Studies on the mechanism of melanosome secretion

膜运输描述了细胞内隔室之间的脂类和蛋白质的流动。这一过程对于这些细胞器的独特性质的建立和维持是至关重要的，并且是由小泡介导的。此外，细胞中合成的蛋白质可以通过小泡与质膜融合的分泌途径被挤出。这种分泌的蛋白质可以被邻近的细胞吸收，就像黑色素一样。这种色素是在称为黑素细胞的特殊细胞中合成的，并储存在一个称为黑素小体的独特细胞器中。这些细胞器被运输到细胞外围，并以迄今尚不清楚的机制将它们的黑色素转移到邻近的角质形成细胞。黑色素用于保护角质形成细胞DNA免受紫外线辐射的有害影响。黑色素从黑素细胞转移到角质形成细胞的确切机制尚不清楚，也不清楚是否真的转移了黑色素或黑素小体。本申请建议使用双管齐下的方法来解决这个问题，方法是(I)检查这一过程中涉及的已知因素，以及(Ii)通过建立基于RNA干扰的筛查来采取公正的方法。这两种方法都依赖于观察到的结果，即B16黑素细胞株未能释放黑色素会导致黑色素在细胞内积累，并且可以通过流式细胞术从混合细胞群中选择性地分离出这种变暗的细胞。筛选将包括随机干扰RNA的表达，通过流式细胞仪对黑色素积累细胞进行分类，然后使用生物信息学方法来识别被击倒的蛋白质。预计这种方法将识别参与黑素小体生物学许多不同阶段的成分，因为任何阻止它们移动或释放的缺陷都将导致细胞变暗。通过筛查或通过更直接的方法鉴定的蛋白质将通过各种生化和细胞生物学方法进行验证。此外，这种筛选还可以适用于其他分泌途径过程的研究，使其广泛适用于蛋白质分泌的许多方面的研究。

Membrane traffic describes the flow of both lipids and proteins between intracellular compartments. This process is crucial for the establishment and maintenance of the distinct nature of these organelles and is mediated by small vesicles. In addition, proteins synthesized in the cell can be extruded via the secretory pathway where vesicles fuse with the plasma membrane. Such secreted proteins can be taken up by neighboring cells as is the case with the pigment melanin. This pigment is synthesized in specialized cells called melanocytes and stored in a unique organelle called the melanosome. These organelles are transported to the cell periphery and by as as yet unidentified mechanism transfer their melanin to neighboring keratinocytes. The melanin serves to protect the keratinocyte DNA from the harmful effects of ultraviolet radiation. The exact mechanism whereby melanin is transferred from melanocytes to keratinocytes is unknown and it remains unclear if melanin or melanosomes are actually transferred. This application proposes to use a two-pronged approach to address this problem by (i) examining factors known to be involved in this process as well as by (ii) taking an unbiased approach by setting up an RNA interference-based screen. Both approaches rely on the observation that failure of the B16 melanocyte cell line to release melanin results in the accumulation of the pigment within the cells and such "darkened" cells can be selectively isolated from a mixed population of cells by flow cytometry. The screen will involve expression of random interfering RNAs, sorting of the melanin-accumulating cells by flow cytometry and then using a bioinformatics approach to identify the protein that was knocked-down. It is anticipated that this approach will identify components involved in many different phases of melanosome biology as any defect that prevents their movement or release will lead to darkened cells. Proteins identified by the screen or by the more directed approach will be validated by a variety of biochemical and cell biological methods. In addition, this screen can be adapted to the study of other secretory pathway processes making it widely applicable to the study of many aspects of protein secretion.