Figure 8. Expression of GTPase deficient Rab5 (Rab5Q79L) in Pclogt/gt neurons rescues the size of the total recycling pool of vesicles but not FM1-43 unloading kinetics.
(A) Representative images showing FM1-43 levels in Pclowt/wt and Pclogt/gt boutons either positive (arrow) or negative (arrowhead) for mCh-Rab5Q79L after 60 mM KCl stimulation. (B) Quantification of (A). (Pclowt/wt = 1 ± 0.01, n = 528 puncta; Pclogt/gt = 0.76 ± 0.01, n = 654 puncta; Pclowt/wt (mCh-Rab5Q79L)=1.04 ± 0.02, n = 304 puncta; Pclogt/gt (mCh-Rab5Q79L)=1.22 ± 0.02, n = 337 puncta; three independent experiments). (C) Left: Images depicting FM1-43 intensities in Pclowt/wt and Pclogt/gt boutons either positive (arrow) or negative (arrowhead) for mCh-Rab5Q79L. Right: Images depicting FM1-43 unloading kinetics in Pclowt/wt and Pclogt/gt boutons either positive (arrow) or negative (arrowhead) for mCh-Rab5Q79L. (D) Quantification of (C). The presence of mCh-Rab5Q79L is not sufficient to rescue slowed FM1-43 unloading kinetics and total amount of FM1-43 dye released in Pclogt/gt boutons (Pclogt/gt = 47.24 ± 0.68, n = 361 synapses; Pclogt/gt(Rab5Q79L)=48.33 ± 0.53, n = 479 synapses; four independent experiments). In Pclowt/wt boutons expressing mCh-Rab5Q79L, FM1-43 unloading kinetics are slowed and total amounts of dye released are reduced similar to what is observed in Pclogt/gt boutons (Pclowt/wt = 56.28 ± 0.81, n = 301 synapses; Pclowt/wt(Rab5Q79L)=50.15 ± 0.85, n = 342 synapses; four independent experiments). Scale bars represent 5 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM. ANOVA with Tukey multi comparison test. * denotes p<0.05, **** denotes p<0.0001.