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First published online 15 January 2003
doi: 10.1242/jcs.00293

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Expression of the nidogen-binding site of the laminin 1 chain disturbs basement membrane formation and maintenance in F9 embryoid bodies

¹ Center for Biochemistry, Medical Faculty, University of Cologne, D-50931 Cologne, Germany
² Institute for Neurophysiology, Medical Faculty, University of Cologne, D-50931 Cologne, Germany

View larger version (15K): [in a new window]   Fig. 1. Production of nidogen-binding site constructs and controls. The site for interaction between laminin-1 and nidogen-1 has been localized to LE module 4 of domain III of the laminin 1 chain (Mayer et al., 1993). Two different FLAG fusion proteins (indicated with flags) were constructed and modified with the BM40 signal peptide to ensure secretion to the extracellular space, 1III3-5 coding for LE modules 3-5 and 1V1-3 comprising three LE modules of domain V.

View larger version (43K): [in a new window]   Fig. 2. Levels of exogenous protein expression: western blot analysis of supernatants of stably transfected F9 teratocarcinoma cells with a mouse monoclonal antibody detecting the FLAG tag shows approximately equal expression of both constructs, 1III3-5 and 1V1-3. As a control, F9 cells carrying the empty expression vector were analysed. For normalization of the loading of secreted proteins, a rabbit polyclonal antibody against the mouse BM40 protein was used. The two blots shown are derived from the same gel.

View larger version (71K): [in a new window]   Fig. 3. Immunohistochemical localization of basement membrane proteins in 12-day-old F9-cell-derived embryoid bodies: cells stably expressing the constructs 1III3-5, 1III3-5mut and 1V1-3 were differentiated into embryoid bodies with 5x10-8 M retinoic acid, cryosectioned and stained for basement membrane components. As a control, F9 cells carrying the empty expression vector were used. 1III3-5/A and 1III3-5/B represent two different clones from one transfection with the 1III3-5 construct. Bar, 50 µm. Upper panels: staining for perlecan shows disruption of basement membrane formation only in embryoid bodies derived from clones expressing the nidogen binding site (1III3-5/A and /B). Middle panels: double immunofluorescence for laminin-1 and nidogen-1. Cryosections of differentiated 12-day-old embryoid bodies were incubated simultaneously with a rabbit polyclonal antibody against laminin-1 (green) and a rat monoclonal antibody against nidogen-1 (red). Embryoid bodies from clones 1III3-5/A and 1III3-5/B show a separation in the laminin-1 and nidogen-1 staining not seen in those derived from control or 1III3-5 expressing cells. Lower panels: double immunofluorescence for nidogen-1 and nidogen-2. Sections of differentiated 12-day-old embryoid bodies stained with a rabbit polyclonal antibody against nidogen-2 (green) and the rat monoclonal antibody against nidogen-1 (red). Both nidogen isoforms colocalize and nidogen-2 is found in the basement membrane of F9-derived embryoid bodies.

View larger version (81K): [in a new window]   Fig. 4. Immunoblot for laminin-1 and nidogen-1 in embryoid body total lysates. A rabbit polyclonal antibody against laminin-1 and nidogen-1 was used to detect these proteins in total lysates prepared from 12-day-old retinoic-acid-treated embryoid bodies in SDS. Equal loading and transfer to nitrocellulose were confirmed by Ponceau Red staining of the membrane (not shown).

View larger version (20K): [in a new window]   Fig. 5. Comparison of diffusion coefficients obtained from incubation of differentiated F9-cell-derived embryoid bodies with rhodamine-labelled dextrans of 10 kDa and 70 kDa. Embryoid bodies stably expressing the constructs 1III3-5, 1V1-3 and a control clone were incubated with 10 µM dextrans of either size. Fluorescence intensities were measured inside the embryoid bodies after 5 minutes of diffusion. (A) Representative curves of single embryoid bodies (control and 1III3-5/A). incubated with 10 kDa and 70 kDa fluorescent dextrans. (B) From at least six traces of individual embryoid bodies, mean diffusion coefficients plus s.d. were calculated. Two independent experiments were performed with comparable results.

View larger version (46K): [in a new window]   Fig. 6. Immunofluorescence detection of TROMA-1. Cryosections of differentiated 12-day-old embryoid bodies derived from control, 1V1-3, 1III3-5/A and 1III3-5/B expressing F9 cells were stained with a rat monoclonal antibody against TROMA-1 (Kemler et al., 1981). Bar, 50 µm.

View larger version (68K): [in a new window]   Fig. 7. Laminin-1 localization in F9 wild-type embryoid bodies treated with the FLAG fusion protein 1III3-5. Embryoid bodies were transferred from a spinner culture system into single wells at day 2 and then either supplied with additional FLAG fusion protein 1III3-5 at a concentration of 10 µg ml-1 (A) or not (B). At day 8 of culture, whole-mount staining with a rabbit polyclonal antiserum against laminin-1 was performed. Representative images show either the embryoid body surface in the upper panel (two images; bar, 100 µm) or part of a section through the embryoid body periphery in the lower panel. Images from 12 different embryoid bodies from each culture condition are shown. Bar, 25 µm.