Rungger-Brändle E , Achtstätter T , Franke WW .

	Figure 1. Immunofluorescence microscopy of frozen sections through sciatic (a and b) and optic (c-e) nerves of X. laevis (a-c) and R. ridibunda (d and e). (a, c, and d) Stained for desmosomes with monoclonal antibody to desmoplakins: DP 1&2-2.19 alone (a and c) or in a mixture with DP 1&2-2.15 (d). (b and e) Stained for cytokeratins with monoclonal antibody K,pan 1.-8.136 (b) or guinea pig antibodies (e). In the sciatic nerve (a and b), the reactions of both desmoplakins (a) and cytokeratins (b) are restricted to the perineurial cell layers (brackets) and are absent from the nerve interior (asterisks). In addition, the endothelial layer of a nerve-associated blood vessel is positive for cytokeratins (b). In the optic nerve (c and d), desmosomes are abundant in the perineural cell layers of the arachnoid (brackets) but also occur throughout the entire interior of the nerve (central portion in c and below the arachnoidal cell layers in d). Note the higher frequency of desmosomes in the perineural meninges and the subjacent region corresponding to the gila limitans. Reaction for cytokeratins in the optic nerve head (e) is often particularly strong in the arachnoidal cells (upper bracket) and in the glial elements of the nerve interior. Here a relatively weak reaction is seen in the adjacent retinal pigment epithelium (lower right brackeT). Bars: (a-c and e) 50 Um; (d) 25 Um.
	Figure 2. Immunofluorescence microscopy of subsequent frozen sections ("step sections ~) through the optic nerve head of X. laevis using monoclonal antibodies to desmoplakins (DP 1&2-2.19; [a] epifluorescence; [b] phase contrast optics) and cytokeratins (K,pan b8.136; [c] epifluorescence; [d] phase contrast). Note intense labeling with both antibodies in the perineural meningeal sheath and in the glial structures of the nerve interior. Arrows denote the ends of the subarachnoidal space; i.e. the transition region into the retina. Note melaninrich tissue elements. Bars, 50 um.
	Figure 3. Immunofluorescence microscopy of frozen sections through the optic nerve ofX. laevis after reaction with antibodies to cytokeratins (a and d), vimentin (b and c), and neurofilament protein NF-L (e). (a and b) Double-label staining of the optic nerve head comparing the distribution of cytokeratins (a, guinea pig antibodies) with vimentin (b; murine antibody VIM 3134). Note that the perineural meninges and certain cell tracts of the nerve interior are stained with both antibodies. (c) Specificity of vimentin staining (VIM 3B4) within the optic nerve (NO) is shown by comparison with the staining of the surrounding interstitial tissue (IT) as well as the erythrocytes, the endothelium, and the smooth muscle wall (brackeO of a blood vessel (BV). (d and e) Double-label staining of an oblique section through the optic nerve for cytokeratins (d; antibody lu-5) and neurofilament protein NF-L (e; guinea pig antibodies). Note that the two patterns are not superimposable. The perineural meningeal cell layer (brackets) is not stained by neurofilament antibodies (e). Arrows point to some cells with prominent neurofilament contents. Bars, 50 um.
	Figure 4. Electron micrographs of sections through the interior of the optic nerve of the frog, R. ridibunda, showing glial filaments and desmosomes. (a) Survey picture showing the abundance of IFs in axonal processes of neurons (N), including myelinated ones, and of glial elements (G), which are exclusively astrocytes in this region. The arrow denotes a desmosome to which IF bundles attach. (b) Higher magnification picture showing an extended junction with typical desmosomal organization; i.e., two membranes and the central midline structure (parallel bars), the two cytoplasmic plaques (brackets), and attached IF bundles (i.e., tonofibrils; T). (c) Cross section of a small desmosome showing the two plaques (brackets) and the numerous IFs that are associated with the plaques, mostly abutting at a low angle. Bars: (a) 0.5/zm; (b and c) 0.25 um.
	Figure 5. Immunoelectmn microscopy of sections showing the astrocyte processes in the interior of the optic nerve of X. laevis after reaction with cytokeratin antibodies and secondary antibodies coupled to colloidal gold particles. (a) Survey picture showing a desmosome between two astrocytes (bracket) and the myelin sheath-covered axon of a neuronal element (N). Note that the gold particles are exclusively associated with the IF bundles of the astrocytes. (b) Higher magnification of peripheral regions of two adjacent astrocytes connected by a desmosome (bracket) showing immunogold decoration of IF bundles. V, intracytoplasmic vesicle with desmosomal plaques, probably originated by endocytosis. Bars: (a) 0.5 #m; (b) 0.2 um.
	Figure 6. Identification of amphibian basic (type II) cytokeratin(s) related to human cytokeratin 8 among the cytoskeletal proteins of microdissected amphibian nerves by SDS-PAGE and immunoblotting. (a) Coomassie blue staining of SDS-PAGE-separated cytoskeletal polypeptides from cultured kidney epithelial cells of X. laevis of line A6 (lane 2) in comparison with those of microdissected optic (lane 3) and sciatic (lane 4) nerves of R. ridibunda and reference proteins (lane 1 shows from top to bottom: myosin heavy chain, B-galactosidase, phosphorylase A, BSA, ovalbumin, carbonic anhydrase). (b) Autoradiography of an immunoblot of a parallel SDSPAGE (with a much lower protein loading in lane 2' compared with a, lane 2) obtained after reaction with monoclonal cytokeratin antibody K~pan 1-8.136. The reactive bands in b correspond to a component of ,~56,000 Mr (indicated by dots in a).
	Figure 7. Identification of IF proteins in microdissected nerve tissue of R. ridibunda (a-d) and X. laevis (e-g) by two-dimensional gel eleetrophoresis of cytoskeletal proteins (horizontal arrow, direction of isoelectric focusing; downward arrow, direction of SDS-PAGE) followed by immunoblotting. Proteins added for reference: B, BSA; A, skeletal muscle ot-actin. (a-d) Identification of cytokeratins in optic nerve tissue of R. ridibunda. (a) Ponceau S staining of proteins blotted on nitrocellulose filter. Major cytoskeletal polypeptides are denoted by triple bracket, bracket, fork, and arrow. (b-d) Autoradiographs showing cytokeratins positively identified by immunoblot with different solutions of guinea pig antibodies to cytokeratins (b and c) and monoclonal antibody K,pan 1-8.136, which reacts only with cytokeratins of the basic (type H) subfamily (d). The major component of ~56,000 Mr, which appears as a series of isoeleetric variants, (denoted by the triple bracket on the left hand side) is a type II cytokeratin related to human cytokeratin 8 and probably corresponds to the 56,000-M~ protein described in R. catesbeiana (see text). It is not clear whether the minor reactive component denoted by the left arrow (c and d) is a genuine minor cytokeratin polypeptide or a degradation product of the major type II cytokeratin. In addition, the guinea pig antibodies (c) react with two of the other major cytoskeletal polypeptides (denoted by the right arrow and the fork), which are probably acidic (type I) cytokeratins. (e and f) Identification of cytokeratins in optic nerve tissue of X. laevis by immunoblotting. (e) Ponceau S staining of proteins blotted on nitrocellulose filter. (f) Autoradiography showing the corresponding immunoblot after reaction with guinea pig cytokemtin antibodies. Major cytokeratins are denoted by brackets. The uppermost component of ~56,000 Mr was a type II cytokeratin as shown by its reaction with antibody K, pan 1-8.136 (not shown). (g) Ponceau S staining of cytoskeletal polypeptides present in microdissected sciatic nerve tissue of X. laevis. Note the high complexity of polypeptide composition. None of the components stained by the dye reacted with any of the antibodies to cytokeratins used. The positions of the three minor components that did react with cytokeratin antibodies are denoted by the arrowheads and are not visible by protein staining with Coomassie blue or Ponceau S. These are probably components of the perineurial epithelium (for details see Fig. 3). Note the numerous Ponceau S-stained proteins, most of which are probably noncytokeratinous IF proteins, which in this species have not yet been positively identified by two-dimensional gel electrophoresis. (h and i) Identification of cytokeratins among the cytoskeletal proteins of the optic nerve of X. laevis (h) by coelectrophoresis with l~SS]methionine-labeled cytoskeletal proteins from cultured kidney epithelial cells (line A6) of the same species (i; same gel electrophoretic system as in a and e). Brackets in h denote the same components as in e and fi, those in i denote the four major cytokeratins of A6 cells. Cytokeratin polypeptides denoted by an open circle comigrate; those denoted by filled circles are exclusive to the specific cytoskeleton. V, vimentin (minor component in the nerve tissue shown in h); a, endogenous nonmuscle actin.

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