FIGURE 2.

Neurons depleted of p600 by RNAi undergo a rapid dyshomeostasis characterized by the aggregation of CaMKIIα and fragmentation of the ER. A, in control neurons, treatment with glutamate (supplemented with glycine, Glu/Gly, 10:1) for 3 min generates a sigmoid dose-response curve with a EC₅₀ of 50–60 μm. Depletion of p600 by RNAi shifts this dose-response curve leftward, indicating greater response for the same dose of glutamate. An average of 500 neurons were scored in total for each condition (control RNAi, n = 18; p600 RNAi, n = 9). B, neurons transfected with either one of the two shRNA constructs targeting p600 were found to be more susceptible to Glu/Gly-induced dyshomeostasis, as indicated by the 2–3-fold increase in CaMKIIα aggregation. n = 5 for control/p600 RNAi 1; n = 6 for control/p600 RNAi 2; more than 500 neurons were analyzed per condition. C, representative confocal pictures of control RNAi-treated and p600 RNAi-treated primary hippocampal neurons (DIV10) transfected with GFP-CaMKIIα following treatment with 30 μm Glu, 3 μm Gly for 3 min. Bar, 20 μm. D, representative confocal pictures of ER morphology visualized with an ER-targeted mCherry protein in control RNAi/GFP-CaMKIIα/mCherry-ER and p600 RNAi/GFP-CaMKIIα/mCherry-ER transfected neurons following treatment with 50 μm Glu, 5 μm Gly for 3 min. The RNAi/GFP-CaMKIIα/mCherry-ER ratio for transfection is 4:1:1. Bar, 10 μm. E, within single neurons, the degree of CaMKIIα aggregation correlates strongly with the degree of fragmentation of the ER (r(17) = 0.89, p < 0.0001). b.b, bimodal bias (see “Materials and Methods”). Error bars, S.E.