Studies on the mechanism of melanosome secretion

膜运输描述了脂质和蛋白质在细胞内区室之间的流动。这一过程对于这些细胞器的独特性质的建立和维持至关重要，并且由小泡介导。此外，在细胞中合成的蛋白质可以通过分泌途径挤出，其中囊泡与质膜融合。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。这种色素在称为黑素细胞的专门细胞中合成，并储存在称为黑素体的独特细胞器中。这些细胞器被运输到细胞周边，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，也不清楚黑色素或黑素体是否真的转移。本申请提出使用双管齐下的方法来解决这个问题，通过（i）检查已知参与这个过程的因素以及通过（ii）通过建立基于RNA干扰的筛选来采取无偏的方法。这两种方法都依赖于观察到B16黑素细胞系不能释放黑色素导致色素在细胞内积累，并且可以通过流式细胞术从混合的细胞群中选择性地分离这种“变暗”的细胞。筛选将涉及随机干扰RNA的表达，通过流式细胞术分选黑色素积累细胞，然后使用生物信息学方法来鉴定被敲低的蛋白质。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，因为任何阻止它们移动或释放的缺陷都会导致黑素体细胞变暗。通过筛选或更直接的方法鉴定的蛋白质将通过多种生物化学和细胞生物学方法进行验证。此外，该筛选可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.