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. 2015 Jul 20;210(2):303–318. doi: 10.1083/jcb.201411001

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© 2015 Gomez-Lamarca et al.

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Figure 4. — Notch associates with enlarged endosomes and recovers more slowly at the membrane in Rme-8–depleted cells. (A) Enlarged puncta containing NICD, Rab4, and Rab7 are detected in Rme8-Ri, where NICD accumulates in proximity to Rab4 (see insets). (B) Enlarged puncta containing NICD, Rab4, and Rab11 were detected in Rme-8-Ri cells. Some puncta contain all three proteins (insets, yellow arrowheads), others contain Rab11 only (insets, green arrowheads) or Rab4+NICD (insets, red arrowheads). (C–F) Superresolution images of endosomes from WT (C and E) or Rme-8-Ri (D and F) wing disc cells stained for NICD and Rab4 (C and D) or Rab7 (E and F). Notch localizes adjacent to Rab4 in protrusions (arrows, C). In Rme-8–depleted cells, tubule-like protrusions appear more extensive (arrows, D). Rab7 labels a compartment adjacent to NICD accumulations, both in WT and Rme-8-Ri cells (arrowheads, E and F). (G) Measurements of protruding Rab4-labeled tubule-like structures on endosomes from WT and Rme-8 knockdown (≥50 tubules/disc; 5 discs per genotype). Asterisks indicate significance according to Mann–Whitney test: ****, P < 0.0001. (H and I) Sections from WT (H) or Rme-8-Ri (I) regions of a wing disc analyzed by TEM, with endosomes highlighted in yellow. (J) Quantification of endosome perimeters from TEM images. (WT n = 28; Rme-8-i+w-Ri n = 76.) Asterisks indicate significance according to t test; ****, P < 0.0001. (K and L) FRAP experiments, measuring recovery of Notch-GFP after photobleaching. (K) Plot of averaged recovery curves (mean ± SEM) with best-fit curves as solid lines. Boxes indicate 25th to 75th percentiles, whiskers represent maximum and minimum values in the dataset, and lines show the median values. (L) Single FRAP examples with red circles showing bleach spots. (P, prebleach). The recovery curves could be described by a biexponential equation (WT, P < 0.0001; Rme-8, P = 0.02; Bulgakova et al., 2013; see Materials and methods) suggestive of two recovery processes, fast diffusion within the membrane and slower endocytic trafficking. Both appear affected by Rme-8 depletion.