Figure 1.
Figure 1. Myo6 releases tubules from melanosome. (A and B) Live imaging frames of mCh-VAMP7 expressing MNT-1 cells. VAMP7 localizes to melanosomes (A, arrows). Magnified views (B; 3× boxed area in A) of consecutive time-lapse images show VAMP7+ tubule (arrowheads) emerging and detaching (red arrow) from melanosome (arrows) over time (s). (C) Quantification of the time of release of mCh-VAMP7+ tubules from melanosomes (n = 4 independent experiments). (D) Live imaging frames of mCh-VAMP7 expressing siCtrl or siMyo6 MNT-1 cells. Magnifications (4×) of boxed area in siMyo6 cells show VAMP7+ tubules (arrowheads) associated with melanosomes (arrows). (E) Relative number of mCh-VAMP7+ melanosomal tubules during 40-s movies per 256-µm2 area of cells treated with siCtrl or siMyo6 expressing or not GFP-Myo6R (siCtrl, n = 4 independent experiments; siMyo6, n = 3 independent experiments; siMyo6+GFP-Myo6R, n = 3 independent experiments). (F) Percentage of mCh-VAMP7+ tubules detaching from melanosomes in cells treated as in E (E andF; siCtrl, n = 4 independent experiments; siMyo6, n = 3 independent experiments; siMyo6 + GFP-Myo6R, n = 3 independent experiments). (G and H) Live imaging frames of mCh-VAMP7 expressing Myo6-depleted MNT-1 cells. Magnified views (H; 4× boxed area in G) depict VAMP7+ tubule (arrowheads) emerging from a melanosome (arrows) without detaching over time (s). (I) Live cell imaging analysis of mCh-VAMP7 expressing MNT-1 cells treated with DMSO or 5 µM TIP. Magnified areas (4×) of boxed regions show tubules (arrowheads) associated with melanosomes (arrows). (J) Relative number of mCh-VAMP7+ melanosomal tubules during 40-s movies per 256-µm2 area of cells treated as in I (DMSO, n = 3 independent experiments; TIP, n = 3 independent experiments). (K) Percentage of mCh-VAMP7+ tubules released from melanosomes in DMSO- or TIP-treated cells (J and K; DMSO, n = 3 independent experiments; TIP, n = 3 independent experiments). (L) Live imaging frame of GFP-Myo6-CBD and mCh-VAMP7 expressing MNT-1 cells. Magnified boxed areas (4×) show a VAMP7+ tubule (arrowhead) associated with a GFP-Myo6-CBD+ melanosome (arrow). (M) Pearson’s correlation coefficient of cells (n = 26) as in L. Control values were obtained by 30° rotation of the GFP layer of the same images. (N and O) Live SR-FM of GFP-Myo6-CBD and mCh-VAMP7 expressing MNT-1 cells (N) and linear pixel values (normalized to maximal fluorescence) across double positive melanosomes associated with tubules (O). GFP-Myo6-CBD peak of fluorescence intersects the core of n melanosome (arrow) and the tubule (arrowhead; n = 30). Data are presented as the mean ± SEM. Bars: (A, D, G, I, and L) 10 µm; (B, H, N and magnifications in D, I, and L) 1 µm. ****, P < 0.0001; *, P < 0.05; n.s., not significant (unpaired t test).

Myo6 releases tubules from melanosome. (A and B) Live imaging frames of mCh-VAMP7 expressing MNT-1 cells. VAMP7 localizes to melanosomes (A, arrows). Magnified views (B; 3× boxed area in A) of consecutive time-lapse images show VAMP7+ tubule (arrowheads) emerging and detaching (red arrow) from melanosome (arrows) over time (s). (C) Quantification of the time of release of mCh-VAMP7+ tubules from melanosomes (n = 4 independent experiments). (D) Live imaging frames of mCh-VAMP7 expressing siCtrl or siMyo6 MNT-1 cells. Magnifications (4×) of boxed area in siMyo6 cells show VAMP7+ tubules (arrowheads) associated with melanosomes (arrows). (E) Relative number of mCh-VAMP7+ melanosomal tubules during 40-s movies per 256-µm2 area of cells treated with siCtrl or siMyo6 expressing or not GFP-Myo6R (siCtrl, n = 4 independent experiments; siMyo6, n = 3 independent experiments; siMyo6+GFP-Myo6R, n = 3 independent experiments). (F) Percentage of mCh-VAMP7+ tubules detaching from melanosomes in cells treated as in E (E andF; siCtrl, n = 4 independent experiments; siMyo6, n = 3 independent experiments; siMyo6 + GFP-Myo6R, n = 3 independent experiments). (G and H) Live imaging frames of mCh-VAMP7 expressing Myo6-depleted MNT-1 cells. Magnified views (H; 4× boxed area in G) depict VAMP7+ tubule (arrowheads) emerging from a melanosome (arrows) without detaching over time (s). (I) Live cell imaging analysis of mCh-VAMP7 expressing MNT-1 cells treated with DMSO or 5 µM TIP. Magnified areas (4×) of boxed regions show tubules (arrowheads) associated with melanosomes (arrows). (J) Relative number of mCh-VAMP7+ melanosomal tubules during 40-s movies per 256-µm2 area of cells treated as in I (DMSO, n = 3 independent experiments; TIP, n = 3 independent experiments). (K) Percentage of mCh-VAMP7+ tubules released from melanosomes in DMSO- or TIP-treated cells (J and K; DMSO, n = 3 independent experiments; TIP, n = 3 independent experiments). (L) Live imaging frame of GFP-Myo6-CBD and mCh-VAMP7 expressing MNT-1 cells. Magnified boxed areas (4×) show a VAMP7+ tubule (arrowhead) associated with a GFP-Myo6-CBD+ melanosome (arrow). (M) Pearson’s correlation coefficient of cells (n = 26) as in L. Control values were obtained by 30° rotation of the GFP layer of the same images. (N and O) Live SR-FM of GFP-Myo6-CBD and mCh-VAMP7 expressing MNT-1 cells (N) and linear pixel values (normalized to maximal fluorescence) across double positive melanosomes associated with tubules (O). GFP-Myo6-CBD peak of fluorescence intersects the core of n melanosome (arrow) and the tubule (arrowhead; n = 30). Data are presented as the mean ± SEM. Bars: (A, D, G, I, and L) 10 µm; (B, H, N and magnifications in D, I, and L) 1 µm. ****, P < 0.0001; *, P < 0.05; n.s., not significant (unpaired t test).

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