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Laminin assembles into separate basement membrane and fibrillar matrices in Schwann cells

Maria V. Tsiper and Peter D. Yurchenco*

Department of Pathology & Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA



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  		Fig. 1. Laminins assemble into two distinct extracellular matrices on Schwann cell surfaces. Laminin-1 and laminin-2/4 were each incubated with Schwann cells. Surface-bound laminin was visualized following fixation and immunostaining with anti-laminin primary and FITC-conjugated secondary antibodies. (a,b) Laminin-1 incubated with Schwann cells for 4 hours at 10 µg/ml accumulated on cell surface showing dense reticular distribution. Insert shows higher magnification view of the boxed area. Phase image of the same field is shown. (c,d) Level of endogenous laminin was negligible. (e) Laminin-1 at lower concentration (2 µg/ml) showed less coverage with two distinct patterns clearly resolved: reticular (boxes) and fibrillar (arrows). (f) Laminin-2/4 assembled similar structures but had different concentration dependency. Shown is laminin-2/4 at 20 µg/ml. (e',f') High magnification view of boxed areas on e and f shown as examples of reticular pattern. Boxed areas are shown magnified in insets e' and f' to define the reticular pattern. (g) Example of a fibular structure is shown. Bars, 10 µm.

 


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  		Fig. 2. Formation of reticular and fibrillar laminin patterns. (a-b) Schwann cells were incubated with fragments and stained with antibodies specific for the corresponding fragments following washing and fixation. Only E3 was found to bind to cell surfaces, appearing in a punctate pattern (a). Fragments E8, E4 and E1' had levels corresponding to background (b). (c-h) Schwann cells were pre-incubated for 20 minutes with blocking or activating reagents followed by the addition of 2 µg/ml of laminin-1. The laminin distribution was detected 8 hours after incubation with EHS laminin-specific antibody (c,d,e,h) or fragment E4-specific antibody (f,g). Laminin-1 (c) formed both fibrillar (arrows) and reticular patterns (box, with corresponding twofold magnification inset) without blocking reagents. In the presence of ß1-integrin blocking antibody Ha2/5 (10 µg/ml), laminin assembled almost exclusively into reticular patterns. In the presence of {alpha}-dystroglycan blocking antibody IIH6 (e), laminin was detected in fibrillar structures (arrows) with only few small surviving reticular arrays. Coincubation of laminin with E3 (50 µg/ml) substantially decreased formation of reticular, but not fibrillar (arrows) arrays. Addition of both E3 fragment and anti ß1-integrin blocking antibody inhibited formation of both reticular and fibrillar structures, with surviving laminin largely in a punctate distribution (g). Treatment with 0.5 mM MnSO4 increased the ratio of the area occupied by fibrillar structures compared with reticular structures (h). All images were taken at the same exposure settings and are shown at the same magnification (bar, 10 µm). (i) Laminin fragment map. (j) Relative surface area covered by laminin determined for the conditions described in c-h.

 


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  		Fig. 3. Cell surface binding of non-polymerizing laminin-1. Laminin-1, untreated (+LM, left panel) or treated with AEBSF to selectively inhibit polymerization (+AEBSF-LM), were incubated at 5 µg/ml for 4 hours with Schwann cells. The cell surface distribution of laminin-1 was determined by immunofluorescence microscopy. Non-polymerizing laminin bound to the cell surface with a diffuse distribution.

 


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  		Fig. 4. Dystroglycan topography in response to laminin-1. Schwann cells were incubated with 2 µg/ml of laminin-1 for 8 hours with (g,h,i) and without (d,e,f) anti-ß1 integrin blocking antibody. Schwann cells incubated in the same media but without exogenous laminin-1 shown as control (a,b,c). Non-permeabilizing fixation conditions were used. (a,b,c) In the absence of laminin, dystroglycan was distributed along the Schwann cell surface in a fine punctuate pattern. The level of endogenous laminin immunostaining was negligible. (d,e,f) Surface dystroglycan was observed to be distributed into focal areas on cell surface following 8 hours of incubation with laminin-1. Cell borders are indicated with lines (determined from the corresponding phase images) to aid in the analysis of dystroglycan rearrangement. Laminin distribution from the corresponding area is shown. Merged image reveals zonal co-localization of dystroglycan with the reticular structures of laminin. (g,h,i) In the presence of ß1-integrin blocking antibody, which blocks formation of fibrillar matrix, laminin and dystroglycan co-localization is now seen almost exclusively within plaque-like reticular matrices. Bar, 10 µm.

 


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  		Fig. 5. Distribution of ß1-integrin in the absence and presence of laminin. Cells were incubated with 2 µg/ml of laminin for 8 hours. Cells were fixed, immunostained with laminin-specific antibody, then permeabilized with Triton X-100 and co-stained with anti-ß1 integrin antibody. (a) ß1-integrin was distributed in fibrillar arrays prior to treatment with laminin. Panels b and c reveal integrin and laminin within the same field. Following laminin incubation, co-localization on a subset of integrin fibrils with laminin (arrows indicate examples) was observed. Bar, 10 µm.

 


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  		Fig. 6. Distributions of cytoskeletal components. Laminin at 5 mg/ml was incubated with Schwann cells for 18 hours. Effect of laminin cell surface assembly on several cytoskeletal proteins was evaluated. (a-c) The distribution of utrophin was detected with monoclonal antibody MANCHO3 with and without laminin treatment. Utrophin reorganization was noted with laminin treatment (compare a and b, border shown with arrows). Laminin-utrophin co-localizations were noted after 18 hours of laminin incubation. (d-g) Vinculin (Vn; d,e) and paxillin (Pax; f,g) were noted to be present on the cell/plastic interface; neither co-localized with a laminin matrix. Bar, 10 µm

 


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  		Fig. 7. (a) Electron microscopy. Schwann cells were incubated with and without 10 µg/ml of laminin-1 for 18 hours. In addition, cells incubated with laminin were treated with E3 fragment or blocking antibodies. Three types of cell membrane-associated ECM were detected: continuous electron-dense cell surface associated structure (lamina densa, frame A, >2 µm); or short stretches of electron-dense matrix (frame B, <2 µm); cells cultured without laminin revealed a matrix-free plasma membrane surface (frame C), with infrequent small amorphous deposits such as that in the middle of frame C. By contrast, cells incubated with laminin showed the presence of a lamina densa. Inhibition of laminin-ß1-integrin interaction by antibody Ha2/5 (50 µg/ml) did not abolish the appearance of a membrane-associated electron dense structure. Fragment E3 (100 µg/ml), in contrast to ß1-integrin blocking antibody, prevented formation of an electron dense structure. Bar, 500 nm.

(b) Quantitation of morphology. The distribution of non-polymerizable laminin and its ability to induce a membrane-associated electron dense structure was analyzed after 8 hours of incubation with 10 µg/ml of polymerizing or AEBSF-laminin-1. Non-polymerizing laminin generated little continuous matrix. The graph shows the relative number of events according to analysis of three random cross-sections, as described in Materials and Methods.

 


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  		Fig. 8. Contributions of type IV collagen. EHS type IV collagen was incubated with Schwann cells for 4 hours with or without laminin. Cells were washed, fixed and double-stained with anti-laminin (a,c,e) and anti-collagen type IV (b,d,f) antibodies and corresponding Cy3-or FITC-conjugated secondary antibodies. Accumulation of exogenous laminin on the cell surface occurred without the presence of type IV collagen (a). Exogenous type IV collagen accumulated on the cell surface (d,f), even in the absence of laminin (d), forming mostly fibrillar structures (arrowheads). In the presence of laminin, exogenous type IV collagen also assembled into the reticular type of pattern (f, arrows, compare laminin in e). Bar, 10 µm.

 


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  		Fig. 9. Laminin-dependent type IV collagen assembly. Cells were either untreated (a,b) or incubated with type IV collagen with (e,f) or without (c,d) laminin in the presence of anti-ß1 blocking antibody (all panels), which prevents the formation of fibrillar structures. Cells were fixed and stained as described in Fig. 8. In the absence of laminin, type IV collagen did not accumulate on the cell surface (d). In the presence of laminin, type IV collagen assembled into reticular structures (f) that co-localized with those of laminin (e). Bar, 10 µm. (g) The graph compares surface coverage by type IV collagen (gray) or laminin (black) with or without exogenous laminin.

 


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  		Fig. 10. Schwann cell passage-dependency of laminin binding capacity and receptor expression. Laminin (10 µg/ml) was incubated in serum-free culture medium with high-passage (HPC, P27) and low-passage (LPC, P12) Schwann cells for 2 hours. Cell surface-bound laminin was visualized by immunofluorescence microscopy with laminin-specific antibody (a,b) and analyzed by flow cytometry (e, thick line shows the distribution of cell surface-bound laminin intensities for HPC, while the thin solid line is the distribution for the LPC). The level of laminin bound to the cell surface of HPC was more than 100-times higher than that bound to LPC. Flow cytometry of ß1-integrin (e) and {alpha}-dystroglycan (f) reveal that ß1-integrin levels are about threefold higher in HPC, whereas {alpha}-dystroglycan levels remain nearly the same in HPC and LPC. High-passaged cells continue to express the Schwann cell-specific marker S-100 (e). Bar, 10 µm (a-d).

 

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© The Company of Biologists Ltd 2002