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An endoskeleton in intestinal brush-border cells
770
Cell Biology
International
Reports,
Vol. 4, No. 8, August
1980
AN ENDOSKELETONIN INTESTINAL BRUSH-BORDER CELLS J.W.Porteous*,G.P.Pugh-Humphreys**,C.M.Towler*, & Fiona Watt***, Department of Biochemistry*,Marischal College, University of Aberdeen, Aberdeen AB9 1AS; Department of Zoology**, King'sCollege,University of Aberdeen, Aberdeen AB9 1UB; Sir William Dunn School of Pathology***, University of Oxford, Oxford OX1 3RE Porteous & Clark2 failed to separate nuclei from the brush-borders of columnar absorptive (brush-border) cells of rabbit or golden hamster small intestine and suggested the presence of some physical connection between the two subcellular structures. This physical connection is a profuse network of 1Onm filaments physically continuous with but chemically distinct from the terminal web of the brushborders; the filaments suspend the nucleus-between the brush-border and the basolateral membranes of the brushborder cell. These structures were obtained by homogenization of isolated rat jejunal brush-border cells435 (Fig. 1) in 320 mM-sucrose/O.5 mM-MgCl /l mM-KH PO4/O.2% Fhase-congrast Triton NlOl, pH 6.8.
microscopy of resuspensions of pelleted material (5000 g, 10 min) revealed the brush-border/filamentous endoskeleton/ nucleus structure (Fig. 2). Fluorescent antibody staining identified actin in the terminal web only (Fig. 3); tubulin, when seen, was confined to a fine ring circumscribing the terminal web (Fig.4,5). Transmission electron microscopic examination of surface relicas of the isolated endoskeletal structure showed that the filaments are about 10 nm thick, that they surround and are apoarentlv attached to the nucleus (Fig.6): The endoskeleton of these brush-border cells thus belongs to the "intermediate filaments" class. Analogous endoskeletal structures exist in chick and auinea oia intestinal brushborder cells but clean: free nuclei can be isolated from these cellsf33.
Fig-l: Isolated, intact, rat jejunal brush-border cells (Nomarski-interference Nucleus (N), filaments (F) and residual brush-border (rBB) microscopy). w: Fi -3. As Fig. 2, with actin staining after homogenlzlng cells (phase-contrast). 2, showing "orifice" of terminal -A?+$ of terminal web (UV-fluorescence). staining for tubulin; cf Fig.4 web (Nomarski-interference). w: As Fig. 2, after Transmission electron micrograph of surface replica of (UV-fluorescence). filaments connecting v:t e terminal web to the nucleus(N). REFERENCES 1. 2. 3. 4. 5.
0309-l
Morrison, A. & Porteous, J.W. (1980) Biochem. J. 186, in press. Porteous, J.W. & Clark, B. (1965) Biochem. J. 96, 159-171. Porteous, J.W., Furneaux, H., Pearson, C.K., Lake, C.M. & Morrison, A. (1979) Bi0chem.J. 180, 455-463. Porteous, J.W. (1980) Biochem. J. 188, in press. Towler,C.M., Pugh-Humphreys, G.P. & Porteous, J.W. (1978) J.Cell Sci.29, 52-75.
651/%0/080770-01/$02.00/0
01980
Academic
Press Inc. (London)
Ltd.
Cell Biology
International
Reports,
Vol. 4, No. 8, August
1980
AN ENDOSKELETONIN INTESTINAL BRUSH-BORDER CELLS J.W.Porteous*,G.P.Pugh-Humphreys**,C.M.Towler*, & Fiona Watt***, Department of Biochemistry*,Marischal College, University of Aberdeen, Aberdeen AB9 1AS; Department of Zoology**, King'sCollege,University of Aberdeen, Aberdeen AB9 1UB; Sir William Dunn School of Pathology***, University of Oxford, Oxford OX1 3RE Porteous & Clark2 failed to separate nuclei from the brush-borders of columnar absorptive (brush-border) cells of rabbit or golden hamster small intestine and suggested the presence of some physical connection between the two subcellular structures. This physical connection is a profuse network of 1Onm filaments physically continuous with but chemically distinct from the terminal web of the brushborders; the filaments suspend the nucleus-between the brush-border and the basolateral membranes of the brushborder cell. These structures were obtained by homogenization of isolated rat jejunal brush-border cells435 (Fig. 1) in 320 mM-sucrose/O.5 mM-MgCl /l mM-KH PO4/O.2% Fhase-congrast Triton NlOl, pH 6.8.
microscopy of resuspensions of pelleted material (5000 g, 10 min) revealed the brush-border/filamentous endoskeleton/ nucleus structure (Fig. 2). Fluorescent antibody staining identified actin in the terminal web only (Fig. 3); tubulin, when seen, was confined to a fine ring circumscribing the terminal web (Fig.4,5). Transmission electron microscopic examination of surface relicas of the isolated endoskeletal structure showed that the filaments are about 10 nm thick, that they surround and are apoarentlv attached to the nucleus (Fig.6): The endoskeleton of these brush-border cells thus belongs to the "intermediate filaments" class. Analogous endoskeletal structures exist in chick and auinea oia intestinal brushborder cells but clean: free nuclei can be isolated from these cellsf33.
Fig-l: Isolated, intact, rat jejunal brush-border cells (Nomarski-interference Nucleus (N), filaments (F) and residual brush-border (rBB) microscopy). w: Fi -3. As Fig. 2, with actin staining after homogenlzlng cells (phase-contrast). 2, showing "orifice" of terminal -A?+$ of terminal web (UV-fluorescence). staining for tubulin; cf Fig.4 web (Nomarski-interference). w: As Fig. 2, after Transmission electron micrograph of surface replica of (UV-fluorescence). filaments connecting v:t e terminal web to the nucleus(N). REFERENCES 1. 2. 3. 4. 5.
0309-l
Morrison, A. & Porteous, J.W. (1980) Biochem. J. 186, in press. Porteous, J.W. & Clark, B. (1965) Biochem. J. 96, 159-171. Porteous, J.W., Furneaux, H., Pearson, C.K., Lake, C.M. & Morrison, A. (1979) Bi0chem.J. 180, 455-463. Porteous, J.W. (1980) Biochem. J. 188, in press. Towler,C.M., Pugh-Humphreys, G.P. & Porteous, J.W. (1978) J.Cell Sci.29, 52-75.
651/%0/080770-01/$02.00/0
01980
Academic
Press Inc. (London)
Ltd.