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. 2019 May 10;8:e46629. doi: 10.7554/eLife.46629

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© 2019, Ackermann et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 7. — (A–B) Rab5 and EEA1 levels at GFP-2x-FYVE organelles along axons in Pclo^wt/wt (A) and Pclo^gt/gt (B) neurons and those expressing mCh-Rab5^Q79L. (B) In Pclo^gt/gt neurons, mCh-Rab5^Q79L expression increases Rab5 and EEA1 level at GFP-2x-FYVE organelles towards Pclo^wt/wt levels. (C–D) Quantification of (A and B). (C) mCh-Rab5^Q79L expression in Pclo^wt/wt neurons slightly reduces Rab5 levels at GFP-2x-FYVE membranes (Pclo^wt/wt = 1 ± 0.02, n = 1066 puncta; Pclo^wt/wt _{(mCh-Rab5^Q79L)}=0.93 ± 0.02, n = 979 puncta; three independent experiments). mCh-Rab5^Q79L expression in Pclo^gt/gt neurons increases Rab5 levels at PI3P-positive organelles (Pclo^gt/gt = 0.74 ± 0.01, n = 840 puncta; Pclo^gt/gt _{(mCh-Rab5^Q79L)}=0.87 ± 0.01, n = 1185 puncta; three independent experiments). (D) In Pclo^wt/wt neurons mCh-Rab5^Q79L expression causes EEA1 levels to drop (Pclo^wt/wt = 1 ± 0.04, n = 1066 puncta; Pclo^wt/wt _{(mCh-Rab5^Q79L)}=0.77 ± 0.04, n = 979 puncta; three independent experiments). mCh-Rab5^Q79L expression rescues EEA1 levels in Pclo^gt/gt neurons (Pclo^gt/gt = 0.53 ± 0.03, n = 840 puncta; Pclo^gt/gt _{(mCh-Rab5^Q79L)}=0.97 ± 0.05, n = 1185 puncta; three independent experiments). (E) mCh-Rab5^Q79L expression in Pclo^wt/wt neurons increases Synaptophysin intensities. (F) Quantification of (E). Synaptophysin puncta intensity slightly increases upon mCh-Rab5^Q79L expression in Pclo^wt/wt neurons. In Pclo^gt/gt neurons, mCh-Rab5^Q79L expression rescues Synaptophysin levels higher than Pclo^wt/wt levels (Pclo^wt/wt = 1 ± 0.02, n = 620 synapses; Pclo^gt/gt = 0.86 ± 0.02, n = 526 synapses; Pclo^wt/wt _{(mCh-Rab5^Q79L)}=1.07 ± 0.03, n = 473 synapses; Pclo^gt/gt _{(mCh-Rab5^Q79L)}=1.26 ± 0.03, n = 446 synapses; three independent experiment). Scale bars represent 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM. C, D and H ANOVA with Tukey multi comparison test. ** denotes p<0.01, *** denotes p<0.001 and **** denotes p<0.0001.

Figure 7—source data 1. This spreadsheet contains the normalized values used to generate the bar plots shown in Figure 7C,D and F.

elife-46629-fig7-data1.xlsx^{(187.3KB, xlsx)}

DOI: 10.7554/eLife.46629.020