PIKfyve regulates melanosome biogenesis

PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes.


Introduction
Melanin, a pigment produced within uveal and epidermal melanocytes, absorbs UV radiation, protecting the eyes and skin from UV-induced DNA damage [1]. Melanin is synthesized in a a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Loss of PIKfyve leads to pigment loss in vivo
PIKfyve forms a complex with VAC14 and FIG4 [32,37] which then phosphorylates PI(3)P to PI(3,5)P 2 [38] and PIP to PI(5)P [39,40]. VAC14 and FIG4 mutants are characterized by early lethality and accumulation of vacuoles in the CNS with accompanying coat color defects [37,41,42]. PIKfyve knockout mice die during embryonic development [43], making it difficult to assess the effects of PIKfyve on melanogenesis. To better elucidate the role of PIKfyve in melanosome biogenesis, we generated melanocyte-specific, inducible PIKfyve knockout mice by crossing an established PIKfyve Flox/Flox strain [44] with an established melanocyte-specific, inducible Cre strain under a tyrosinase promoter [45] on a pure C57B6 background. Induction of Cre recombination with tamoxifen results in the excision of the kinase domain of PIKfyve producing a slightly smaller protein than the full length PIKfyve (Fig 1A). Previously published studies suggested that truncated PIKfyve is unstable-infecting PIKfyve Flox/Flox mouse embryonic fibroblasts with adenovirus expressing Cre recombinase resulted in the loss PIKfyve protein [44]. Initial experiments verified that treating TyrCreER T2 PIKfyve Flox/Flox melanocytes with tamoxifen also inhibited the accumulation of PIKfyve protein ( Fig 1B). Notably, tamoxifen treatment did not result in the complete deletion of PIKfyve as was described previously [44], indicating that Cre recombination in this model system is less efficient. In addition, PIKfyve deletion inhibited the accumulation of pigmented melanosomes (Fig 1C and 1D), while also resulting in the accumulation of full length PMEL protein (Fig 1B).
To examine the effect of PIKfyve knockout on melanogenesis, TyrCreER T2 PIKfyve Flox/Flox mice were administered tamoxifen-containing chow for 29 days beginning at P21 (Fig 2A) to induce Cre-mediated excision of the kinase domain of PIKfyve. Mice were photographed initially (S1A Fig), shaved and depilated on a region of their back at P50, and hairs were allowed to regrow. TyrCreER T2 PIKfyve Flox/Flox mice fed tamoxifen for 29 days accumulated numerous white hairs that initially presented in the shaved and depilated area and were visually apparent at P85 (Figs 2B and S1B). The same hair phenotype was not observed in Cre negative PIKfyve Flox/Flox mice fed tamoxifen for 29 days or TyrCreER T2 PIKfyve Flox/Flox mice that were administered a normal diet (Figs 2B, S1B and S1C). Additional experiments revealed that the weights of PIKfyve Flox/Flox mice that were fed tamoxifen were similar regardless of whether they expressed TyrCreER T2 (S1D Fig), indicating that the smaller relative size of the TyrCreER T2 PIKfyve Flox/Flox mice fed tamoxifen chow as compared to mice fed normal chow was a consequence of diet and not PIKfyve loss. Hair from the backs of experimental mice was solubilized and the relative accumulation of melanin was quantified using standard spectrophotometric methods [46,47]. Hairs from the TyrCreER T2 PIKfyve Flox/Flox mice that were fed tamoxifen accumulated 50% less melanin as compared to mice that were not fed tamoxifen or Cre negative controls (Fig 2C).
To better assess whether this phenotype was exclusively related to an effect on melanogenesis, we allowed the hairs to regrow after shave depilation and observed whether there was an increased accumulation of white hair after the mice were switched off tamoxifen feed. More white hairs were visually apparent after the mice were fed a normal diet for an additional 20 days (p105, Figs 2B, S1B and S1C). This phenotype appeared to be progressive initially as even less melanin accumulated in the hair of PIKfyve Flox/Flox mice after they were taken off tamoxifen chow (Fig 2B). Over the course of a year, PIKfyve Flox/Flox mice continued to accumulate numerous white hairs on the head and upper back, areas that had never been shave depilated (S1B and S1C Fig), indicating that the phenotype was accelerated but not induced by shave depilation. The observed phenotype did not progress completely, as the mice continued to maintain the same relative level of depigmentation over the course of a year (S1C Fig). Taken together, these results indicate that PIKfyve loss inhibits melanin accumulation in the mouse Cells were treated with 4-hydroxytamoxifen (4-OHT) and mice were administered feed containing tamoxifen (TAM) to remove exon 38 resulting in the inactivation of the PIKfyve kinase. B) Six different batches of primary melanocytes isolated from neonatal Tyrosinase::Cre ERT2 ; PIKfyve Flox/Flox were treated with 4-OHT or vehicle control for 48hrs. Protein lysates were collected and immunoblotted with the indicated Abs to measure PIKfyve and Pmel levels. C) Cells from Tyrosinase::Cre ERT2 ; PIKfyve Flox/Flox or wild type mice treated with 4-OHT or vehicle were then fixed and imaged using phase contrast microscopy, scale bar = 10μm. D) The number of melanosomes per cell in Tyrosinase::Cre ERT2 ; PIKfyve Flox/Flox melanocytes treated with 4-OHT or vehicle was counted and quantified using ImageJ. ÃÃÃ , p < 0.001 using a two-tailed Student's paired T test.
https://doi.org/10.1371/journal.pgen.1007290.g001 hair and this phenotype is not progressive as would be expected if the phenotype were related to stem cell depletion [48].
To better understand how PIKfyve loss influences melanin accumulation, we next sought to characterize the PIKfyve knockout mice at the cellular level. Initial studies sought to better understand why TyrCreER T2 PIKfyve Flox/Flox mice fed tamoxifen were not completely white but instead accumulated sporadic white hairs. TyrCreER T2 PIKfyve Flox/Flox littermates fed either a control diet or tamoxifen diet were shave depilated at P50. In both groups, mice had entered the 3 rd anagen by ; PIKfyve Flox/Flox mice were fed a control diet throughout the course of the experiment. All mice were shave depilated at p50 and subsequently fed normal chow beginning at p50 for the subsequent days. Gray bar denotes the duration that mice, with the exception of the control group, were on tamoxifen feed. B) Littermates were photographed at P85, P105, and P365. C) Mouse hair from the genotypes indicated was dissolved in solune-350 and melanin quantitation was performed as described. The relative amount of melanin in the hair was calculated relative to Cre-controls at P85, P105,and P365. Data shown are mean ± S.D. (n = 5 or 3 as indicated by error bars. ÃÃÃ , p < 0.001 using a two-tailed Student's paired T test. Histology on skin collected at P60 revealed that mice fed tamoxifen had both pigmented and unpigmented hair growing in follicles while mice fed the control diet had fully pigmented hair growth (S2B Fig). Since pigmented and unpigmented hairs are present in neighboring follicles, this suggests that loss of PIKfyve does not affect the growth of the hair. Further studies examined the structure of the hairs histologically and verified that the hairs from PIKfyve knockout mice had the same morphology as wild-type mice with the exception that a number of the hairs in the knockout mouse lacked pigment (Fig 3A). High magnification images of these hair follicles revealed that some of them exhibited the accumulation of cells with intracellular vesicles with some residual pigment ( that not all hair follicles in the mice exhibit a vacuolated phenotype is consistent with in vitro results indicating that deletion of PIKfyve was not 100% efficient ( Fig 1B). To further verify that the observed phenotypes were not a consequence of stem cell loss, TyrCreER T2 PIKfyve Flox/Flox mice were crossed with ROSA mTmG/mTmG mice, a Cre reporter strain in which Cre expressing cells would express GFP [49]. Resulting animals (TyrCreER T2 PIKfyve Flox/Flox ROSA mTmG/+ ) or controls (TyrCreER T2 ROSA mTmG/+ ) were fed tamoxifen from p21 to p50, animals were sacrificed at p100 and frozen sections were examined by fluorescence microscopy. Both control and PIKfyve knockout mouse skin had GFP positive cells associated with hair follicles (Fig 3C) while the PIKfyve knockouts accumulated white hairs (Fig 3D), suggesting that PIKfyve deletion does not significantly affect the survival of melanocytes in vivo.
Published studies have demonstrated that PIKfyve is essential for vesicular trafficking as loss or inhibition results in severe trafficking defects and vacuolization [21,28,33,38]. Other studies have also demonstrated that PIKfyve is essential for lysosomal trafficking [24,50,51] and in lysosomal reformation from endolysosomes [36]. To further characterize PIKfyve's role in melanosome biogenesis, skin biopsies were taken at p60 and assessed at the ultrastructural level by electron microscopy and DOPA histochemistry electron microscopy (EM). Intriguingly, melanocytes from knockout mice exhibit three morphological phenotypes when compared to controls ( Fig 4A, subpanel a). Some of the melanocytes were phenotypically normal (N) (Fig 4A,  Similarly, after DOPA incubation, morphologically normal cells contain minimal melanin reaction product in the Golgi area ( Fig 4B, subpanel a), while uncharacteristic deposition was observed in intermediate cells ( Fig 4B, subpanel b). The abnormal cells had less TYR reaction product (Fig 4B, subpanel c). Closer examination of the grossly abnormal melanocytes revealed the accumulation of single membrane vesicles that had multiple vesicles within them and some TYR reaction product, reminiscent of multivesicular endosomes ( Fig 4B, subpanel d). To understand whether the variable phenotypes observed in vivo were a consequence of partial/incomplete loss of PIKfyve, we cultured melanocytes from TyrCreER T2 PIKfyve Flox/Flox mice, treated them with tamoxifen or vehicle, and examined the structure of melanosomes in the cultured cells by electron microscopy. Electron microscopy analysis indicated that some tamoxifen treated TyrCreER T2 PIKfyve Flox/Flox melanocytes accumulated single membrane structures resembling multivesicular endosomes with few stage IV melanosomes ( Fig 4C, subanel a,b inset). Other treated melanocytes accumulated early and late stage melanosomes with a few single membrane structures (Fig 4C, subpanel c,d).

Pharmacologic inhibition of PIKfyve blocks melanosome maturation
Initial studies verified that loss of PIKfyve inhibits the normal maturation of melanosomes in vivo (Figs 1-4). To more accurately determine how PIKfyve modulates melanosome maturation, we examined the consequences of pharmacologic inhibition of PIKfyve in vitro. Published studies have verified that pharmacologic inhibition of PIKfyve potently and acutely blocks enzymatic activity [38,50] and significantly reduces phosphoinositide levels [38]. MNT-1 melanoma cells that produce higher amounts of melanin than normal melanocytes in vitro were treated with two PIKfyve inhibitors, YM-201636 (YM) and apilimod, the latter of which has been noted for increased potency and specificity [30]. MNT-1 cells treated with YM or apilimod accumulated less melanin as compared to vehicle treated cells (Fig 5A and 5B), similar to what was observed in experimental mice and cells from experimental mice (Figs 1 and 2).
Once we determined that pharmacologic inhibition of PIKfyve resulted in decreased melanin production, we sought to determine how inhibition of PIKfyve blocks melanosome biogenesis at the ultrastructural level. Established cultures of darkly pigmented human melanocytes (DP melanocytes) were treated with various dosages of YM and processed for routine and DOPA histochemistry electron microscopy. YM (1000 nM) treated melanocytes were dramatically hypopigmented in comparison to vehicle treated cells (Fig 5C). Upon DOPA incubation, melanosomes in the treated melanocytes contained melanin reaction product almost to the extent of control melanocytes ( Fig 5C). However, the melanin deposition within melanosomes in treated melanocytes appeared irregular and less homogeneous. Higher magnification images of vehicle versus YM treated cells demonstrated that within the Golgi zone, melanosomes of all stages existed in the control, whereas primarily Stage I and a few Stage II melanosomes existed in the YM treated melanocytes (Fig 5D). In regions lateral to the Golgi zone and within dendrites, all stages of melanosomes existed in the YM treated melanocytes as opposed to predominantly Stage IV in the control treated melanocytes (Fig 5D). Quantification of melanosome stages treated with YM demonstrated that high doses lead to an increase in stage I melanosomes and a decrease in stage IV melanosomes (Figs 5E and S3A). Taken together, these results suggest that PIKfyve influences the maturation of stage I and II melanosomes.
Once we realized that the percentage of "stage I" melanosomes increased with YM treatment, these primitive organelles were subjected to further scrutiny. It has been demonstrated that tyrosinase exits the Golgi in 50 nm trafficking vesicles. En route to the stage II melanosome, tyrosinase enters the multivesicular endosome (MVE) and then is rapidly re-recruited by a complex containing AP-3 into vesicles that ultimately transports this cargo to Stage II melanosomes [18]. Without DOPA incubation it is difficult to ultrastructurally discern these MVEs from Stage I melanosomes particularly when melanofibrils are not apparent. After DOPA incubation, the MVEs appear with DOPA reaction peripherally around their limiting membranes due to the fact that tyrosinase is a transmembrane enzyme whose catalytic carboxy end protrudes into the lumen (S3B Fig). These MVEs can occasionally appear in the Golgi zone of control melanocytes however subjectively many more appear in the YM treated melanocytes and possibly in the dendrites (S3B Fig). Melanosome density within both the cell body and the dendrites of control and YM treated melanocytes with and without DOPA treatment was quantified. There was a statistically significant increase in melanosome density in the cell body of YM treated melanocytes versus control in both the non-DOPA and DOPA treated group (S1 and S2 Tables). In contrast, no difference in melanosome density was observed in the dendrites. The density of 50 nm vesicles containing tyrosinase cargo was quantitated in DOPA processed melanocytes. In the Golgi area, the density of DOPA positive 50 nm vesicles was significantly increased in the YM versus the vehicle treated melanocytes (S3C Fig), indicating the accumulation of stage I melanosomes.
Taken together, these studies suggest that PIKfyve inhibitors block melanosome maturation and the trafficking of tyrosinase from the MVE to the melanosome.
Next we sought to verify that PIKfyve regulates the maturation of the stage I to the stage IV melanosome by inhibiting the trafficking of proteins out of an intermediate endosomal compartment/MVE. To do this, we examined the accumulation of stage I and stage III/IV melanosome markers and the processing or accumulation of melanosome proteins. Previous studies demonstrated that stage I melanosomes containing MART-1 are primarily localized to the perinuclear region [52]. Immunofluorescence microscopy revealed that MART-1 positive vesicles accumulate in PIKfyve inhibitor treated MNT-1 cells (Fig 6A and 6B), consistent with the electron microscopy observation that stage I melanosomes accumulate in PIKfyve inhibitor treated primary melanocytes (Fig 5). Conversely, PIKfyve inhibitor treatment blocked the normal trafficking of TYRP1, resulting in the accumulation of TYRP1 containing vesicles in the perinuclear region and a lack of TYRP1 staining at the cell periphery (Fig 6A and 6B). Published studies have revealed that mature Tyrp1 containing melanosomes are localized to the cell periphery, while disruption of melanosome maturation results in the accumulation of Tyrp1 in an early endosome, which is located adjacent to the nucleus [16,53]. These imaging results suggest that after PIKfyve inhibition, Tyrp1 is trapped in an early endosomal compartment (perinuclear localization), unable to reach the mature melanosome.
PMEL is cleaved during the formation of the stage II melanosome [7], resulting in the formation of PMEL fibrils that serve as a platform for melanin polymerization [12]. In order to determine whether PIKfyve affects the formation of the stage II melanosome, we determined whether PIKfyve inhibition or depletion inhibited the accumulation of full length PMEL. Increasing concentrations of PIKfyve inhibitor blocked the cleavage of PMEL protein resulting in the accumulation of full length PMEL protein while having little effect on tyrosinase accumulation (Fig 6C). Similarly, we observed that PIKfyve deletion in primary mouse melanocytes also resulted in the accumulation of full length PMEL protein (Fig 1B). In addition, PIKfyve inhibition blocked the accumulation of mature cathepsin D (Fig 6C), consistent with previously published studies indicating that PIKfyve inhibition blocks lysosomal processing of cathepsin [24] and lyososomal reformation [36].
PIKfyve inhibition is known to induce endosomal vacuolation within hours of treatment [54]. In this study, we sought to measure the effects of PIKfyve inhibition on melanosomes, vesicles that are generated from endosomes [3] that turn over slowly within the cell. To get a better understanding of the kinetics of the effect of PIKfyve inhibition on melanosome biogenesis, we measured the effects of PIKfyve inhibition on PMEL cleavage after treatment for 30 minutes, 1 hour, 2 hours, or 4 hours (S4A Fig). These studies indicated that the accumulation of full length PMEL could be observed 4 hours after treatment. Recently published studies indicated that PIKfyve inhibition can disrupt lysosome reformation, a phenotype that could be observed 6 hours after inhibitor treatment [36]. The observation that PIKfyve inhibition could induce changes in PMEL accumulation 4 hours after treatment is on par with the time scale required for lysosome disruption [36]. Previously published studies demonstrate that PIKfyve inhibitor treatment induced cell death [55]. To verify that the PIKfyve inhibitors used here had similar cytotoxic effects, we examined the effect of PIKfyve inhibitors on MNT-1 cell viability. PIKfyve inhibitor treatment induced cell death in MNT-1 cells, an effect that could be rescued by the addition of PI(3,5)P 2 but not PI(5)P or PI(3)P (S4B Fig). Taken together, these studies demonstrate that PIKfyve regulates the processing of PMEL and transport TYRP1 from an intermediate vesicle to the melanosome. In the absence of functional PIKfyve, PMEL, TYRP1, and TYR accumulate in a vesicular structure that resembles a MVE and are unable to get to the melanosome (Fig 7).

Discussion
Published studies revealed that VAC14 ingls and FIG4 pale tremor mice had lightening of the coat and severe neurologic disease, resulting in early lethality and the accumulation of autophagosomes within the CNS [35]. These phenotypes made it difficult to determine how exactly these mutations resulted in the coat color defects observed. Similarly, the PIKfyve gene-trap mouse also had severe neurologic disease, which ultimately resulted in early lethality [43], making it difficult to assess whether PIKfyve regulates melanogenesis. To circumvent these limitations and study the effects on adult melanocytes, we generated a melanocyte lineage specific PIKfyve-knockout mouse model that utilized a tyrosinase driven Cre recombinase [45]. Melanocytes were cultured from TyrCreER T2 PIKfyve Flox/Flox mice and incubated in the presence and absence of tamoxifen (Fig 1). Tamoxifen treated melanocytes accumulated less melanosomes and less PIKfyve protein as compared to untreated cells (Fig 1B). While these cells accumulated less PIKfyve, PIKfyve was not completely absent as was observed in experiments where PIKfyve Flox/Flox MEFs were infected with adenovirus expressing Cre recombinase [44].
Melanocyte-specific PIKfyve knockout mice accumulated grey hairs that were initially more obvious in shave depilated areas but that eventually were observed in hairs that were not shave depilated (Fig 2). This phenotype did not progress as the mice aged in contrast to other models of stem cell depletion [48]. TyrCreER T2 PIKfyve Flox/Flox mice were crossed with a Cre reporter strain (ROSA mTmG ) and treated with tamoxifen to determine whether PIKfyve deletion affected melanocyte viability in vivo (Fig 3C). Both PIKfyve knockout skin and control skin had GFP expressing melanocytes (Fig 3C), indicating that the observed phenotypes were not secondary to complete melanocyte loss. High magnification light microscopy images of mouse hair follicles revealed that not all of the hair follicles from PIKfyve knockout mice contained vacuolated cells (Fig 4C). Similarly, EM studies revealed that not all melanocytes from the animal accumulated abnormal endosomal vesicles (Fig 4A and 4B). Taken together, these results indicate that the mouse phenotype observed was not the result of complete deletion of PIKfyve but instead a partial loss of function phenotype. This allowed us to study the effects of PIKfyve depletion on melanosome biogenesis as melanocytes remained in the PIKfyve knockout animal.
As PIKfyve deficient melanocytes were still detectable in the adult animal (Fig 3C), we were able to then examine the consequences of PIKfyve depletion on melanosome biogenesis in B) The number of GFP positive puncta in each MNT-1 cell was counted, and at least 20 cells were included for each group. The data are presented as the mean ± S.D. based on three independent experiments, data shown as percent signal relative to vehicle. For all experiments, data shown are mean ± S.D Ã , p < 0.05; ÃÃ , p < 0.01; or ÃÃÃ , p < 0.001 using a Student's paired T test versus vehicle treated control. C) MNT-1 cells were treated with PIKfyve inhibitors YM-201636, apilimod or vehicle control for 72 hours. The relative accumulation of TYR, and unprocessed PMEL (100 kD) and Cathepsin D was measured by immunoblotting. Protein accumulation relative to GAPDH levels was quantified by densitometry. Pre pro cathepsin D and pro cathepsin D was quantified relative to GAPDH level (black) as well as relative to mature Cathepsin D (gray). Representative experiment of three independent experiments is shown.
https://doi.org/10.1371/journal.pgen.1007290.g006 vivo. Epidermal melanocytes from tamoxifen fed TyrCreER T2 PIKfyve Flox/Flox mice accumulated abnormal single membrane vesicles with smaller vesicles within them reminiscent of MVEs that also contained tyrosinase reaction product (Fig 4A and 4B). A similar phenotype was observed when melanocytes from TyrCreER T2 PIKfyve Flox/Flox were cultured and treated with tamoxifen (Fig 4C), and MVEs containing DOPA reaction product were also shown to accumulate in PIKfyve inhibitor treated primary melanocytes (S3 Fig). Similarly, some of the MVE-like structures in the animal had DOPA reaction product within them, suggesting that they contain TYR, which may be trapped within this compartment (Fig 4B). Consistent with these observations, immunofluorescence studies in MNT-1 cells demonstrated that PIKfyve inhibition blocked PMEL processing and the trafficking of TYRP1 to the melanosome (Fig 6).
Other studies have shown that PI(3,5)P 2 plays an essential role in MVE protein sorting and retrograde trafficking [31,56,57]. Specifically, PI(3,5)P 2 has been shown to regulate ESCRT-III function and MVE trafficking [57,58] downstream of ESCRT-I [59], which has been shown to be required for TYRP1 transport [16]. Interestingly, PIKfyve inhibition also affected the trafficking of MART-1 (Fig 6), whose proper trafficking requires ESCRT-I. Taken together, these results are consistent with a model where PIKfyve regulates the delivery of TYRP1 and TYR from the endosome/MVE to the terminal melanosome (Fig 7).
Recently published studies revealed that PI(3,5)P 2 depletion inhibits the process of lysosome reformation from endolysosomes [36]. While we observed that PIKfyve inhibition affected lysosomal enzyme processing (Fig 6C), we observed a distinct phenotype in melanocytes-MVE accumulation. These results indicate that PI(3,5)P 2 or PI(5)P that is generated from PI(3,5)P 2 modulates the biogenesis of melanosomes in a way that is distinct from its effect on lysosomes. PI(3,5)P 2 or PI(5)P could control the biogenesis of melanosomes in several different ways. These lipids could control the budding of vesicle cargo from the MVE en route to the melanosome or the fusion of vesicle cargo with the developing melanosomes. Alternatively, PI(3,5)P 2 could be specifically required for ESCRT-I and ESCRT-III based trafficking of melanosome proteins. Finally, PI(3,5)P 2 may regulate melanosome biogenesis by influencing conductance regulators required for pigmentation. Two families of cation channels, the TPCs and the TRPMLs, act as PI(3,5)P 2 effectors and function in vesicular fusion [26,60,61]. In particular, TPC2 was found to be activated by PI(3,5)P 2 and regulate pigmentation in vitro in an expression dependent context [25,62,63]. Furthermore, single-nucleotide polymorphisms in TPC2 have also been identified in humans that are correlated with skin, eye, and hair pigment variation [64]. Finally, mice mutant for TRPML3, another putative PI(3,5)P 2 regulated channel, exhibit hypopigmentary phenotypes [65]. Future studies will define the specific phosphoinositide that regulates melanogenesis, determine how and when these phosphoinositides regulate melanosome maturation, and identify phosphoinositide effectors protein present on the melanosome that participate in this process.

Ethics statement
All experiments involving mice conform to the NIH guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, approval number 2011-3020.

Antibodies and primers
All antibodies used in experimental assays are listed in S3 Table. PIKfyve genotyping primers and PCR parameters are described in [44]. Other genotyping primers and PCR settings were taken from the mouse mutant resource website, (Jackson Laboratory)).

Drug treatment
MNT-1 cells were plated in 6-well plates at a concentration of 2 x 10 5 cells per well and allowed to adhere overnight. Cells were then incubated with varying concentrations of YM-201636 (Cayman Chemical), Apilimod (US Biological), or vehicle control (DMSO) in normal MNT-1 media. At the conclusion of the treatment, cells were lysed with RIPA buffer for downstream analysis. In treatment assays that exceeded 48 hours, media containing drug was refreshed every 48 hours.

Pigment measurement
MNT-1 cells were plated in 96-well plates at a concentration of 1.5 x 10 4 cells per well and allowed to re-attach overnight. Media was refreshed for drug treated cells every 48 hours. After five days of treatment cells were lysed with Cell-Titer-Glo reagent (Promega). Relative melanin accumulation was quantified by measuring absorbance at 405 nm and normalizing this value to luminescence to determine cell number as determined by the Cell-Titer-Glo assay as previously described [66]. Pigment percent was quantified relative to vehicle control. A Student's two-tailed t-test was used to calculate the statistical significance in comparison to vehicletreated control.

PI Lipid treatment
All carriers and phospholipids were obtained from Echelon Biosciences. Unlabeled PI(5)P, PI (3)P and PI(3,5)P 2 were reconstituted in DMSO:H 2 O (10:1); carrier 2 and carrier 3 were reconstituted in H 2 O. Carriers and lipids were combined at 1:1 molar ratio and incubated for 15 minutes at room temperature. Charge matched carriers were used to optimize lipid delivery-carrier 3 was used as a control for PI(5)P; carrier 2 was used as a control PI(3,5)P 2 . The mixture was then diluted in MNT-1 media and incubated with cells for 48 hours. The relative survival of cells in the presence and absence of lipid or carrier and PIKfyve inhibitor was measured using a Cell Titer-Glo assay. MNT-1 cells were plated in 96-well plates at a concentration of 1.5 x 10 4 cells per well and allowed to re-attach overnight. Media containing drug and lipid was refreshed on cells every 48 hours. After five days of treatment cells were lysed with Cell-Titer-Glo reagent (Promega). The luminescence value was used to determine cell survival as determined by the Cell-Titer-Glo assay as previously described [67]. A Student's two-tailed ttest was used to calculate the statistical significance in comparison to vehicle-treated control.

Electron microscopy
Cultured melanocytes or whole mouse skin (n = 2 per genotype) harvested from either anesthetized or euthanized mice using a 4-mm round punch biopsy were fixed in half-strength Karnovsky's fixative (Karnovsky, 1965) for 24 hours before being transferred to sodium cacodylate buffer, 0.2M, pH 7.4 (Electron Microscopy Sciences). Tissue was then postfixed with 1% osmium tetroxide containing 1.5% potassium ferrocyanide. After being dehydrated, tissues were embedded in EPON and sections were obtained using a RMC-MT6000XL ultramicrotome and stained with uranyl acetate and lead citrate. Sections were viewed and selected images were digitally photographed using a JEOL JEM-1230 transmission electron microscope. For DOPA histochemistry and prior to postfixation, cells or tissues were incubated in a 0.1%solution of l-DOPA twice for 2.5 hours. The cells and tissues were washed and processed as described above.
Darkly pigmented melanocytes were treated with various dosages of YM-201636 (0-1000 nM) for 72 hours. Cells were then fixed for 4 hours in Karnovsky's fixative, pH 7.2, before being washed with sodium cacododylate buffer (0.2 M). Primary melanocytes were fixed for 30 minutes in Karnosky's fixative, pH 7.2, before being washed with sodium cacododylate buffer (0.2M). Samples were then processed for routine DOPA histochemistry electron microscopy. Melanosome stages (I-IV) were quantified visually in the electron micrographs and melanosome stage percentage was assessed versus vehicle treated controls. Electron microscopy on whole mouse skin was obtained and processed as previously described by (48).

Immunofluorescence microscopy
MNT-1 cells were plated in 12-well plates on coverslips at a concentration of 1 x 10 4 cells per well and allowed to adhere overnight. Cells were then treated with 100 nM Apilimod, 1000 nM YM-201636, or vehicle control overnight. Alternatively, cells were treated with charge matched carrier or PI(3,5)P 2 for 8 hours. Cells were fixed with 4% paraformaldehyde for 1 hour. Coverslips were rinsed with PBS and permeablized with 0.1% Triton X-100 (Fisher Scientific) and subsequently blocked in 2% BSA in PBS containing 0.1% Tween 20 for 1 hour. Cells were then incubated with primary antibodies (S3 Table) followed by secondary antibodies conjugated to Alexa Fluor 594 (Invitrogen) and were mounted in a solution containing DAPI. Confocal images were acquired using a LSM 780 confocal multiphoton microscope and images were processed in Zen lite (Zeiss). For imaging mouse skin from TyrCreER T2 PIKfyve Flox/Flox ROSA mTmG/+ , mice was harvested, skin was frozen in OCT blocks and cryosectioned. 4μm sections were imaged using a Nikon Eclispse Ti fluorescent microscope.

Mouse strains and genotyping
All experiments involving mice conform to the NIH guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, approval number 2011-3020. C57BL/6 PIKfyve Flox/Flox mice on a pure C57BL/6 background were obtained from Dr. Takehiko Sasaki (Akita University, Akita, Japan). PIKfyve Flox/Flox were crossed to Tyrosinase::CreER T2 (JAX stock no: 012328) on a pure C57BL/6 background. The resulting Tyrosinase::CreER T2, PIKfyve Flox/+ progeny were backcrossed to PIKfyve Flox/Flox to generate Tyrosinase::CreER T2, PIKfyve Flox/Flox mice. Upon weaning, mice were placed on tamoxifen feed (Harlan Laboraties, 250 mg/kg) for 29 days. Genomic DNA was isolated from mouse tail biopsies using the Quick Genotyping DNA Preparation Kit (Bioland Scientific, LLC) according to the manufacturer's instructions. Tyrosinase::CreER T2, PIKfyve Flox/Flox mice were crossed with ROSA mTmG/mTmG mice obtained from Jackson laboratories. Genotypes of progeny was determined using specific genotyping primers (S4 Table) using guidelines provided by Jackson laboratories. Resulting TyrCreER T2 PIKfyve Flox/Flox ROSA mTmG/+ progeny were similarly placed on tamoxifen feed for 29 days as has been described above.

Primary melanocyte isolation
Primary mouse melanocytes were collected based on methods described by Godwin et al. [68]. In brief, newborn mice less than 3 days old were sacrificed and sterilized. The skin was removed and cleaned of muscle. To dissociate the epidermis, the skin was incubated for 1 hour in 5mg/ml trypsin (Sigma-Aldrich) at 37˚C. The skin was washed and the epidermis was split off. The epidermis was chopped in 0.25% trypsin-EDTA solution (Gibco) and resuspended in growth media. The resuspended cells were filtered using an 100μm cell strainer (Falcon) and plated on 10 cm dishes. Once melanocytes were established, TPA concentration was increased to 400nM to treat fibroblast contamination and increase pigmentation. To increase purity of melanocytes for later experiments, cells isolated from epidermis were incubated on CD117 MicroBeads (Miltenyi Biotec) and sorted on MACS LS columns (Miltenyi Biotec). CD117+ melanocytes were then plated in 24-well dishes and incubated in media containing 5% FBS, SCF, END3, FGF, α-MSH, Phosphoethanolamine, ethanolamine, and insulin.

Primary melanocyte treatment and microscopy
Primary melanocytes were plated on 4-chambered coverglass (Thermo Fisher Scientific) and treated with 1μg/ml 4-hydroxytamoxifen (4-HTA) for 48hrs. Cells were fixed in 2% PFA for 1 hour at room temperature and imaged. Phase contrast images were acquired using a Nikon Eclipse Ti fluorescent microscope.

Mouse hair
Dorsal hairs of mice at P50, P100, or P365 were shaved and 1 mg was dissolved overnight in 1 mL of 9:1 Soluene-350 (PerkinElmer) and water. Quadruplicate 150 μL aliquots for each mouse hair sample were then analyzed for absorbance values at 405 nm as previously described [47]. Ganesan.