Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.
Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.
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DOI：
10.1007/978-1-4939-2330-4_14
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发表时间：
2015
影响因子：
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通讯作者：
Adam W. Smith
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文献类型：
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作者：
Adam W. Smith
视紫红质在细胞膜中自我缔合。在低浓度下，相互作用与单体-二聚体平衡一致（Comar等人，J Am Chem Soc 136（23）：8342-8349，2014）。在天然组织中的高浓度下，已经观察到更高阶的簇（Fotiadis等人，Nature 421：127-128，2003）。视紫红质二聚化的生理作用仍在研究中，但很明显，定量评估对于确定视紫红质簇在视觉中的功能至关重要。为了量化视紫红质的相互作用，我将概述一个专门的时间分辨荧光光谱测量膜蛋白质-蛋白质相互作用称为脉冲交错激发荧光互相关光谱（PIE-FCCS）的理论和方法。该技术的优势在于其能够原位定量视紫红质相互作用（即，活细胞质膜）。有两个原因限制了活细胞膜的范围。首先，质膜的组成异质性产生了具有数千种脂质、蛋白质和碳水化合物种类的复杂环境。这使得难以从洗涤剂溶解的样品中推断四级相互作用或构建概括天然膜中存在的所有相互作用的模型磷脂双层。其次，组织结构和动力学是质膜的关键特征，并且固定技术如甲醛交联和玻璃化将调节相互作用JIE-FCCS基于具有时间相关单光子计数（TCSPC）的双色荧光成像（Becker et al.，Rev Sci Instrumum 70：1835-1841，1999）。通过对每个检测到的光子进行时间标记，可以将数据分析为荧光强度分布、荧光寿命直方图或荧光（交叉）相关光谱（FCS/FCCS）（Becker，Advanced time-correlated single-photon counting techniques，Springer，柏林，2005）。然后，这些分析工具可用于量化蛋白质浓度、迁移率、聚类和Förster共振能量转移（FRET）。在本文中，我将集中在PIE-FCCS，它交错两个波长的激发事件的时间，使光谱串扰和FRET的影响可以被隔离。以这种方式，可以高精度地表征单体-二聚体-低聚物平衡（Müller等人，Biophys J 89：3508-3522，2005）。目前，PIE-FCCS需要定制的设备配置，这将在下面描述。有一个极好的方案概述了在市售仪器上的传统FCCS（巴西亚和Schwille，Nat Protoc 2：2842-2856，2007）。PIE-FCCS方法是FCCS中相对较新的进展，其已用于活细胞测定中以量化脂质锚定蛋白质聚类（Triffo等人，J Am Chem Soc 134：10833-10842，2012）、表皮生长因子受体二聚化（Endres等人，Cell 152：543-556，2013），以及最近的视蛋白二聚化（Comar et al. J Am Chem Soc 136（23）：8342-8349，2014）。本文将概述PIE-FCCS的理论和仪器要求，以及数据收集和分析过程。
Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342–8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127–128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions.PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835–1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508–3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842–2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833–10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543–556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342–8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.