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Fig. 5. Clustering and colocalization of TrkA and/or β1 integrin induced by laminin-1. (A) TrkA clustering. DRG cells were treated with 5 µg/ml of laminin-1 for 10 minutes. Cells were then fixed and stained with anti-TrkA antibody and secondary antibody conjugated to 5-nm gold particles. Labeled cells were analyzed by electron microscopy. Boxed areas in left panels are shown enlarged in the middle panels. Gold-particle-positive areas are boxed and labeled 1 and 2 and are further enlarged in right panels. The number of gold particles in each cluster was counted within a 100-nm area of the luminal surface of the cells, as shown in the `calculation method' below, giving the percentage of clusters that contain the same number of gold particles. Total numbers of gold particles were not significantly different between cells treated with NGF only [59.07 +6.48 (s.d.)] and cells treated with NGF+laminin-1 [63.06 + 6.58 (s.d.)]; n=30. The graph gives the percentage of each cluster. Asterisks indicate significant differences of clusters containing 4 or >5 particles comparing NGF only and NGF + laminin-1 treatments (*P<0.00001; two-sided t-test). (B) TrkA and β1 integrin clustering. DRG cells were treated with 5 µg/ml of laminin-1 for 10 minutes. Cells were fixed and incubated with anti-TrkA antibody and anti-β1 integrin antibody. The cells were incubated with secondary antibody conjugated to 5-nm gold particles (TrkA, arrows) and 10-nm gold particles (β1 integrin, arrowheads). NGF+laminin-1 induced clustering of β1 integrin as well as TrkA, and both type of clusters localized closely to each other. Bar, 100 nm.
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