INTERMEDIATE FILAMENTS IN NON-NEURONAL CELLS OF INVERTEBRATES - ISOLATION AND BIOCHEMICAL-CHARACTERIZATION OF INTERMEDIATE FILAMENTS FROM THE ESOPHAGEAL EPITHELIUM OF THE MOLLUSK HELIX-POMATIA

To screen invertebrate tissues for the possible expression of intermediate filaments (IF), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used. In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris and H. pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, H. pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in EM of esophagus impressive arrays of IF as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and 2-dimensional gels show 3 components, all of which are recognized by anti-IFA in immunoblotting: 66 kD[kilodalton]/pI[isoelectric point] 6.35, 53 kD/pI 6.05 and 52 kD/pI 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the 2 more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all 3 polypeptides. The results establish firmly the existence of invertebrate IF outside neurons and demonstrate that the esophagus of H. pomatia displays IF which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.