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Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells

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Abstract

Background

Melanosomes are lysosome-related organelles that contain melanogenic factors and synthesize melanin as they mature. FYVE finger-containing phosphoinositide kinase (PIKfyve) regulates late endosome and lysosome morphology, vesicle trafficking, and autophagy. In melanocytes, PIKfyve inhibition has been reported to induce hypopigmentation due to impairments in the metabolism of early-stage melanosomes.

Methods and results

Here, we report a new type of melanosome metabolism: post-PIKfyve inhibition, which was found during the characterization of the endosome/lysosome fluoroprobe GIF-2250. In B16F10 mouse melanoma cells, GIF-2250 highlighted vesicles positive for lysosomal-associated membrane protein 1 (lysosome marker) and other endosome/lysosome markers (CD63 and Rab7/9). When cells were continuously treated with PIKfyve inhibitors, intracellular vacuoles formed, while GIF-2250 fluorescence signals diminished and were diffusely distributed in the vacuoles. After removal of the PIKfyve inhibitors, the GIF-2250 signal intensity was restored, and some GIF-2250-positive vesicles wrapped the melanosomes, which spun at high speed. In addition, intermittent PIKfyve inhibition caused melanin diffusion in the vacuoles and possible leakage into the cytoplasmic compartments, and melanosome degradation was detected by a transmission electron microscope. Melanosome degradation was accompanied by decreased levels of melanin synthesis enzymes and increased levels of the autophagosome maker LC3BII, which is also associated with early melanosomes. However, the protein levels of p62, which is degraded during autophagy, were increased, suggesting an impairment in autophagy flux during intermittent PIKfyve inhibition. Moreover, the autophagy inhibitor 3-methyladenine does not affect these protein levels, suggesting that the melanosome degradation by the intermittent inhibition of PIKfyve is not mediated by canonical autophagy.

Conclusions

In conclusion, disturbance of PIKfyve activity induces melanosome degradation in a canonical autophagy-independent manner.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. The original blots of western analyses are in Supplementary Materials.

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Acknowledgements

We thank Ms. Akiko Tsujimoto and Mr. Yasuaki Hotta for their support of the TEM analyses. 

Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) (Grant No. 20K05703); Japan Science and Technology Agency (JST) A-Step (Grant No. JPMJTM20E8); START University Ecosystem Promotion Type (Supporting Creation of Startup Ecosystem in Startup Cities) (Grant No. JPMJST2183), Japan; the AIST-Gifu Univ. Joint Project; the Nakatani Foundation; the Ogawa Foundation; and the Kobayashi Foundation.

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Authors and Affiliations

  1. Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kyoka Kawaguchi, Miyu Watanabe, Saho Furukawa, Kenichi Koga, Miwa Maeda, Kentaro Oh-Hashi, Yoko Hirata, Kyoji Furuta & Hiroshi Takemori

  2. Department of Cardiology, Gifu University Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Hiromitsu Kanamori

  3. Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, 305-8566, Ibaraki, Japan

    Mitsushi J. Ikemoto

  4. Graduate School of Science, Toho University, 2-2-1 Miyama, Funabashi, 274-8510, Chiba, Japan

    Mitsushi J. Ikemoto

  5. Advanced Research Initiative for Human High Performance (ARIHHP), Faculty of Health and Sports Sciences, University of Tsukuba, Tsukuba, 305-8574, Japan

    Mitsushi J. Ikemoto

  6. Institute for Glycocore Research (iGCORE), Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Shigeo Takashima

  7. The United Graduate School of Drug Discovery and Medical Information Sciences of Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Shigeo Takashima, Kentaro Oh-Hashi, Yoko Hirata, Kyoji Furuta & Hiroshi Takemori

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  1. Kyoka Kawaguchi
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Contributions

Experiments were performed by KK, MW, SF, HK, MJI, ST, and MMK GIF-2250 was synthesized by K and KF. Data analysis was performed by KO-H and YH. The manuscript was written by HT. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Hiroshi Takemori.

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The title compound is protected by PCT application no. PCT/JP2022/1692, submitted by the authors of this paper. The authors have no other relevant financial or non-financial interests to declare.

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Kawaguchi, K., Watanabe, M., Furukawa, S. et al. Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells. Mol Biol Rep 50, 5917–5930 (2023). https://doi.org/10.1007/s11033-023-08536-9

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