RLC T18, S19 diphosphorylation and cofilin S3 phosphorylation regulate adhesion and PSD maturation. (A) Knockdown of either ROCK1 or ROCK2 reduces adhesion size but can be rescued by coexpression of either diphosphomimetic RLC-T18D, S19D or phosphomimetic cofilin S3D, respectively. CHO.K1 cells cotransfected with either control empty vector or ROCK shRNAs and the indicated GFP construct and plated on 2 µg/ml fibronectin for 2 h were stained for actin and the adhesion marker vinculin. (B) Quantification of mean adhesion size per cell. (C) Nonphosphorylatable RLC-T18A, S19A significantly reduces adhesion size in control and ROCK2 knockdown cells, but not ROCK1 knockdown cell. Quantification of mean adhesion size per cell in CHO.K1s expressing GFP or GFP-tagged RLC-T18A, S19A (RLC-AA–GFP). (D) Diphosphomimetic RLC-T18D, S19D rescues adhesion size in ROCK1 shRNA cells and increases adhesion size in control and ROCK2 shRNA cells. Quantification of mean adhesion size per cell in CHO.K1s expressing either GFP or GFP-tagged RLC-T18D, S19D (RLC-DD–GFP). (E) Phosphomimetic cofilin S3D rescues adhesion size in ROCK2 shRNA cells, but also increases adhesion size in control and ROCK1 shRNA cells. Quantification of mean adhesion size per cell in CHO.K1s expressing either GFP or GFP-tagged cofilin S3D. n for adhesion analysis ≥ 9 cells per condition with a mean of >100 adhesions analyzed per cell. (F) Cofilin inactivation increases PSD size. Immunofluorescence of DIV 14 rat hippocampal neurons transfected with either the active, nonphosphorylatable cofilin-S3A–GFP or the inactive phosphomimetic mutant, cofilin-S3D–GFP, and costained for the presynaptic marker, VGlut-1, and postsynaptic marker, PSD-95. Arrowheads indicate synapses in the two conditions. Arrows indicate a dendritic, as opposed to spine-associated, synapse, in a cofilin-S3D–expressing neuron. (G) Quantification of mean PSD area per neuron in rat hippocampal neurons expressing either GFP (control [Ctrl]) or GFP-tagged cofilin constructs (S3A, WT, and S3D). n = 12 cofilin S3D-expressing neurons, 11 cofilin S3A-expressing neurons, 12 WT cofilin-expressing neurons, and 5 control neurons. Error bars indicate SEM.