Diphosphomimetic RLC-T18D, S19D rescues spine actin enrichment and synapse formation in ROCK1 knockdown neurons. (A) DIV 19 rat hippocampal neurons were fixed and stained for diphosphorylated RLC (RLC-PP). Arrowheads indicate RLC-PP enrichment in postsynaptic spines of control and ROCK2 knockdown neurons, whereas arrows highlight the lack of RLC-PP in spines of ROCK1 knockdown neurons. (B) DIV 22 rat hippocampal neurons were fixed and stained for actin with rhodamine phalloidin (magenta) and the presynaptic marker, Synaptophysin (Sphysin). Control neurons (top) and ROCK2 knockdown neurons (sequence A) exhibit actin-enriched spines that colocalize with the presynaptic marker, Synaptophysin. ROCK1 knockdown neurons exhibit a loss of spine actin enrichment and synapse formation that was rescued by coexpressing diphosphomimetic RLC-T18D, S19D-GFP (bottom). (C) Quantification of spine actin enrichment, quantified as the ratio of the mean intensity of rhodamine phalloidin in spines to the mean intensity in the adjacent dendrite segment. Actin enrichment was calculated for n ≥ 40 spines for all conditions. Error bars indicate SEM. Ctrl, control.