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Figure 5. Newly synthesized eIF3A and RPS17 are required for visual experience-dependent structural plasticity.(A) Schematic of different steps of regulation in gene and protein expression. (B–D’’) VE-dependent changes in tectal neuron dendritic arbor growth rate over 4 hr in dark and VE in tecta electroporated with control MO (B, C, D), designated CPP MOs (B’, B’’, C’, D’), or CPP MOs and rescue constructs (C’’, D’’). Gray lines connect data points for individual neurons and black lines are average changes in TDBL in dark and VE. (B–B”) VE-dependent changes in growth rate in tecta treated with control MO (B, eIF3A MO (B’), or RPS17 MO (B’’). Control MO: n = 22 cells; eIF3A MO: n = 17 cells; RPS17 MO: n = 10 cells. Both eIF3A MO and RPS17 MO blocked the VE-dependent increase in dendritic arbor growth rate observed in controls. (C–C’’) Co-expression of eIF3A MO and exogenous eIF3A rescued the deficit in VE-induced structural plasticity seen with eIF3A knockdown. Control MO: n = 10 cells; eIF3A MO: n = 14 cells; eIF3A MO + eIF3A: n = 7 cells. (D–D’’) Co-expression of RPS17 MO and exogenous RPS17 rescued the deficit in VE-induced dendritic structural plasticity seen with RPS17 knockdown. Control MO: n = 9 cells; RPS17 MO: n = 10 cells; RPS17 MO + RPS17: n = 9 cells. (E–H) Validation of eIF3A and RPS17 knockdown and OE. (E, F) Left: representative WB of eIF3A and β-tubulin from tecta electroporated with eIF3A MO (E) or MO-insensitive eIF3A expression construct (F) compared to controls. Right: eIF3A MO significantly decreased synthesis of eIF3A protein (eIF3A MO: 0.5 ± 0.05, p=0.0003; n = 5 independent experiments) and the eIF3A expression construct generated significantly more eIF3A protein (eIF3A-OE: 1.89 ± 0.18, p=0.0198; n = 3 independent experiments). (G) Left: WB of RPS17 and β-tubulin from tecta electroporated with control or RPS17 MO. Right: Normalized RPS17 expression levels in tecta electroporated with control or RPS17 MO. RPS17 MO significantly reduced synthesis of RPS17 protein (RPS17 MO: 0.54 ± 0.16, p=0.0309; n = 4 independent experiments). (H) Left: Images of GFP (top), RPS17 (middle) expression and DAPI (bottom) labeling in HEK cells expressing GFP alone (Control, left) or GFP and RPS17 (right). Right: RPS17 expression in GFP+ ROI, normalized to RPS17 expression in GFP- ROI. The RPS17 expression construct increased RPS17 immunolabeling. Control: 1.6 ± 0.16; RPS17-OE: 2.43 ± 0.19; p=0.0029; n = 7 different fields imaged from two independent experiments for each experimental condition. *p<0.05, **p<0.01, two-tailed paired Student’s t test were used to compare between two matched pairs (B–D) and one-tailed Student’s t test for comparisons of two independent groups (E–H). Error bars represent ±SEM (E–H).10.7554/eLife.33420.015Figure 5—source data 1. Values of VE-dependent changes in tectal neuron growth rate over 4 hr in dark and VE presented in Figure 5B–B’’, Figure 5C–C’’, and Figure 5D–D’’.Raw values of normalized protein expression of eif3A or RPS17 in tecta electroporated with control/eif3A MO, control/eIF3A expression construct, or control/RPS17 MO presented in Figure 5E–G. Raw values of normalized protein expression of RPS17 in HEK cells transfected with control or RPS17 expression construct presented in Figure 5H. Image published in: Liu HH et al. (2018) © 2018, Liu et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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