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. 2018 Aug 6;217(8):2709–2726. doi: 10.1083/jcb.201709055

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© 2018 Ripoll et al.

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Figure 2. — Optineurin associates with Myo6 and releases melanosomal tubules. (A and B) SR-IFM on MNT-1 cells costained for endogenous Myo6 and TYRP1 (A) and linear pixel values across n melanosomes (B; n = 130) show Myo6 (A; arrowheads, 4× boxed regions) localization to TYRP1⁺ melanosomal subdomain. (C and D) Percentage of Myo6/TYRP1⁺ melanosomes (C; n = 15 cells), and relative number of Myo6⁺ spots per n melanosome (D; n = 441). (E and F) SR-IFM of MNT-1 cells costained for endogenous optineurin and TYRP1 (E) and linear pixel values across n melanosomes (F; n = 95) show optineurin (E; arrowheads, 4× boxed regions) localization to TYRP1⁺ melanosomal subdomain. (G and H) Percentage of optineurin/TYRP1⁺ melanosomes (G; n = 20 cells) and relative number of optineurin⁺ spots per n melanosome (H; n = 400). (I) SR-IFM on GFP-Myo6-CBD–expressing MNT-1 cells labeled for optineurin and TYRP1. Magnified views (4×) show Myo6 and optineurin codistribution on TYRP1⁺ melanosome subdomains. (J) Quantification of the percentage of melanosomes with overlapping GFP-Myo6-CBD and optineurin subdomains as in I (n = 14 cells). (K) Western blot analysis of lysates and GFP IPs of GFP- or GFP-Myo6-CBD–expressing MNT-1 cells probed for optineurin or GFP antibodies. (L) Live imaging frames of MNT-1 cells expressing mCh-VAMP7 and treated with control or optineurin (Optn) siRNAs. Magnified areas (4×) of boxed regions in siOptn cells show tubules (arrowheads) associated with melanosomes (arrows). (M) Relative number of mCh-VAMP7⁺ melanosomal tubules during 40-s movies per 256-µm² area of cells treated as in L (siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). (N) Percentage of mCh-VAMP7⁺ tubules detaching from melanosomes in control- or optineurin-depleted cells (M and N; siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). Molecular masses are in kilodaltons. Data are presented as the mean ± SEM. Bars: (A, E, I, and L) 10 µm; (A, B, E, F, and L, magnifications) 1 µm; (I, magnifications) 500 nm. **, P < 0.01; *, P < 0.05 (unpaired t test).