Figure 1.
Figure 1. Depletion of AP-1 inhibits melanogenesis and results in Tyrp1 accumulation in endosomal vacuoles. (A) MNT-1 cells treated with control siRNA (si Ctrl) were analyzed by ultracryomicrotomy (a) or conventional EM (b). (a) In control-inactivated cells, Tyrp1 is detected in vesicles close to the Golgi apparatus in vacuolar endosomes (arrowheads) and in AP-1–positive vesicles and tubules in the vicinity of endosomes (arrows) and pigmented melanosomes (inset, arrows). (b) Control cells show different melanosomal stages including unpigmented (stage I) and pigmented melanosomes (stages III and IV). G, Golgi apparatus; M, melanosome; PM, plasma membrane; End, endosome; I–IV, stages I–IV. (B) Western blot analysis of lysates of MNT-1 cells treated with control, AP-1 (µ1A), and AP-3 (β3A) siRNAs using SA4 (δ subunit of AP-3), anti-µ1A, or anti–β-tubulin antibodies as a loading control. (C) MNT-1 cells treated with control (a and b) or µ1A/γ-adaptin (c and d) siRNAs were labeled for AP-1 (γ-adaptin subunit) and observed by IFM (a and c) and bright field microscopy (b and d). Note the decrease of pigmented melanosomes in AP-1–depleted cells (arrows). (D) Evaluation of melanin content of cells treated with control, AP-1 (µ1A), or AP-3 (β3A) siRNAs. Data represent four replicates ± SD; *, P < 0.05. (E) Cells treated with AP-1 siRNAs were analyzed as described in A. (a) In inactivated cells, the bulk of Tyrp1 localizes to vacuolar endosomes (arrows). (b) Pigmented melanosomes are barely observed in AP-1–depleted cells. These cells accumulate vacuolar endosomes often displaying cytosolic coats (arrows). Characteristic stage II premelanosomes are observed in AP-1–depleted cells (b, inset). (F) Western blot analysis of lysates of MNT-1 cells treated with control or AP-1 siRNAs using anti-µ1A, Tyrp1, or anti–β-tubulin antibodies as a loading control. (G) Quantification of immunogold labeling for Tyrp1 on ultrathin cryosections. Localization of an equivalent number of gold particles in each sample was assessed relative to the indicated cell compartments. Data are presented as mean ± SD. Vac Endo, vacuolar endosome; TVE Endo, tubulovesicular endosomes associated to endosomes; MVB, multivesicular body; Mel, melanosome; TVE Mel, tubulovesicular endosomes associated to melanosomes; Lyso, lysosome; Vesic, vesicle. (B and F) Molecular masses are given in kilodaltons. Bars: (A and E) 200 nm; (C) 10 µm.

Depletion of AP-1 inhibits melanogenesis and results in Tyrp1 accumulation in endosomal vacuoles. (A) MNT-1 cells treated with control siRNA (si Ctrl) were analyzed by ultracryomicrotomy (a) or conventional EM (b). (a) In control-inactivated cells, Tyrp1 is detected in vesicles close to the Golgi apparatus in vacuolar endosomes (arrowheads) and in AP-1–positive vesicles and tubules in the vicinity of endosomes (arrows) and pigmented melanosomes (inset, arrows). (b) Control cells show different melanosomal stages including unpigmented (stage I) and pigmented melanosomes (stages III and IV). G, Golgi apparatus; M, melanosome; PM, plasma membrane; End, endosome; I–IV, stages I–IV. (B) Western blot analysis of lysates of MNT-1 cells treated with control, AP-1 (µ1A), and AP-3 (β3A) siRNAs using SA4 (δ subunit of AP-3), anti-µ1A, or anti–β-tubulin antibodies as a loading control. (C) MNT-1 cells treated with control (a and b) or µ1A/γ-adaptin (c and d) siRNAs were labeled for AP-1 (γ-adaptin subunit) and observed by IFM (a and c) and bright field microscopy (b and d). Note the decrease of pigmented melanosomes in AP-1–depleted cells (arrows). (D) Evaluation of melanin content of cells treated with control, AP-1 (µ1A), or AP-3 (β3A) siRNAs. Data represent four replicates ± SD; *, P < 0.05. (E) Cells treated with AP-1 siRNAs were analyzed as described in A. (a) In inactivated cells, the bulk of Tyrp1 localizes to vacuolar endosomes (arrows). (b) Pigmented melanosomes are barely observed in AP-1–depleted cells. These cells accumulate vacuolar endosomes often displaying cytosolic coats (arrows). Characteristic stage II premelanosomes are observed in AP-1–depleted cells (b, inset). (F) Western blot analysis of lysates of MNT-1 cells treated with control or AP-1 siRNAs using anti-µ1A, Tyrp1, or anti–β-tubulin antibodies as a loading control. (G) Quantification of immunogold labeling for Tyrp1 on ultrathin cryosections. Localization of an equivalent number of gold particles in each sample was assessed relative to the indicated cell compartments. Data are presented as mean ± SD. Vac Endo, vacuolar endosome; TVE Endo, tubulovesicular endosomes associated to endosomes; MVB, multivesicular body; Mel, melanosome; TVE Mel, tubulovesicular endosomes associated to melanosomes; Lyso, lysosome; Vesic, vesicle. (B and F) Molecular masses are given in kilodaltons. Bars: (A and E) 200 nm; (C) 10 µm.

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