The role of multivesicular endosomes and OA1 in melanosome biogenesis

皮肤中黑色素的主要功能之一是充当防晒霜，吸收紫外线辐射，防止DNA损伤和皮肤癌。黑色素被合成并储存在膜结合的细胞器中，称为黑素小体。黑素小体存在于皮肤的黑素细胞中，也存在于眼睛的细胞中，包括视网膜色素上皮(RPE)细胞。RPE细胞位于视杆细胞和视锥细胞(眼睛中探测和传递光的细胞)的正下方。在各种类型的白化病中发现的黑色素缺乏会导致严重的视力损害。一些类型的白化病是由合成黑色素的能力缺陷引起的，而另一些类型的白化病是由制造黑素小体的能力缺陷引起的。黑素小体形成的机制尚未完全确定，但它们涉及内吞途径，这是病毒和营养物质等细胞外分子被吸收到细胞内的途径。细胞外分子被输送到内小体，在那里它们与细胞新合成的蛋白质相遇。来自内体的蛋白质可能会被输送到许多不同的地方，如溶酶体进行降解或返回到细胞表面。我们已经证明，这一关键决定是在多囊内体/小体(MVB)中做出的。被运送到溶酶体进行降解的蛋白质被分选到MVB的内部小泡上，而要返回到质膜的蛋白质则停留在MVB的周膜上。在黑色素合成细胞中，MVB也可能参与将黑素小体蛋白输送到黑素小体，或者MVB的一个亚群可能发展成黑素小体。在黑色素产生细胞中，一种名为PMEL 17的蛋白质是在黑色素沉积的黑素小体内产生内部条纹所必需的。在黑素小体形成的非常早期阶段，pMel17在MVB的内部小泡上被发现，当足够的蛋白质积累在MVB的内部小泡上时，开始形成条纹。如果经过基因改造以产生pMel17，正常情况下不会产生黑色素的细胞的微血管内甚至会开始形成条纹。这意味着MVB内的内向囊泡可能在内纹的形成和黑素小体的生物发生中发挥关键作用。最近，在MVB内和MVB形成中涉及蛋白质分选的分子机制的组件已经被确定。我们一直在使用电子显微镜(EM)来确定其中一些蛋白质的功能，因为EM是显示MVB非常小(直径50 nm)内部小泡的唯一方法。到目前为止，我们已经确定了其中一些蛋白质在不产生黑素小体的细胞中的功能。我们现在建议确定这些蛋白质在黑素细胞和RPE细胞中黑素小体形成中的作用。除了确定MVB机制的核心组件的作用外，我们还将确定一种只在制造黑素小体的细胞中表达的蛋白质的作用。OA1是一种在最常见的眼部白化病中存在缺陷的蛋白质，这种疾病的患者可以产生黑色素，但会减少异常大的黑素小体的数量。该蛋白已被发现与MVB相关，并被认为在MVB水平上在黑小体形成中发挥作用。我们将使用我们的EM分析来确定OA1在MVB中的作用。这些研究将共同阐明调节细胞器形成的基本机制，细胞器是人体抵御太阳影响的天然防御机制，在视觉中也起着重要作用。这将有助于我们理解这些机制在人类疾病中是如何失败的。

One of the main functions of melanin pigment in the skin is to act as a sunscreen to absorb ultraviolet radiation and prevent DNA damage and skin cancer. Melanin is synthesised and stored in membrane bound organelles called melanosomes. Melanosomes are found in melanocytes of the skin and also in cells of the eye, including in retinal pigment epithelial (RPE) cells. RPE cells lie immediately beneath the rods and cones (the cells of the eye that detect and transduce light). Deficit of melanin pigment, as is found in the various types of albinism, leads to severe visual impairment. Some types of albinsm are caused by defects in the ability to synthesise melanin while others are caused by defects in the ability to make melanosomes. The mechanisms whereby melanosomes are formed are not fully characterised but they involve the endocytic pathway, which is the route where by extracellular molecules like viruses and nutrients are taken up into the cell. Extracellular molecules are delivered to endosomes where they meet proteins that have been newly synthesised by the cell. From the endosome proteins may be delivered to many different places, such as the lysosome for degradation or back to the cell surface. We have shown that this critical decision is taken in multivesicular endosomes/bodies (MVB). Proteins that are to be delivered to the lysosome for degradation are sorted onto the internal vesicles of MVB while proteins that are to be returned to the plasma membrane stay on the perimeter membrane of the MVB. In melanin synthesising cells the MVB may also be involved in delivering melanosomal proteins to the melanosome or a subpopulation of MVBs may develop into melanosomes. In melanin producing cells a protein called pmel 17 is required for the generation of internal striations within the forming melanosome upon which melanin is deposited. In the very early stages of melanosome formation pmel17 is found on the internal vesicles of MVBs and when sufficient protein has accumulated on the internal vesicles of MVB striations begin to form. Striations will even begin to form within the MVBs of cells which do not normally make melanin if they are genetically modified so that they make pmel17. This implies that inward vesiculation within MVB may play a critical role in formation of internal striations and in melanosome biogenesis. Recently components of the molecular machinery that are involved in sorting of proteins within MVB and in MVB formation have been identified. We have been determining the function of some of these proteins using electron microscopy (EM), as EM is the only way to visualise the very small (50nm diameter) internal vesicles of MVB. So far we have determined the functions of some of these proteins in cells which do not make melanosomes. We now propose to determine the role of these proteins in the formation of melanosomes in melanocytes and RPE cells. In addition to determining the role of the core components of the MVB machinery we will determine the role of a protein that is only expressed in cells that make melanosomes. OA1 is the protein which is defective in the most common type of ocular albinism and patients with this disease can make melanin but make a reduced number of unusually large melanosomes. This protein has been found associated with MVBs and has been proposed to play a role at the level of the MVB in melanosome formation. We will use our EM assays to determine what OA1 does within the MVB. Together these studies will elucidate the fundamental mechanisms regulating the formation of an organelle which is the body's natural defence mechanism against the effects of the sun and which also plays an important role in vision. This will help us to understand how these mechanisms fail in human disease.