Figure 3: Shrm4 links GABABRs to the dynein/dynactin complex.
(a) Immunofluorescence of GABABR intensity in the cell body of 18DIV cultured hippocampal neurons transfected with Shrm4 scrambled#1, knockdown (shRNA#1) or rescue constructs at 8DIV. All data in bar graphs throughout are means±s.e.m.; n=10, 10, 15 *P=0.0057; F(2,32)=6.094, one-way ANOVA-Mann–Whitney. (b) Co-immunoprecipitation experiments on brain extracts using polyclonal anti-Shrm4 show that Shrm4, GABAB1, GABAB2 and DIC are associated. By contrast, KIF5A is not present in this complex. (c) Co-immunoprecipitation of both GABAB1 isoforms in HEK293 cells transfected with Shrm4-V5 and GABAB1a-myc (top) or GABAB1b-myc (bottom). GABAB1a, GABAB1b and the endogenous DIC co-immunoprecipitated with Shrm4-V5. (d) Endogenous DIC co-immunoprecipitates with expressed HA-Shrm4 in HEK293 cells in the absence of GABAB1a or GABAB1b subunits. Monoclonal antibodies (anti-V5, anti-myc and anti-DIC) were used for western blot. (e) Western blot from in vitro pull-down assay of purified Histidine-tagged DIC using GST-PDZ and mutant GST-PDZ (AA). The Shrm4-PDZ domain is able to directly bind DIC even when its common binding site is mutated. Ponceau Red staining of the blot has been presented at the bottom. (f) GST pull-down experiments on brain extracts show that the Shrm4-PDZ domain pulls down DIC while the mutant GST-PDZΔ14 does not. Ponceau red staining of the blot has been presented at the bottom (full blot in Supplementary Fig. 11). (g) Conventional (top left) and super-resolution direct stochastic optical reconstruction microscopy (dSTORM) (bottom left) imaging of Tubulin-ATTO 488 (shown in red) and Shrm4-Alexa 647 (shown in green). Shrm4 positive puncta are localized along microtubule-positive filaments, as evidenced in the details of the super-resolution image (top right). The super-resolution data has been quantified by cross-correlation analysis. The positive, higher than 1 cross-correlation, indicates co-clustering of the two fluorescent signals (Error-bars are s.e.m.s, number of regions for the cross-correlation measurement: 30, number of fields: 3. Scale bar, 0.4 μm). (h) Schematic illustrating unilateral local injection of AAV5-scrambled#1 (left hemisphere) and AAV5-shRNA#1 (right hemisphere) into rat brain CA1 hippocampus, with time line for recovery and experiments. (i) Western blots and histograms showing monoclonal anti-DIC immunoprecipitation from scrambled and shRNA lysates of infected hippocampus extracts (n=6 rats). Co-precipitated GABAB1 levels were normalized to DIC immunoprecipitation levels and the normalized percentage of GABAB1 co-precipitation was calculated. Histograms show mean±s.e.m.; *P<0.05, t-test.
