Hume Alistair N, Ushakov Dmitry S, Tarafder Abul K, Ferenczi Michael A, Seabra Miguel C
Molecular and Cellular Medicine, National Heart and Lung Institute, Imperial College London, London, SW7 2AZ, UK.
J Cell Sci. 2007 Sep 1;120(Pt 17):3111-22. doi: 10.1242/jcs.010207. Epub 2007 Aug 14.
Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plus-end binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for melanosome targeting and stability of Mlph. Mutants and siRNA that affect Mlph-MyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to melanosomes, MyoVa but not EB1 interaction is required for transport of melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and melanosome transfer.
黑素细胞中的黑素小体运输是研究细胞骨架对细胞内运输调控的一个模型系统。黑素亲和素（Mlph）是一种与Rab27a和肌球蛋白Va（MyoVa）结合的蛋白质，它调节这一过程。通过酵母双杂交筛选，我们鉴定出微管正端结合蛋白（EB1）是一种在黑素细胞中表达的与Mlph相互作用的蛋白质。为了探讨EB1与Rab27a和MyoVa相互作用在Mlph靶向和功能中的作用，我们使用小干扰RNA（siRNA）和Mlph突变体来特异性破坏培养的黑素细胞中的每种相互作用。使用阻断Mlph-Rab27a相互作用的Mlph R35W突变体和Rab27a siRNA，我们发现这种相互作用对于黑素小体靶向和Mlph的稳定性是必需的。影响Mlph-MyoVa和Mlph-EB1相互作用的突变体和siRNA表明，虽然MyoVa和EB1都不影响Mlph靶向黑素小体，但黑素小体运输到外周树突需要MyoVa而非EB1的相互作用。我们提出，Rab27a将Mlph靶向黑素小体和/或使其在黑素小体上稳定，然后募集MyoVa，MyoVa为该复合物提供额外的稳定性，并使黑素小体从微管转移到基于肌动蛋白的运输并实现外周分布。在培养的黑素细胞中，EB1似乎对这一过程并非必不可少，这表明它发挥着冗余作用和/或对黑素细胞/角质形成细胞接触及黑素小体转移是必需的。
