Figure 4. Increased RAB-5 activity causes EFF-1 accumulation in large intracellular puncta, which are RAB-5-positive compartments. a–c, Representative maximum projection confocal images of the PLM cell body in eff-1(ok1021) animals expressing RAB-5(DN) (a), RAB-5(WT) (b), or RAB-5(CA) (c) compared with control animals lacking each respective RAB-5 transgene. Displayed for each cell body are the overlay of the red (diffusible mCherry) and green (GFP-tagged EFF-1) channels (top row), the green channel (middle row), and the result of automatic puncta selection using Squassh (bottom row). Scale bars, 1 μm. Below the confocal images are graphs showing quantification of the average EFF-1::GFP puncta size in independent transgenic isolates expressing each version of RAB-5: RAB-5(DN) (a) RAB-5(WT) (b), or RAB-5(CA) (c). Animals with a given RAB-5 transgene (red) were compared with siblings lacking the transgene (black). A significant increase in average puncta size was found for three independent RAB-5(CA) isolates and for two RAB-5(WT) isolates injected at different concentrations (5 ng/μl and 10 ng/μl). Each data point represents one cell body from one animal; n ≥ 14. Bars represent the mean of each group with the SD; exact p-values from t test. AU, Arbitrary units. d, Representative maximum projection confocal images of the PLM cell body in an eff-1(ok1021) animal at 6 h after axotomy compared with an uncut control animal. As for a–c, displayed for each cell body are the overlay of the red (diffusible mCherry) and green (GFP-tagged EFF-1) channels (top row), the green channel (middle row), and the result of automatic puncta selection using Squassh (bottom row). Scale bars, 1 μm. Below the confocal images are graphs showing quantification of the average EFF-1::GFP puncta size. Animals at 3 and 6 h after axotomy (red) are compared with uncut animals (black). No significant difference was noted. Each data point represents one cell body from one animal. Bars represent the mean of each group with the SD. e, Representative maximum projection confocal image of the PLM cell body in an animal coexpressing EFF-1::GFP and BFP::RAB-5; the overlay and individual green and blue channels (left column) are matched with the result of automatic puncta selection using Squassh (right column), demonstrating strong colocalization between the EFF-1::GFP and BFP::RAB-5 puncta. Scale bar, 1 μm. Next to these confocal images is a graph showing quantification of the average EFF-1::GFP puncta size in a transgenic strain carrying EFF-1::GFP and BFP::RAB-5. A significant increase in EFF-1::GFP puncta size occurred when BFP::RAB-5 was also present (+). The BFP::RAB-5 transgene was injected at the same concentration as the other RAB-5 transgenes. Each data point represents one cell body from one animal; n ≥ 17. Bars represent the mean of each group with the SD; exact p-values from t test. AU, Arbitrary units.