Fig. 3

Rbc3a knockdown disrupts endosomal maturation but not acidification in NC.

(A-D) Confocal images of whole mount immunohistochemical staining for endocytic markers in NC cells in sox10:lyn-gfp transgenics, which labels NC cell membranes (green). (A′-D′) 4× insets of (A-D). (A-B′) Anti-EEA1 marks early endosomes (red). rbc3a-MO1-injected embryos show large EEA1+ aggregates in NC cells (white arrowheads in B′). (C-D′) Anti-LAMP1 marks late endosomes/lysosomes (red). No increase in number or size of LAMP1+ vesicles was observed with rbc3a-MO1 injection. (E, F) Automated quantification of average % area (E) and average particle size (F) per NC cell stained positive for EEA1, LAMP1, or Lysotracker Red (Lyso) using ImageJ Particle Analyzer. rbc3a-MO1 injection produced significantly higher EEA1+ and Lysotracker+ relative area and particle size but significantly smaller LAMP1+ relative area and particle size per NC cell. Error bars represent ± SEM. (G, H) Live whole-mount images of sox10:gfp+ NC cells labeled with Lysotracker (red). rbc3a-MO1-injected embryos show many large, acidic Lyso+ vesicles, which colocalize with EEA1 (I′,J′, white arrows) but not LAMP1 (K′, L′). ** p<0.001, *** p<0.0001. Scale bar, 10 µm.