Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation
Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation
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PIP2;1 动力学和分配的单分子分析揭示了拟南芥质膜水通道蛋白调节的多种模式
DOI：
10.1105/tpc.111.091454
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发表时间：
2011-10-01
期刊：
影响因子：
11.6
通讯作者：
Lin, Jinxing
中科院分区：
文献类型：
--
作者：
Li, Xiaojuan;Wang, Xiaohua;Lin, Jinxing
PIP2；1是一种完整的膜蛋白，可以促进水在质膜上的运输。为了在单分子水平上研究拟南芥PIP2；1的动态变化及其在PIP2；1调控中的作用，我们利用可变角消逝波显微镜和荧光相关光谱(FCS)跟踪了绿色荧光蛋白PIP2；1的分子。单粒子跟踪分析表明，PIP2；1存在四种扩散模式，扩散系数弥散较大，表明PIP2；1的分配和动力学是非均相的，更重要的是，PIP2；1可以进入或离开膜微区。盐胁迫下，PIP2；1的扩散系数和限制扩散百分率增大，表明PIP2；1内化作用增强。FCS对质膜上PIP2；1密度的降低进一步支持了这一点。此外，我们还证明了PIP2；1的内化涉及两种途径的结合：酪蛋白A23敏感的clathrin依赖途径和甲基-β-环糊精敏感的膜RAFT相关途径。后者在氯化钠条件下被有效地刺激。综上所述，我们的研究结果表明，PIP2；1分子在质膜上分布不均，并且网状蛋白和膜筏途径协同调节PIP2；1的亚细胞转运，提示动态分配和循环途径可能参与了多种调节水分通透性的方式。
PIP2;1 is an integral membrane protein that facilitates water transport across plasma membranes. To address the dynamics of Arabidopsis thaliana PIP2;1 at the single-molecule level as well as their role in PIP2;1 regulation, we tracked green fluorescent protein-PIP2;1 molecules by variable-angle evanescent wave microscopy and fluorescence correlation spectroscopy (FCS). Single-particle tracking analysis revealed that PIP2;1 presented four diffusion modes with large dispersion of diffusion coefficients, suggesting that partitioning and dynamics of PIP2;1 are heterogeneous and, more importantly, that PIP2;1 can move into or out of membrane microdomains. In response to salt stress, the diffusion coefficients and percentage of restricted diffusion increased, implying that PIP2;1 internalization was enhanced. This was further supported by the decrease in PIP2;1 density on plasma membranes by FCS. We additionally demonstrated that PIP2;1 internalization involves a combination of two pathways: a tyrphostin A23-sensitive clathrin-dependent pathway and a methyl-beta-cyclodextrin-sensitive, membrane raft-associated pathway. The latter was efficiently stimulated under NaCl conditions. Taken together, our findings demonstrate that PIP2;1 molecules are heterogeneously distributed on the plasma membrane and that clathrin and membrane raft pathways cooperate to mediate the subcellular trafficking of PIP2;1, suggesting that the dynamic partitioning and recycling pathways might be involved in the multiple modes of regulating water permeability.