Studies on the mechanism of melanosome secretion

膜交通描述脂质和蛋白质在细胞内隔室之间的流动。这一过程对这些细胞器的独特性质的建立和维持至关重要，并由小泡介导。此外，细胞内合成的蛋白质可以通过囊泡与质膜融合的分泌途径挤出。这些分泌的蛋白质可以被邻近的细胞吸收，就像黑色素一样。这种色素在称为黑素细胞的特殊细胞中合成，并储存在称为黑素小体的独特细胞器中。这些细胞器被运输到细胞周围，并通过尚未确定的机制将其黑色素转移到邻近的角质形成细胞。黑色素可以保护角质细胞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化细胞的确切机制尚不清楚，也不清楚黑色素或黑素体是否真的被转移。本应用程序建议使用双管齐下的方法来解决这个问题，通过(i)检查已知参与该过程的因素，以及（ii）通过建立基于RNA干扰的筛选采取公正的方法。这两种方法都依赖于观察到B16黑素细胞系释放黑色素的失败会导致细胞内色素的积累，这种“变暗”的细胞可以通过流式细胞术从混合细胞群中选择性地分离出来。筛选将包括随机干扰rna的表达，通过流式细胞术对黑色素积聚细胞进行分类，然后使用生物信息学方法识别被敲除的蛋白质。预计这种方法将识别参与黑素体生物学许多不同阶段的成分，因为任何阻止它们运动或释放的缺陷都会导致细胞变暗。通过筛选或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，该筛选还可适用于其他分泌途径过程的研究，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.