Figure 1. VAMP7 localizes to melanosomes... | Rockefeller University Press

Figure 1.

$Figure 1. VAMP7 localizes to melanosomes and is required for pigmentation and cargo trafficking. (a–c) WT melan-Ink4a melanocytes transiently expressing GFP-VAMP7 (green) were fixed and labeled for TYRP1 (red) and analyzed by deconvolution immuno-FM. Bar, 10 µm. BF (melanin) image in c is pseudocolored blue in insets. Insets of boxed regions are magnified five times; bar, 1 µm. Melanosomes labeled by both VAMP7 and TYRP1 (arrowheads) or VAMP7 only (arrow) are indicated. (d) Ultrathin cryosection of WT melan-a melanocytes transfected with GFP-VAMP7 and labeled for VAMP7 with 10 nm protein A gold (PAG10). Arrows show GFP-VAMP7 on stage IV melanosomes (IV) and adjacent vesicles. Bar, 200 nm. (e–k) MNT-1 melanoma cells treated with control (siCTRL) or VAMP7-specific siRNA (siVAMP7) were analyzed 5 d later. (e) Whole-cell lysates fractionated by SDS-PAGE were immunoblotted for VAMP7 or the AP-1 subunit γ-adaptin as a control. Relevant bands and positions of nearby molecular weight markers are shown. (f and g) Thin sections of fixed cells in epon were analyzed by conventional EM. Bars, 500 nm. (h) Melanin content in cell lysates was assayed by spectrophotometry. Data are normalized to siCTRL and represent mean ± SD from at least three experiments. (i and j) Ultrathin cryosections of fixed cells were labeled for TYRP1 with PAG10. Asterisks show pigmented melanosomes, and arrowheads show TYRP1 labeling on melanosomal membrane or closely adjacent vesicular structures. Bars, 200 nm. (k) Quantification of TYRP1 localization in siCTRL and siVAMP7 cells (mean ± SD from three measurements). Golgi/TGN, Golgi and trans-Golgi network; Lyso, lysosomes; Melan, melanosomes; MVBs, multivesicular bodies; TVE endo, tubulovesicular endosomes associated with endosomes; TVE Melan, tubulovesicular endosomes associated with melanosomes; Vac Endo, vacuolar endosomes; Vesicle, other vesicular structures. *, P < 0.05; **, P < 0.01; ***, P < 0.005; n.s., no significant difference.$

VAMP7 localizes to melanosomes and is required for pigmentation and cargo trafficking. (a–c) WT melan-Ink4a melanocytes transiently expressing GFP-VAMP7 (green) were fixed and labeled for TYRP1 (red) and analyzed by deconvolution immuno-FM. Bar, 10 µm. BF (melanin) image in c is pseudocolored blue in insets. Insets of boxed regions are magnified five times; bar, 1 µm. Melanosomes labeled by both VAMP7 and TYRP1 (arrowheads) or VAMP7 only (arrow) are indicated. (d) Ultrathin cryosection of WT melan-a melanocytes transfected with GFP-VAMP7 and labeled for VAMP7 with 10 nm protein A gold (PAG10). Arrows show GFP-VAMP7 on stage IV melanosomes (IV) and adjacent vesicles. Bar, 200 nm. (e–k) MNT-1 melanoma cells treated with control (siCTRL) or VAMP7-specific siRNA (siVAMP7) were analyzed 5 d later. (e) Whole-cell lysates fractionated by SDS-PAGE were immunoblotted for VAMP7 or the AP-1 subunit γ-adaptin as a control. Relevant bands and positions of nearby molecular weight markers are shown. (f and g) Thin sections of fixed cells in epon were analyzed by conventional EM. Bars, 500 nm. (h) Melanin content in cell lysates was assayed by spectrophotometry. Data are normalized to siCTRL and represent mean ± SD from at least three experiments. (i and j) Ultrathin cryosections of fixed cells were labeled for TYRP1 with PAG10. Asterisks show pigmented melanosomes, and arrowheads show TYRP1 labeling on melanosomal membrane or closely adjacent vesicular structures. Bars, 200 nm. (k) Quantification of TYRP1 localization in siCTRL and siVAMP7 cells (mean ± SD from three measurements). Golgi/TGN, Golgi and trans-Golgi network; Lyso, lysosomes; Melan, melanosomes; MVBs, multivesicular bodies; TVE endo, tubulovesicular endosomes associated with endosomes; TVE Melan, tubulovesicular endosomes associated with melanosomes; Vac Endo, vacuolar endosomes; Vesicle, other vesicular structures. *, P < 0.05; **, P < 0.01; ***, P < 0.005; n.s., no significant difference.