Rab27A-binding protein Slp2-a is required for peripheral melanosome distribution and elongated cell shape in melanocytes

Abstract

The synaptotagmin-like protein (Slp) family is implicated in regulating Rab27A-mediated membrane transport1,2,3, but how it might do this is unknown. Here we report that Slp2-a, a previously uncharacterized Rab27A-binding protein in melanocytes, controls melanosome distribution in the cell periphery and regulates the morphology of melanocytes. Slp2-a is the most abundantly expressed of the Slp- and Slac2-family proteins in melanocytes and colocalizes with Rab27A on melanosomes. Knockdown of endogenous Slp2-a protein by small-interfering RNAs (siRNAs) markedly reduced the number of melanosomes in the cell periphery of mouse melanocytes ('peripheral dilution'). Expression of siRNA-resistant Slp2-a (Slp2-aSR) rescued the peripheral dilution of melanosomes induced by Slp2-a siRNAs, but Slp2-aSR mutants, which failed to interact with either phospholipids or Rab27A, did not. Loss of Slp2-a protein also induced a change in melanocyte morphology, from their normal elongated shape to a more rounded shape, which depended on the phospholipid-binding activity of Slp2-a, but not on its Rab27A-binding activity. By contrast, knockdown of Slac2-a (also called melanophilin), another Rab27A-binding protein in melanocytes4,5, caused perinuclear aggregation of melanosomes alone without altering cell shape. These results reveal the differential and sequential roles of Rab27A-binding proteins in melanosome transport in melanocytes.

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Figure 1: Rab27A-binding proteins expressed in melanocytes.
Figure 2: Localization of Slp2-a deletion and point mutants in melanocytes.
Figure 3: Phospholipid-binding activity of the C2A and C2B domains of Slp2-a.
Figure 4: Slp2-a depletion induced peripheral dilution of melanosomes and round melanocytes.
Figure 5: Rescue of Slp2-a-siRNA-induced peripheral dilution of melanosomes and round cell shape by siRNA-resistant Slp2-a.

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Acknowledgements

We thank D. C. Bennett (St. George's Hospital Medical School, London, UK) for the melan-a cell line, E. Kanno and Y. Ogata for expert technical assistance, and T. Itoh for valuable discussion. This work was supported in part by grants from the Ministry of Education, Culture, Sports and Technology of Japan (15689006 and 16044248 to M.F.). T.S.K. was supported by a Special Postdoctoral Researchers Program in RIKEN.

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Correspondence to Mitsunori Fukuda.

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Kuroda, T., Fukuda, M. Rab27A-binding protein Slp2-a is required for peripheral melanosome distribution and elongated cell shape in melanocytes. Nat Cell Biol 6, 1195–1203 (2004). https://doi.org/10.1038/ncb1197

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