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A melanosomal two-pore sodium channel regulates pigmentation.  

Nicholas W. Bellono#1,2, Iliana E. Escobar#1, Elena Oancea1*  

Supplementary Information

 

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Supplementary Figures  

 

Supplementary Figure 1. IPIP2 properties.  

a) PI(3,5)P2 activates inwardly rectifying IPIP2 in the absence of OCA2-mediated Cl- currents

(representative of n = 2 melanosomes). OCA2 expression was reduced in Oa1-/- melanocytes, with

OCA2-targeted siRNA1.

b) In a RPE melanosome, IPIP2 is inhibited by 150 µM verapamil, similar to dermal melanosome IPIP2

(representative of n = 2 melanosomes).

c) IPIP2 current density (pA/pF) measured from Oa1-/- melanosomes was similar when the luminal (pipette)

pH was 4.6 or 6.8. Average current density (pA/pF) measured at -120 mV (± s.e.m., n = 4

melanosomes).

   

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Supplementary Figure 2. TPC2 does not exhibit significant lysosomal localization in pigment cells.  

a) In melan-a melanocytes GFP-tagged TPC2 (green) localizes to melanin (50.9 ± 3.2%) (bright field) and

TYRP1 (red)-positive compartments, but does not significantly overlap with structures immunolabeled

with antibodies against the lysosomal marker LAMP2 (red) (7.0 ± 2.7%, p < 0.0001 ) . Enlarged images

of outlined regions shown in lower panels. Scale bar = 10 µm.

b) Expression of the lysosomal protein LAMP1 tagged with GFP (green) localizes to LAMP2 (red)-positive

compartments in melan-a cells (71.4 ± 6.2%). LAMP1-GFP (green) does not localize to melanin (bright

field) and TYRP1 (red)-positive compartments LAMP1 (12.1 ± 2.1%, p < 0.0001).  

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Supplementary Figure 3. Efficiency of tpc2-targeted CRISPR-Cas9 and siRNA in melanocytes.  

a) High resolution melt analysis of genomic DNA (gDNA) obtained from control (blue) melan-a, B16-F1, or

Oa1-/- melanocytes and tpc2-targeted CRISPR-Cas9 (red) populations of the same types of cells.

Differences in the relative fluorescence intensity (Δ Rel Fluorescence) between the control and

CRISPR-Cas9 treated cells are due to gDNA mutations in the targeted sequence.

melan-a B16-F1 Oa1-/-

5’

tpc2 CRISPR Site PAM CT GT T ACAT T GGT GGGAT GGCGGCAGAAGAGCAGCCCCT T CT GGGCCGGGACCGAGGCAGT GGCCAGGT CCACT CCGGT GMajority

10 20 30 40 50 60 70 80

CT GT T ACAT T GGT GGGAT GGCGGCAGAAGAGCAGCCCCT T CT GGGCCGGGACCGAGGCAGT GGCCAGGT CCACT CCGGT G 80mTPCN2 Crisper1 Site.seqCT GT T ACAT T GGT GGGAT GGCGGCAGAAGAGCAGCCCCT T CT GGGCCGGGACCGAGGCAGT GGCCAGGT CCACT CCGGT G 80mTPCN2 Crisper1 Site.seq

10 20 30 40 50 60 70 80

CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT - - - - GGCCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_1CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCG- - - - - - - - - - - GGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_2CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT T CT G- - CCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_3CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT T CT G- - CCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_4CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT T CT GG- CCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_5CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT T CGG- - CCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_6CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCC- - - - - - - - - - - GGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_7CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT T CGG- - CCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_8CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT T - - - GGCCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G Melan-a TPC2 Crisper1 TOPO_9CT GT T A CA T T GGT GGGA T GGCGGCA GA A GA GCA GCCCCT T CT GGGCCGGGA CCGA GGCA GT GGCCA GGT CCA CT CCGGT G mTPCN2 Crisper1 Site.seq

bp 1000 750 500

250

a

c

b

Temp (°C) Δ

Rel

Flu

ores

cenc

e

CTL CRISPR CTL CRISPR CTL CRISPR

0.05

0.00

-0.05

-0.15

-0.10

80 82 84 86 0.05

0.00

-0.05

-0.15

-0.10

82 84 86 0.05

0.00

-0.05

-0.15

-0.10

80 82 84 86 Temp (°C) Temp (°C)

f

CTLCRISPRCTL CRISPR

0

20

40

60

mel

an-a

Oa1

-/-

B16

-F1 F c

ut (%

)

d

0.0

0.5

1.0

e

rel.

mR

NA

leve

ls

cont

rol

tpc2

siR

NA

0.0

0.5

1.0

1.5

2.0

norm

. mel

anin

con

tent

cont

rol

tpc2

siR

NA si

RN

A C

RIS

PR/C

as9

Oa1-/-

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b) Representative gels from mutation detection assay using gDNA from melan-a, B16-F1, or Oa1-/-

melanocytes expressing control or tpc2-targeted CRISPR-Cas9. Cleaved DNA fragments are due to

indels caused by CRISPR-Cas9-induced mutations.

c) Average fraction of cleaved fragments (Fcut) from Guide-it Resolvase assay determined from n = 3

independent experiments.

d) Identification of individual mutations in melanocytes treated with tpc2-targeted CRISPR-Cas9 using

single gDNA species cloned in the TOPO vector. The sequences from each clone were aligned with the

wild-type sequence (in bold) revealing a range of deletions and insertions the tpc2 gene (highlighted in

blue) at the CRISPR site.  

e) Mouse tpc2-targeted siRNA stably expressed in Oa1-/- melanocytes reduced the TPC2 mRNA levels by

~30%, compared to control siRNA. (± s.e.m., n = 3, p < 0.01)

f) Melanocytes expressing TPC2-targeted siRNA have ~50% higher melanin content than control siRNA

expressing cells. (± s.e.m., n = 3, p < 0.001)

   

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Supplementary Figure 4. WT and V443I OCA2 variants localize to melanosomes in B16-F1 melanocytes.  

a) In B16-F1 melanocytes mCherry-tagged wild type (WT) OCA2 (red) localizes to TYRP1 immunostained

(green) compartments. Enlarged images of outlined regions shown in lower panels. Scale bar = 10 µm.

b) mCherry-tagged V443I mutant OCA2 (red) localizes to TYRP1 immunostained (green) compartments in

B16-F1 cells.

   

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     Supplementary reference  

1 Bellono, N. W., Escobar, I. E., Lefkovith, A. J., Marks, M. S. & Oancea, E. An intracellular anion

channel critical for pigmentation. eLife 3 (2014).