FIGURE 6.

Silencing ERM protein expression alters cell morphology, neuritogenesis, and growth cone morphology; rescue by wild-type but not AKAP-deficient radixin. A, IMR-32 cells were transfected with empty plasmid (Ctrl) or with shRNA plasmids targeting ezrin, radixin, and moesin (shERM) for 72 h. Cells were then re-plated onto uncoated glass coverslips in regular growth medium containing 10% serum (Glass/10% FBS) or onto poly-l-lysine-coated coverslips (PLL) in serum-free medium containing 5 μm all-trans retinoic acid (atRA) or 500 ng/ml netrin-1 (Ntn). Cells were fixed 12 h after re-plating and stained with Alexa 488-phallodin and DAPI to visualize F-actin and nuclei, respectively, then imaged by epifluorescence microscopy (scale bar = 20 μm; note that the panels in the last column have been magnified 2× to better visualize the growth cones). In some instances the images are purposely overexposed to better visualize the fine details in growth cones and other structures. B, the number of neurites per nucleus, neurite length, and the percent of neurites exhibiting well defined growth cones (% GC⁺ neurites) were determined from three separate transfections and after differentiation in the presence of netrin-1. Data are presented as the averages ± S.E. (n ≥ 40) and were analyzed by a two-tailed, unpaired t test (*, p = 0.022; **, p < 0.001). C, radixin, but not ezrin or moesin, rescues growth cone morphology in ERM-silenced IMR-32 cells. IMR-32 cells were transfected with empty silencing plasmid (shCtrl) or with shRNA plasmids targeting ezrin, radixin, and moesin (shERM) for 24 h, then co-transfected again with a plasmid expressing mCherry (red) along with an empty expression vector (vector) or plasmids expressing wild-type ezrin (Ezr), moesin (Msn), or radixin (Rdx) or an AKAP-deficient point mutant of radixin (Rdx^L421P) for 36 h. Cells were then re-plated onto poly-l-lysine-coated coverslips in serum-free medium containing 500 ng/ml netrin-1 for 12 h before fixing and staining with Alexa 488-phallodin (shown in inverted grayscale) to visualize F-actin and general growth cone morphology. D, quantification of growth cone area (in μm²) in cells transfected as indicated. n ≥ 12 growth cones total from three separate transfections (*, p = 0.0132 versus shCtrl and p < 0.0005 versus all other conditions). E, phospho-mimetic T564D Rdx, but not WT Rdx, anchors PKA RIIα to the DCC cytoplasmic domain in vitro. Purified, recombinant RIIα, and either wild-type (WT) or T564D (⁵⁶⁴D) radixin (Rdx) were combined with purified GST fused to either full-length DCC cytoplasmic domain (WT) or to the cytoplasmic domain lacking the P3 region (ΔP3). Complexes were collected on glutathione beads and analyzed along with a portion of the input mixtures by SDS-PAGE and immunoblotting with the indicated antibodies. F, T564D Rdx, but not L421P/T564D Rdx, anchors PKA RIIα to the DCC cytoplasmic domain in vitro. Purified, recombinant RIIα, and either T564D or L421P/T564 Rdx were combined with purified GST-DCC cytoplasmic domain. Complexes were collected on glutathione beads and analyzed along with a portion of the input mixtures by SDS-PAGE and immunoblotting with the indicated antibodies.