Characterization of a detergent-resistant surface lamina in cultured human fibroblasts

Copyright Q 1983 by Academic Press, inc. ~11 rights of reproduction in any form resened 0014-4827/83!020287-0Rt;S2.0Qi0

Experimental Cell Research 14.3(1983) 2

ARACTERIZATION LAMINA

OF A DETERGENT-RESISTA IN CULTURED

V.-P. LEHTO,‘, * T. VARTIO,’

AC

MU * R. A. BADLEU” and I. VHRTANEN’

Departments of ‘Pathology and ‘Virology, University ofHelsinki, SF-00290 Helsinki 29, Finlrrrzd nnd “Unilever Research, Colworth Laboratory, Sharnbrook, Bedford, UK

SUMMARY Treatment of cultured human fibroblasts with 0.5% Triton X-100 produces substratum-anchored cytoskeletal preparations consisting of cytoplasmic filaments, nucleus and a plasma membrane-derived surface lamina. The lamina was visualized in fluorescence microscopy with fiuor~cbr~mecoupled wheat germ agglutinin (WGA) as a lace-like structure, extending throughout the cell domain. It displayed a different organization at the ventral and dorsal surfaces of the cell, partially coaligning with bundles of actin and myosin filaments at the dorsal cell surface. At the ventral surface vinculin patches appeared to be included in the surface lamina. Polyacrylamide gel electrophoresis, combined with lectin reactivity studies and lectin afftnity chromatography, revealed a 140kD sialoglycoprotein as the major glycoprotein component of the surface lamina.

Treatment of cultured cells with non-ionic detergents, such as Triton X-100, reveals a cytoplasmic, filamentous network, defined as a detergent-resistant cytoskeletal (e.g. [I-3]>. Its main constituents are intermediate filaments, microfilaments and possibly microtrabeculae [l-4]. Recent studies have shown that after non-ionic detergent treatment not only are the cytoskeletal filaments preserved, but also a surface lamina which retains the original morphology of the cell and which contains both cell surface proteins (cf [5, 61) and some glycolipids [7]. In this study we have used fluorochromecoupled wheat germ agglutinin (WGA) to visualize the organization of the surfacelamina in Triton X-loo-treated cultured fibroave also made an attempt to blasts. aracterize the polypeptide composition of e Iamina and show the existence of a 140 19-821815

kD glycoprotein as a majsr co the lamina. MAT~R~A~~ AN Cell cullure and extractions Human embryonic fibroblasts were obtained from a local source and cultured on plastic Petri dishes or on small glass coverslips in Rosweli Park tute (RPMI) 1640 medium, supplemented with 10% fetal calf serum (KS) (Flow Laboratories, Irvine, Scotland) and antibiotics. Detergent-resistant cytoskeletons were produced by treating the cells with 0.5% Triton X-100 (BDH Chemicals Ltd, Poole, UK) in 50 mM Tris-HCI buffer, pH 7.2, with freshly added 1 mM uhenvlmethvl suifonvlfluoride (PMSF) and l-ltosyla~ide-~-p~en~lethylc~lorometbylket~~e (TPCK) (both from Sigma Chemical Co., St Louis, MO) at 0°C for 30 min. Extractions with the ATP-containing low and high-ionic actomyosin-dissociating buffers were performed as described earlier [2].

Fluorescence microscopy For fluorescence microscopy with lectins, the cells were exposed to tetramethyi rhodamine isothiocyanate * To whom offprint requests should be directed.

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(TRITC)- or fluorescein isothiocyanate (FITC)-coupled WGA (Vector Laboratories, Burlingame, CA) at 0°C for 15 min and then fixed in 3.5% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, or, alternatively, first treated with Triton X-100 and the actomyosin-dissociating buffers, fixed with paraformaldehyde and then reacted with the fhrorochrome-coupled lectin. Similar results were obtained also if the cells were first reacted with the lectin-conjugate and then extracted as described above. For some experiments, paraformaldehyde-fixed cells were permeabilized by treating them with 0.05% Nonidet P40 (NP 40, BDH Chemicals) for 10 min. Rabbit antibodies against actin, myosin and vimentin have been characterized in detail previously [8, 93. Vinculin was isolated from chicken gizzard as described by Feramisco & Burridge [lo]. Antibodies were raised in rabbits and they reacted only with a I30 kD polypeptide of cultured tibroblasts, as shown elsewhere [II]. For indirect immunofluorescence microscopy the cells and the cytoskeletal preparations were fixed in -20°C methanol for 1.5min, reacted with the first antibody, washed and then exposed to FITC-coupled goat anti-rabbit IgG (Cappel Laboratories, Cochraneville, PA). For double-staining, the specimens were subsequently reacted with TRITCWGA and then embedded in Veronal-alvcerol. DH 8.4. The specimens were examined in a-Zeiss Universal microscope equipped with an epi-illuminator III RS and filters for FITC- and TRITC-fluorescence.

Electron microscopy For transmission electron microsconv (TEM) the cells or the cytoskeletal preparations were exposed to peroxidase-coualed WGA (POX-WGA. 50 &ml. Sigma) as described above for’ fluorochrome-coupled WGA; fixed and then exoosed to the substrate solution 0.03 % 3,3-diamino-be&din-tetrahydrochloride (DAB) in 50 mM Tris-HCl, pH 7.5, with freshly added 0.06% H,O,). Both whole cells and cytoskeletons were fixed in 2.5% elutaraldehvde. buffered with 0.1 M sodium cacodyla&, pH 7.2,-post-fixed in 1% osmium tetroxide, dehydrated and embedded in Epon 812. Thin sections were studied either unstained or after staining with lead citrate and uranyl acetate in a Jeol 100 CX electron microscope at the Department of Electron Microscopy, University of Helsinki.

Affinity chromatography For lectin affinity chromatography, cytoskeletal preparations from surface-labelled cells were solubilized in 1% Zwittergent (ZwittergentTMR’4, Calbiochem, San Diego, CA), in 25 mM Tris-HC1 buffer, pH 7.5. After centrifugation at 30000 g for 60 min, ahquots of the extract were mixed with 50 ~1 of WGA-coupled Seoharose 4B (Pharmacia. Uoosala) at room temoerature for 60 min. ‘To control the&specificity of the binding, 0.1 M N-acetylglucosamine in NaCI-P buffer (140 mM NaCl, 10 mM phosphate buffer, pH 7.2) supplemented with CaCl, was added in parallel samples. After the incubations, the lectin beads were washed thoroughly with NaCl-P buffer and thereafter 70 hl of electrophoresis sample buffer was applied on the beads which Exp Cell Res 143 (1983)

were then heated at 90°C for 3 min. After a brief centrifugation, the supernatants were collected and subjected to polyacrylamide gel electrophoresis.

Surface labelling For surface labelhng experiments, the galactose oxidaselNaB3H, method of Gahmberg & Hakomori [12] and the metaperiodate/NaB3H, method of Gahmberg & Andersson [ 131were used.

Electrophoretic techniques Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was performed according to Laemmli [14] using 6.5 and 8 % slab gels. For the lectin-blotting experiments the polypeptides of cultured fibroblasts and cytoskeletal preparations were first separated in polyacrylamide gel and then transferred [ 151to a nitrocellulose sheet using a commercial blotting apparatus (Trans-Blot, Bio Rad Laboratories, Richmond, CA). The nitrocellulose sheets were soaked with 3% bovine serum albumin (Sigma) in NaCl-P buffer to saturate unspecific protein binding and then reacted with POX-WGA (50 fig/ml) in NaCl-P buffer. The polypeptides recognized by-the lectin were thereafter visualized by exposing the sheets to DABsolution as described above.

RESULTS Visualization of the surface lamina with lectins Cultured, paraformaldehyde-fixed fibroblasts showed a homogenous surface fluorescence after staining with TRITC-WGA (fig. 1a). After permeabilization with NP 40 also a distinct decoration of the Golgi apparatus was revealed ([ 161,fig. lb). A short exposure of TRITC-WGA-treated cells to Triton X-100 resulted into a distinct porosity in the surface staining pattern (fig. lc). On the other hand, when the cells were extracted with Triton X-100 for a longer time (30 min), TRITC-WGA was seen to decorate a meshwork-like surface lamina (fig. Id). The surface lamina of the Triton X100-treated cells displayed a reticular structure at the substratum-facing side of the cell and a more linear, rim-like organization at the dorsal surface of the cell, as judged by differential focusing in fluorescence mi-

Fig. 1. (a) Human embryonal fibroblasts fixed with paraformaldehyde and surface-stained with TRITCWGA. (b) Cells treated with NP40 after parafonnaldehyde fixation show a distinct, perinuclear reticular staining corresponding to the Golgi apparatus (nr-

I.OMV).When unfixed, spread cells are bhieRj treated with Triton X-100 (c, 15 min), a fine porosity is seen after staining with TRITC-WGA, whereas in ceils exposed to Triton X-100 for 30 min a meshwork-like staining is discernible (d).

croscopy (fig. 2a, Ip). In double-staining experiments, employing anti-a&in (fig. 2c) and anti-myosin (fig. 2~) antibodies and ITC-WGA (fig. 2d, f), coalignment of straight lamellae of the lamina and the actin- and myssin-containing stress fibers e seen at the dorsal surface of the cytoskeleta! preparations. Similarly, parts of the surface lamina, remaining at the substratum-facing side of the cytoskeletons, appeared to colocalize with patches of vin-

culin which were still ~ern~~~~ra~~eafter Triton-treatment of the cefls (fig. 2g, h). Pf the adherent c&s were first extracted with Triton X- 100 and then wit and ~~~b-i~~i~ buffers, they iost both stresssf vinc~Pin, but s~HII fibers and patcks unaltered surface lashowed an apparently mina, as judged by t ing (fig. 2j). In these cytoskeletal preparations, intermediate ~~arne~ts couid bmelocated to areas occupied by the surface lam-

Fig. 2. (N, b) Cultured human fibroblasts treated with Triton X-100 and stained with TRITC-WGA. The surface lamina shows a different organization at the (a) substratum-facing side; (b) dorsal aspect of the cells. The distinctly linear patches of the WGA-binding lamina (d, J) appear to coalign with (c) actin-; (e) myosin-containing bundles of microtilaments, as seen in double-staining. Similarly some of the ventral patches

of vinculin (g) appear to be included in the surface lamina (cf arrows in h), as shown in double staining. In cells extracted with Triton X-100 and the low- and high-ionic, actomyosin-extracting buffers and then reacted with anti-vimentin antibodies (i) and with TRITC-WGA (j), both the lamina and intermediate filaments appear to maintain their normal organization.

ina (fig. 29. Cytoskeletal preparations, first reacted with TRITC-WGA, did not bind anti-rabbit immunoglobulin, ruling out the possibility of lectin-immunoglobulin re-

action as the basis of the colocalization in double-staining experiments (see also [ 161). Electron microscopy of the POX-WGAreacted cytoskeletal preparations demon-

Exp Cell Res 143 (1983)

Fig. 3. Electron microsopy of the cytoskeletal preparations of cui?ured human fibroblasts reacted with POXWGA and DAB. Horizontal sections. Note the ciose association between the plasma membrane remnants

decorated by the peroxidase-reaction prociuclirrri’o~i’.~ ~1 and the cytoskeletai structures, mainly composed fi* inlermediate filaments. ((1) X4OOO@0~) X65 000.

Grated patch-like plasma membrane remnants with peroxidase reaction product associated with them (fig. 30, b). Cytoskelela1 filaments seemed to interact closely with such membrane remnants.

the glycoproteins were able to bind the electrophoretical!y separated poiypeptides of the whole and extracted ccStswere

Su&uce

&c*oprateins

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In order to identify ceIi surface glycoproteins remaining in the detergent-resistant surface lamina, cultured fibroblasts were first labelled using a sialic acid-specific surface iabelling technique [ 131,extracted with Triton X- 100 and then subjected to gel electrophoresis and !&orography. This analysis revealed that many of the cell surface proteins were extracted already in Triton X400 and that only a 220 kD and a 140 kD surface glycoproteins were retained in the ~erna~~i~gsurface lamina after a further extraction with isw and high ionic actomyosin (fig. 4). In our previous study the 220 kD glycoprotein has been identified as fibronectin [17j. Hn order to detect which of

Fig. 4. Polypeptide profile Qluorography) of culture~d

human fibroblasts surface periodate/NaB3H,-techl7ique. 2. Triton X-100 extractable skeletons further extracted ionic, actomyosin-extracting

labelled with the mesaI, Unextracted cells: material: 3, Triton cytowith the low- and bighbuffers.

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6. Detection of the WGA-binding polypeptides of the cvtoskeletal preparations of cultured human fibroblasts by lectin affinity chromatography. Fluorography of the SDS-aolvacrvlamide gel. I, Surface-labelled (as in fig. 4) c&red tibroblasts; 2-4, Zwittergent-extract of the cytoskeletal preparations; 3, material bound to WtiA-Yepharose; 4, material bound to WGA-Sepharose in the presence of 0.2 M N-acetyl glucosamine.

Fig.

Fig. 5. Detection of the WGA-binding polypeptides among the electrophoretically separated polypeptides of cultured fibroblast and their cytoskeletons. I, 3, 5, Coomassie-blue staining of the polyacrylamide gels; 2, 4, 6, electrophoretic blots of the parallel runs reacted with POX-WGA and DAB. 1, 2, Whole cells; 3, 4, Triton X-lOO-extracted cells; 5, 6, Triton X100-extracted cells further extracted with the low- and high-ionic buffers.

transferred to nitrocellulose sheets which were then exposed first to POX-WGA and then to DAB. The results show that after Triton treatment and subsequent extractions with low- and high-ionic buffers there was only one major WGA-binding polypeptide left with an apparent molecular weight (MW) of 140 kD (fig. 5). A glycoprotein of a similar MW could also be recovered from the Zwittergent-extract of the surface-labelled cytoskeletal preparations using WGA-Sepharose beads (fig. 6). DISCUSSION The results of the present study show that cytoskeletal preparations of cultured human Iibroblasts retain a surface lamina Exp Cell Res 143 (1983)

which resists both Triton X-100 treatment and a complete dissociation of the actomyosin system of the cell. A 140 kD surface glycoprotein was revealed as the major glycoprotein component of the lamina. In substratum-attached cells, the surface lamina has been defined as part of a detergent-resistant cytoskeleton derived from plasma membrane proteins [5, 61. Originally it was detected in cultured fibroblasts and in HeLa cells [5], but similar structures have also been discovered in many other cells [6, 7, 18-221 and it is probably an ubiquitous property of cultured cells. Surface lamina has been suggested to be intimately connected to the underlying cytoskeleton, mainly to actin-containing microfilaments [l&-22]. The results of the present study show that the surface lamina of cultured human

~br~b~a§t$ can be visualized by means of a sialic acid-recognizing lectin, WGA. Flusrescence microscopic findings, usingfluoroWGA, indicate that the e original morphology of elI, suggesting that it is not snly conto specialized areas of the cell surface) as sites of stress fiber-cell surfaceinteraction [23j or focal adhesion sites whichrepresent termination sites of stress fibres 124, Pi]. On the other hand, the organization of the reticular lamina seems to be different on the ventral and dorsal surfaces of the cell possibly reflecting different constraints imposed on the cell surface components by adjacent struc%ures, such as cytoskeleton and pericellular matrix. Interestingly, stress ich were visualized both with antianti-my&n antibodies, appeared to imteract closely with straight parts of the Iamina at the dorsal face of the cells, whereas parts of the iamina at the ventral aspect of the cells coaligned with vinculin plaques, suggesting that the lamina may play a role in organizing the act yosin system of the surface iamina with cell. Tnteractions of the actomyosin system, however, do not appear $0 be an essential requirement for the integrity of the iamina, since a similar Ian-ha could be visualized also after a complete actomyosin dissolution. Previous studies with fibroblasts have inat most of the surface glyco ~~t~r~e~t-r~~~~tant Beta%preparations [S results with iymphoid ells have suggested a selective persistence of only some of the swface ~~y~~~~Qte~~~in the lamina, pro ably in association with the actin meshwork cyteplasmic aspect of cell surrane [ 1%211. Some surface glye also been found to remain in such cy%Q§ke~et~~~ [7j.

lectin blotting, that only mme of the cex surface glyco@roteins are retained in the cytoskeietal preparations. A 140 kD g@coprotein appeared as a major compoweni: c’ the actomynssn-free surface iamina. we have previously descri ed *b-heenrichmen: coproiein in cytoof a similar skeletons of cuhured human fibreblasts pg. It may be analogous %@ a g@xqxo:eiri ’ recently described by Cak-. ] and suggested.la! form ded, detergent-iv solub!e glycoprotein matrix at the surface G? cultured Rbroblast5. I The surface iamina has been sugges::ed to be connected tc the cytoptasmic cyts-

fsund to copurify with plasma membranes or cell surface g!ycoprotesns [B1 211. our results ako support the ysica?, de;ergent-resistant association bebveen swface iamina and these cyruskektal elements. T’P?iscn:;i*ci ik Wel’: provide one mecha~~~~~~~~~~~~~~i~ estab!ished synchrony between cell sur%cice and cytoskeletal phenomena [X3]. The skilful technical assistance of MS Pipsa jI4aipaii:en. MS Raili Taavela and MS Tuire Km3 is kindly acknowledged. This study was supported by the Picirish Medical Research Council, the Association of the Firrnish Life Insurance Companies, the Sigrid Ju~eligs Foundanion and Ike FirlIliSh Catlce; RescaKh Fiend.

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Exp Cell Res 143 (1983)

Received Mav 5, 1982 Revised version received July 27, 1982 Accepted July 28, 1982

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