J. Cell Sci. 5a, 313-325 (1981) 313 Printed in Great Britain © Company of Biologists Limited 1981

A BIOCHEMICAL DISSECTION OF THE CARDIAC INTERCALATED DISK: ISOLATION OF SUBCELLULAR FRACTIONS CONTAINING FASCIA ADHERENTES AND GAP JUNCTIONS

C. A. L. S. COLACO AND W. HOWARD EVANS* National Institute for Medical Research, Mill Hill, London NW-j \AA

SUMMARY In view of our limited knowledge of the biochemical composition of intercellular junctions, a method was developed for the preparation from rats and mice of plasma membranes containing cardiac intercalated disks. When these membranes were extracted with deter- gents, e.g. AMauryl sarcosinate or deoxycholate, the detergent-insoluble material contained structures derived mainly from fascia adherentes junctions, but a few gap junctions and maculae adherentes were also present. When the detergent extraction was carried out at an alkaline pH, the maculae adherentes junctions were dissolved. Fractionation of the detergent- insoluble extract on a sucrose gradient yielded a fraction containing fascia adherentes junction of density 1-20-1-26 g/cm'. Gap junctions banded at a lower density, 1-16-1-20 g/cm3. Poly- acrylamide gel electrophoresis showed that the major polypeptide bands in the fascia adherentes-enriched fraction were of molecular weights 134000, 108000, 62-64000, 58000, 47000 and 43000. Although fractions with the gap junctions were contaminated by fascia adherentes junctions, the major polypeptides were calculated by subtraction to be of mol. wt 37000, 26000 and 19000. Two glycoproteins corresponding to minor polypeptides visualized by Coomassie Blue staining were present in the fascia adherentes fraction. Comparison of the fasci aadherentes-ennched fraction with a Z-disc fraction prepared from rabbit indicated a different morphology and polypeptide composition.

INTRODUCTION A distinctive feature of cells organized into tissues and organs is the presence on the plasma membrane of various types of junctional specializations on the mediating cell-cell adhesion and communication. Although well-characterized morphologically, little is known of their composition and molecular organization. Methods for the isolation and biochemical characterization of intercellular junctions have been described for cow nose epidermis maculae adherentes (Skerrow & Matolsty, 1974; Drochmans et al. 1978; Skerrow, 1979), synaptic junctions (Matus, 1978) and gap junctions (Evans & Gurd, 1972; Goodenough & Stoeckenius, 1972). The present paper extends our knowledge of the composition of mammalian intercellular junctions by describing procedures for dissecting the junctional components of the cardiac plasma membrane. The cardiac plasma membrane incorporates a specialized region termed the • Address correspondence to this author, u CKL52 314 C. A. L. S. Colaco and W, H. Evans intercalated disk, which facilitates intercardiocytic adhesion and communication. The intercellular junctions present are the macula adherens, a junction mediating cell-cell adhesion, the fascia at which the attach to the sarco- lemma; and the nexus or , which provides the intercellular channels mediating intercardiocytic communication (McNutt & Fawcett, 1969; Simpson, Rayns & Ledingham, 1974; McNutt, 1975; De Haan, Williams, Ypey & Clapham, 1981; Page, 1978). Using a cardiac plasma membrane fraction containing intact and fragmented intercalated disks, we describe procedures utilizing detergents that remove the non-junctional membranes and amorphous material, and leave a residue highly enriched in junctional components. The most numerous junctions present j n the preparations were fascia adherens junctions, and these were shown to have a different composition from isolated Z disks. A fraction containing gap junctions was also isolated.

MATERIALS AND METHODS Preparation of intercellular junctions Intercalated disks were isolated from mouse or rat hearts as described previously (Colaco & Evans, 1981) with the following modifications. Hearts (15 g) were disrupted using an Ultra- turrax tissue homogenizer (3x53, at setting 2-5) followed by homogenization in a loose- fitting Dounce homogenizer. The homogenate was filtered through 2 layers of muslin and centrifuged at 500 g for 5 min. The pelleted material was extracted overnight at 4 °C with o-6 M-KC1 dissolved in 8% (w/v) sucrose and was then centrifuged at 500 g for 5 min. The pelleted material was resuspended in 25 % (w/v) sucrose, layered onto 37 %, 45 %, 50% and 54% (w/v) sucrose solutions (6 ml of each) and centrifuged at 98000 £ for 2 h. The intercalated disk-containing sarcolemmal fraction was collected at the 45% to 50% (w/v) sucrose interface. The intercalated disks were collected and resuspended in JV-lauryl sarco- sinate at a concentration of 1 g detergent per 4 g protein. The iV-lauryl sarcosinate was prepared 83 a 5% (w/v) solution dissolved in C225 M-Tris-HCl and the final pH was 78. In some experiments, the JV-lauryl sarcosinate was adjusted to pH 10. The fractions were dispersed using a Dounce homogenizer fitted with a tight-fitting pestle, left on ice with occasional stirring for 30 min and then centrifuged at 98000 £ for 20 min. The pelleted detergent- insoluble material was resuspended in 25% (w/v) sucrose containing 0-5% Triton Xioo sonicated (Branson Sonicator, 3x53 bursts, setting 4) and introduced onto a discontinuous, density gradient containing 6 ml of 37%, 45%, 54% and 72% (w/v) sucrose dissolved in o-i% Triton Xioo. After centrifugation at 98000 j* for 2 h, the interfaces were collected, diluted with 10 mM-Tris-HCl (pH 10) and centrifuged at ioooo# for 15 min in plastic tubes (Sterilin). The pellets were analysed by sodium dodecyl sulphate/polyacrylamide gel electro- phoresis and electron microscopy. All density-gradient centrifugations were carried out in a Beckman SW27 rotor and all the solutions used in the fractionation procedure contained 1 mM phenylmethylsulphonyl fluoride.

Preparation of cardiac Z discs Rabbit hearts (40 g) were homogenized using an Ultraturrax (3x53, at setting 2^5) followed by homogenization using a loose-fitting Dounce homogenizer in 10 mM-Tris-histidine (pH 7'8), o-i mM-EDTA and 20 mM-sodium pyrophosphate. The homogenate was filtered through 2 layers of muslin and centrifuged at 2000 g for 5 min. The pellet was resuspended in 8 % (w/v) sucrose and loaded onto an MSE A XII zonal rotor containing linear gradients of 8 % to 36 % (300 ml) and 36% to 60% (600 ml) (w/v) sucrose. The 2 linear gradients were separated by 36% (w/v) sucrose (150 ml) and underlayered by a cushion of 60% (w/v) sucrose. After centrifugation at 3000 rev./min for 30 min, the rotor was unloaded and material banding Composition of intercellular junctions 315 at 36% (w/v) sucrose was collected, extracted with Sarcosyl (see above) and the composition of the pellet was examined by electron microscopy and shown to contain material resembling Z-disk remnants.

Electrophoresis SDS/polyacrylamide gel electrophoresis was carried out using the method of Laemmli (1971). The molecular weight markers used were: /?-galactosidase (130000), phosphorylase a (94000), catalase (60000), aldolase (40000), carbonic anhydrase (29000) and myoglobin (17500). Glycoprotein bands in the gels wer edetected by exploiting their property of binding specifically to iodinated concanavalin A (Gurd & Evans, 1976). Polypeptide patterns in poly- acrylamide gels were traced by using a Joyce-Lobel densitometer connected to a CALCOMP printer. Electron microscopy Fractions were routinely examined by negative staining with 1% sodium silicotungstate. Fractions for conventional transmission electron microscopy were processed as a pellet. The pelleted material was fixed overnight in 4% glutaraldehyde buffered in 50 mM-sodium cacodylate (pH 7-5) and post-fixed in 1% buffered osmium tetroxide. Samples were stained en bloc with buffered 1 % uranyl acetate, dehydrated using graded ethanol and embedded in Araldite using propylene oxide as a penetrating agent. Sections were cut using an LKB microtome, stained with lead citrate and uranyl acetate and examined in a Phillips 300 electron microscope.

RESULTS Fractionation of homogenates yielded a sarcolemmal fraction containing large numbers of intercalated disks (Fig. 1). This fraction was used to prepare the constituent intercellular junctions of the intercalated disk by solubilization of the non-junctional membranes in various detergents followed by a sucrose-density centrifugation step.

Detergent extraction and subfractionation of the intercalated disk-containing sarcolemmal fraction Extraction of the intercalated disk-containing sarcolemmal fraction with 1-5% N- lauryl sarcosinate at pH 10 solubilized approximately 95% of the protein. Morpho- logical examination of the detergent-insoluble fraction showed paired filamentous matrices, some gap junctions, structures resembling fascia adherentes junctions and amorphous material (Fig. 2). After extraction with detergent, the structures resembling fascia adherentes junctions showed no unit membrane profile but the cytoplasmic filamentous mats always remained paired (Fig. 2). When the detergent extraction of the parent fraction was carried out at pH 7-9, some maculae adherentes junctions were also observed in the detergent-insoluble fraction (Fig. 2). Analysis by SDS/ polyacrylamide gel electrophoresis of the detergent-insoluble fraction prepared from rat and mouse hearts showed 2 major doublets with average molecular weights of 62-64000 and 58000 as well as many other minor components (Fig. 3 A). Many of the preparations also showed minor bands of 29000 and 37000 mol. wt and a 43000 mol. wt -component was also present. Extraction of intercalated disks with deoxy- cholate at pH 10 (used at the same concentration as iV-lauryl sarcosinate) solubilized approximately 90% of the protein and the detergent-insoluble material contained 316 C.A.L. S. Colaco and W. H. Evans major bands of 54000, 43000 and 29000 mol. wt, as well as a prominent band of 34000 mol. wt (Fig. 3 A). Using both detergents to extract the intercalated disk fraction, a doublet of average mol. wt of 64000 was prominent. On the basis of the specific binding of iodinated ligand none of the major polypeptides of the detergent- insoluble fractions was found to be glycosylated, but 2 minor polypeptides of 180000

Fig. 1. Electron micrograph of mouse intercalated disk-containing plasma membrane fraction. Arrowheads point to strips of intercalated disks; arrows point to gap-junction areas, x 29000. and 38000 mol. wt were identified as glycoproteins in fractions from both rat and mouse hearts (Fig. 3B). The major glycoprotein of the deoxycholate-insoluble- material of approx. 88000 mol. wt was also prominent in the parent intercalated disk-containing fraction. Since Z disks have also been reported to be insoluble in detergents (Sainsbury & Bullard, 1980), the polypeptide composition of the detergent-insoluble fraction prepared from the intercalated disks was compared to that of a fraction containing

Fig. 2. Electron micrograph of intercalated disk-containing plasma membrane fraction after extraction with 1-5 % AMauryl sarcosinate, pH 10. x 20750. Inset: higher magnification electron micrograph ( x 97 500) showing differences between macula (arrowhead) and fascia adherentei junctions (arrows) after extraction with detergent at pH 7'8. Composition of intercellular junctions 317 318 C. A. L. S. Colaco and W. H. Evans Composition of intercellular junctions 319 material closely resembling Z disk isolated from cardiac muscle by zonal centri- fugation followed by a detergent extraction step (Fig. 4B). The polypeptide com- ponents of the detergent-insoluble fraction were found to be different to those present in the fraction containing cardiac Z disks (Fig. 5 A and Table 1). Fractionation of the detergent-insoluble material was carried out on sucrose gradients in an attempt to reduce the complexity of the detergent-extracted material. This required the inclusion of o-i % Triton X100 in the sucrose solutions to minimize reaggregation of the sonicated material. Electron microscopic examination of all the

Table 1. Major polypeptides identified in the gap junction, fascia adherentes and Z disk fractions prepared from heart tissue and their comparison with major structural proteins of cardiac muscle

Cardiac muscle Gap Fascta adherentes junctions (mol1. WtX IO"1) Z disks Component Reference

— — 180 (heavy chain) — — 150 — — — — 134 — Geiger et al. (1980) — 108 100 a-actinin Mooseker & Stephens (1980) — 81 — — — — 62-64* — Fimbrin Bretscher & Weber (1980) — 58* — Lazarides (1980) — — 55 Z disk protein Ohashi & Maruyama (1979) — 47 — — — — 43 43 — fTropomyosin Sainsbury & Bullard 37 37 IZ disk protein (1980) 34 — — — — 29 — — — — 26 — — — — 19 — — Myosin (light chain) — • These bands appear as cioublets. The mol. wts were estimated from Figs. 5A and 9. In identifying the gap-junction pOly- peptides, the polypeptides of the contaminating fascia adherentes junctions were subtracted. subfractions collected at the sucrose-gradient interface showed the presence of varying amounts of the paired filamentous matrices and amorphous material. The fraction collected at the 54% to 72% (w/v) sucrose interface (Fig. 6) contained large areas filled with paired filamentous matrices, and although the vesicular profiles present in the parent fraction (Fig. 2) were absent, some amorphous material was still present. The gap junctions were found mainly at the 37% to 45 % (w/v) sucrose interface (Fig. 5) and when examined by negative staining showed a hexagonal lattice of subunits with a 7-8 nm lattice constant (not shown). Freeze-fracture of the isolated cardiac gap junctions also showed a hexagonal lattice of intramembranous particles 8-9 nm in diameter (not shown). C.A.L. S. Colaco and W. H. Evans 4A B

Mol. wts X10"3 Composition of intercellular junctions 321 Analysis of the subfractions collected from the sucrose interfaces by SDS/poly- acrylamide gel electrophoresis showed 2 major polypeptide bands of average molecular weights of 150000 and 58000 as well as a band of 62-64000 mol. wt common to all the subfractions (Fig. 7, Table 1). The 62-64000 mol. wt component was the major

Fig. 6. Electron micrograph of fraction collected from the 54 % to 72 % (w/v) sucrose gradient interface (see Fig. 1). x 45 300. component in the 54% to 72% (w/v) sucrose interface fraction (Fig. 7), which contained predominantly the paired filamentous matrices. Prominent polypeptides of approx. 58000 and 43000 mol. wt were also seen in this fraction. The doublet of average molecular weight 58000 was the major component of the material found at

Fig. 4. A. Polyacrylamide gel electrophoresis of: (a) N-lauryl sarcosinate-extracted (pH 10) intercalated disk-containing plasma membrane fraction. (6) iV-lauryl sarco- sinate-extracted Z-disk-containing fraction. B. Electron micrograph of Z-disk fraction prepared by extraction with detergent of the material collected from the zonal rotor (see Materials and methods), x 97500. Fig. 5. Electron micrograph of fraction collected from the 37 % to 45 % (w/v) sucrose gradient interface showing gap junctions present (arrows), x 25000. Inset: gap junction, x 250000. 322 C. A. L. S. Colaco and W. H. Evans

Mol.wtX 10-3 94 60 40 29 17-5

94 60 40 29 17-5 Mol.wtX 10"3 Fig. 7. Densitometer tracings of polyacrylamide gel electrophoresis separation stained with Coomassie Blue of gap junction-enriched fraction {A), fascia adherentes- enriched fraction (B), and pellet (C). the bottom of the gradient, which contained by far the highest proportion of amorphous material in the fractions collected from the sucrose gradient.

DISCUSSION In cardiac tissue, specialized junctional complexes occur as a conglomerate in the intercalated disk: a plasma membrane region that maintains the cardiocytes in close apposition for mechanical and electrical purposes. In working myocardium, the fascia adherens is by far the most abundant of the 3 types of intercellular junctions present (Sommer & Johnson, 1979). The maculae adherentes and the gap junctions (nexus) are the 2 other junctional specializations present, the latter representing about 10% of the intercalated disk area in rat ventricular muscle (Page & McCallister, 1973). In a previous paper (Colaco & Evans, 1981), a method for the isolation of a sarcolemmal fraction containing the intercalated disk region of the cardiac plasma membrane was described. Although the intercalated disks present in the sarcolemmal fraction varied in length, they appeared to be representative of those seen in intact cardiac muscle. All 3 junction specializations were retained, with the fascia adherentes being the most abundant (Fig. 1). Extraction of the intercalated disk-containing Composition of intercellular junctions 323 sarcolemmal fraction using detergents solubilized most of the non-junctional mem- branes and left a residue of junctional remnants and amorphous material. The main structural components of the detergent-insoluble material were paired filamentous matrices and the assignment of the origin of these structures was required. Since Z disks have been reported to be insoluble in most detergents (Sainsbury & Bullard, 1980), a procedure was developed for their isolation from cardiac tissue and their structure and composition was compared with the paired filamentous matrices present in the detergent-extracted intercalated disk fraction. The results showed that Z disks isolated from cardiac muscle showed a distinct unpaired morphology as well as a different polypeptide composition from the paired fiilamentous matrices derived from intercalated disk fractions. The 2 adherens-type junctions present in intercalated disks emerged as the likely origin of the paired filamentous matrices. The maculae adherentes junctions were unlikely candidates on the basis of their ultrastructural appearance (Fig. 2). Furthermore, maculae adherentes-type junctions isolated from bovine nasal epidermis have been reported to be soluble at alkaline pH (Drochmans et al. 1978; Skerrow, 1979; Skerrow & Matolsty, 1974). In the present work, extraction of the intercalated disk-containing sarcolemmal fraction with detergent at pH 10 resulted in few maculae adherentes junctions being observed, leaving the paired filamentous matrices as the major components in the detergent-insoluble material. The paired filamentous matrices were thus identified as detergent-extracted fascia adherentes structures. Although little unit membrane structure could be recognized after extraction with detergent, the filamentous matrices were invariably arranged in pairs, suggesting that the intercellular links had persisted. The nature of the intercellular links connecting the junction halves is not known, but these components may comprise the mechanism for transmission of the contractile force across the fascia adherens between cardiocytes in intact muscle. Fractionation of the detergent-insoluble material on sucrose gradients initially gave poor yields of the subfractions, owing to the aggregation of the detergent- insoluble material leading to the recovery of large amounts of material at the bottom of the gradient. The use of sonication to disperse the aggregates and the inclusion of o-i % Triton X100 in the gradients minimized reaggregation and greatly improved the yield of the subfractions recovered from the gradient. The gap junctions now isolated from cardiac tissue showed a similar morphology to those isolated from liver (Evans & Gurd, 1972; Goodenough & Stoeckenius, 1972). However, the cardiac gap junctions generally showed a less-ordered lattice arrangement and in thin sections more cytoplasmic elaboration was apparent. Similar results have been reported by Kensler & Goodenough (1980), who described a procedure for the isolation of gap junctions from mouse myocardium. These authors identified 4 major polypeptides of 47000, 38000, 33500 and 31000 mol. wt as possible components of the cardiac gap junctions. In the present work, the gap junction-enriched fraction, equilibrated at the 37% to 45%(w/v) sucrose interface, and polypeptides of approx. 37000, 29000, 26000 and 19000 mol. wt were identified (Table 1). It was reasoned that the other major polypeptides also present in this fraction were probably contributed by the fasciae adherentes that were present. 324 C.A.L. S. Colaco and W. H. Evans The fraction enriched in fasciae adherentes junctions was collected at the 54% to 72% (w/v) sucrose interface. This fraction contained a major band (possibly a doublet) of approx. 64000 mol.'wt and 2 prominent doublets of average molecular weights of 150000 and 58000 (Table 1). Polypeptides corresponding in electrophoretic mobility to actin and a-actinin were also present. Recently, 2 polypeptides postulated as being involved in actin-membrane linkages have been located to the cardiac intercalated disk by using immunocytochemical techniques. These are vinculin, a 130000 mol. wt polypeptide located in cardiac junctions using antibodies against material prepared from chick gizzard (Geiger, Tokuyasu, Dutton & Singer, 1980), and a 68000 mol. wt polypeptide fimbrin, located using antibodies raised against material from intestinal epithelial microvilli (Bretscher & Weber, 1980). Polypeptides with similar electrophoretic mobilities to both vinculin and fimbrin were present in the fascia adherentes-enriched fraction and their identity with the authentic poly- peptides is currently being investigated. An component termed desmin, of approx. 58000 mol. wt, has also been localized immunohistochemically to the cardiac intercalated disk (Lazarides, 1980) and the possible co-identification of desmin with the major 58000 mol. wt component prominent in both the fascia adherentes- and gap junction-enriched fractions also needs to be established. The present paper thus identifies some of the major polypeptide components in the fraction containing cardiac fascia adherentes and gap junctions. However, caution must still be exercised in attributing all these polypeptides to the fascia adherentes and gap junctions present in the respective fractions, for regions of amorphous material were present in all fractions. In this respect, antibodies raised against the individual polypeptides in the fractions now isolated should provide further inform- ation on the molecular organization of the junctions within the intercalated disk- containing region of the plasma membrane of the working myocardium.

C. A. L. S. Colaco thanks the MRC for a research studentship. We thank Kate Sullivan and Elaine Brown for help with the electron microscopy, and Dr Milan Nermut for the freeze- fracture.

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