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First published online 31 May 2005
doi: 10.1242/jcs.02395

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Laminin-6 assembles into multimolecular fibrillar complexes with perlecan and participates in mechanical-signal transduction via a dystroglycan-dependent, integrin-independent mechanism

¹ Division of Pulmonary Medicine, Feinberg School of Medicine, Northwestern University, Morton 4-616, 303 East Chicago Avenue, Chicago, IL 60611, USA
² Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Morton 4-616, 303 East Chicago Avenue, Chicago, IL 60611, USA
³ Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, Department of Physiology and Biophysics, University of Iowa, 400 Eckstein Medical Research Building, Iowa City, IA 52242, USA

View larger version (96K): [in a new window]   Fig. 1. Matrix organization of AECs. AECs at 2 and 4 days after isolation were processed for double-label indirect immunofluorescence microscopy using antibodies against perlecan (A,D) in combination with an antiserum against the ß1 laminin subunit (B,E). Images of cells were generated using confocal laser scanning microscopy. The focal plane was as close as possible to the substratum-attached surface of the cells. Areas of co-localization are indicated by the yellow color in the overlay images shown in C,F. Bar, 10 µm.

View larger version (120K): [in a new window]   Fig. 2. AECs maintained in culture secrete a laminin-6-rich matrix. AECs at 4 days after isolation were processed for double-label indirect immunofluorescence using antibodies against the 3 laminin subunit (A,D) in combination with antibodies against either ß1 laminin (B) or 1 laminin (E). (G-I) Cells were processed with a mix of antibodies against 1 and ß1 laminin subunits, as indicated. (J-L) Cells were processed with a mix of antibodies against 3 and ß2 laminin subunit, as indicated. Specimens were viewed by confocal laser-scanning microscopy with the focal plane being as close as possible to the substrate-attached surface of the cells. Yellow color in the overlays in C,F,I marks colocalized protein. (L) The lack of yellow indicates the absence of colocalization of laminin subunits. Bar, 10 µm.

View larger version (50K): [in a new window]   Fig. 3. Perlecan and nidogen colocalize in the matrix of AECs maintained in vitro. AECs at 4 days after isolation were processed for double-label indirect immunofluorescence using a monoclonal antibody against perlecan (A) and an antiserum against nidogen (B). Confocal laser-scanning images of the cells were generated at a focal plane as close as possible to the substrate-attached surface of the cells. The yellow color in the overlay in C indicates where perlecan and nidogen co-distribute. Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 4. Laminin subunit and dystroglycan expression by AECs in vitro. (A) Extracts of AECs (at 4 days after plating) and the rat bladder-cell line 804G were prepared for western immunoblotting using antibodies against ß1, 1, 3, ß3, 2 and ß2 laminin subunits. (B) A matrix preparation derived from AECs at 4 days after plating was processed for immunoblotting using antibodies against the 3, ß1and 1 laminin subunits. (C) An immunoblot of an AEC extract at 4 days after plating was processed using the ß-dystroglycan antibody 8D5. Molecular weight standards are marked at the left of each series of blots.

View larger version (96K): [in a new window]   Fig. 5. Laminin matrix organization of AECs maintained on a deformable substrate in vitro. AECs, maintained for 4 days on elastomer membranes, were prepared for confocal indirect immunofluorescence microscopy using antibodies against the 3 laminin subunit (A). Notice the fibrillar arrays of matrix proteins recognized by the antibodies. (B) Phase-contrast image of the cells shown in A. Bar, 10 µm.

View larger version (49K): [in a new window]   Fig. 6. Laminin fibers in the matrix of AECs undergo deformation upon stretching. (A) The 3 laminin subunit in native matrix of AECs maintained on an elastomer membrane was immunostained and then the membrane was mounted on the stage of a fluorescent microscope. The membrane was then subjected to cyclic stretching for 10 minutes and then allowed to relax. Images of matrix were captured before, during and after stretching, and colorized in green, red and blue, respectively, as indicated in A. An overlay of the color images is shown. (B) Graphical representation of the changes in area enclosed by the matrix fiber network and fiber length during stretching and following stretching (relaxed). Each measurement is relative to that of unstretched matrix and represents eight different lengths and areas in three independent trials. *, P<0.001. Bar, 10 µm (A).

View larger version (19K): [in a new window]   Fig. 7. The role of matrix molecules and their associated proteins in stretching-mediated signaling. AECs maintained for 4 days on elastomer membranes were subjected to 10% stretching at 30 cycles per minute for 10 minutes. (A) Immunoblots of extracts of AECs, either unstretched (–) or subjected to the stretch regimen (+), probed first with antibodies against phosphorylated MAPK. The same blots were then reprobed with antibodies against total MAPK. The blots are representative of at least three separate trials. The static and stretched cells were incubated in the presence (+) or absence (–) of antibodies that functionally inhibit the 3 laminin subunit (50 µg ml–1), 3 integrin (50 µg ml–1), ß1 integrin (50 µg ml–1) or dystroglycan (40 µg ml–1), as indicated, for 18 hours before stretching. The bar graphs in B are quantifications of the densitometric scans of immunoblots of extracts derived from AECs in three separate trials following 10% stretching at 30 cycles per minute for 10 minutes in the presence of various cell-surface and matrix antibody antagonists or reagents, or following infection with adenovirus encoding either control or dystroglycan shRNA. Blots were probed first with antibodies against activated p42/p44 MAPK and then with antibodies against total p42/p44 MAPK. In each case, the extent of phosphorylation of p42/p44 MAPK in stretched AECs was normalized to the total level of MAPK in the same sample. The percentage phosphorylation was calculated relative to untreated, stretched values in each set of studies. Error bars indicate standard deviations. (C) AECs were infected with adenovirus encoding either control or dystroglycan shRNA at 2 days after plating. 24 hours later, the cells were washed and incubated for 18 hours in serum-free medium. Extracts of uninfected and infected cells were then prepared for SDS-PAGE and immunoblotting using a monoclonal antibody against ß-dystroglycan. The same immunoblot was then reprobed with an antibody against actin.

View larger version (18K): [in a new window]   Fig. 8. The organization of matrix components and matrix receptors that mediates outside-in mechanical signaling in AECs. Stretching deflects matrix fibers that (as our data indicate) transduce signals via dystroglycan at the surface of AECs.

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