Melanoregulin Regulates a Shedding Mechanism That Drives

Melanoregulin regulates a shedding mechanism that PNAS PLUS drives melanosome transfer from melanocytes to keratinocytes Xufeng S. Wua,1, Andreas Masedunskasb, Roberto Weigertb, Neal G. Copelandc, Nancy A. Jenkinsc,1, and John A. Hammera,1 aLaboratory of Cell Biology, National Heart, Lung, and Blood Institute and bOral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892; and cMethodist Cancer Research Program, Methodist Hospital Research Institute, Houston, TX 77030 Contributed by Nancy A. Jenkins, June 6, 2012 (sent for review April 2, 2012) Mammalian pigmentation is driven by the intercellular transfer of Non-agouti black mice that are homozygous for the dilute l20j pigment-containing melanosomes from the tips of melanocyte functional null allele d appear gray (3). When these mice also dendrites to surrounding keratinocytes. Tip accumulation of mel- are made homozygous for a functional null allele at the dilute anosomes requires myosin Va, because melanosomes concentrate suppressor (dsu) locus, their coat color is restored almost com- in the center of melanocytes from myosin Va-null (dilute) mice. pletely (i.e., from gray back to nearly black) (3, 4). In other This distribution defect results in inefficient melanosome transfer words, the coat color defect resulting from the loss of expression and a dilution of coat color. Dilute mice that simultaneously lack of myosin Va is rescued by the loss of expression of melanor- melanoregulin, the product of the dilute suppressor locus, exhibit egulin, the product of the dsu locus. dsu is inherited in a semi- a nearly complete restoration of coat color, but, surprisingly, mel- dominant fashion, because the presence of one mutant dsu allele l20j l20j anosomes remain concentrated in the center of their melanocytes. partially suppresses the diluted coat color of d /d mice, Here we show that dilute/dsu melanocytes, but not dilute mela- whereas the presence of two mutant dsu alleles suppresses the nocytes, readily transfer the melanosomes concentrated in their coat color to nearly WT (i.e., black) (4). Studies performed using center to surrounding keratinocytes in situ. Using time-lapse im- chimeric mice argue that melanoregulin functions cell autono- CELL BIOLOGY aging of WT melanocyte/keratinocyte cocultures in which the mously within melanocytes to rescue coat color (5). plasma membranes of the two cells are marked with different Positional cloning of the dsu locus revealed that melanor- colors, we define an intercellular melanosome transfer pathway egulin is a vertebrate-specific, highly charged protein of 22 kDa that involves the shedding by the melanocyte of melanosome-rich with no obvious sequence similarity to any currently character- packages, which subsequently are phagocytosed by the keratino- ized protein (3). Northern blots suggest that the protein is highly cyte. Shedding, which occurs primarily at dendritic tips but also expressed in melanocytes, heart, liver, testes, and thymus and at from more central regions, involves adhesion to the keratinocyte, lower levels in other tissues. Moreover, Western blots indicate thinning behind the forming package, and apparent self-abscis- that melanoregulin is abundant in primary melanocytes. Con- sion. Finally, we show that shedding from the cell center is sixfold sistent with the large deletion present in the sole extant mutant dilute/dsu dilute more frequent in cultured melanocytes than in dsu allele, melanocytes from homozygous dsu mice were shown melanocytes, consistent with the in situ data. Together, these to be completely devoid of melanoregulin (3). dsu dilute results explain how restores the coat color of mice with- Very surprisingly, coat color rescue by dsu occurs without out restoring intracellular melanosome distribution, indicate that restoration of normal peripheral melanosome distribution within melanoregulin is a negative regulator of melanosome transfer, dilute melanocytes, based on images of melanocytes present in l20j l20j and provide insight into the mechanism of intercellular melano- the Harderian glands of d /d , dsu/dsu mice (3). Specifically, some transfer. Harderian gland melanocytes from these rescued black mice exhibit an accumulation of melanosomes in their central cyto- he coloration of mammalian skin and hair requires the transfer plasm that is indistinguishable from that exhibited by Harderian l20j l20j Tof melanin pigment from melanocytes, which create the pig- gland melanocytes from gray d /d mice. This key observa- ment, to keratinocytes, which make up the bulk of skin and hair tion, which we confirm and extend here in a variety of ways, flies (1). Although the exact mechanism of intercellular pigment directly in the face of the currently accepted mechanism thought transfer is unknown, efficient transfer is thought to require that to drive mammalian pigmentation, according to which the ac- melanosomes, the pigment-producing organelles generated within cumulation of melanosomes in peripheral regions of the mela- the melanocyte’s central cytoplasm, be accumulated at the tips of nocyte’s dendrites is a prerequisite for their effective transfer to the melanocyte’s long dendritic extensions, the likely major site of keratinocytes and subsequent pigmentation. How then does dsu transfer. The accumulation of melanosomes at dendritic tips is rescue the coat color of dilute mice without rescuing the un- generated by a cooperative transport mechanism that couples derlying defect in intracellular melanosome distribution exhibi- long-range, bidirectional, microtubule-dependent transport within ted by dilute melanocytes? Although this conundrum remains dendritic extensions with myosin Va-dependent capture of the completely unresolved, O’Sullivan et al. (3) reasonably could organelles at tips (the Cooperative Capture model) (2). In melanocytes from dilute mice, which lack myosin Va (Myo5a), the connection between the melanosome and F-actin is broken, Author contributions: X.S.W. and J.A.H. designed research; X.S.W. performed research; resulting in melanosomes redistributing according to microtubule X.S.W., A.M., R.W., N.G.C., N.A.J., and J.A.H. contributed new reagents/analytic tools; distribution alone. The net result of this redistribution is the X.S.W. and J.A.H. analyzed data; and X.S.W., N.G.C., N.A.J., and J.A.H. wrote the paper. pronounced accumulation of the organelles in the central cyto- The authors declare no conflict of interest. plasm, in striking contrast to their normal accumulation in the cell 1To whom correspondence may be addressed. E-mail: [email protected], njenkins2@ periphery. In the mouse, this defect in intracellular melanosome tmhs.org, or [email protected]. distribution results in a striking reduction in the intensity of the See Author Summary on page 12276 (volume 109, number 31). ’ fi animal s coat color, most likely because of a signi cant reduction This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in the transfer of pigment from melanocytes to keratinocytes. 1073/pnas.1209397109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1209397109 PNAS | Published online July 2, 2012 | E2101–E2109 Downloaded by guest on September 27, 2021 conclude that melanoregulin functions in a myosin Va-indepen- delineate the shape of melanocytes (Fig. 2 E–H). Note that in dent pathway to restore coat pigmentation. For example, mela- these images the melanocyte’s shape is shown in red, the distri- noregulin might serve as a negative regulator of a myosin Va- bution of black pigment is shown in green (by pseudo coloring), independent intercellular melanosome transfer pathway. Here we and the distribution of melanosomes inside of melanocytes is show that this is indeed the case, identifying dsu as a mouse coat shown in blue using an antibody to the melanosomal membrane color mutant that specifically influences the intercellular transfer protein TRP1 (yielding a blue/white color when superimposed component of the pigmentation pathway. on red and green). Comparison of the images for ear skins from dilute (Fig. 2G) and dilute/dsu (Fig. 2H) mice indicates clearly Results that pigment has escaped readily from the center of dilute/dsu dsu Does Not Rescue the Defect in Intracellular Melanosome melanocytes but not from the center of dilute melanocytes. Distribution Exhibited by Dilute Melanocytes. First, using primary l20j l20j melanocytes cultured from d /d , DSU/DSU mice (null at Pigment that Escapes the Center of dilute/dsu Melanocytes Is Inside myosin Va, WT at dsu; referred to hereafter as “dilute”) (Fig. 1B) Adjacent Keratinocytes. To demonstrate that this escaped pigment l20j l20j and from d /d , dsu/dsu mice (null at both myosin Va and actually is inside keratinocytes immediately surrounding the cell dsu; referred to hereafter as “dilute/dsu”) (Fig. 1C), we con- body of dilute/dsu melanocytes, we stained dilute/dsu ear skin firmed that the loss of melanoregulin does not rescue the defect with anti-Kit to visualize the shape of melanocytes (green signals in intracellular melanosome distribution caused by the lack of in Fig. 3 A, C, E, and F), with anti-keratin 14 to see the distri- myosin Va (compare these cells with the cells in Fig. 1A, which bution of keratin filaments within keratinocytes (red signals in are from a D/D, DSU/DSU mouse, i.e., WT at both loci; referred Fig. 3 B, C, and F), and with DAPI to identify the positions of to hereafter as “WT”). As expected, reintroduction of a full- nuclei (blue signals in Fig 3 E and F). In the corresponding length, GFP-tagged version of the melanocyte-spliced isoform of transmitted-light image in Fig. 3D, we circled in yellow the myosin Va into dilute/dsu melanocytes results in the complete dramatic accumulations of pigment that surround the two dilute/ restoration of peripheral melanosome distribution (Fig. 1D; di- dsu melanocyte cell bodies present in this field (which are R lute /dsu). Similarly, primary melanocytes from D/D, dsu/dsu marked with asterisks in Fig.

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