Figure 1.
Shank1B and Homer1b recruit calreticulin, calbindin, and SERCA2b to dendritic spines but not mGluRs1/5. Neurons were transfected (Trx) at 11 d in vitro (DIV) with HA-Shank1B plus Homer1b, with the HA-Shank1 (1-1440) fragment, or with an empty vector, as indicated on the left side of the panels, and the neurons were stained at 18 DIV for Shank (on the red channel; left column) together with calreticulin (on the green channel) (A2, B2, C2, D2; middle column), calbindin (E2, F2, G2; middle column), SERCA2b (H2, I2, J2; middle column), or mGluR5 (K2, L2; middle column). Merged images are shown in the right column. In Shank1B plus Homer1b-cotransfected neurons, the calreticulin and SERCA2b colocalize with Shank. Only a few spines of transfected neurons show intense staining for calbindin (F1-F3, arrow heads), although most show a moderate staining (F1-F3, arrows). Endogenous mGluR5 staining is colocalized with Shank partially (K1-K3) but is not recruited further to synapses by Shank1B and Homer1b overexpression (L1-L3). B1-B3, F1-F3, and I1-I3 are higher magnifications of A1-A3, E1-E3, and H1-H3, respectively. Scale bars: (in H3) A1-A3, E1-E3, H1-H3, 10 μm; (in J3) B1-D3, F1-G3, I1-L3, 2.8 μm. M, Quantification of the changes in synaptic staining of the indicated proteins induced by the overexpression of Shank1B plus Homer1b. At least six neurons were analyzed for each endogenous protein (30-50 synapses scored per neuron). Histograms and error bars show the means ± SD, normalized to the staining intensity in nontransfected and vector-transfected neurons; *p < 0.05.