Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution
Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution
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DOI：
10.1016/j.bpj.2009.07.059
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发表时间：
2009-11-04
影响因子：
3.4
通讯作者：
Gerritsen, Hans C.
中科院分区：
文献类型：
--
作者：
Bader, Arjen N.;Hofman, Erik G.;Gerritsen, Hans C.
基于荧光各向异性的同源 FRET 检测方法可用于研究细胞中相同蛋白质的聚类。在这里，研究了荧光各向异性显微镜在亚细胞分辨率的蛋白质簇定量成像方面的潜力。比较了稳态和时间分辨各向异性检测以及一光子和双光子激发方法。该方法在表达绿色荧光蛋白 (GFP) 构建体的细胞上进行评估，该构建体包含一种或两种 FK506 结合蛋白，这使得控制构建体的二聚化和寡聚化成为可能，并产生各向异性和簇大小之间的实验关系。结果表明，与实验方法无关，通常提出的在单个能量转移步骤后完全去极化的假设在这里是无效的。这是由于荧光蛋白的非随机相对方向。我们的实验表明，这种相对方向受到 GFP 桶之间相互作用的限制。我们描述了如何在其他 GFP 融合的定量簇大小成像实验中采用各向异性和簇大小之间的实验关系。对糖基磷脂酰异醇 (GPI) 锚定蛋白的实验表明，GPI 形成平均大小超过两个亚基的簇。对于表皮生长因子受体 (EGFR)，我们观察到大约 40% 的未刺激受体作为预先存在的受体存在于质膜中。这两个例子都揭示了簇大小和分布的亚细胞异质性。
Fluorescence-anisotropy-based homo-FRET detection methods can be employed to study clustering of identical proteins in cells. Here, the potential of fluorescence anisotropy microscopy for the quantitative imaging of protein clusters with subcellular resolution is investigated. Steady-state and time-resolved anisotropy detection and both one- and two-photon excitation methods are compared. The methods are evaluated on cells expressing green fluorescent protein (GFP) constructs that contain one or two FK506-binding proteins, This makes it possible to control dimerization and oligomerization of the constructs and yields the experimental relation between anisotropy and cluster size. The results show that, independent of the experimental method, the commonly made assumption of complete depolarization after a single energy transfer step is not valid here. This is due to a nonrandom relative orientation of the fluorescent proteins. Our experiments show that this relative orientation is restricted by interactions between the GFP barrels. We describe how the experimental relation between anisotropy and cluster size can be employed in quantitative cluster size imaging experiments of other GFP fusions. Experiments on glycosylphosphatidylinisotol (GPI)-anchored proteins reveal that GPI forms clusters with an average size of more than two subunits. For epidermal growth factor receptor (EGFR), we observe that similar to 40% of the unstimulated receptors are present in the plasma membrane as preexisting dinners. Both examples reveal subcellular heterogeneities in cluster size and distribution.