Analysis of Aquaporin-Mediated Diffusional Water Permeability by Coherent Anti-Stokes Raman Scattering Microscopy
Analysis of Aquaporin-Mediated Diffusional Water Permeability by Coherent Anti-Stokes Raman Scattering Microscopy
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DOI：
10.1016/j.bpj.2011.08.045
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发表时间：
2011-11-02
影响因子：
3.4
通讯作者：
Yasui, Masato
中科院分区：
文献类型：
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作者：
Ibata, Keiji;Takimoto, Shinichi;Yasui, Masato
水可以通过两种途径通过生物膜：通过脂质双层的简单扩散和通过水通道蛋白(AQPs)的水选择性促进扩散。虽然水通道蛋白在渗透水渗透率(P(F))中起着重要的作用，但由于扩散水渗透系数(P(D))的测量困难，水通道蛋白在扩散水通道中的作用尚不清楚。在这里，我们报道了一种使用相干反斯托克斯拉曼散射(CARS)显微镜和快速灌流装置进行H(2)O/D(2)O交换的精确和瞬时测量单个HeLaS3细胞的P(D)的方法。每隔0.488毫秒获取一次超高速线条扫描汽车图像。外液H(2)O/D(2)O交换的平均CARS强度衰减时间常数(tau(CARS))为16.1ms，细胞内H(2)O/D(2)O交换的平均衰减时间常数为100.7+/-19.6ms。在HeLa S3细胞中瞬时表达AQP4和增强型绿色荧光蛋白(AQP4-EGFP)，以探讨AQP在水扩散通透性中的作用。AQP4-EGFP-HeLa S3细胞内H(2)O/D(2)O交换的平均tau(CARS)为43.1+/-15.8ms。我们还计算了细胞体积和细胞表面积，以计算P(D)。AQP4-EGFP-HeLa S3细胞和对照细胞的平均P(D)值分别为2.7+/-1.0×10(-3)和8.3+/-2.6×10(-4)cm/S。AQP4介导的水扩散与温度无关，但依赖于质膜上蛋白质的表达水平。这些结果表明，利用CARS成像研究单个哺乳动物细胞的流体动力学以及水通道蛋白的调节是可能的。
Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P(f)), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P(d)). Here, we report an accurate and instantaneous method for measuring the P(d) of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H(2)O/D(2)O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (tau(CARs)) for the external solution H(2)O/D(2)O exchange was 16.1 ms, whereas the intracellular H(2)O/D(2)O exchange was 100.7 +/- 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average tau(CARS) for the intracellular H(2)O/D(2)O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 +/- 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P(d). The average P(d) values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 +/- 1.0 x 10(-3) and 8.3 +/- 2.6 x 10(-4) cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.