Figure 3: Net4 disrupts pre-existing laminin networks.

(a) Inhibition of laminin 111 polymerization (Lm111) by human laminin β1 (β1LN-LEa1-4), mouse laminin γ1 (γ1LN-LEa1-4), mouse Net1 (Net1-ΔC), mouse Net4-ΔC and the laminin-binding mutant mouse Net4-ΔC^E195A,R199A. For the experimental scheme depicting the laminin 111 polymerization assay see Supplementary Fig. 7a. SDS–PAGE analysis of the pellet (P) and the supernatant fraction (S). The position of the laminin chains (arrow heads) and the recombinant proteins (stars) are indicated. (b) Densitometric analysis of three independent polymerization experiments and the percentage of the pellet fraction are displayed. (mean±s.d.; n=3; ****P<0.00006 (ctrl vs β1LN-LEa1-4, Net4-ΔC and Net4-ΔC^E195A,R199A) and ***P=0.0004 (ctrl vs γ1LN-LEa1-4); ns, not significant). (c) For the method procedure see Supplementary Fig. 7b. The resulting fractions (supernatant of laminin polymerization (S), supernatant after addition of different proteins (S+) and polymer (P+)) were separated by SDS–PAGE, followed by Coomassie Brilliant-Blue staining. Different laminin chains are colour labelled (α1 (yellow), β1 (green) and γ1 (blue)) and added recombinant proteins are indicated with asterisks. (d) The ratio between polymer (P+) and supernatant (S+) was determined by densitometric analysis of at least three independent experiments (mean±s.d.; n=3; ****P=0.00005). (e) N-terminal laminin fragments (LN-LEa1-4) of α1 (yellow), β1 (green) and γ1 (blue) as well as MBP-Net4-ΔC (red) single proteins were analysed using analytical size-exclusion chromatography (top). SEC profile of the laminin fragment γ1LN-LEa1-4 (γ1) in complex with MBP-Net4-ΔC (green, middle). Complexes [α1-β1-γ1 (blue) and α1-β1-γ1+MBP-Net4-ΔC (red)] were analysed by SEC (bottom) revealing that the MBP-Net4-ΔC is able to disrupt the α1-β1-γ1 complex. Asterisks indicate the single proteins within the complex. (f) Atomic force microscopy anaylsis of a polymerized Matrigel matrix treated with Net4-ΔC or with the laminin-binding mutant Net4-ΔC^E195A,R199A. (g) Model depicting inhibition of laminin polymerization via laminin β1 (β1LN-LEa1-4), laminin γ1 (γ1LN-LEa1-4), mouse Net4-ΔC, and the laminin-binding mutant mouse Net4-ΔC^E195A,R199A but not via mouse Net1 (Net1-ΔC). The pre-existing laminin network can only be solubilized through Net4. Error bars, s.d. (n=3 independent technical replicates).