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Dispersed and Aggregated Gap Junction Channels Identified by Immunogold Labeling of Freeze-Fractured Membranes
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
233, 240–251 (1997)
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Dispersed and Aggregated Gap Junction Channels Identified by Immunogold Labeling of Freeze-Fractured Membranes Dieter F. Hu¨lser,1 Beate Rehkopf, and Otto Traub* Biologisches Institut, Abt. Biophysik, Universita¨t Stuttgart, 70550 Stuttgart, Germany; and *Institut fu¨r Genetik, Abt. Molekulargenetik, Universita¨t Bonn, 53117 Bonn, Germany
An indirect immunogold labeling technique was applied to replicas of freeze-fractured membranes of rapidly frozen unfixed cells. The endogenous gap junction protein Cx43 of BICR/M1Rk rat mammary tumor cells was preferentially identified in quasi-crystalline gap junction plaques as were the transfected connexins Cx40, Cx43, and Cx45 in HeLa (human cervical carcinoma) cells. With this method we also detected contact areas with dispersed gap junction channels which are the only structural correlation for endogenous Cx45 in HeLa wild-type cells where no gap junction plaques exist. In double-transfected HeLa cells a colocalization of Cx40 and Cx43 was occasionally detected in quasicrystalline gap junction plaques, whereas in contact areas with dispersed particles only one Cx type was present. Our results indicate that functional gap junction channels exist outside the quasi-crystalline plaques. q 1997 Academic Press
INTRODUCTION
Gap junction channels permit direct signal transfer between most vertebrate cells; they are essential for the functional coordination of tissues and organs. In contact regions of adjacent cells up to several hundred of these channels are aggregated into gap junction plaques (for recent reviews see Shivers and McVicar [1] and Wolburg and Rohlmann [2]). Thin sections through contacting cells reveal gap junction areas as pentalaminar membrane structures where the intercellular gap of about 2–3 nm is bridged by dense material. Freeze fracturing of plasma membranes also cleaves these cell–cell channels (for a summary of freeze-fracture techniques, see Severs and Shotton [3]). A replicated gap junction plaque is identified by distinct particles on the protoplasmic fracture face (P-face) and by pits (extracted particles) on the extracellular fracture face 1 To whom correspondence and reprint requests should be addressed at Biologisches Institut, Abt. Biophysik, Pfaffenwaldring 57, D-70550 Stuttgart, Germany. Fax: 0049711/6855096. E-mail: [email protected].
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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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(E-face). Particles and pits represent individual hemichannels, the connexons. A connexon is a hexamer of membrane spanning proteins, the connexins (Cx), which are characterized by four transmembrane regions, two extracellular loops, and intracellular domains which mainly account for the differences in the connexin types (for reviews see Kumar and Gilula [4] and Goodenough et al. [5]). Whether heteromeric connexons will be formed is still controversial [6, 7] but heterotypic coupling between different connexons is well documented [8, 9]. With immunofluorescence a colocation of gap junctions containing more than one connexin was detected in mouse liver cells and in cultured mouse embryonic hepatocytes [10]. The resolution of this light microscopical technique allows no discrimination between gap junction plaques containing different Cx channels and several adjoining gap junction plaques, each containing identical Cx channels. However, immunogold labeling of ultrathin sections revealed a mixture of Cx26 and Cx32 in gap junction plaques of mouse liver cells [10], a colocation which was recently confirmed with a modified freeze-fracture cytochemical technique [11]. Conventional preparation methods for freeze fracturing require chemical fixation of the cells; most often glutaraldehyde is used, which, however, closes gap junction channels irreversibly within 1–2 min [12, 13]. In addition, fixed material is often infiltrated with glycerol as a cryoprotectant, which reduces the size of ice crystals formed during the freezing procedure but also increases the fluidity of cell membranes and may facilitate a rearrangement of membrane proteins. An alternative preparation method is rapid freezing of unfixed biological material. This preserves cellular structures in their native state only when the freezing rate is sufficiently high [14]. We have previously demonstrated that unfixed monolayer cells placed between gold specimen carriers and manually plunged into liquid propane have the same center to center spacings of gap junction channels as cells which were rapidly frozen by freon jet [15]. With the liquid propane technique we found polymorphic gap junction structures in cultured mammalian cells: dispersed particles were
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detected in addition to quasi-crystalline plaques, which are commonly observed with conventional freezing techniques [13]. These data are consistent with the idea of a structure–function relationship: dispersed gap junction channels could represent communication-competent channels, whereas quasi-crystalline ordered plaques may correspond to closed channels [16–19] (for a recent review see [2]). However, a clear identification of dispersed particles as gap junction channels is difficult, especially when the fracture plane does not change from one face to the other. Utilizing a combination of rapid freezing and immunogold labeling [11, 20], we tested whether besides gap junction plaques other membrane areas are also labeled with anti-connexin antibodies. Membrane replicas were cleaned with sodium dodecyl sulfate instead of sodium hypochlorit solution; thus, cellular material and unfractured portions of the membrane were solubilized, whereas integral membrane proteins were protected by the Pt/C cast. They remained in the replica and could be labeled with antibodies directed against cytoplasmic epitopes of these proteins [11]. With this indirect immunogold labeling technique we investigated permanently growing cell lines as well as Cxtransfected HeLa cells. Quasi-crystalline gap junction plaques were found to be labeled but so too were dispersed particles in areas which did not have the typical appearance of gap junctions. In double-transfected HeLa cells these contact areas predominantly contained only one Cx type of gap junction channels, whereas in quasi-crystalline gap junction plaques a mixture of Cx types was also observed. Gap junction plaques were not detected in HeLa wild-type cells [21, 8], but electrophysiological investigations revealed few gap junction channels with a low single channel conductance [22]. After probing these cells with antibody directed against Cx45, we have now identified gap junction channels as dispersed particles in nontypical gap junction areas. MATERIALS AND METHODS Cell culture. HeLa is a permanently growing epithelioid cell line derived from a human cervical carcinoma (for characterization see [22]). BICR/M1Rk is a neoplastic cell line derived from a spontaneous mammary tumor of the Marshall rat [23]. Cells were cultivated in Dulbecco’s modified Eagle’s medium (plus 3.7 g/liter NaHCO3 , 100 mg/liter streptomycin sulfate, 150 mg/liter penicillin G Å DMEM, supplemented with 10% newborn calf serum) at pH 7.4 and 377C in a humidified incubator with an 8% CO2/air mixture. HeLa cells were transfected with cDNA from several connexins [9]. Transfectants were also cultivated in DMEM, but supplemented with 10% fetal calf serum. Transfectants Cx40 and Cx45 were kept with 1 mg/ml puromycin, Cx43 with 1 mg/ml geneticin, and Cx40/43 with 0.5 mg/ ml puromycin and 0.3 mg/ml geneticin. Medium was renewed at 2to 3-day intervals, and cultures were passaged at confluency by a treatment with 0.25% trypsin in phosphate-buffered saline (PBS) without calcium and magnesium.
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Antibodies. Antibodies were prepared by immunizing rabbits with synthetic peptides conjugated to keyhole limpet hemocyanin. These specific peptide antibodies were directed against the last 22 amino acids of the COOH terminus of either Cx40 or Cx43 [24]. Antibodies directed against Cx45 were prepared as described by Butterweck et al. [25]. Briefly, rabbits were immunized by a fusion protein of glutathion S-transferase (GST) and the COOH terminus of Cx45 (Cx45c) which was synthesized in Escherichia coli. The resulting antiserum was purified by affinity chromatography: GST and GST–Cx45c were coupled to cyanobromide-activated Sepharose and the antiserum was affinity purified on a GST column and subsequently on a GST–Cx45c column. Antibodies were eluted with 3 M KSCN and dialyzed against PBS. Prior to use, these antibodies were diluted in PBS with 2% bovine serum albumin (BSA, Sigma A 4503): anti-Cx40 or anti-Cx43 1:80 and anti-Cx45 1:10. The following antibodies were purchased from Biotrend (Ko¨ln, Germany): Mouse antiCx43 (Nr. CT 1359), prior to use diluted 1:25 in PBS with 2% BSA; goat anti-mouse IgG (H / L), 15 nm gold (Nr. 115.022); goat antimouse IgG (H / L), 10 nm gold (Nr. 110.022); goat anti-rabbit IgG (H / L), 6 nm gold (Nr. 106.011); and goat anti-rabbit IgG (H / L), 10 nm gold (Nr. 110.011). Prior to use, these antibodies were diluted 1:30 in PBS with 2% BSA. Electron microscopy. For conventional freeze fracturing, monolayer cells in plastic petri dishes were washed in PBS and fixed with 2% glutaraldehyde (electron microscopy grade, Nr. 4239, E. Merck, Darmstadt, Germany) in PBS for 30 min, washed several times in PBS, and infiltrated with glycerol (Nr. 4093, E. Merck) solutions in PBS with increasing concentrations to a final glycerol concentration of 30%. After infiltration overnight at 47C, the cells were collected with a rubber policeman, pelleted, placed between two gold specimen carriers (4.6 mm diameter, Balzers, Liechtenstein), and frozen in liquid propane (about 90 K). Carrier sandwiches were mounted on a double replica table (BB 172 137-T, Balzers) and transferred into a Balzers 301 instrument. Cells were fractured under high vacuum and immediately replicated by shadowing with platinum/carbon (Pt/ C) at an angle of 457 to about 2 nm thickness by a high-voltage evaporation device (EVM 052A, Balzers) and stabilized by perpendicular carbon (C) evaporation (É20 nm). Replicas were cleaned overnight in 12% sodium hypochlorit solution, washed three times in distilled water, and transferred onto copper grids. For labeling with immunogold, cells were washed in PBS, collected, pelleted, and placed between two gold specimen carriers. The remaining fluid was drained off with a filter paper, and the carrier sandwich was immediately plunged into liquid propane. After fracturing and replicating of the unfixed material, replicas were floated on PBS, and treated for 1 h or overnight under continuous stirring in 2.5% sodium dodecyl sulfate (SDS, BDH Chemicals Ltd., Poole, England) containing 10 mM Tris–HCl plus 30 mM sucrose, pH 8.3 [11]. After washing 10 times in PBS, nonspecific reactions were blocked with 2% BSA and the replicas were put for 1 h on a droplet of primary antibody directed against connexin. Prior to use, both primary and secondary antibodies were centrifuged for 1 min at 5000g. After washing 10 times in PBS and a second treatment with BSA, replicas were put on a droplet of secondary antibody for 1 h. For double labeling the same procedure was followed with an appropriate mixture of diluted primary and secondary antibodies. Controls were treated with secondary antibodies only; in addition, Cx40-containing replicas were probed with antibodies directed against Cx43 and vice versa. After washing with PBS (10 times) and distilled water (2 times) replicas were positioned on copper grids, dried, and examined in a transmission electron microscope (EM 10, Zeiss, Oberkochen, Germany). When a membrane is freeze fractured, different faces are exposed and also a gap junction plaque is cleaved, as is schematically shown in Fig. 1. The fracture plane is preset by the lipid leaflets (solid line between lipids in Fig. 1A) and may change between the membranes of two attached cells. Thus, a gap junction plaque with the P-face of
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RESULTS
Conventionally Replicated Membranes Freeze fracturing of glutaraldehyde-fixed and glycerol-infiltrated cells that were coupled by gap junctions revealed smoothly contoured membrane areas with typical quasi-crystalline gap junction plaques, where both E- and P-face leaflets can be distinguished, as illustrated with BICR/M1Rk cells in Fig. 2A. In HeLa wild-type cells no gap junction plaques were identified even in the close neighborhood of tight junctions (Fig. 2B). Transfected HeLa cells regularly formed gap junction plaques, as is shown for Cx40 (Fig. 2C) and Cx43 transfectants (Fig. 2D). Immunogold-Labeled Gap Junction Plaques
FIG. 1. (A) Schematic section through a gap junction plaque with a given breaking line between the lipid leaflets. (B) Schematic section through a replica of a freeze-fractured and immunogold-labeled gap junction plaque. Connexons (9 nm), antibodies (24 nm), and immunogold (left side, 10 nm; right side, 15 nm) are drawn to the same scale. In the bottom cell, connexons in the P-face are cast with Pt/C and C and thus are protected against SDS solubilization, as are the bottom cell connexons under the E-face of the top cell. The P-surface cast of the top cell does not prevent solubilization of the counterpart connexons in the bottom cell.
the bottom cell and the E-face of the top cell may be replicated (Fig. 1B). A gap junction channel consists of two hemichannels, which are separated by the fracturing procedure. The P-face is then characterized by connexons which remain integrated in the protoplasmic half of the membrane and the Pt/C and C cast partially covers these proteins. In the E-face, holes of the extracted connexons are cast; the underlying connexons of the bottom cell come in contact with the evaporated material only at their extracellular part. In both cases connexons are protected from being solubilized by SDS [11]. The situation is different for the P-surface; here the connexons in the bottom cell are not cast and thus are not protected against solubilization by SDS. The extracellular parts of the connexons in the top cell will not be identified by our primary antibodies. In Fig. 1B the sizes of proteins, antibodies, and immunogold are drawn to scale. A connexon is about 8–9 nm long, with a 5-nm transmembrane segment and extracellular (gap) and intracellular segments of about 2 nm [26]. For tightly packed connexons the lattice constant is not less than 8 nm [26], so that we consider the connexons in a first approximation as cubes. When antibodies and immunogold particles (left side, 10 nm; right side, 15 nm) are drawn to the same scale, it is seen that steric hindrance prevents a high labeling efficiency, which may be further reduced by solubilization of connexons which were not sufficiently cast or by denaturating their COOH ends by the SDS washing procedure. Gold particles can be located in a
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Fractured membranes of unfixed cells are often not smooth but appear as a ruffled surface; however, typical quasi-crystalline gap junction plaques are easily identified. Figure 3 gives examples of gap junction plaques in membranes of BICR/M1Rk cells which were labeled with mouse anti-Cx43 antibody. The presented areas exhibit mainly E-faces tagged with 15-nm immunogold (Figs. 3A, 3B, and 3C) but also P-faces were labeled (C). In (D) labeled E- and P-faces of a gap junction plaque are replicated together with an unlabeled cytoplasmic organelle. Albeit the labeling efficiency is not high, the unequivocal specificity of labeling is striking, which can especially be seen in a higher magnification of (A) where two gold particles label a tiny Eface gap junction on the right side (B). As has been tested with Cx43-transfected HeLa cells, labeled gap junction plaques were found with both rabbit (Fig. 4A) and mouse (Fig. 4B) antibodies directed against Cx43. Concave or convex plaques of channels (Figs. 4A and 5B) suggest invaginations which precede the internalization and digestion of gap junction plaques. The labeling efficiency does not vary with the replicated membrane half (E- or P-face) of a gap junction plaque or with the size of the immunogold. With 6 nm, however, it is difficult to discriminate immunogold from replicated connexons. This can be seen in Fig. 5A, where an extended gap junction area in the membranes of Cx40-transfected HeLa cells is labeled with rabbit anti-Cx40 antibody and with 6-nm immunogold. These gold particles can easily be detected on the E-face but they are also present on the P-face. With the same rabbit anti-Cx40 antibody but with 10-nm immunogold secondary antibody it is obvious that both E- and Pfaces show a similar labeling efficiency (Figs. 5B–5D). Treatment with SDS for 10 min (Fig. 5A) or overnight (Fig. 3C) did not alter the labeling efficiency, indicating
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FIG. 2. Freeze-fractured membranes of cells which were chemically fixed with glutaraldehyde and infiltrated with glycerol. (A) BICR/ M1Rk cells. Gap junction plaque with P- and E-faces. (B) HeLa wild-type cells. Tight junctions, but no gap junction plaques. (C) Cx40transfected HeLa cells. Gap junction plaques with P- and E-faces surrounded by tight junctions. (D) Cx43-transfected HeLa cells. Gap junction plaques with P- and E-face in a tight junction area. Bars, 250 nm.
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FIG. 3. Gap junction plaques between BICR/M1Rk cells are labeled with 15-nm immunogold, which indicates Cx43. (A) Several gap junction plaques where mainly E-face is present. (B) Segment of (A) at higher magnification. Note the specificity of labeling. (C) Gap junction plaque where E- and P-faces are present. (D) Gap junction plaque with E- and P-faces and an attached cytoplasmic organelle. Bars, 250 nm.
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FIG. 4. Immunogold-labeled gap junction plaques between Cx43-transfected HeLa cells. (A) Rabbit antibody directed against Cx43. Invaginated plaque where mainly the P-face is exposed and labeled with 10-nm immunogold. (B) Mouse antibody directed against Cx43. Elongated P-face plaque is labeled with 15-nm immunogold. Bars, 250 nm.
that a sufficient portion of connexins was well protected. Variations in the concentrations of both primary and secondary antibodies did not notably influence the labeling. The indirect labeling of wild-type or transfected connexins in quasi-crystalline gap junction plaques revealed a negligible background and a high specificity. However, uncontrolled deposition of single immunogold particles or unspecific binding of both primary and secondary antibodies can never be excluded. Sometimes aggregates of immunogold were found in different regions of the replicas. These cases, however, could clearly be discriminated from specific binding in nontypical gap junction areas which was in most cases associated with replicated particles in a P-face. In the P-face areas of Figs. 4B and 5B particle aggregates are present which might not have been unequivocally identified as gap junction channels without their labeling. The problem of identification is more difficult for Cx45. In Cx45-transfected HeLa cells always typical quasi-crystalline gap junction plaques were present (Figs. 6A and 6B) and were as clearly labeled as were contact areas with dispersed particles (Figs. 6C and 6D).
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Immunogold-Labeled Contact Areas in HeLa Cells Gap junction plaques were not detected in HeLa wild-type cells, but electrophysiological measurements with high resolution revealed that they are deficiently coupled through channels which are characterized by a low single channel conductance [22]. Channels with similar electrical properties connect SkHep1 cells where they were identified as Cx45 gap junction channels [27]. We, therefore, probed HeLa wild-type cells with antibody directed against Cx45. Some results are presented in Fig. 7 where label is seen with dispersed particles in a tight junctional area (A), in an area of close contact (B), or with an invagination (C). Quasi-crystalline gap junction plaques, however, were never detected. As a control, these HeLa wild-type cells were probed with antibody directed against Cx43 and no label was found, as is shown for a tight junctional area (D). Double-Labeled Contact Regions To test whether the endogenous Cx45 channels are colocated with channels of a transfected connexin, we
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FIG. 5. Cx40-transfected HeLa cells labeled with rabbit anti-Cx40 antibody. (A) Gap junction plaques with E- and P-faces. The 6-nm immunogold is best seen with the E-face, but is also present with the P-face. (B) Two gap junction plaques labeled with 10-nm immunogold. (C) A gap junction plaque where mainly the E-face is labeled with 10-nm immunogold. (D) Elongated gap junction area where both faces are labeled with 10-nm immunogold. Bars, 250 nm.
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FIG. 6. Immunogold (10-nm)-labeled gap junction plaques and contact areas between Cx45-transfected HeLa cells. (A) Gap junction plaque with both E- and P-faces. (B) Three gap junction plaques. (C) Four contact areas with dispersed particles. (D) Two contact areas with dispersed particles. Bars, 250 nm.
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FIG. 7. Immunogold (10-nm)-labeling of HeLa wild-type cells. (A) Tight junctional area without typical gap junction structures but labeled with antibody directed against Cx45. (B) Area of close contact where dispersed particles are labeled with antibody directed against Cx45. (C) Invaginated area labeled with antibody directed against Cx45. Neither the E- nor the P-face of typical gap junctions is seen. (D) Control: In a tight junction area no label was found with antibody directed against Cx43. Bars, 250 nm.
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FIG. 8. Double-labeling of Cx43-transfected HeLa cells. (A) An area with small gap junction plaques is labeled for Cx43 (15 nm) and a contact area with separate label for Cx43 (15 nm) and Cx45 (10 nm). Some smaller contact sites are preferentially labeled for Cx43. (B) Contact region with two typical gap junction plaques labeled for Cx43 (15 nm) and a separate Cx45 contact area (10 nm). Bars, 250 nm.
double-labeled Cx43-transfected HeLa cells with antibodies directed against Cx43 and Cx45. As can be seen from Figs. 8A and 8B, membrane regions were tagged with 10-nm (Cx45) and 15-nm (Cx43) immunogold, but the label is separately clustered. In (A) an area with small gap junction plaques is labeled as Cx43—the transfected connexin—whereas in a contact area dispersed particles are discriminated as Cx43 and Cx45. A few other small spots are exclusively labeled as Cx43. In (B) a contact region is depicted which is labeled as Cx43, but well separated from a small Cx43 gap junction plaque a contact area is present with Cx45 labeled particles. The problem of colocalization was further investigated with double-transfected (Cx40 and Cx43) HeLa cells. Both homomeric channel types form gap junction plaques in single-transfected HeLa cells and these structures were also present in the double-transfected cells. Figures 9A and 9B show that they can be colocated in quasi-crystalline gap junction plaques, but some plaques and contact areas with dispersed particles contained only one channel type.
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DISCUSSION
A reliable structural identification of connexons in freeze-fractured and replicated membranes of vertebrate cells has always been limited to membrane areas where tightly packed particles on a P-face and an array of pits on an E-face were found in the same plaque (Figs. 2A, 2C, 2D, 3C, 5A, 5C, 6A, and 9B). However, a nonordered arrangement of membrane proteins with different particle to particle distances may also still be considered as a typical gap junction area (Figs. 4B and 5B, left side). Whether this variation in pattern reflects different physiological states of cells and/or cell-specific differences is controversially discussed [15–19, 28–31] (for review see [2]). When only dispersed particles are present (Figs. 6C and 6D), an indisputable identification is impossible. Membrane patches of attached cells and tight junctions can help to identify close cellular contacts (Figs. 7A and 7D); they do not certify, however, that particles in these contact areas represent gap junction channels. In our fixed preparations, tight junction structures were abundant in membranes of HeLa cells, but were not often apparent in rapidly frozen unfixed
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FIG. 9. Immunogold labeling of double-transfected (Cx40 and Cx43) HeLa cells. (A) Two gap junction plaques are preferentially labeled for Cx40 (10 nm), one plaque is clearly double labeled (10 and 15 nm). (B) A double-labeled gap junction plaque. A small gap junction plaque and contact areas with dispersed particles contain only Cx40. Bars, 250 nm.
cells (compare Figs. 2B, 2C, and 2D with 7A and 7D), since the strands are broken and unequally partitioned between E- and P-faces, a well-known phenomenon with unfixed material [18]. Indirect immunogold labeling with anti-connexin antibodies improves the identification of gap junction proteins in freeze-fractured membranes and will allow new insights in the life cycle of these membrane proteins. In HeLa wild-type cells gap junction plaques were neither detected in thin sections [8] nor on freeze-fracture replicas [8, 21], a result consistent with early electrophysiological measurements where no coupling could be detected [32]. However, from studies of metabolic coupling, HeLa wild-type cells were characterized by an intermediate uridine nucleotide transfer [33], and with high resolution patch clamp measurements gap junction channels with a single channel conductance of about 26 pS were resolved [22]. Similar electrical properties were found for Cx45 gap junction channels in SkHep1 cells [27]. From these findings we assumed that Cx45 gap junction channels connect HeLa wild-type cells. Indeed, after probing with antibody directed against Cx45 some nontypical gap junction areas were clearly labeled (Figs. 7A, 7B,and 7C and 8A
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and 8B). These results leave no doubt that functional gap junction channels are present as dispersed channels in contact areas. In addition, in double-labeling experiments such contact areas contain only one Cxtype, whereas in quasi-crystalline plaques both Cx types were present. Whether these structures represent different functional states in the life cycle of gap junction channels is an unresolved problem. Connexins are inserted as connexons in the plasma membrane via secretory vesicles [34–36] and labeled areas were also found at vesicle fusion sites (our unpublished results). It must be clarified, therefore, if labeling reveals contact sites with gap junction channels or noncontacting membranes with connexons. This might be answered with future experiments when extracellular and intracellular epitopes of connexons will be labeled separately with a freeze-fracture replica immunolabeling technique before [37] and after replicating. Interestingly, a calculation of access resistances revealed that the most favorable configuration of gap junction plaques is one in which channels are spaced as far from one another as possible [38]. Even when this assumption does not consider other requirements for the formation of gap junctions, it is tempt-
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ing to extend these estimates. Only a few percent of the electronmicroscopically identified gap junction channels are necessary to maintain the communicating paths since the total junctional conductances for the investigated Cx40- and Cx43-transfected Hela cell clones were 140 and 70 nS at comparable open probabilities. The single channel conductance depends on the phosphorylation state and is 120 and 150 pS for Cx40 and 40, 60, and 90 pS for Cx43 [7]. From these figures one can conclude that on average about 1000 gap junction channels were open between two contacting cells. In Cx40-transfected HeLa cells several large quasi-crystalline gap junction plaques were very often found, each containing up to 1000 and more channels (see Fig. 5), whereas in Cx43transfected HeLa cells smaller plaques were regularly found (see Fig. 4). Since the number and the size of gap junction plaques vary with the total amount and the life cycle of connexins, the degree of coupling is only indirectly reflected by plaques. It might well be that dispersed gap junction channels in contact areas are sufficient to maintain the necessary communication between contacting cells. REFERENCES 1. Shivers, R. R., and McVicar, L. K. (1995) Micro. Res. Tech. 31, 437–445. 2. Wolburg, H., and Rohlmann, A. (1995) Int. Rev. Cytol. 157, 315– 373. 3. Severs, N. J., and Shotton, D. M., Eds. (1995) Rapid Freezing, Freeze Fracture, and Deep Etching, Wiley–Liss, New York. 4. Kumar, N. M., and Gilula, N. B. (1996) Cell 84, 381–388. 5. Goodenough, D. A., Goliger, J. A., and Paul, D. L. (1996) Annu. Rev. Biochem. 65, 475–502. 6. Stauffer, K. A. (1995) J. Biol. Chem. 270, 6768–6772. 7. Eckert, R., and Hu¨lser, D. F. (1995) in Progress in Cell Research, Vol. 4, Intercellular Communication through Gap Junctions (Kanno, Y., Kataoka, K., Shiba, Y., Shibata, Y., and Shimazu, T., Eds.), pp. 423–426, Elsevier, Amsterdam. 8. Bra¨uner, T., Schmid, A., and Hu¨lser, D. F. (1990) Invasion Metastasis 10, 18–30. 9. Elfgang, C., Eckert, R., Lichtenberg-Frate´, H., Butterweck, A., Traub, O., Klein, R. A., Hu¨lser, D. F., and Willecke, K. (1995) J. Cell Biol. 129, 805–817. 10. Traub, O., Look, J., Dermietzel, R., Bru¨mmer, F., Hu¨lser, D., and Willecke, K. (1989) J. Cell Biol. 108, 1039–1051. 11. Fujimoto, K. (1995) J. Cell Sci. 108, 3443–3449. 12. Spray, D. C., Harris, A. L., and Bennett, M. V. L. (1981) Biophys. J. 33, 108a.
13. Hu¨lser, D. F., Paschke, D., and Greule, J. (1989) in Electron Microscopy of Subcellular Dynamics (Plattner, H., Ed.), pp. 33– 49, CRC Press, Boca Raton. 14. Moor, H. (1986) in Cryotechniques in Biological Electron Microscopy (Steinbrecht, R. A., and Zierold, K., Eds.), p. 175, Springer-Verlag, Berlin. 15. Mu¨ller, A., Blanz, E. W., Laub, G., and Hu¨lser, D. F. (1985) Studia Biophys. 110, 185–192. 16. Peracchia, C., and Dulhunty, A. F. (1974) J. Cell Biol. 63(2, Pt 2), 263a. 17. Peracchia, C., and Dulhunty, A. F. (1976) J. Cell Biol. 70, 419– 439. 18. Raviola, E., Goodenough, D. A., and Raviola, G. (1980) J. Cell Biol. 87, 273–279. 19. Sikerwar, S., and Malhotra, S. (1981) Eur. J. Cell Biol. 25, 319– 323. 20. Gruijters, W. T. M., Kistler, J., Bullivant, S., and Goodenough, D. A. (1987) J. Cell Biol. 104, 565–572. 21. Hu¨lser, D. F., and Demsey, A. (1973) Z. Naturforsch. 28c, 603– 606. 22. Eckert, R., Dunina-Barkovskaya, A., and Hu¨lser, D. F. (1993) Pflu¨egers Arch. Eur. J. Physiol. 424, 335–342. 23. Rajewsky, M. F., and Gru¨neisen, A. (1972) Eur. J. Immunol. 2, 445–447. 24. Traub, O., Eckert, R., Lichtenberg-Frate´, H., Elfgang, C., Bastide, B., Scheidtmann, K. H., Hu¨lser, D. F., and Willecke, K. (1994) Eur. J. Cell Biol. 64, 101–112. 25. Butterweck, A., Gergs, U., Elfgang, C., Willecke, K., and Traub, O. (1994) J. Membr. Biol. 141, 247–256. 26. Sosinsky, G. E. (1992) Electron Microsc. Rev. 5, 59–76. 27. Moreno, A. P., Laing, J. G., Beyer, E. C., and Spray, D. C. (1995) Am. J. Physiol. 268, C356–C365. 28. Hanna, R. B., Reese, T. S., Ornberg, R. L., Spray, D. C., and Bennett, M. V. L. (1981) J. Cell Biol. 91, 125a. 29. De´le`ze, J., and Herve´, J. C. (1986) J. Membr. Biol. 93, 11–21. 30. De´le`ze, J., and Herve´, J. C. (1983) J. Membr. Biol. 74, 203– 215. 31. Green, C. R., and Severs, N. J. (1984) J. Cell Biol. 99, 453–463. 32. Hu¨lser, D. F., and Webb, D. J. (1973) Exp. Cell Res. 80, 210– 222. 33. Pitts, J. D., and Simms, J. W. (1977) Exp. Cell Res. 104, 153– 163. 34. Musil, L. S., and Goodenough, D. A. (1995) in Progress in Cell Research, Vol. 4, Intercellular Communication through Gap Junctions (Kanno, Y., Kataoka, K., Shiba, Y., Shibata, Y., and Shimazu, T., Eds.), pp. 327–330, Elsevier, Amsterdam. 35. Laird, D. W. (1996) J. Bioenerg. Biomembr. 28, 311–318. 36. Shivers, R. R., and Bowman, P. D. (1985) J. Submicrosc. Cytol. 17, 199–203. 37. Severs, N. J. (1995) in Rapid Freezing, Freeze Fracture, and Deep Etching (Severs, N. J., and Shotton, D. M., Eds.), pp. 173– 208, Wiley–Liss, New York. 38. Hall, J. E., and Gourdie, R. G. (1995) Micros. Res. Techn. 31, 446–451.
Received November 25, 1996 Revised version received March 4, 1997
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EX973568
Dispersed and Aggregated Gap Junction Channels Identified by Immunogold Labeling of Freeze-Fractured Membranes Dieter F. Hu¨lser,1 Beate Rehkopf, and Otto Traub* Biologisches Institut, Abt. Biophysik, Universita¨t Stuttgart, 70550 Stuttgart, Germany; and *Institut fu¨r Genetik, Abt. Molekulargenetik, Universita¨t Bonn, 53117 Bonn, Germany
An indirect immunogold labeling technique was applied to replicas of freeze-fractured membranes of rapidly frozen unfixed cells. The endogenous gap junction protein Cx43 of BICR/M1Rk rat mammary tumor cells was preferentially identified in quasi-crystalline gap junction plaques as were the transfected connexins Cx40, Cx43, and Cx45 in HeLa (human cervical carcinoma) cells. With this method we also detected contact areas with dispersed gap junction channels which are the only structural correlation for endogenous Cx45 in HeLa wild-type cells where no gap junction plaques exist. In double-transfected HeLa cells a colocalization of Cx40 and Cx43 was occasionally detected in quasicrystalline gap junction plaques, whereas in contact areas with dispersed particles only one Cx type was present. Our results indicate that functional gap junction channels exist outside the quasi-crystalline plaques. q 1997 Academic Press
INTRODUCTION
Gap junction channels permit direct signal transfer between most vertebrate cells; they are essential for the functional coordination of tissues and organs. In contact regions of adjacent cells up to several hundred of these channels are aggregated into gap junction plaques (for recent reviews see Shivers and McVicar [1] and Wolburg and Rohlmann [2]). Thin sections through contacting cells reveal gap junction areas as pentalaminar membrane structures where the intercellular gap of about 2–3 nm is bridged by dense material. Freeze fracturing of plasma membranes also cleaves these cell–cell channels (for a summary of freeze-fracture techniques, see Severs and Shotton [3]). A replicated gap junction plaque is identified by distinct particles on the protoplasmic fracture face (P-face) and by pits (extracted particles) on the extracellular fracture face 1 To whom correspondence and reprint requests should be addressed at Biologisches Institut, Abt. Biophysik, Pfaffenwaldring 57, D-70550 Stuttgart, Germany. Fax: 0049711/6855096. E-mail: [email protected].
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(E-face). Particles and pits represent individual hemichannels, the connexons. A connexon is a hexamer of membrane spanning proteins, the connexins (Cx), which are characterized by four transmembrane regions, two extracellular loops, and intracellular domains which mainly account for the differences in the connexin types (for reviews see Kumar and Gilula [4] and Goodenough et al. [5]). Whether heteromeric connexons will be formed is still controversial [6, 7] but heterotypic coupling between different connexons is well documented [8, 9]. With immunofluorescence a colocation of gap junctions containing more than one connexin was detected in mouse liver cells and in cultured mouse embryonic hepatocytes [10]. The resolution of this light microscopical technique allows no discrimination between gap junction plaques containing different Cx channels and several adjoining gap junction plaques, each containing identical Cx channels. However, immunogold labeling of ultrathin sections revealed a mixture of Cx26 and Cx32 in gap junction plaques of mouse liver cells [10], a colocation which was recently confirmed with a modified freeze-fracture cytochemical technique [11]. Conventional preparation methods for freeze fracturing require chemical fixation of the cells; most often glutaraldehyde is used, which, however, closes gap junction channels irreversibly within 1–2 min [12, 13]. In addition, fixed material is often infiltrated with glycerol as a cryoprotectant, which reduces the size of ice crystals formed during the freezing procedure but also increases the fluidity of cell membranes and may facilitate a rearrangement of membrane proteins. An alternative preparation method is rapid freezing of unfixed biological material. This preserves cellular structures in their native state only when the freezing rate is sufficiently high [14]. We have previously demonstrated that unfixed monolayer cells placed between gold specimen carriers and manually plunged into liquid propane have the same center to center spacings of gap junction channels as cells which were rapidly frozen by freon jet [15]. With the liquid propane technique we found polymorphic gap junction structures in cultured mammalian cells: dispersed particles were
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detected in addition to quasi-crystalline plaques, which are commonly observed with conventional freezing techniques [13]. These data are consistent with the idea of a structure–function relationship: dispersed gap junction channels could represent communication-competent channels, whereas quasi-crystalline ordered plaques may correspond to closed channels [16–19] (for a recent review see [2]). However, a clear identification of dispersed particles as gap junction channels is difficult, especially when the fracture plane does not change from one face to the other. Utilizing a combination of rapid freezing and immunogold labeling [11, 20], we tested whether besides gap junction plaques other membrane areas are also labeled with anti-connexin antibodies. Membrane replicas were cleaned with sodium dodecyl sulfate instead of sodium hypochlorit solution; thus, cellular material and unfractured portions of the membrane were solubilized, whereas integral membrane proteins were protected by the Pt/C cast. They remained in the replica and could be labeled with antibodies directed against cytoplasmic epitopes of these proteins [11]. With this indirect immunogold labeling technique we investigated permanently growing cell lines as well as Cxtransfected HeLa cells. Quasi-crystalline gap junction plaques were found to be labeled but so too were dispersed particles in areas which did not have the typical appearance of gap junctions. In double-transfected HeLa cells these contact areas predominantly contained only one Cx type of gap junction channels, whereas in quasi-crystalline gap junction plaques a mixture of Cx types was also observed. Gap junction plaques were not detected in HeLa wild-type cells [21, 8], but electrophysiological investigations revealed few gap junction channels with a low single channel conductance [22]. After probing these cells with antibody directed against Cx45, we have now identified gap junction channels as dispersed particles in nontypical gap junction areas. MATERIALS AND METHODS Cell culture. HeLa is a permanently growing epithelioid cell line derived from a human cervical carcinoma (for characterization see [22]). BICR/M1Rk is a neoplastic cell line derived from a spontaneous mammary tumor of the Marshall rat [23]. Cells were cultivated in Dulbecco’s modified Eagle’s medium (plus 3.7 g/liter NaHCO3 , 100 mg/liter streptomycin sulfate, 150 mg/liter penicillin G Å DMEM, supplemented with 10% newborn calf serum) at pH 7.4 and 377C in a humidified incubator with an 8% CO2/air mixture. HeLa cells were transfected with cDNA from several connexins [9]. Transfectants were also cultivated in DMEM, but supplemented with 10% fetal calf serum. Transfectants Cx40 and Cx45 were kept with 1 mg/ml puromycin, Cx43 with 1 mg/ml geneticin, and Cx40/43 with 0.5 mg/ ml puromycin and 0.3 mg/ml geneticin. Medium was renewed at 2to 3-day intervals, and cultures were passaged at confluency by a treatment with 0.25% trypsin in phosphate-buffered saline (PBS) without calcium and magnesium.
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Antibodies. Antibodies were prepared by immunizing rabbits with synthetic peptides conjugated to keyhole limpet hemocyanin. These specific peptide antibodies were directed against the last 22 amino acids of the COOH terminus of either Cx40 or Cx43 [24]. Antibodies directed against Cx45 were prepared as described by Butterweck et al. [25]. Briefly, rabbits were immunized by a fusion protein of glutathion S-transferase (GST) and the COOH terminus of Cx45 (Cx45c) which was synthesized in Escherichia coli. The resulting antiserum was purified by affinity chromatography: GST and GST–Cx45c were coupled to cyanobromide-activated Sepharose and the antiserum was affinity purified on a GST column and subsequently on a GST–Cx45c column. Antibodies were eluted with 3 M KSCN and dialyzed against PBS. Prior to use, these antibodies were diluted in PBS with 2% bovine serum albumin (BSA, Sigma A 4503): anti-Cx40 or anti-Cx43 1:80 and anti-Cx45 1:10. The following antibodies were purchased from Biotrend (Ko¨ln, Germany): Mouse antiCx43 (Nr. CT 1359), prior to use diluted 1:25 in PBS with 2% BSA; goat anti-mouse IgG (H / L), 15 nm gold (Nr. 115.022); goat antimouse IgG (H / L), 10 nm gold (Nr. 110.022); goat anti-rabbit IgG (H / L), 6 nm gold (Nr. 106.011); and goat anti-rabbit IgG (H / L), 10 nm gold (Nr. 110.011). Prior to use, these antibodies were diluted 1:30 in PBS with 2% BSA. Electron microscopy. For conventional freeze fracturing, monolayer cells in plastic petri dishes were washed in PBS and fixed with 2% glutaraldehyde (electron microscopy grade, Nr. 4239, E. Merck, Darmstadt, Germany) in PBS for 30 min, washed several times in PBS, and infiltrated with glycerol (Nr. 4093, E. Merck) solutions in PBS with increasing concentrations to a final glycerol concentration of 30%. After infiltration overnight at 47C, the cells were collected with a rubber policeman, pelleted, placed between two gold specimen carriers (4.6 mm diameter, Balzers, Liechtenstein), and frozen in liquid propane (about 90 K). Carrier sandwiches were mounted on a double replica table (BB 172 137-T, Balzers) and transferred into a Balzers 301 instrument. Cells were fractured under high vacuum and immediately replicated by shadowing with platinum/carbon (Pt/ C) at an angle of 457 to about 2 nm thickness by a high-voltage evaporation device (EVM 052A, Balzers) and stabilized by perpendicular carbon (C) evaporation (É20 nm). Replicas were cleaned overnight in 12% sodium hypochlorit solution, washed three times in distilled water, and transferred onto copper grids. For labeling with immunogold, cells were washed in PBS, collected, pelleted, and placed between two gold specimen carriers. The remaining fluid was drained off with a filter paper, and the carrier sandwich was immediately plunged into liquid propane. After fracturing and replicating of the unfixed material, replicas were floated on PBS, and treated for 1 h or overnight under continuous stirring in 2.5% sodium dodecyl sulfate (SDS, BDH Chemicals Ltd., Poole, England) containing 10 mM Tris–HCl plus 30 mM sucrose, pH 8.3 [11]. After washing 10 times in PBS, nonspecific reactions were blocked with 2% BSA and the replicas were put for 1 h on a droplet of primary antibody directed against connexin. Prior to use, both primary and secondary antibodies were centrifuged for 1 min at 5000g. After washing 10 times in PBS and a second treatment with BSA, replicas were put on a droplet of secondary antibody for 1 h. For double labeling the same procedure was followed with an appropriate mixture of diluted primary and secondary antibodies. Controls were treated with secondary antibodies only; in addition, Cx40-containing replicas were probed with antibodies directed against Cx43 and vice versa. After washing with PBS (10 times) and distilled water (2 times) replicas were positioned on copper grids, dried, and examined in a transmission electron microscope (EM 10, Zeiss, Oberkochen, Germany). When a membrane is freeze fractured, different faces are exposed and also a gap junction plaque is cleaved, as is schematically shown in Fig. 1. The fracture plane is preset by the lipid leaflets (solid line between lipids in Fig. 1A) and may change between the membranes of two attached cells. Thus, a gap junction plaque with the P-face of
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RESULTS
Conventionally Replicated Membranes Freeze fracturing of glutaraldehyde-fixed and glycerol-infiltrated cells that were coupled by gap junctions revealed smoothly contoured membrane areas with typical quasi-crystalline gap junction plaques, where both E- and P-face leaflets can be distinguished, as illustrated with BICR/M1Rk cells in Fig. 2A. In HeLa wild-type cells no gap junction plaques were identified even in the close neighborhood of tight junctions (Fig. 2B). Transfected HeLa cells regularly formed gap junction plaques, as is shown for Cx40 (Fig. 2C) and Cx43 transfectants (Fig. 2D). Immunogold-Labeled Gap Junction Plaques
FIG. 1. (A) Schematic section through a gap junction plaque with a given breaking line between the lipid leaflets. (B) Schematic section through a replica of a freeze-fractured and immunogold-labeled gap junction plaque. Connexons (9 nm), antibodies (24 nm), and immunogold (left side, 10 nm; right side, 15 nm) are drawn to the same scale. In the bottom cell, connexons in the P-face are cast with Pt/C and C and thus are protected against SDS solubilization, as are the bottom cell connexons under the E-face of the top cell. The P-surface cast of the top cell does not prevent solubilization of the counterpart connexons in the bottom cell.
the bottom cell and the E-face of the top cell may be replicated (Fig. 1B). A gap junction channel consists of two hemichannels, which are separated by the fracturing procedure. The P-face is then characterized by connexons which remain integrated in the protoplasmic half of the membrane and the Pt/C and C cast partially covers these proteins. In the E-face, holes of the extracted connexons are cast; the underlying connexons of the bottom cell come in contact with the evaporated material only at their extracellular part. In both cases connexons are protected from being solubilized by SDS [11]. The situation is different for the P-surface; here the connexons in the bottom cell are not cast and thus are not protected against solubilization by SDS. The extracellular parts of the connexons in the top cell will not be identified by our primary antibodies. In Fig. 1B the sizes of proteins, antibodies, and immunogold are drawn to scale. A connexon is about 8–9 nm long, with a 5-nm transmembrane segment and extracellular (gap) and intracellular segments of about 2 nm [26]. For tightly packed connexons the lattice constant is not less than 8 nm [26], so that we consider the connexons in a first approximation as cubes. When antibodies and immunogold particles (left side, 10 nm; right side, 15 nm) are drawn to the same scale, it is seen that steric hindrance prevents a high labeling efficiency, which may be further reduced by solubilization of connexons which were not sufficiently cast or by denaturating their COOH ends by the SDS washing procedure. Gold particles can be located in a
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Fractured membranes of unfixed cells are often not smooth but appear as a ruffled surface; however, typical quasi-crystalline gap junction plaques are easily identified. Figure 3 gives examples of gap junction plaques in membranes of BICR/M1Rk cells which were labeled with mouse anti-Cx43 antibody. The presented areas exhibit mainly E-faces tagged with 15-nm immunogold (Figs. 3A, 3B, and 3C) but also P-faces were labeled (C). In (D) labeled E- and P-faces of a gap junction plaque are replicated together with an unlabeled cytoplasmic organelle. Albeit the labeling efficiency is not high, the unequivocal specificity of labeling is striking, which can especially be seen in a higher magnification of (A) where two gold particles label a tiny Eface gap junction on the right side (B). As has been tested with Cx43-transfected HeLa cells, labeled gap junction plaques were found with both rabbit (Fig. 4A) and mouse (Fig. 4B) antibodies directed against Cx43. Concave or convex plaques of channels (Figs. 4A and 5B) suggest invaginations which precede the internalization and digestion of gap junction plaques. The labeling efficiency does not vary with the replicated membrane half (E- or P-face) of a gap junction plaque or with the size of the immunogold. With 6 nm, however, it is difficult to discriminate immunogold from replicated connexons. This can be seen in Fig. 5A, where an extended gap junction area in the membranes of Cx40-transfected HeLa cells is labeled with rabbit anti-Cx40 antibody and with 6-nm immunogold. These gold particles can easily be detected on the E-face but they are also present on the P-face. With the same rabbit anti-Cx40 antibody but with 10-nm immunogold secondary antibody it is obvious that both E- and Pfaces show a similar labeling efficiency (Figs. 5B–5D). Treatment with SDS for 10 min (Fig. 5A) or overnight (Fig. 3C) did not alter the labeling efficiency, indicating
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FIG. 2. Freeze-fractured membranes of cells which were chemically fixed with glutaraldehyde and infiltrated with glycerol. (A) BICR/ M1Rk cells. Gap junction plaque with P- and E-faces. (B) HeLa wild-type cells. Tight junctions, but no gap junction plaques. (C) Cx40transfected HeLa cells. Gap junction plaques with P- and E-faces surrounded by tight junctions. (D) Cx43-transfected HeLa cells. Gap junction plaques with P- and E-face in a tight junction area. Bars, 250 nm.
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FIG. 3. Gap junction plaques between BICR/M1Rk cells are labeled with 15-nm immunogold, which indicates Cx43. (A) Several gap junction plaques where mainly E-face is present. (B) Segment of (A) at higher magnification. Note the specificity of labeling. (C) Gap junction plaque where E- and P-faces are present. (D) Gap junction plaque with E- and P-faces and an attached cytoplasmic organelle. Bars, 250 nm.
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FIG. 4. Immunogold-labeled gap junction plaques between Cx43-transfected HeLa cells. (A) Rabbit antibody directed against Cx43. Invaginated plaque where mainly the P-face is exposed and labeled with 10-nm immunogold. (B) Mouse antibody directed against Cx43. Elongated P-face plaque is labeled with 15-nm immunogold. Bars, 250 nm.
that a sufficient portion of connexins was well protected. Variations in the concentrations of both primary and secondary antibodies did not notably influence the labeling. The indirect labeling of wild-type or transfected connexins in quasi-crystalline gap junction plaques revealed a negligible background and a high specificity. However, uncontrolled deposition of single immunogold particles or unspecific binding of both primary and secondary antibodies can never be excluded. Sometimes aggregates of immunogold were found in different regions of the replicas. These cases, however, could clearly be discriminated from specific binding in nontypical gap junction areas which was in most cases associated with replicated particles in a P-face. In the P-face areas of Figs. 4B and 5B particle aggregates are present which might not have been unequivocally identified as gap junction channels without their labeling. The problem of identification is more difficult for Cx45. In Cx45-transfected HeLa cells always typical quasi-crystalline gap junction plaques were present (Figs. 6A and 6B) and were as clearly labeled as were contact areas with dispersed particles (Figs. 6C and 6D).
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Immunogold-Labeled Contact Areas in HeLa Cells Gap junction plaques were not detected in HeLa wild-type cells, but electrophysiological measurements with high resolution revealed that they are deficiently coupled through channels which are characterized by a low single channel conductance [22]. Channels with similar electrical properties connect SkHep1 cells where they were identified as Cx45 gap junction channels [27]. We, therefore, probed HeLa wild-type cells with antibody directed against Cx45. Some results are presented in Fig. 7 where label is seen with dispersed particles in a tight junctional area (A), in an area of close contact (B), or with an invagination (C). Quasi-crystalline gap junction plaques, however, were never detected. As a control, these HeLa wild-type cells were probed with antibody directed against Cx43 and no label was found, as is shown for a tight junctional area (D). Double-Labeled Contact Regions To test whether the endogenous Cx45 channels are colocated with channels of a transfected connexin, we
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FIG. 5. Cx40-transfected HeLa cells labeled with rabbit anti-Cx40 antibody. (A) Gap junction plaques with E- and P-faces. The 6-nm immunogold is best seen with the E-face, but is also present with the P-face. (B) Two gap junction plaques labeled with 10-nm immunogold. (C) A gap junction plaque where mainly the E-face is labeled with 10-nm immunogold. (D) Elongated gap junction area where both faces are labeled with 10-nm immunogold. Bars, 250 nm.
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FIG. 6. Immunogold (10-nm)-labeled gap junction plaques and contact areas between Cx45-transfected HeLa cells. (A) Gap junction plaque with both E- and P-faces. (B) Three gap junction plaques. (C) Four contact areas with dispersed particles. (D) Two contact areas with dispersed particles. Bars, 250 nm.
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FIG. 7. Immunogold (10-nm)-labeling of HeLa wild-type cells. (A) Tight junctional area without typical gap junction structures but labeled with antibody directed against Cx45. (B) Area of close contact where dispersed particles are labeled with antibody directed against Cx45. (C) Invaginated area labeled with antibody directed against Cx45. Neither the E- nor the P-face of typical gap junctions is seen. (D) Control: In a tight junction area no label was found with antibody directed against Cx43. Bars, 250 nm.
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FIG. 8. Double-labeling of Cx43-transfected HeLa cells. (A) An area with small gap junction plaques is labeled for Cx43 (15 nm) and a contact area with separate label for Cx43 (15 nm) and Cx45 (10 nm). Some smaller contact sites are preferentially labeled for Cx43. (B) Contact region with two typical gap junction plaques labeled for Cx43 (15 nm) and a separate Cx45 contact area (10 nm). Bars, 250 nm.
double-labeled Cx43-transfected HeLa cells with antibodies directed against Cx43 and Cx45. As can be seen from Figs. 8A and 8B, membrane regions were tagged with 10-nm (Cx45) and 15-nm (Cx43) immunogold, but the label is separately clustered. In (A) an area with small gap junction plaques is labeled as Cx43—the transfected connexin—whereas in a contact area dispersed particles are discriminated as Cx43 and Cx45. A few other small spots are exclusively labeled as Cx43. In (B) a contact region is depicted which is labeled as Cx43, but well separated from a small Cx43 gap junction plaque a contact area is present with Cx45 labeled particles. The problem of colocalization was further investigated with double-transfected (Cx40 and Cx43) HeLa cells. Both homomeric channel types form gap junction plaques in single-transfected HeLa cells and these structures were also present in the double-transfected cells. Figures 9A and 9B show that they can be colocated in quasi-crystalline gap junction plaques, but some plaques and contact areas with dispersed particles contained only one channel type.
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DISCUSSION
A reliable structural identification of connexons in freeze-fractured and replicated membranes of vertebrate cells has always been limited to membrane areas where tightly packed particles on a P-face and an array of pits on an E-face were found in the same plaque (Figs. 2A, 2C, 2D, 3C, 5A, 5C, 6A, and 9B). However, a nonordered arrangement of membrane proteins with different particle to particle distances may also still be considered as a typical gap junction area (Figs. 4B and 5B, left side). Whether this variation in pattern reflects different physiological states of cells and/or cell-specific differences is controversially discussed [15–19, 28–31] (for review see [2]). When only dispersed particles are present (Figs. 6C and 6D), an indisputable identification is impossible. Membrane patches of attached cells and tight junctions can help to identify close cellular contacts (Figs. 7A and 7D); they do not certify, however, that particles in these contact areas represent gap junction channels. In our fixed preparations, tight junction structures were abundant in membranes of HeLa cells, but were not often apparent in rapidly frozen unfixed
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FIG. 9. Immunogold labeling of double-transfected (Cx40 and Cx43) HeLa cells. (A) Two gap junction plaques are preferentially labeled for Cx40 (10 nm), one plaque is clearly double labeled (10 and 15 nm). (B) A double-labeled gap junction plaque. A small gap junction plaque and contact areas with dispersed particles contain only Cx40. Bars, 250 nm.
cells (compare Figs. 2B, 2C, and 2D with 7A and 7D), since the strands are broken and unequally partitioned between E- and P-faces, a well-known phenomenon with unfixed material [18]. Indirect immunogold labeling with anti-connexin antibodies improves the identification of gap junction proteins in freeze-fractured membranes and will allow new insights in the life cycle of these membrane proteins. In HeLa wild-type cells gap junction plaques were neither detected in thin sections [8] nor on freeze-fracture replicas [8, 21], a result consistent with early electrophysiological measurements where no coupling could be detected [32]. However, from studies of metabolic coupling, HeLa wild-type cells were characterized by an intermediate uridine nucleotide transfer [33], and with high resolution patch clamp measurements gap junction channels with a single channel conductance of about 26 pS were resolved [22]. Similar electrical properties were found for Cx45 gap junction channels in SkHep1 cells [27]. From these findings we assumed that Cx45 gap junction channels connect HeLa wild-type cells. Indeed, after probing with antibody directed against Cx45 some nontypical gap junction areas were clearly labeled (Figs. 7A, 7B,and 7C and 8A
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and 8B). These results leave no doubt that functional gap junction channels are present as dispersed channels in contact areas. In addition, in double-labeling experiments such contact areas contain only one Cxtype, whereas in quasi-crystalline plaques both Cx types were present. Whether these structures represent different functional states in the life cycle of gap junction channels is an unresolved problem. Connexins are inserted as connexons in the plasma membrane via secretory vesicles [34–36] and labeled areas were also found at vesicle fusion sites (our unpublished results). It must be clarified, therefore, if labeling reveals contact sites with gap junction channels or noncontacting membranes with connexons. This might be answered with future experiments when extracellular and intracellular epitopes of connexons will be labeled separately with a freeze-fracture replica immunolabeling technique before [37] and after replicating. Interestingly, a calculation of access resistances revealed that the most favorable configuration of gap junction plaques is one in which channels are spaced as far from one another as possible [38]. Even when this assumption does not consider other requirements for the formation of gap junctions, it is tempt-
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IMMUNOGOLD LABELING OF FREEZE-FRACTURED GAP JUNCTIONS
ing to extend these estimates. Only a few percent of the electronmicroscopically identified gap junction channels are necessary to maintain the communicating paths since the total junctional conductances for the investigated Cx40- and Cx43-transfected Hela cell clones were 140 and 70 nS at comparable open probabilities. The single channel conductance depends on the phosphorylation state and is 120 and 150 pS for Cx40 and 40, 60, and 90 pS for Cx43 [7]. From these figures one can conclude that on average about 1000 gap junction channels were open between two contacting cells. In Cx40-transfected HeLa cells several large quasi-crystalline gap junction plaques were very often found, each containing up to 1000 and more channels (see Fig. 5), whereas in Cx43transfected HeLa cells smaller plaques were regularly found (see Fig. 4). Since the number and the size of gap junction plaques vary with the total amount and the life cycle of connexins, the degree of coupling is only indirectly reflected by plaques. It might well be that dispersed gap junction channels in contact areas are sufficient to maintain the necessary communication between contacting cells. REFERENCES 1. Shivers, R. R., and McVicar, L. K. (1995) Micro. Res. Tech. 31, 437–445. 2. Wolburg, H., and Rohlmann, A. (1995) Int. Rev. Cytol. 157, 315– 373. 3. Severs, N. J., and Shotton, D. M., Eds. (1995) Rapid Freezing, Freeze Fracture, and Deep Etching, Wiley–Liss, New York. 4. Kumar, N. M., and Gilula, N. B. (1996) Cell 84, 381–388. 5. Goodenough, D. A., Goliger, J. A., and Paul, D. L. (1996) Annu. Rev. Biochem. 65, 475–502. 6. Stauffer, K. A. (1995) J. Biol. Chem. 270, 6768–6772. 7. Eckert, R., and Hu¨lser, D. F. (1995) in Progress in Cell Research, Vol. 4, Intercellular Communication through Gap Junctions (Kanno, Y., Kataoka, K., Shiba, Y., Shibata, Y., and Shimazu, T., Eds.), pp. 423–426, Elsevier, Amsterdam. 8. Bra¨uner, T., Schmid, A., and Hu¨lser, D. F. (1990) Invasion Metastasis 10, 18–30. 9. Elfgang, C., Eckert, R., Lichtenberg-Frate´, H., Butterweck, A., Traub, O., Klein, R. A., Hu¨lser, D. F., and Willecke, K. (1995) J. Cell Biol. 129, 805–817. 10. Traub, O., Look, J., Dermietzel, R., Bru¨mmer, F., Hu¨lser, D., and Willecke, K. (1989) J. Cell Biol. 108, 1039–1051. 11. Fujimoto, K. (1995) J. Cell Sci. 108, 3443–3449. 12. Spray, D. C., Harris, A. L., and Bennett, M. V. L. (1981) Biophys. J. 33, 108a.
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Received November 25, 1996 Revised version received March 4, 1997
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