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Figure 2.
Calpain activity regulates adhesion protein localization and signaling in filopodia. a–i, Immunocytochemistry experiments were performed with cultured Xenopus spinal neurons plated on laminin. The fluorescence intensity within filopodia was quantified in the following three conditions: control, with 1 μm CPI (calpain inhibition), or without AGAs (−AGAs, calpain activation). a–i, Representative images of growth cones (left) and filopodia (right) shown for selected adhesion proteins, as follows: talin (a–c), phospho-tyrosine 397 FAK (FAK Y397; b–h), and phospho-tyrosine 118 paxillin (PXN Y118; c–i). Immunolabeled adhesion proteins are in green, and phallodin-labeled F-actin is in red. Arrows indicate localization to filopodial tips. j, Quantification of filopodial fluorescence intensity of all adhesion markers tested, including vinculin and total phospho-tyrosine (pY99), with inhibition of calpain or Ca2+-dependent activation of calpain by removal of AGAs. k, Quantification of filopodial fluorescence intensity of talin, FAK Y397, and PXN Y118 in growth cones expressing GFP-tagged dominant-negative calpain1 (H272A) normalized to cocultured wild-type neurons. Scale bar, 5 μm. n > 60 growth cones and n > 3 cultures for all conditions. *p < 0.05, Mann–Whitney U test. |
