chick cardiac muscle cells

Proc. Nail. Acad. Sci. USA Vol. 74, No. 2, pp. 662-665, February 1977

Cell Biology

Localization of bovine brain filament antibody on intermediate (100 A) filaments in guinea pig vascular endothelial cells and chick cardiac muscle cells (antibody localization/demecolcine)

STEPHEN H. BLOSE*, MICHAEL L. SHELANSKIt, AND SAMUEL CHACKO* * Department of Pathobiology, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, Pa. 19174; and t Department of Neurosciences of the Mental Retardation Research Center, Children's Hospital Medical Center, and Department of Neuropathology, Harvard Medical School, Boston, Massachusetts 02115

Communicated by Alex B. Notlkoff, December 3,1976

ABSTRACT Guinea pig vascular endothelial cells contain naturally occurring rings of intermediate filaments that completely encircle the nucleus. Indirect immunofluorescence staining showed that these perinuclear rings bound antibody prepared against protein from bovine brain 9-nm filaments. In endothelial cells grown in the presence of 1 MM demecolcine (Colcemid) the perinuclear ring "coils"' into a juxtanuclear "cap." Throughout this process we could demonstrate staining of the intermediate filaments. Chick cardiac muscle cells in culture stained diffusely with the antibody. After treatment for 24 hr with 1 MAM demecolcine the cardiac cells accumulated large bands- of intermediate filaments. These bands stained intensely with the antibody. Our findings sugest that interand those inmediate filaments is guinea pig endothelial duced in chick cardiac muscle cells are antigenically similar to bovine brain filaments. The staining of these filaments is not affected by treatment with demecolcine. Intermediate filaments ("100 A filaments," 8-10 nm in diameter) represent a distinct class of cytoplasmic filaments that are different from actin filaments ("microfilaments," about 6 nm in diameter), myosin filaments ("thick filaments," about 15 nm in diameter), and microtubules (about 24 nm in diameter) (1, 2). Intermediate filaments are ubiquitous, to various eukaryotic cells (1-10) although their precise function is not known. They have been implicated with the slow component of axopasmic transport (11-13), with pigment granule movement (14, 15), and with cell spreading (7) and as a cytoskeleton in smooth muscle cells (8). Intermediate filaments are observed in large numbers in several neurologic diseases (12, 16) and are conspicuous in thermally injured lymphatic capillaries (17) and damaged smooth muscle (18). They are found as large aggregates in cells treated with colchicine or demecolcine (Colcemid) (19,20) or with vinblastine (20,21) and in neurons treated with AIC13 (16, 22) or nerve growth factor (23). The presence of intermediate filaments in a various cells in both normal and abnormal conditions raises the question of whether these filaments of -similar diameter may be biochemicilly related. In order to answer this question, we made use of antibody prepared against bovine brain 9-nm-filament protein (24) to determine if there was any antigenic similarity between intermediate filaments in neural and non-neural tissue. Two types of tissue were studied-cultured guinea pig vascular endothelial cells that contain naturally occurring perinuclear rings of intermediate filaments (25), and cultured chick cardiac muscle cells in which large bands of intermediate filaments were induced by treatment with demecolcine (26). By indirect immunofluorescence we demonstrated that this antibody specifically bound to intermediate filaments in both types of cells.

MATERIALS AND METHODS Cell Preparation. Endothelial cells were obtained by sterile technique from the thoracic aortas and portal veins of young adult guinea pigs as described before (25, 27). Briefly, the vessels were turned inside-out, to expose the endothelial surface, and the ends were ligated. After several washings with Hanks' balanced salt solution to remove erythrocytes, the vessels were immersed in dissociation medium (0.125% trypsin and 0.4% collagenase in Ca2+- and Mg2+-free balanced salt solution). At the end of 20-30 min, the dissociation medium with liberated endothelial cells was collected in a centrifuge tube and an equal volume of cold (100) Eagle's minimal essential medium supplemented with 15% fetal calf serum was added to neutralize the enzymes. After centrifugation the endothelial cells were resuspended in nutrient medium and plated out at 1 to 5 X 103 cells per 60-mm Falcon Cooper dish. The cultures were fed on alternate days with E.agle's minimal essential medium containing 15% fetal calf serum, 1% L-glutamine solution, and 1% penicillin-streptomycin solution (all from GIBCO, Grand Island, N.Y.). Endothelial cells to be examined by polarized light and immunofluorescence were grown on sterile glass cover slips (24 X 40 mm). In selected cultures, demecolcine (1 WM) was added to "coil" the perinuclear ring of intermediate filaments into the juxtanuclear "cap" as previously reported (25). Chick cardiac muscle cells were obtained from 5-day embryos and cultured by the method described by Chacko (28). The cardiac muscle cells were grown on collagen-coated glass cover slips. Selected cultures were grown in the presence of 1-3 MM demecolcine for 24 hr in order to induce the accumulation of large bands of intermediate filaments (26). Preparation of Antibody Against Bovine Brain Filaments. Calf brain filaments were purified from white matter as previously described (24, 29). Electrophoretic analysis showed that > 90% of this material was a protein with a molecular weight of 52,000. This major component migrated as a single band on various detergent-containing gel systems and on 8 M urea gels which separate on a charge basis Rabbits were immunized with the major filament protein band (cut out of sodium dodecyl sulfate gels) and complete Freund's adjuvant (24). The antiserum had a titer of 1:1250 against the major band by radioimmunoassay and by microcomplement fixation. Detergent-solubilized major band protein was also completely precipitable by the antiserum. The antiserum had no detectable titer against actin, tubulin, a-tubulin, #-tubulin, or the high-molecularweight proteins associated with tubulin. Conversely, antiserum against tubulin did not react against the major band or against the purified filament preparation (R. Liem, S. Yen, and M. Shelanski, unpublished data).

Abbreviation: Pi/NaCl, phosphate-buffered saline. 662

Cell Biology: Blose et al.

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FIG. 1. (A) Phase-contrast micrograph of living guinea pig vascular endothelial cells near confluence at day 14 of culture. Many of these cells contained a phase-lucent ring (arrows) that encircled the nucleus (N). (B) When these same cells are viewed with polarized light the rings (arrows) appear to be birefringent. (Phase microscopy with Zeiss 40X phase 2 water immersion lens; X489.)

Indirect Immunofluorescence. Cells were prepared for immunofluorescence by a slight modification of the method of Lazarides and Weber (30). Endothelial cells grown on cover slips were fixed in 4% formalin in phosphate-buffered saline (Pi/NaCI) at pH 7.4 for 1 hr at room temperature. The cover slips were then washed with 10 changes of Pi/NaCI, extracted in absolute acetone at -20° for 10 min, and air dried. The cover slips were incubated with rabbit antibody against bovine brain filament protein (1:3 or 1:30 dilution in Pi/NaCl) or with the appropriate control serum for 1 hr at 370 in a humidified atmosphere. The cover slips were washed 10 times with Pi/NaCl and incubated with fluorescein-labeled goat antiserum to rabbit IgG (diluted 1:3 or 1:30). The cover slips were then washed 10 times with Pi/NaCl and briefly with deionized water. Cover slips were mounted on glass slides with polyvinyl alcohol (El-

vanol). Control studies were performed with rabbit IgG, with rabbit antiserum against bovine brain filaments and addition of either brain filament major band or tubulin, and with fluorescein-labeled goat antiserum to rabbit IgG alone. Photomicrographs were made on Tri-X 135 film (El, 400). Preparation of Cells for Electron Microscopy. Cells were grown on a collagen substrate in carbon-coated Cooper dishes. The cells were fixed in situ and embedded in Epon 812 as previously described by Blose and Chacko (25). RESULTS Endothelial cells are easily identified, 3 days after they are cultured, as flat polygons as previously described (27). By day 10 to day 14 of culture, when endothelial cells approached confluency, many cells contained phase-lucent rings (Fig. 1A) that were birefringent when examined with polarized light (Fig. IB). These rings encircled the nucleus and were composed

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Am FIG. 2. Electron micrographs of cultured endothelial cells. (A) Sectioned en face. A perinuclear bundle of intermediate filaments (arrows) encircles the nucleus (N). The location of this filament bundle corresponds to the phase-lucent or birefringent rings in Fig. 1 (X1861). (B) In cross section. The perinuclear bundle consisted of 200 to 500 filaments in cross section (arrow). These filaments were about 10 nm (9 ± 1.3 nm) in diameter. Through-focus series of electron micrographs revealed the filaments to have a less dense core (X41,360).

FIG. 3. Indirect immunofluorescence of an endothelial cell stained with the antibody to brain filament. The antibody specifically stained the perinuclear ring (arrow) of intermediate filaments around the nucleus (N); (Dark-field fluorescence; X764).

FIG. 4. Endothelial cells incubated with 1 AM demecolcine, fixed, and stained with the brain filament antibody. Throughout this process of coiling the intermediate filaments continued to be stained by the brain filament antibody. N, nucleus, (X1239). (A) At 16 hr of incubation. The ring is coiling into a juxtanuclear cap (arrow indicates direction of coiling). (B) At 24 hr of incubation. The perinuclear ring has completely coiled into the cap (arrow).

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Cell Biology: Blose et al.

Proc. Natl. Acad. Sc4. USA 74 (1977)

FIG. 5. Phase-contrast micrograph of a living chick cardiac muscle cells. (A) Control culture. These cells contained glycogen (G) and welldifferentiated myofibrils (arrow) (28) (X440). (B) Culture treated with demecolcine for 24 hr. These cells continued to contract and exhibit phase-lucent bands (arrows) of intermediate filaments (19, 26) (X440).

of a bundle of intermediate filaments (Fig. 2) as previously reported (25). When these cells were fixed and incubated with antibody prepared against bovine brain filament protein (24), the ring of intermediate filaments was intensely stained (Fig.

FIG. 6. Indirect immunofluorescence studies of cardiac muscle cells stained with the brain filament antibody. N, nucleus (X484). (A) Cell not treated with demecolcine exhibits diffuse staining over the cytoplasm. (B) In demecolcine-treated cardiac muscle cell, the antibody specifically stained the bands of intermediate foments. (C) After demecolcine treatment, binucleated cardiac muscle cell (28) exhibited extensive staining of the band formations.

3). When the antiserum was absorbed by the addition of purified brain filament major band or total filament preparation (1 Mg of protein per Ml of antiserum), all staining of the filament bundle was abolished. Tubulin had no effect on the immunofluorescent staining reaction. No staining was seen with rabbit control IgG or with the fluorescent goat antiserum to rabbit serum alone. When endothelial cells were grown in the presence of 1tM demecolcine and then fixed and stained sequentially over 24 hr, throughout the entire process of coiling of the perinuclear ring into a cap the intermediate filaments were intensely stained by the antibody (Fig. 4). As in the case of untreated cells, no staining was seen with the absorbed serum or with control serum. Because the demecolcine treatment did not affect antibody staining of intermediate filaments in endothelial cells, we were interested to learn if the antibody would bind to demecolcine-induced intermediate filament bands in chick cardiac muscle cells. When cardiac muscle cells were cultured as described above, by the second day of culture, vigorously beating cells were observed. (Fig. 5A). In sister cultures grown in the presence of demecolcine, the cells contained twisting phaselucent bands of intermediate filaments (Fig. SB) (19, 26). During the formation of these bands the heart cells continued to beat. The untreated cardiac muscle cells stained diffusely with the antibody (Fig. 6A). However, after demecolcine treatment the induced filament bands were intensely stained by antibody (Fig. 6B and C). Control studies with absorbed and control antisera gave no staining of these bands. DISCUSSION Published reports point out that the intermediate-size filaments are related primarily by their diameter (1, 2, 6-8). Even though the molecular weights of the putative intermediate filament protein in several cells are similar (51,000-55,000) (8, 24, 31, 32), immunological "relatedness" is unknown. The availability of antibody (24) against intermediate filaments from the nervous system made it possible for us to see if there was antigenic similarity between intermediate filaments in different cells. Guinea pig vascular endothelial cells uniquely localize their intermediate filaments to a perinuclear ring. The antibody prepared against bovine brain 9-nm filaments specifically stained this ring. Staining of these filaments persisted even when the perinuclear ring coiled into a juxtanuclear cap after demecolcine treatment. Untreated cardiac muscle cells stained diffusely with the antibody. This was expected because muscle cells have intermediate filaments scattered throughout the cytoplasm (1, 5). Demecolcine treatment of the cardiac muscle cells caused the intermediate filaments to aggregate into bands that demonstrated intense antibody staining. These observations strongly suggest that intermediate filaments of various cell types and species are related. Their anti-

Cell Biology: Blose et al. genicity is not affected by demecolcine treatment, and the demecolcine-induced filament bundles are also antigenilly related to normally occurring filaments. Thus, it appears that, like actin and tubulin, the intermediate filaments show a high degree of biochemical as well as morphological conservation.

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