Membrane protein targeting and regulation by exocytosis-

NIAAA分子生理学实验室细胞生物光子学组成立于2003年4月。本节的主要目的是使用成像技术来研究蛋白质复合物，特别是由完整的膜蛋白组成的复合物，是如何在活细胞中形成和维持的。膜蛋白，如受体，通道，和他们的监管合作伙伴，通常是由核糖体合成？与内质网相关，但最终在细胞表面起作用。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正常生理功能和与人类疾病相关的异常生理状态的关键。此外，将膜蛋白复合物递送至细胞表面微区必须涉及胞吐和胞吞反应的协调。胞吐病变与某些形式的肌营养不良症有关，也可能与酒精诱导的肌病有关。Srinagesh Koushik博士和Christopher Thaler博士于2003年5月加入该部门，分别担任研究员和博士后IRTA。Fernando Covian-Nares博士于2004年5月作为博士后IRTA加入该部门。 在过去的一年里，我们主要参与组装和测试一种新的显微镜，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。所有这三种成像模式都可以运行。 我们目前正在进行几个研究项目，利用这种新的成像系统的能力。过去一年的研究重点包括：1。完成了我们的研究，研究了使用光谱解混来定量活细胞中CFP和YFP浓度的有效性。我们发现，只要参考光谱是基线校正的，并且供体（CFP）和受体（YFP）分子之间不发生荧光共振能量转移（FRET），光谱解混就可以产生定量结果。2.接下来，我们开发并测试了一种新的光谱去混合算法，该算法在存在和不存在FRET的情况下都有效。这种新方法可用于测量CFP和YFP标记分子的细胞内丰度，而不管标记蛋白质之间的分子相互作用如何，它也可用于测量活细胞中的FRET效率，这是分子相互作用的指标。3.我们已经表明，海胆卵至少有两种不同的机制的补偿性内吞作用。接下来，我们证明了这些细胞机制检索细胞表面膜的特定和独特的补丁。这些机制之一，特别是检索膜插入钙触发胞吐作用，而第二种机制检索膜补丁插入组成性胞吐作用。 此外，我们还参与了与该部分主要目标相关的两项合作。与Stephen Ikedas博士合作，我们一直参与开发和评估基因工程试剂，以监测活细胞中G蛋白亚基的定位和激活。玛格丽特·戴维斯博士（Dr. Dave Lovinger）我们已经参与了开发活细胞中可光活化的GFP构建体的双光子释放方法。该方法将用于研究酪氨酸激酶激活的动力学和神经细胞表面离子通道的调节。

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. Dr. Fernando Covian-Nares joined the section in May 2004 as a postdoctoral IRTA. Over the past year 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. All three of these imaging modes are operational. We are currently pursuing several research projects that utilized the capabilities of this new imaging system. Research highlights over this past year include: 1. The completion of our study that investigated the validity of using spectral un-mixing for quantifying CFP and YFP concentrations in living cells. We found that spectral un-mixing can yield quantitative results as long as reference spectra are baseline corrected, and fluorescence resonance energy transfer (FRET) is not occurring between donor (CFP) and acceptor (YFP) molecules. 2. We next developed and tested a new spectral un-mixing algorithm that works in the presence and absence of FRET. This new method can be used to measure the intracellular abundance of CFP and YFP tagged molecules regardless of molecular interactions between the tagged proteins, and it can also be used to measure FRET efficiencies in living cells, an indicator of molecular interactions. 3. We have shown that sea urchin eggs have at least two different mechanisms of compensatory endocytosis. We next demonstrated that these cellular mechanisms retrieve specific and unique patches of the cell surface membrane. One of these mechanisms specifically retrieved membranes inserted by calcium triggered exocytosis, while the second mechanism retrieved membrane patches inserted by constitutive exocytosis. Additionally, we have also been involved in two collaborations that are associated with the primary aims of the section. In collaboration with Dr. Stephen Ikedas 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.