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      TPC2 controls pigmentation by regulating melanosome pH and size

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          Abstract

          <p id="d15160447e165">Melanin pigments are synthesized in skin and hair cells called melanocytes and provide color to skin and hair and protection against UV rays. Inadequate protection poses the risk of accumulating genetic mutations in the DNA of skin cells, which can lead to skin cancer. It can also reduce folate levels, which then causes birth defects. Therefore, understanding pigmentation is important for human health. There are several protein components of the machinery that regulates human pigmentation that work in unknown ways. Two-pore channel 2 (TPC2) is one of them. Here we found that TPC2 is located in compartments inside melanocytes known as melanosomes, where melanin is synthesized. TPC2 regulates the pH and size of melanosomes, thus controlling the amount of melanin produced. </p><p class="first" id="d15160447e168">Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 ( <i>TPCN2</i>) gene is strongly linked to pigmentation variations. <i>TPCN2</i> encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. <i>TPCN2</i> knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca <sup>2+</sup> release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca <sup>2+</sup> sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size. </p>

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          Most cited references23

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          NAADP mobilizes calcium from acidic organelles through two-pore channels

          Ca2+ mobilization from intracellular stores represents an important cell signaling process 1 which is regulated, in mammalian cells, by inositol 1,4,5-trisphosphate (InsP3), cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cADPR release Ca2+ from sarco / endoplasmic reticulum (S/ER) stores through activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). By contrast, the nature of the intracellular stores targeted by NAADP and molecular identity of the NAADP receptors remain controversial 1,2, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments 3,4. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with TPC1 and TPC3 being expressed on endosomal and TPC2 on lysosomal membranes. Membranes enriched with TPC2 exhibit high affinity NAADP binding and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release via InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but only attenuated by depleting ER Ca2+ stores or blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger additional Ca2+ signals via S/ER. TPCs therefore provide new insights into the regulation and organization of Ca2+ signals in animal cells and will advance our understanding of the physiological role of NAADP.
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            Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment.

            Ebola virus causes sporadic outbreaks of lethal hemorrhagic fever in humans, but there is no currently approved therapy. Cells take up Ebola virus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebola virus entry into host cells requires the endosomal calcium channels called two-pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs, or small-molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule that we tested, inhibited infection of human macrophages, the primary target of Ebola virus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebola virus infection and may be effective targets for antiviral therapy.
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              Near-infrared fluorescent proteins for multicolor in vivo imaging

              Near-infrared fluorescent proteins are in high demand for in vivo imaging. We developed four spectrally distinct fluorescent proteins, iRFP670, iRFP682, iRFP702, and iRFP720, from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging in living cells and mice using standard approaches. Five iRFPs including previously engineered iRFP713 allow multicolor imaging in living mice with spectral unmixing.
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                Author and article information

                Journal
                Proceedings of the National Academy of Sciences
                Proc Natl Acad Sci USA
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                May 17 2016
                May 17 2016
                : 113
                : 20
                : 5622-5627
                Article
                10.1073/pnas.1600108113
                4878521
                27140606
                4795923e-4953-418d-b285-40dc7b2187ca
                © 2016
                History

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