Figure 5. Depletion of KIF13A inhibits... | Rockefeller University Press

Figure 5.

$Figure 5. Depletion of KIF13A inhibits melanogenesis and results in Tyrp1 accumulation in endosomal vacuoles. (A) Cells treated with control (a) or KIF13A (b) siRNAs were analyzed by conventional EM. KIF13A-depleted cells accumulate vacuoles with internal membrane vesicles (arrows). Stage II premelanosomes are not affected (arrowhead). (B) KIF13A-depleted cells were analyzed by IEM. Ultrathin cryosections were labeled for AP-1 (PAG15) and Tyrp1 (PAG10). Tyrp1 localizes to the limiting membrane of endosomal vacuoles (a and b, arrows). An enrichment of AP-1–positive vesicles are observed in the vicinity of the Golgi apparatus (a, arrowheads) and close to the vacuoles (b, arrowheads). (C) Quantification of immunogold labeling for AP-1 (left) or Tyrp1 (right) on ultrathin cryosections. Localization of an equivalent number of gold particles in each sample was assessed relative to the indicated cell compartments. (D) Evaluation of melanin content of cells treated with control or KIF13A siRNAs. (E) MNT-1 cells were cross-linked with DSP, lysed, and immunoprecipitated with Tyrp1 antibody. Immunoprecipitates were reduced in sample buffer to cleave the cross-linker, fractionated by SDS-PAGE, and immunoblotted using antibodies to Tyrp1, γ-adaptin, or KIF13A. Black lines indicate that intervening lanes have been spliced out. Molecular masses are given in kilodaltons. G, Golgi apparatus; I–IV, stages I–IV; Vac Endo, vacuolar endosome; TVE Endo, tubulovesicular endosomes associated to endosomes; MVB, multivesicular body; Mel, melanosome; TVE Mel, tubulovesicular endosomes associated to melanosomes; Lyso, lysosome; Vesic, vesicle. Data are presented as mean ± SD. Bars, 200 nm.$

Depletion of KIF13A inhibits melanogenesis and results in Tyrp1 accumulation in endosomal vacuoles. (A) Cells treated with control (a) or KIF13A (b) siRNAs were analyzed by conventional EM. KIF13A-depleted cells accumulate vacuoles with internal membrane vesicles (arrows). Stage II premelanosomes are not affected (arrowhead). (B) KIF13A-depleted cells were analyzed by IEM. Ultrathin cryosections were labeled for AP-1 (PAG15) and Tyrp1 (PAG10). Tyrp1 localizes to the limiting membrane of endosomal vacuoles (a and b, arrows). An enrichment of AP-1–positive vesicles are observed in the vicinity of the Golgi apparatus (a, arrowheads) and close to the vacuoles (b, arrowheads). (C) Quantification of immunogold labeling for AP-1 (left) or Tyrp1 (right) on ultrathin cryosections. Localization of an equivalent number of gold particles in each sample was assessed relative to the indicated cell compartments. (D) Evaluation of melanin content of cells treated with control or KIF13A siRNAs. (E) MNT-1 cells were cross-linked with DSP, lysed, and immunoprecipitated with Tyrp1 antibody. Immunoprecipitates were reduced in sample buffer to cleave the cross-linker, fractionated by SDS-PAGE, and immunoblotted using antibodies to Tyrp1, γ-adaptin, or KIF13A. Black lines indicate that intervening lanes have been spliced out. Molecular masses are given in kilodaltons. G, Golgi apparatus; I–IV, stages I–IV; 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. Data are presented as mean ± SD. Bars, 200 nm.