Abstract
We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys- 324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right- handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody.
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- Aebi U., Cohn J., Buhle L., Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature. 1986 Oct 9;323(6088):560–564. doi: 10.1038/323560a0. [DOI] [PubMed] [Google Scholar]
- Aebi U., Fowler W. E., Rew P., Sun T. T. The fibrillar substructure of keratin filaments unraveled. J Cell Biol. 1983 Oct;97(4):1131–1143. doi: 10.1083/jcb.97.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bennett G. S., Fellini S. A., Toyama Y., Holtzer H. Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. J Cell Biol. 1979 Aug;82(2):577–584. doi: 10.1083/jcb.82.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Danto S. I., Fischman D. A. Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin. J Cell Biol. 1984 Jun;98(6):2179–2191. doi: 10.1083/jcb.98.6.2179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischman D. A., Danto S. I. Monoclonal antibodies to desmin: evidence for stage-dependent intermediate filament immunoreactivity during cardiac and skeletal muscle development. Ann N Y Acad Sci. 1985;455:167–184. doi: 10.1111/j.1749-6632.1985.tb50411.x. [DOI] [PubMed] [Google Scholar]
- Gard D. L., Lazarides E. The synthesis and distribution of desmin and vimentin during myogenesis in vitro. Cell. 1980 Jan;19(1):263–275. doi: 10.1016/0092-8674(80)90408-0. [DOI] [PubMed] [Google Scholar]
- Geisler N., Kaufmann E., Fischer S., Plessmann U., Weber K. Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins. EMBO J. 1983;2(8):1295–1302. doi: 10.1002/j.1460-2075.1983.tb01584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geisler N., Kaufmann E., Weber K. Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments. J Mol Biol. 1985 Mar 5;182(1):173–177. doi: 10.1016/0022-2836(85)90035-x. [DOI] [PubMed] [Google Scholar]
- Geisler N., Kaufmann E., Weber K. Proteinchemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments. Cell. 1982 Aug;30(1):277–286. doi: 10.1016/0092-8674(82)90033-2. [DOI] [PubMed] [Google Scholar]
- Geisler N., Weber K. Comparison of the proteins of two immunologically distinct intermediate-sized filaments by amino acid sequence analysis: desmin and vimentin. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4120–4123. doi: 10.1073/pnas.78.7.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geisler N., Weber K. The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins. EMBO J. 1982;1(12):1649–1656. doi: 10.1002/j.1460-2075.1982.tb01368.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Georgatos S. D., Blobel G. Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments. J Cell Biol. 1987 Jul;105(1):105–115. doi: 10.1083/jcb.105.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldman R., Goldman A., Green K., Jones J., Lieska N., Yang H. Y. Intermediate filaments: possible functions as cytoskeletal connecting links between the nucleus and the cell surface. Ann N Y Acad Sci. 1985;455:1–17. doi: 10.1111/j.1749-6632.1985.tb50400.x. [DOI] [PubMed] [Google Scholar]
- Granger B. L., Lazarides E. Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy. Cell. 1982 Aug;30(1):263–275. doi: 10.1016/0092-8674(82)90032-0. [DOI] [PubMed] [Google Scholar]
- Heuser J. E., Keen J. H., Amende L. M., Lippoldt R. E., Prasad K. Deep-etch visualization of 27S clathrin: a tetrahedral tetramer. J Cell Biol. 1987 Nov;105(5):1999–2009. doi: 10.1083/jcb.105.5.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heuser J. E. Procedure for freeze-drying molecules adsorbed to mica flakes. J Mol Biol. 1983 Sep 5;169(1):155–195. doi: 10.1016/s0022-2836(83)80179-x. [DOI] [PubMed] [Google Scholar]
- Huiatt T. W., Robson R. M., Arakawa N., Stromer M. H. Desmin from avian smooth muscle. Purification and partial characterization. J Biol Chem. 1980 Jul 25;255(14):6981–6989. [PubMed] [Google Scholar]
- Ip W., Heuser J. E., Pang Y. Y., Hartzer M. K., Robson R. M. Subunit structure of desmin and vimentin protofilaments and how they assemble into intermediate filaments. Ann N Y Acad Sci. 1985;455:185–199. doi: 10.1111/j.1749-6632.1985.tb50412.x. [DOI] [PubMed] [Google Scholar]
- Jacobson G. R., Schaffer M. H., Stark G. R., Vanaman T. C. Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues. J Biol Chem. 1973 Oct 10;248(19):6583–6591. [PubMed] [Google Scholar]
- Jones J. C., Goldman R. D. Intermediate filaments and the initiation of desmosome assembly. J Cell Biol. 1985 Aug;101(2):506–517. doi: 10.1083/jcb.101.2.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaufmann E., Weber K., Geisler N. Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues. J Mol Biol. 1985 Oct 20;185(4):733–742. doi: 10.1016/0022-2836(85)90058-0. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lasek R. J., Phillips L., Katz M. J., Autilio-Gambetti L. Function and evolution of neurofilament proteins. Ann N Y Acad Sci. 1985;455:462–478. doi: 10.1111/j.1749-6632.1985.tb50429.x. [DOI] [PubMed] [Google Scholar]
- Lazarides E., Granger B. L., Gard D. L., O'Connor C. M., Breckler J., Price M., Danto S. I. Desmin- and vimentin-containing filaments and their role inthe assembly of the Z disk in muscle cells. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):351–378. doi: 10.1101/sqb.1982.046.01.036. [DOI] [PubMed] [Google Scholar]
- Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature. 1980 Jan 17;283(5744):249–256. doi: 10.1038/283249a0. [DOI] [PubMed] [Google Scholar]
- Lazarides E. Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem. 1982;51:219–250. doi: 10.1146/annurev.bi.51.070182.001251. [DOI] [PubMed] [Google Scholar]
- McKeon F. D., Kirschner M. W., Caput D. Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature. 1986 Feb 6;319(6053):463–468. doi: 10.1038/319463a0. [DOI] [PubMed] [Google Scholar]
- Parry D. A., Steven A. C., Steinert P. M. The coiled-coil molecules of intermediate filaments consist of two parallel chains in exact axial register. Biochem Biophys Res Commun. 1985 Mar 29;127(3):1012–1018. doi: 10.1016/s0006-291x(85)80045-0. [DOI] [PubMed] [Google Scholar]
- Potschka M. The structure of intermediate filaments. Biophys J. 1986 Jan;49(1):129–130. doi: 10.1016/S0006-3495(86)83621-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pruss R. M., Mirsky R., Raff M. C., Thorpe R., Dowding A. J., Anderton B. H. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell. 1981 Dec;27(3 Pt 2):419–428. doi: 10.1016/0092-8674(81)90383-4. [DOI] [PubMed] [Google Scholar]
- Quinlan R. A., Cohlberg J. A., Schiller D. L., Hatzfeld M., Franke W. W. Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells. J Mol Biol. 1984 Sep 15;178(2):365–388. doi: 10.1016/0022-2836(84)90149-9. [DOI] [PubMed] [Google Scholar]
- Quinlan R. A., Hatzfeld M., Franke W. W., Lustig A., Schulthess T., Engel J. Characterization of dimer subunits of intermediate filament proteins. J Mol Biol. 1986 Nov 20;192(2):337–349. doi: 10.1016/0022-2836(86)90369-4. [DOI] [PubMed] [Google Scholar]
- Sauk J. J., Krumweide M., Cocking-Johnson D., White J. G. Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly. J Cell Biol. 1984 Nov;99(5):1590–1597. doi: 10.1083/jcb.99.5.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steinert P. M., Idler W. W., Zhou X. M., Johnson L. D., Parry D. A., Steven A. C., Roop D. R. Structural and functional implications of amino acid sequences of keratin intermediate filament subunits. Ann N Y Acad Sci. 1985;455:451–461. doi: 10.1111/j.1749-6632.1985.tb50428.x. [DOI] [PubMed] [Google Scholar]
- Steinert P. M., Steven A. C., Roop D. R. The molecular biology of intermediate filaments. Cell. 1985 Sep;42(2):411–420. doi: 10.1016/0092-8674(85)90098-4. [DOI] [PubMed] [Google Scholar]
- Steven A. C., Hainfeld J. F., Trus B. L., Wall J. S., Steinert P. M. Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments. J Cell Biol. 1983 Dec;97(6):1939–1944. doi: 10.1083/jcb.97.6.1939. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tokuyasu K. T., Maher P. A., Singer S. J. Distributions of vimentin and desmin in developing chick myotubes in vivo. I. Immunofluorescence study. J Cell Biol. 1984 Jun;98(6):1961–1972. doi: 10.1083/jcb.98.6.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Traub P., Nelson W. J. Occurrence in various mammalian cells and tissues of the Ca 2+ activated protease specific for the intermediate-sized filament proteins vimentin and desmin. Eur J Cell Biol. 1981 Dec;26(1):61–67. [PubMed] [Google Scholar]
- Tyler J. M., Branton D. Rotary shadowing of extended molecules dried from glycerol. J Ultrastruct Res. 1980 May;71(2):95–102. doi: 10.1016/s0022-5320(80)90098-2. [DOI] [PubMed] [Google Scholar]
- Wang E. Intermediate filament associated proteins. Ann N Y Acad Sci. 1985;455:32–56. doi: 10.1111/j.1749-6632.1985.tb50402.x. [DOI] [PubMed] [Google Scholar]
- Weber K., Geisler N. Intermediate filaments: structural conservation and divergence. Ann N Y Acad Sci. 1985;455:126–143. doi: 10.1111/j.1749-6632.1985.tb50408.x. [DOI] [PubMed] [Google Scholar]
- Woodcock C. L. Nucleus-associated intermediate filaments from chicken erythrocytes. J Cell Biol. 1980 Jun;85(3):881–889. doi: 10.1083/jcb.85.3.881. [DOI] [PMC free article] [PubMed] [Google Scholar]