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Response of Basal Epithelial Cell Surface and Cytoskeleton to Solubilized Extracellular Matrix Molecules

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Response of Basal Epithelial Cell Surface and Cytoskeleton to Solubilized Extracellular Matrix Molecules Response of Basal Epithelial Cell Surface and Cytoskeleton to Solubilized Extracellular Matrix Molecules STEPHEN P . SUGRUE and ELIZABETH D. HAY Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface . If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ . This study examines the effect of soluble extracellular matrix components on the basal surface . Corneal epithelia from 9- to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp ., Bedford, Mass .), and cultured at the medium-air interface . Media were prepared with no serum, with 10% calf serum, or with serum from which plasma fibronectin was removed . Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-24 h) . If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat . Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb . Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium . The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments ; some, if not all, of these filaments point away from the plasmalemma . The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasma- lemma . In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical of epithelia in situ . Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells . The basal cell surface and cortical cytoskeleton of the embry- lagens (e .g., type V) may be concentrated in the lamina densa onic corneal epithelium are intimately related to the underlying (4) but might also extend in the form of fine filaments (1) to extracellular matrix (ECM) . The cortical cytoskeleton forms a the cell surface in concentrations too dilute to be detected by dense mat next to the basal epithelial plasmalemma, and the immunohistochemistry . latter is relatively smooth or flat in configuration . The basal The existing evidence that the organization of the basal plasmalemma seems to be connected to the lamina densa of epithelial cell cytoplasm is dependent on the presence of the the underlying basal lamina (basement membrane) by extra- underlying ECM is mainly circumstantial. In corneal epithelia cellular filaments that traverse the lamina rara externa (1). The or epidermis with well-developed hemidesmosomes, tonofibrils laminae rarae externa and interna of the corneal, and most, if insert in cytoplasmic plaques that seem to be attached to not all, other epithelial basal laminae (2-5) contain proteogly- specialized regions of ECM (1) . In younger corneal epithelia can granules rich in glycosaminoglycans (GAG), such as he- lacking hemidesmosomes, the mat of cortical filaments in the paran and/or chondroitin sulfates (HS, CS) and hyaluronic basal cytoplasm seem to follow the contour of the basal lamina acid (HA) . Laminin and fibronectin occur in the laminae rarae (1) . More direct evidence that the configuration of the basal of the glomerular basement membrane (4), and both are found cell surface and cortical cytoplasm depends on ECM derives in the embryonic avian corneal basal lamina (Sugrue and Hay, from studies using enzymes or EDTA to remove the epithelial unpublished observations) . Type IV collagen is also present in basal lamina; such epithelia extend cytoplasm-filled blebs from the corneal basement membrane (6) . Type IV and other col- their naked basal surfaces (see references 5 and 7) . When THE JOURNAL OF CELL BIOLOGY " VOLUME 91 OCTOBER 1981 45-54 ©The Rockefeller University Press " 0021-9525/81/10/0045/10 $1 .00 45 grown on solid collagenous substrata, isolated embryonic avian the basal epithelial surface was exposed to the underlying medium without epithelia withdraw these blebs, become smooth-surfaced again, physical support on the basal side. Still other epithelia were grown on Millipore and step up synthesis of filters that had been soaked for 18 h in collagen (type 1, 100 !rg/ml, see below) both collagen and GAG (7-9) . and washed briefly in three changes of Hanks' solution. In the present paper, we ask whether the reaction of the Purified collagens were dissolved in 0.5 M acetic acid at a concentration of 1 basal corneal cytoplasm to ECM is due merely to the physical mg/ml, then dialyzed against 0.1 M phosphate-buffered saline (PBS), pH 7 .4. support of the collagenous substratum, or whether solubilized Type I collagen was denatured by heating at 50°C for 20 min . Type I collagen ECM molecules themselves can induce isolated epithelia to was purified from rat tail tendons by our laboratory (8, 9), and type II collagen from sternal cartilage by Dr . Thomas Linsenmayer (Department of Anatomy, withdraw the blebs . Blebbing epithelia impaled on Millipore Harvard Medical School, Boston, Mass.) (10) . Type IV collagen and laminin filters (Millipore Corp ., Bedford, Mass .) are exposed to solu- from a murine tumor (1l, 12) were provided by Dr. George Martin (National tions of various collagens, glycosaminoglycans, fibronectin, Institute of Dental Research, Bethesda, Md .). Rat tendon a2(1) chains were also and laminin to judge the effect of these molecules on the obtained from Dr. Martin. Cellular fibronectin was extracted from NIL8 cells, structure of the basal epithelial appearance and plasma fibronectin was purified from human plasma by Dr. Richard Hynes surface. The of the (Massachusetts Institute of Technology, Cambridge, Mass.) (13) . HA and CS basal surface on solid but inert substrata is also examined . mixed chains (Sigma Chemical Co .), HS chains (Dr. J. A. Cifonelli, University Finally, we address the question of the organization and nature of Chicago, Chicago, III.), and heparin (Upjohn Co ., Kalamazoo, Mich.) were of the cortical cytoskeleton in blebbing and ECM-treated em- dissolved double-strength in H2O and diluted in double-strength media. Bovine bryonic corneal epithelia by examining the actin components serum albumin (Sigma Chemical Co .) and nonspecific rabbit 1gG (Miles Labo- ratories Inc., Elkhart, Ind .) were dissolveddirectly into medium in concentrations labeled with S-1 fragments of heavy meromyosin . as stated in Results and in Table 1 . After incubation, cultures were routinely fixed for 30 min in 2% paraformal- MATERIALS AND METHODS dehyde, 2.5% glutaraldehyde in cacodylate buffer (0 .1 M), and postfixed in I% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4, at 4°C for 30-60 min . They were stained en block in 1% uranyl acetate, dehydrated, and embedded in Spurr Corneas harvested from chick embryos after 9-11 d of incubation were treated at (D . E. R. 736 embedding kit, Tousimis Research Corp ., Rockville, Md .) . (Sigma Chemical Co ., St . Louis, Mo .) and 0.1% collagen- 25°C with 0.1% trypsin Myosin S-1 was prepared by the method of Margossian and Lowey (see .) in solution, 7.4, for 9-12 min ase (Sigma Chemical Co Hanks' pH or were reference 14) and stored at a concentration of 30 mg/ml in 50% glycerol. Epithelia 0.04% EDTA (Sigma Chemical Co .) in calcium-magnesium-free Hanks' soaked in were extracted with Triton X100 in 0.05-0.1% in 0.1 M PIPES buffer (Sigma solution, pH 7.4, for 30 min . The epithelia were removed with forceps from the Chemical Co .), 2 mM MgC12, 2 mM EGTA, l mM phenylmethyl sulfonyl a transferred a Millipore filter (HATF 0.45-pm pore size) stroma as sheet, to . fluoride (PMSF), 1 MM P-tosyl-L-argone methyl ester-HCI . S-1 decoration was Such epithelia are completely clean of contaminating ECM (8, 9). Epithelia on performed in 0.3 M PIPES buffer containing S-1 fragments, 0.5-1 mg/ml, for 10 cultured the air-medium dishes filters were then at interface of Falcon culture min at room temperature. Fixation was carried out subsequently in 1% glutaral- (Falcon Plastics, Oxnard, Calif) in Ham's F-12 medium (Gibco Laboratories, dehyde with 0.2% tannic acid (Mallinckrodt, St. Louis, Mo., batch AR 1764) in Grand Island, N. Y.) without serum or with 10% Grand Island Biological Co ., 0.1 MPIPES buffer (14) for 30 min . Afterthorough washing, tissue was postfixed McLean, .) fetal calf serum(Flow Laboratories, Va from which plasma fibronectin in 1% OsO, in 0.1 M phosphate buffer, pH 6.0, for 30 min at 4°C, and prepared chromatography Medium was (CIG) was removed by affinity with gelatin . as described above for electron microscopy . supplemented with 50 itg/ml ascorbic acid and 1% antibiotic-antimycotic (peni- cillin, fungizone, and streptomycin, Gibco Laboratories). Each isolated corneal epithelium was usually placed basal-side down on a disk RESULTS (3-mm Diam) made of Millipore filter and transferred within 2 h after isolation to standard medium (above) or to medium containing soluble ECM molecules, The avian corneal epithelium at 9-11 d of embryonic devel- albumin, or IgG. Some epithelia were cultured at the air-medium interface on opment is two to three cells thick. The cells of the basal glass cover slips coated with 0.1% poly-L-lysine (Sigma Chemical Co .) or tissue layer culture plastic (Falcon Plastics). Other epithelia were attached by their apical are cuboidal to columnar in shape and possess extensive rough surfaces to Millipore filter disks coated with poly-L-lysine .
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