THE DYNAMIC CELLULAR LOCALIZATION OF TRPM1

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 恶性黑色素瘤是最致命的皮肤癌，其发病率以每年3%的速度持续增加。 Melastatin（TRPM 1）是一种在小鼠和人类高转移性肿瘤中下调的基因，表明TRPM 1表达可能是黑色素瘤侵袭性的指标。这一发现提出了一种有趣的可能性，即TrpM 1可能是一种肿瘤抑制因子，这是一种前所未有的离子通道功能。 尽管是离子通道TrpM家族的创始成员，但对TrpM 1的分子特性和细胞功能知之甚少。 这项研究的目的是了解TrpM 1的功能以及TrpM 1与黑色素瘤之间的联系。确定TrpM 1参与正常和恶性细胞的分子机制可能会导致黑色素瘤诊断和治疗的重大进展。 为了了解TrpM 1在细胞功能中的作用，表征其亚细胞定位和细胞动力学是至关重要的。为了在原代人黑素细胞和黑素瘤细胞中表达荧光标记的TRPM 1，我们将使用慢病毒系统。 然后，我们将采用共聚焦成像，全内反射荧光（TIRF）和电子显微镜（EM），以确定细胞内与质膜分布的TrpM 1，含有TRPM 1和它们的身份的细胞内结构的动态特性。 拟议的实验的结果将代表一个重要的一步了解TrpM 1在黑素细胞中的功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Malignant melanoma is the deadliest form of skin cancer that continues to increase in incidence at a rate of 3% per year. Melastatin (TRPM1) was discovered as a gene down-regulated in highly metastatic tumors in mice and humans, suggesting that TRPM1 expression may be an indicator of melanoma aggressiveness. This finding raised the interesting possibility that TrpM1 might be a tumor suppressor, an unprecedented function for an ion channel. Despite being the founding member of the TrpM family of ion channels, very little is known about the molecular properties and cellular functions of TrpM1. The goal of the proposed research is understanding the function of TrpM1 and the connection between TrpM1 and melanoma. Determining the molecular mechanisms by which TrpM1 is involved in normal and malignant cells could lead to significant progress in the diagnostic and treatment of melanoma. To understand the role of TrpM1 in cellular function, it is critical to characterize its sub-cellular localization and cellular dynamic. To express fluorescently tagged TRPM1 in primary human melanocytes and melanoma cells we will use a lenti-viral system. We will then employ confocal imaging, total internal reflection fluorescence (TIRF) and electron microscopy (EM) to determine the intracellular versus plasma membrane distribution of TrpM1, the dynamic properties of the intracellular structures containing TRPM1 and their identity. The results of the proposed experiments will represent a significant step towards understanding the function of TrpM1 in melanocytes.