweave to form a dense “subapical disk” immediately beneath the actinand myosin-rich terminal web (17,181. Goblet cells also have a lateral, desmosomeassociated system of tonofilament bundles, but this system seems to be separate from those that form the theta. From our thin sections we were not able to follow the entire three-dimensional course of the circumferential and interwoven filament bundles of the theta so we cannot rule out attachments of this system to lateral desmosomes. Evidence from many cell types indicates that intermediate filaments provide an intracellular scaffold that influences the arrangement of organelles but that does not directly participate in cellular or organellar motility (27,28). These concepts are consistent with our data showing that the goblet cell theta seems to be structurally stable during accelerated secretion. Mucin is apparently expelled by the rapid expansion that accompanies hydration and decondensation of the mucin granule contents, and
not by collapse or contraction of the theta. A recently emptied goblet cell resembles an empty cup. The cup shape of the theta is maintained, even after 6 h of colchicine treatment when the theta is largely depleted of microtubules. The elaborate architecture of the intermediate filament networks, resembling a reinforced basket, suggests that these elements play
.. I._
Figure
-
9. Autoradiographs of goblet cells in the surface epitheliurn of rabbit colonic mucosa, maintained in organ culture and pulse-labeled with [“Hlglucosamine. o. Control mucosa, pulse-labeled for 30 min and fixed 2 h later. Labeled secretory granules (arrow!) are present in the supranuclear region and at the base of the theta. x1020. b. Control mucosa, pulse-labeled for 30 min and fixed 6 h later. Most labeled granules have moved out of the supranuclear region and are located along the periphery of the apical granule mass. x 1020. c. Colchtine-treated mucosa. fixed 6 h after pulse-labeling. Labeled granules remain in the supranuclear region and at the base of the theta. x1020.
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Figure
1984
10. Apical portion of a rabbit colonic goblet cell, maintained for 3 h on colchicine-containing medium and then exposed to acetylcholine and eserine in the presence of colchicine. The apical cell surface is deeply cavitated by compound exocytosis of many mucin granules. Some intact granules (mg) remain in the base of the theta. This secretory response is identical to that seen in control cells that were not exposed to colchicine. x 15,330.
the major role in segregating the apical granule mass and in maintaining the distinctive shape of the cell apex. The intestinal goblet cell theta thus provides a dramatic example of the importance of intermediate filaments in the organization of intracellular space (271.
An intact, functional system of microtubules is generally believed to be necessary for the directed movement of secretion granules toward the cell surface. Although the precise modes of interaction between secretory granule membranes and polymerized tubulin are not yet clear, there is evidence to suggest that microtubuleand perhaps membraneassociated proteins play roles in the movement of granules along microtubule paths (30). In any case, inhibition of tubulin polymerization by drugs such as colchicine is now well documented and the dependence of normal microtubule functions on tubulin polymerization/depolymerization, or tread-
CYTOSKELETON OF INTESTINAL GOBLET CELLS
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milling, is supported by compelling evidence (31,321. Colchicine has been shown to inhibit secretion by halting granule movement in many secretory cell types, including endocrine cells, connective tissue cells, and a variety of exocrine epithelial cells (l-6). Transport of membrane-associated glycoprotein to the luminal (but not basal-lateral) cell surfaces of intestinal columnar cells was also blocked by colchicine (33-37). Inhibition of mucin glycoprotein secretion by colchicine treatment of the respiratory mucosa was detected biochemically by some authors (38) although not by others (39). It is thus not surprising that inhibition of secretory granule movement was observed in the colchicine-treated goblet cells in this study. The observation is underscored in this cell, however, by the unusual barrel-stave arrangement of the microtubules in the theta and the preferential upward migration of peripheral secretory granules, apparently along these orderly microtubular pathways. Microtubules were generally not observed within the mass of stored granules, and central granules did not seem to participate in baseline secretion. In a preliminary autoradiographic study of goblet cells continuously labeled with [3H]glucosamine for 12 h (40), we observed that central granules remained unlabeled despite heavy labeling of the peripheral layer of granules. This suggests that central granules are synthesized early in the life of the goblet cell and that in the absense of secretagogues, they remain unmoved and unsecreted through most of the cell’s 4-6-day lifetime. In acetylcholine-stimulated cells, in contrast, compound exocytosis begins with the uppermost central granules, spreads sequentially through granules at the center of the apical granule mass, and finally includes peripheral granules (8). Studies on mucin secretion from the respiratory epithelium in explants of tracheal mucosa reported that colchicine inhibited the accelerated release of glycoconjugates normally observed in response to methacholine (39) and cholera toxin (41). Tracheal goblet cells are ultrastructurally very similar to their intestinal counterparts (42). These findings were thus inconsistent with our studies of accelerated intestinal goblet cell secretion, in which compound exocytosis in response to acetylcholine did not seem to require granule movement (8). Unlike most other secretory cells, in which rapid exocytosis of stored secretory product requires movement of many granules toward the cell surface and toward each other, the goblet cell need only close the narrow gap between the plasma membrane and a single apical granule membrane to initiate a long series of exocytotic events. This cytoplasmic gap contains no microtubules; it is comparable in width and in compo-
1324
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sition to that described between peripheral secretory granules and plasmalemma in Limulus amebocytes (43). Through the fusion of multiple granule membranes, the luminal plasma membrane in effect moves down to progressively deeper granules until the theta is empty. The present study confirms that functional microtubules are not needed for this process to occur.
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