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。费尔南多·科维安-纳雷斯博士于2004年5月加入该部门，担任博士后IRTA。 在过去的一年里，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。这三种成像模式都可以运行。 我们目前正在进行几个利用这种新成像系统能力的研究项目。过去一年的研究亮点包括：1.我们的研究完成了使用光谱分解来量化活细胞中CFP和YFP浓度的有效性。我们发现，只要参考光谱被基线校正，并且给体(CFP)和受体(YFP)分子之间不发生荧光共振能量转移(FRET)，光谱混合就可以得到定量结果。2.接下来，我们开发并测试了一种新的谱分解算法，该算法在存在和不存在FRET的情况下都可以工作。这种新的方法可以用来测量CFP和YFP标记的分子在细胞内的丰度，而不需要考虑标记蛋白质之间的分子相互作用，它还可以用来测量活细胞中的FRET效率，这是分子相互作用的指标。3.我们已经证明，海胆卵至少有两种不同的代偿内吞作用机制。接下来，我们演示了这些细胞机制检索细胞表面膜的特定和独特的斑块。其中一种机制专门回收由钙离子插入的膜，引发胞吐作用，而第二种机制回收由结构性胞吐插入的膜补片。 此外，我们还参与了与该科的主要目标有关的两项合作。与Stephen Ikedas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. 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.