Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution

Abstract

Mutant alleles at the dilute unconventional myosin heavy chain locus cause diluted coat color, opisthotonic seizures, and death. The dilute coat color phenotype is caused by irregular clumping of pigment in the hair, but amounts of melanin are unchanged from wild-type controls. The melanocyte phenotype has been described as adendritic, since hair bulb and Harderian gland melanocytes appear to be rounded in tissue sections. These observations do not exclude the possibility that the processes lack pigment, since the melanocyte shape was judged by the distribution of melanin. We have tested this hypothesis by culturing primary melanocytes from dilute mutant and wild-type mice. The mutant melanocytes do not lack processes; instead, they exhibit a concentrated perinuclear distribution of melanosomes, while wild-type melanocytes have a very uniform cytoplasmic distribution of melanosomes. Electron micrographs show no detectable differences in melanosome morphology or maturation between dilute and wild-type melanocytes. Immunofluorescence experiments indicate that the dilute protein is concentrated in regions of the cytoplasm that contain melanosomes. These experiments show that the dilute myosin is necessary for the localization of melanosomes, either by active transport or tethering.

Figure 1

Phase-contrast micrograph of wild-type (A) and d^v/d^v (B) primary melanocytes, shown at the same magnification. The regions between the accumulated melanosomes in the d^v/d^v cells (B) contain cytoplasm lacking melanosomes. (Bar = 50 μm.)

Figure 2

Thin-section electron micrograph of d^v/d^v melanocyte showing the presence of Stage 2, 3, and 4 melanosomes. (Bar = 500 nm.)

Figure 3

Bright-field (showing melanosomal distribution) (A and E) and immunofluorescence (B–D, F, and G) photomicrographs of wild-type (A–D) and d^v/d^v (E–G) primary melanocytes. Antiserum to a dilute tail peptide was used as the primary antibody in all panels except D, for which an antibody to the carboxyl terminus of tyrosinase (αPEP7) was used. G is an underexposure of the same negative shown in F; it shows the background labeling and the extent of the cell’s cytoplasm. The cell at the middle of the left margin of B is a fibroblast that cannot be seen in A. (Bars: A, 10 μm; C, 5 μM. C and D have the same magnification; the remaining panels have the same magnification as A.) The difference between the punctate staining of the anti-dilute antibody and the ring-like staining of the melanosome periphery by αPEP7 (arrowheads) can be seen in at higher magnification in C and D.

Figure 4

Bright-field (showing melanosomal distribution) (A and D) and immunofluorescence (B–F) photomicrographs of dilute suppressor (a/a d^v/d^v dsu/dsu) (A–C) and leaden (C57BL/6J fz ln/ln) (D–F) primary melanocytes, using the anti-dilute tail peptide antiserum as primary antibody. C and F are underexposures of the same negatives shown in B and E, respectively, to show the extent of the cytoplasm of the cells. (Bar = 10 μm.)