Fig. 4

Ribosome responsiveness to the BDNF/TrkB signaling as well as ribosome/ER tethering are impaired in Smn-deficient axon terminals. a BDNF-stimulated Smn+/+;SMN2tgtg and Smn−/−;SMN2tgtg motoneurons expressing mCherry-KDEL were stained against RPL24, RPS6 and mCherry-KDEL. Growth cones were imaged by SIM. White boxes indicate enlarged ROIs within growth cones. In control but not Smn−/−;SMN2tgtg motoneurons, BDNF induces formation of RPL24/RPS6 co-clusters that colocalize with the axonal ER at 10 s and 1-min poststimulation. b Smn−/−;SMN2tgtg motoneurons do not exhibit increased number of RPL24/RPS6 co-clusters upon stimulation (n.s., P = 0.9999; n = 21–23 cells), in contrast to Smn+/+;SMN2tgtg (*P = 0.0147 for no BDNF vs. 10-s BDNF; *P = 0.0210 for no BDNF vs. 1-min BDNF; n = 13–16 cells from 3 independent experiments). In control experiments, either RPL24 or RPS6 antibodies were omitted to exclude the crosstalk between channels. Colocalization analysis detected almost no co-clusters in the growth cones of control neurons (see also Additional file 1: Fig. S3). c In Smn+/+;SMN2tgtg motoneurons, the number of RPL24/RPS6 co-clusters that colocalize with ER increases significantly within 10 s and 1-min of stimulation (**P = 0.0089 for no BDNF vs. 10 s BDNF; **P = 0.0092 for no BDNF vs. 1-min BDNF; n = 13–16 cells from 3 independent experiments). In Smn−/−;SMN2tgtg motoneurons the number of RPL24/RPS6 co-clusters that associate with ER does not increase upon stimulation (n.s., P = 0.7601; n = 21–24 cells from 3 independent experiments). In all graphs, the average number of co-clusters are shown per growth cone. All representative images are from maximum projection of five 0.12-µm z-stacks. Data are presented in scatter dot plot; error bars represent mean ± SEM. Statistical analyses were done by one-way ANOVA with Dunn’s post-test