Membrane protein targeting and regulation by exocytosis-

NIAAA分子生理学实验室细胞生物光学组成立于2003年4月。这一部分的主要目的是利用成像技术研究蛋白质复合体，特别是由完整的膜蛋白组成的复合体，是如何在活细胞中形成和维持的。膜蛋白，如受体、通道和它们的调节伙伴，通常由与内质网相关的核糖体-S合成，但最终在细胞表面发挥作用。因此，它们的生产、组装、运输和胞吐插入细胞表面的物流是理解与人类疾病相关的正常生理功能和异常生理状态的关键。此外，将膜蛋白复合体输送到细胞表面微区必须涉及胞吐和内吞反应的协调。胞吐的损害与某些形式的肌营养不良有关，也可能与酒精引起的肌病有关。斯里纳盖什·库希克博士和克里斯托弗·泰勒博士分别于2003年5月作为研究员和博士后IRTA加入该部门。 在过去的六个月里，我们主要致力于组装和测试一种能够进行多光子激光扫描成像、光谱成像和时间分辨荧光成像的新型显微镜。在这三种成像模式中，前两种目前正在运行，我们预计时间分辨荧光将在未来两个月内运行。在此期间，我们还在该部门开展了以下研究项目： 1.细胞外段绿色荧光蛋白突变体标记的整合膜蛋白构建体的产生。这些构建物将被用作细胞表面微域的标记，细胞外猝灭剂对其固有荧光的猝灭将被用于监测它们向细胞表面输送的动力学。 2.使用纯化的GFP、CFP、YFP和CFP-YFP结构，对Zeiss 510在多光子和共焦模式下用于CFP-YFP荧光能量转移(FRET)应用的元光谱解混算法进行了评估。 3.确定酪氨酸磷酸酶在海胆发育早期调节组成性胞吐和代偿性内吞的解偶联中的作用。 此外，我们还参与了与该科的主要目标有关的两项合作。与池田S博士合作，我们参与了基因工程试剂的开发和评估，以监测活细胞中G蛋白亚单位的定位和激活。与玛格丽特·戴维斯博士(戴夫·洛文格？S博士)一起，我们一直致力于开发活细胞中可光激活绿色荧光蛋白结构的双光子去功能化方法。该方法将用于研究神经细胞表面酪氨酸激酶的激活动力学和离子通道的调节。

The Section on Cellular Biophotonics, of the Laboratory of Molecular Physiology, NIAAA, was established in April 2003. The principle aim of this section is to use imaging techniques to study how protein complexes, with special emphasis on complexes comprised of integral membrane proteins, are formed and maintained in living cells. Membrane proteins, such as receptors, channels, and their regulatory partners, are typically synthesized by ribosome?s associated with the endoplasmic reticulum, but ultimately function at the cell surface. Thus, the logistics of their production, assembly, transport, and exocytotic insertion into the cell surface is key to understanding both normal physiological function, and abnormal physiological states associated with human maladies. Furthermore, the delivery of membrane protein complexes to cell surface micro-domains must involve the coordination of exocytotic and endocytotic reactions. Lesions in exocytosis have been tied to some forms of muscular dystrophy, and may also be involved in alcohol induced myopathy. Drs. Srinagesh Koushik , and Christopher Thaler, joined the section in May 2003 as a Research Fellow, and as a postdoctoral IRTA respectively. Over the past six months we have primarily been involved in assembling and testing a new microscope capable of multi-photon laser scanning imaging, spectral imaging, and time-resolved fluorescence imaging. Of these three imaging modes, the first two are currently operational, and we expect the time-resolved fluorescence to be operational within the next two months. During this period, we have also initiated the following research projects in the section: 1. The generation of integral membrane protein constructs, labeled with green fluorescent protein variants on extracellular segments. These constructs will be used as markers for cell surface micro-domains, and the quenching of their intrinsic fluorescence by extracellular quenchers, will be used to monitor the dynamics of their delivery to the cell surface. 2. The evaluation of Zeiss 510 META spectral un-mixing algorithms in multi-photon and confocal modes for CFP-YFP fluorescence energy transfer (FRET) applications using purified GFP, CFP, YFP, and a CFP-YFP construct. 3. Determining the role of tyrosine phosphatase in regulating the uncoupling of constitutive exocytosis and its compensatory endocytosis during early sea urchin development. Additionally, we have also been involved in two collaborations that are associated with the primary aims of the section. In collaboration with Dr. Stephen Ikeda?s section, we have been involved in developing and evaluating genetically engineered reagents to monitor G-protein subunit localization and activation in living cells. With Dr. Margaret Davis (in Dr. Dave Lovinger?s Laboratory) we have been involved in developing methods for two-photon uncaging of photactivatible GFP constructs in living cells. This method will be used to study the dynamics of tyrosine-kinase activation and regulation of ion channels on the neuronal cell surface.