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Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

Paterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, Eby V, Boissy RE, Ganesan AK - PLoS ONE (2015)

Bottom Line: Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair.Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis.These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California Irvine, Irvine, CA, United States of America.

ABSTRACT
The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

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Tyr depletion inhibits the normal deposition of melanin within the melanosome.(A) Fresh whole mouse skin was excised from the indicated mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. DOPA histochemistry was performed to assess the relative activity of tyrosinase within the melanosome. The top row contains the relevant control images for the images listed in the middle and bottom rows, respectively. Images are representative of 15 melanocytes from 2 mice per group. (B) The relative accumulation of stage I-IV melanosomes in each coat color background was quantified as described in the methods. For black and yellow agouti coat colors, the rtTA3 driver was used. For white-bellied agouti coat color, the rtTA2 driver was used. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05, or ** = p ≤0.005, Bars = standard deviation (n = 15 melanocytes from 2 mice per group).
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pone.0143702.g003: Tyr depletion inhibits the normal deposition of melanin within the melanosome.(A) Fresh whole mouse skin was excised from the indicated mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. DOPA histochemistry was performed to assess the relative activity of tyrosinase within the melanosome. The top row contains the relevant control images for the images listed in the middle and bottom rows, respectively. Images are representative of 15 melanocytes from 2 mice per group. (B) The relative accumulation of stage I-IV melanosomes in each coat color background was quantified as described in the methods. For black and yellow agouti coat colors, the rtTA3 driver was used. For white-bellied agouti coat color, the rtTA2 driver was used. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05, or ** = p ≤0.005, Bars = standard deviation (n = 15 melanocytes from 2 mice per group).

Mentions: Having determined how efficiently the Tyr-shRNA could inhibit TYR expression, we next sought to examine whether loss of endogenous Tyr affects the structure of the melanosome. Fresh skin harvested from Tyr-knockdown mice and appropriate littermate controls on both the white-bellied agouti background and the yellow agouti background were analyzed by transmission electron microscopy (TEM, Fig 3). The respective littermate controls had normal early and late-stage melanosomes that were round with smooth, fibrillar deposits of melanin, consistent with the normal melanosome maturation pattern observed in mice (Fig 3A, top row, center & right) [51]. In contrast, the majority of melanosomes originating from Tyr-knockdown mouse skin accumulated melanosome structures characterized by an irregular pattern of melanin deposition, similar to the stage II and III melanosomes of MNT-1 melanoma cells [24]) but different from the stage II and III melanosomes observed in mouse skin (Fig 3A, middle row, center and right images) [51]. Finally, depletion of Tyr quantitatively inhibited the maturation of the melanosome, as more stage III, but less stage IV melanosomes were present in Tyr-knockdown mice on both agouti coat color backgrounds (Fig 3B, center & right graphs).


Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

Paterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, Eby V, Boissy RE, Ganesan AK - PLoS ONE (2015)

Tyr depletion inhibits the normal deposition of melanin within the melanosome.(A) Fresh whole mouse skin was excised from the indicated mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. DOPA histochemistry was performed to assess the relative activity of tyrosinase within the melanosome. The top row contains the relevant control images for the images listed in the middle and bottom rows, respectively. Images are representative of 15 melanocytes from 2 mice per group. (B) The relative accumulation of stage I-IV melanosomes in each coat color background was quantified as described in the methods. For black and yellow agouti coat colors, the rtTA3 driver was used. For white-bellied agouti coat color, the rtTA2 driver was used. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05, or ** = p ≤0.005, Bars = standard deviation (n = 15 melanocytes from 2 mice per group).
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pone.0143702.g003: Tyr depletion inhibits the normal deposition of melanin within the melanosome.(A) Fresh whole mouse skin was excised from the indicated mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. DOPA histochemistry was performed to assess the relative activity of tyrosinase within the melanosome. The top row contains the relevant control images for the images listed in the middle and bottom rows, respectively. Images are representative of 15 melanocytes from 2 mice per group. (B) The relative accumulation of stage I-IV melanosomes in each coat color background was quantified as described in the methods. For black and yellow agouti coat colors, the rtTA3 driver was used. For white-bellied agouti coat color, the rtTA2 driver was used. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05, or ** = p ≤0.005, Bars = standard deviation (n = 15 melanocytes from 2 mice per group).
Mentions: Having determined how efficiently the Tyr-shRNA could inhibit TYR expression, we next sought to examine whether loss of endogenous Tyr affects the structure of the melanosome. Fresh skin harvested from Tyr-knockdown mice and appropriate littermate controls on both the white-bellied agouti background and the yellow agouti background were analyzed by transmission electron microscopy (TEM, Fig 3). The respective littermate controls had normal early and late-stage melanosomes that were round with smooth, fibrillar deposits of melanin, consistent with the normal melanosome maturation pattern observed in mice (Fig 3A, top row, center & right) [51]. In contrast, the majority of melanosomes originating from Tyr-knockdown mouse skin accumulated melanosome structures characterized by an irregular pattern of melanin deposition, similar to the stage II and III melanosomes of MNT-1 melanoma cells [24]) but different from the stage II and III melanosomes observed in mouse skin (Fig 3A, middle row, center and right images) [51]. Finally, depletion of Tyr quantitatively inhibited the maturation of the melanosome, as more stage III, but less stage IV melanosomes were present in Tyr-knockdown mice on both agouti coat color backgrounds (Fig 3B, center & right graphs).

Bottom Line: Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair.Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis.These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California Irvine, Irvine, CA, United States of America.

ABSTRACT
The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

Show MeSH