Cx47 regional expression and subcellular distribution
The regional expression of Cx47 in adult mouse brain was assessed by immunoblotting whole homogenates of various brain areas using 12.5% polyacrylamide gels (). In all brain regions examined, anti-Cx47 Ab36-4700 detected a protein band migrating at about 49 kDa. Additional faint bands detected at higher and lower molecular weights, particularly in thalamus and medulla, are of unknown identity. The 49 kDa band observed in brain corresponded to the migration profile of Cx47 detected by Ab36-4700 in HeLa cells stably transfected with Cx47. In comparison, homogenates from control HeLa cells transfected with empty vector showed absence of Cx47 detection, indicating specificity of the anti-Cx47 antibody. Very little variation in Cx47 levels was observed along the rostro-caudal axis of brain, including forebrain cerebral cortex to subcortical areas to hindbrain medullary regions, although hypothalamus contained slightly lower Cx47 levels than other structures. Immunobloting showed an absence of Cx47 in sciatic nerve, even with high levels of protein loading and long immunoblot-to-film exposures (not shown).
Fig. 1 Regional expression of Cx47 protein in adult mouse brain. Immunoblots show a migration profile of Cx47 in brain (lanes 1–7) comparable to that observed in Cx47-transfected HeLa cells (lane 9), and an absence of Cx47 in control empty vector-transfected (more ...)
The subcellular distribution of Cx47 in comparison with that of Cx32 and Cx29 in adult mouse brain is shown in . In immunoblots of subcellular fractions examined using 10% polyacrylamide gels, Cx47 migrated slightly slower at 51–53 kDa than seen in 12.5% gels, and appeared as a doublet of bands, possibility due to differential mobilities of Cx47 arising from post-translational modifications, similar to the altered migration patterns of various phosphorylated forms of Cx43 (Musil and Goodenough, 1991
; Hossain et al., 1994
). In subcellular fractions examined on 12.5% gels, the relative levels of Cx47 in the various fractions was similar to that shown in , but only a single band was evident with migration profile corresponding to that seen in brain regions and in HeLa cells (not shown). The highest levels of Cx47 appeared in the microsomal P3 fraction, and in the synaptosomal fraction which is known to contain glial membrane contaminants as well as elevated levels of astrocytic Cxs (Lynn et al., 2001b
). Curiously, and unexpected given the membrane localization of Cxs, moderate levels were also observed in the soluble fraction, suggesting a readily releasable, possibly intracellular pool of Cx47 that may have gone undetected by immunohistochemistry.
The lowest levels of Cx47 were observed in the crude synaptosomal–mitochondrial fraction, and in the myelin fraction where it was barely detectable. In contrast, Cx32 and Cx29 were most concentrated in the myelin fraction, with little evidence of their presence in the soluble fraction. In addition to recognition of the monomeric forms of Cx32 and Cx29, the anti-Cx32 and anti-Cx29 antibodies also detected bands migrating at 51 kDa and at 50 kDa, respectively, which likely correspond to the dimeric forms of these Cxs, as previously confirmed for Cx32 using WT and Cx32 knockout mice (Nagy et al., 2003b
), and Cx29 transfected HeLa cells (Li et al., 2002
). Designation of these bands as the dimeric forms of Cx32 and Cx29 is further supported by their similar distribution profile in subcellular fractions, and similar levels of enrichment in the myelin fraction as the monomeric forms of these Cxs. Confirmation that membrane elements of myelin were successfully isolated and enriched during subcellular fractionation was provided by immunoblots showing that the myelin compared with all other fractions contained substantially greater levels of CNPase, which was previously reported to be a highly concentrated, insoluble component of myelin.
Immunofluorescence labeling of Cx47
In sections throughout adult mouse CNS, anti-Cx47 antibody produced robust immunolabelling of cell bodies having a distribution pattern and morphological appearance of oligodendrocytes (as confirmed ultrastructurally in results presented below). At low magnification, the minimal non-specific background labeling evident with this antibody readily revealed immunopositive cells having a moderate and even distribution in cerebral cortex, and a more dense distribution in gray matter of the thalamus and spinal cord (). Oligodendrocytes in white matter were also labeled for Cx47 (), but generally more weakly than those in gray matter. Similar broadly distributed labeling of Cx47 in association with oligodendrocyte somata was observed in all CNS regions examined, including olfactory bulb, hippocampus, striatum, hypothalamus, midbrain and medulla (not shown). Optimal immunolabelling for Cx47 was achieved in brains of mice perfused with 2% formaldehyde/picric acid fixative with no post-fixation, and labeling was slightly or substantially suppressed using 4% formaldehyde without post-fixation or 4% formaldehyde with a 1.5 h post-fixation, respectively.
Fig. 3 Low magnification immunofluorescence micrographs showing the distribution of Cx47-immunopositive cells in various regions of adult mouse CNS. Labeling for Cx47 is seen associated with oligodendrocyte cell bodies in all layers of the cerebral cortex (A), (more ...)
In sections double-labeled for the oligodendrocyte-myelin marker CNPase and for Cx47, it appeared that virtually all CNPase-immunopositive cells were also immunopositive for Cx47, as illustrated in the hippocampus () and the hypothalamus (). While the low magnification fields shown in these areas contained an abundance of myelinated fibers as revealed by their immunolabelling for CNPase, these fibers were either unlabelled or displayed only faint, fine punctate labeling for Cx47. Other brain areas also displayed a sparse association of Cx47 with myelinated fibers in both gray and white matter (). Similar results were obtained in sections double-labeled for Cx47 and the oligodendrocyte-myelin marker MAG (not shown).
Fig. 4 Double immunofluorescence micrographs showing relationships of cells labeled for CNPase, Cx32 and Cx47 in adult mouse brain. (A–D) The same fields in the hippocampus (A, B) and hypothalamus (C, D) showing correspondence of cells (arrows) labeled (more ...)
The cellular localization of Cx47 was further examined in relation to that of Cx32, which was previously shown to be concentrated in both oligodendrocyte somata and along myelinated fibers (Li et al., 1997
; Nagy et al., 2003a
). After double-labeling of sections derived from diverse areas of mouse brain, many oligodendrocyte somata found to be immunolabelled for Cx32 were also labeled for Cx47, as shown in cerebral cortex () and globus pallidus (). Cx32 was also distributed along myelinated fibers in these () and other regions, whereas the same fibers displayed only scant labeling for Cx47 ().
The percentage of CNPase-positive oligodendrocytes that were also positive for Cx29, Cx32 and Cx47 was quantitatively examined to determine whether subpopulations of oligodendrocytes differentially express the three Cxs. Greater than 9300 CNPase-positive oligodendrocytes in various brain regions were counted in sections simultaneously labeled and displaying the robust immunofluorescence for Cx47 or Cx32 shown in and , and in those exhibiting the typically low but still identifiable labeling for Cx29 associated with oligodendrocyte somata (Nagy et al., 2003a
). As shown in , 93% to 98% of CNPase-positive oligodendrocytes in the brain regions listed were found to be immunolabelled for Cx29, Cx32 and Cx47, suggesting that virtually all oligodendrocytes express all three Cxs.
Percentage of CNPase-positive oligodendrocytes immunolabelled for Cx29, Cx32 or Cx47 in various brain regions of adult mice
Laser scanning confocal microscopy () was used for detailed analyses of Cx47 immunolabelling associated with oligodendrocyte somata, and to determine Cx47 relationships with oligodendrocytic Cx32 and as-trocytic Cx43. As can be gleaned from lower magnification images (), and readily evident by confocal immunofluorescence, labeling for Cx47 appeared exclusively as puncta decorating the periphery of oligodendrocyte somata and their initial processes. The density and spatial distribution of these immunopositive puncta on cells can be best appreciated in composite images of multiple Z-scans through single oligodendrocytes, with rotation of the image in the x axis. This is shown in the case of a Cx47-labeled oligodendrocyte, rotated 135° at 45° intervals (), displaying greater than fifty Cx47-positive puncta associated with its somata and along short stretches of its initial processes.
Fig. 5 Laser scanning confocal immunofluorescence of Cx47-immunopositive puncta on CNPase-labeled oligodendrocyte somata, and their co-localization with Cx32 and Cx43. (A) Z-stack of 34 scans showing the same oligodendrocyte rotated through 135° at 45° (more ...)
In single confocal scans of oligodendrocytes labeled by triple immunofluorescence for Cx47 and Cx32, combined with labeling of CNPase for identification of oligodendrocytes, the vast majority of Cx47-positive and Cx32-positive puncta associated with these cells throughout mouse brain consistently displayed a high degree of co-localization, as shown in the cerebral cortex () and hypothalamus (). As shown in cerebral cortex () and hippocampus (), similar single confocal scans of triple immunofluorescence for Cx47 and astrocytic Cx43, combined with labeling for CNPase, indicated substantial co-association of Cx47-positive puncta with Cx43-positive puncta along the periphery and initial processes of oligodendrocytes, presumably reflecting localization of these Cxs to gap junctions between oligodendrocytes and astrocytes. However, due to the lack of Cx47 expression in astrocytes, together with the much greater abundance of Cx43 associated with gap junctions between astrocytes, only a small proportion of Cx43-positive puncta were co-associated with Cx47-positive puncta.
Co-localization of Cx47 with ZO-1 in brain and HeLa cells
Confocal double immunofluorescence for CNPase and ZO-1 revealed that CNPase-positive oligodendrocytes throughout mouse CNS were decorated with ZO-1-positive puncta, as shown by an example of an oligodendrocyte in the cerebral cortex (). Low magnification double immunofluorescence for Cx47 and ZO-1, illustrated in hypothalamus (), indicated that the vast majority if not all Cx47-positive oligodendrocytes were also immunopositive for ZO-1. Higher magnification confocal analysis demonstrated near total co-localization of Cx47-positive puncta with ZO-1 positive puncta on oligodendrocytes in cerebral cortex (), as well as in other brain regions shown only by image overlays of Cx47/ZO-1 double labeling (). Fine, punctate labeling for ZO-1 () was more widely and more densely distributed than labeling for Cx47 () in all brain regions, suggesting association of ZO-1 with other cell types and cellular structures. However, oligodendrocytes were often identifiable based simply on the greater size, labeling intensity and density of their ZO-1 associated immunopositive puncta, which was evident in sections double-labeled for CNPase and ZO-1 ().
Fig. 6 Immunofluorescence co-localization relationships of ZO-1 with CNPase, Cx47 and Cx32. (A) Confocal double immunofluorescence of a CNPase-positive oligodendrocyte somata (A1, arrow) displaying punctate labeling for ZO-1 (A2, arrow) as shown in overlay (A3). (more ...)
As expected from the above results demonstrating near total co-localization of Cx47 with both Cx32 and ZO-1, triple immunofluorescence labeling examined by confocal microscopy indicated substantial overlap of punctate labeling for Cx32 and ZO-1 along the periphery of CNPase-positive oligodendrocytes, shown in hippocampus () and the thalamus ().
Double immunofluorescence labeling for Cx47 and ZO-1 in cultured HeLa cells is shown in . In cells stably transfected with Cx47, both Cx47 () and ZO-1 () immunoreactivity appeared as immunopositive puncta distributed around the periphery of cells, particularly at points of cell-cell contact. Low magnification image overlay () and confocal analysis () of cells in culture indicated near total co-localization of punctate labeling for Cx47 and ZO-1. HeLa cells transfected with empty vector were devoid of Cx47-immunopositive puncta (), and displayed only sparse, weak punctate labeling for ZO-1, suggesting that transfection with Cx47 results in an induction of ZO-1 expression. High background fluorescence was seen in transfected as well as control HeLa cells even after primary antibody omission (not shown), indicating non-specific attachment of secondary antibody, which was likely due to the relatively weak fixation conditions required for visualization of ZO-1.
Fig. 7 Immunofluorescence labeling of Cx47 and ZO-1 in cultured HeLa cells. (A) Double immunofluorescence of the same field showing punctate labeling of Cx47 (A1) and ZO-1 (A2) at points of cell-to-cell contact (arrows) in HeLa cells stably transfected with (more ...)
Cx47 at oligodendrocyte gap junctions by FRIL
By FRIL, Cx47 was abundant in gap junctions on oligodendrocyte somata (), and was present in gap junctions of all sizes, from those having < 30 connexons to those having > 500 connexons (inscribed boxes, ). In contrast to data resolvable by immunofluorescence microscopy, Cx47 was also observed in numerous small gap junctions on the outer layer of myelin () in adult rat spinal cord. This discrepancy between LM and FRIL data may be due to the ability of FRIL to detect the 0.03– 0.1 μm diameter gap junctions consisting of 10 –100 connexons on myelin surfaces. These minute junctions may be too small to have been detected by light microscope immunofluorescence. Also, as in the case of low Cx29 levels associated with oligodendrocyte cell bodies (Nagy et al., 2003a
), sparsely distributed, fine Cx47-positive puncta seen along fibers in optimally fixed material were largely eliminated with increasing fixation strength, indicating narrow fixation thresholds and disproportionately greater suppression of Cx47 detection by immunofluorescence in very small gap junctions along myelinated fibers compared with large gap junctions on oligodendrocyte somata.
Fig. 8 FRIL labeling of Cx47 in oligodendrocytes in adult rat spinal cord. (A) In a small area of oligodendrocyte soma, Cx47-immunoreactivity (12 nm gold) is seen in each of five gap junctions, which range from about 100 to more than 500 connexons. (B) At higher (more ...)
ZO-1 in tight junctions and gap junction by FRIL
By FRIL, immunogold beads representing ZO-1 labeling were abundant along margins linking capillary endothelial cells in samples labeled by both monoclonal and polyclonal anti-ZO-1 antibodies (). At high magnification, gold beads were localized to tight junction strands, as previously shown by Fujimoto (1995)
, presumably indicating that cytoplasmic ZO-1 molecules remain firmly bound to tight junction transmembrane proteins, which in turn strongly adhere to the platinum/carbon replica (Fujimoto, 1995
). This positive control for ZO-1 labeling also allows comparison of the relative level of labeling under the conditions used in simultaneous labeling experiments on CNS tissues.
Fig. 9 Capillary in adult rat spinal cord after immunogold labeling for ZO-1. (A) At low magnification, the margins of five endothelial cells (E1–E5) reveal abundant 18 nm gold beads representing ZO-1 immunoreactivity. (B) In higher magnification stereoscopic (more ...)
Polyclonal antibody labeling for ZO-1 was found at oligodendrocyte gap junctions, both on oligodendrocyte somata () and on the outer surface of myelin (). In contrast to our observations by immunofluorescence, monoclonal anti-ZO-1 antibody failed to produce FRIL labeling of oligodendrocyte gap junctions. It should be noted that the sequence against which monoclonal anti-ZO-1 was raised (amino acids 334–634) contains the PDZ3 domain (amino acids 409–490) and the SH3 domain (amino acids 504–572) of ZO-1. Thus, it is possible that the monoclonal anti-ZO-1 epitope lies within one of these domains, which at gap junctions but not tight junctions, remain blocked by interacting proteins in procedures used for FRIL, but become exposed in those used for immunofluorescence.
Fig. 10 FRIL localization of ZO-1 immunoreactivity at oligodendrocyte gap junctions (green overlays), but not at “reciprocal patches” (blue overlays). In images obtained from the same replica as , nm and 18 nm gold beads are (more ...)
Interaction of Cx47 with ZO-1
In order to determine whether Cx47 has direct or indirect molecular interaction capability with ZO-1, ZO-1 was immunoprecipitated from homogenates of various brain regions and from Cx47-transfected HeLa cells, and blots of immunoprecipitate protein were probed with anti-Cx47 antibody. As shown in , Cx47 was detected after IP of ZO-1 from hypothalamus, cerebellum, midbrain and medulla, but not in a control sample of hypothalamus processed for IP with anti-ZO-1 antibody omission. Levels of Cx47 detected in IP material reflect variability of IP with anti-ZO-1 antibody in the various brain regions, rather than comparative levels of Cx47 in these regions. Similar detection of Cx47 was obtained after IP of ZO-1 from Cx47-transfected HeLa cells (, lane 3), whereas control IP with anti-ZO-1 omission showed an absence of Cx47 (, lane 4). Blots of Cx47 immunoprecipitated from empty vector-transfected and Cx47-transfected HeLa cells with anti-Cx47 antibody are shown as negative (, lane 1) and positive (, lane 2) controls for Cx47 detection.
Fig. 11 Immunoblots showing co-IP of ZO-1 and Cx47 from various brain regions and from Cx47-transfected HeLa cells. (A) Homogenates of brain regions were taken for IP with anti-ZO-1 antibody and immunoblots of precipitates were probed with anti-Cx47 antibody. (more ...)
As further controls, Cx47-transfected HeLa cells probed with anti-ZO-1 showed lack of anti-ZO-1 cross-reaction (, lane 1) with a band corresponding to Cx47 in these cells (, lane 2). However, in HeLa cells and in thalamus, anti-ZO-1 antibody detected proteins of unknown identity migrating at about 31 kDa and 60 kDa, respectively, (). No reaction with these proteins was seen with monoclonal anti-ZO-1 antibody (not shown). In addition to detection of ZO-1 in thalamus, a protein migrating at about 125–130 kDa was detected by polyclonal and monoclonal anti-ZO-1 in HeLa cells and thalamus, which may represent a cross-reaction with ZO-3, with which ZO-1 has sequence homology. In view of this potential cross-reaction, it should be noted that anti-ZO-3 antibody, which produced robust immunofluorescence labeling of ZO-3 in liver, showed no labeling comparable to that seen with polyclonal anti-ZO-1 in brain, except along blood vessels (not shown), indicating that immunofluorescence labeling obtained with anti-ZO-1 antibodies was not the result of ZO-3 detection.
Direct binding of Cx47 to ZO-1 was examined by in vitro pull-down assays using homogenates from brain and Cx47-transfected HeLa cells incubated with sepharose beads coupled to fusion proteins containing the GST–first PDZ domain of ZO-1 (PDZ1), GST–second PDZ domain of ZO-1 (PDZ2) or GST–third PDZ domain of ZO-1 (PDZ3). As shown in (lanes 4–9), Cx47 was detected on immunoblots of brain and Cx47-transfected HeLa cell homogenate proteins eluted from beads coupled to the GST-PDZ2 domain of ZO-1, but was absent on blots of proteins eluted from beads coupled to the GST-PDZ1 and GST-PDZ3 domains of ZO-1, indicating direct and selective binding of Cx47 to the second PDZ domain of ZO-1. Immunoblots of Cx47 in homogenates of Cx47-transfected HeLa cells and brain were used as positive controls (, lanes 2 and 3), and empty vector-transfected cells as negative controls (, lane 1), for Cx47 detection. Immunoblot membranes shown in (lanes 4–6 and 7–9) were stripped and reprobed with anti-GST antibody (, corresponding lanes 1–3 and 4–6) to confirm the presence and equal loading of GST fusion proteins derived from the pull-down assays.
Fig. 12 Pull-down assays showing Cx47 interaction with the second PDZ domain of ZO-1. (A) Immunoblots of lysates from empty vector-transfected HeLa cells (lane 1), Cx47-transfected HeLa cells (lane 2) and homogenate from brain tissue (lane 3) show negative and (more ...)