|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 8. The marker of neuronal macroendocytosis, Pincher, regulates constitutive plasma-membrane retrieval at the growth cone. Growth cones of neurons at 3 DIV that were infected with adenoviruses driving the simultaneous expression of GFP and either HA-Pincher or HA–Pincher-G68E are shown. (A) Growth cones expressing low levels of HA-Pincher or HA–Pincher-G68E that were stained with anti-HA antibody (green) after basal FM4-64 (red) uptake (1 minute) to reveal the distribution of the exogenous proteins. The overlay between the fluorescence and DIC images is shown in the right panels. (B) Growth cone expressing HA-Pincher at low levels exposed to 20-nm beads (red in the merged image) for 1 minute in KRH, fixed and stained with anti-HA antibody (green in the merged image). The images are maximal projections of deconvoluted z-stacks. Pincher overlaps with the compartments of bulk endocytosis. (C,D) Basal FM4-64 uptake (1 minute) in growth cones overexpressing GFP and either HA-Pincher or HA–Pincher-G68E, or infected with adenoviruses expressing only GFP (control). (C) Shows the overlay between the fluorescence and DIC images of a HA-Pincher/GFP-expressing growth cone (arrowhead) and of an uninfected growth cone (asterisk) located in the same field of view. Overexpression of either Pincher or the dominant-negative mutant Pincher G68E inhibits constitutive bulk plasma-membrane uptake, whereas endocytosis via smaller vesicles is unaffected (arrowheads in D). (E) Quantification of FM4-64 uptake in HA-Pincher- or HA–Pincher-G68E-expressing growth cones (mean ± s.e.m.; n=50 growth cones per condition; *P<0.001, Dunnett's test vs control, i.e. growth cones expressing only GFP). Expression of either mutant does not affect the total growth-cone area (P>0.05, Dunnett's test vs control). (F) Quantification of axon length in 2-DIV neurons that were nucleofected with GFP alone (control) or together with either HA-Pincher or HA–Pincher-G68E. The length of the longest neurite, corresponding to the axonal process, was measured for each transfected neuron (mean ± s.e.m.; n=100-150 growth cones per condition from three independent experiments; **P<0.001, Tukey's test Pincher G68E vs control; *P<0.05, Pincher wild-type vs control; 
P<0.05, Pincher wild-type vs Pincher G68E). Scale bar: 10 µm (A,C,D); 3.3 µm (B).
|
