Single-molecule analysis of PIP2;1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation.
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-β-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.
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
Xiaojuan
X
Li
Xiaohua
X
Wang
Yong
Y
Yang
Ruili
R
Li
Qihua
Q
He
Xiaohong
X
Fang
Doan-Trung
DT
Luu
Christophe
C
Maurel
Jinxing
J
Lin
D020346:Aquaporins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008954:Models, Biological; D010539:Permeability; D018517:Plant Roots / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride / Q000494:pharmacology; D013312:Stress, Physiological; D014867:Water / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism
D020346:Aquaporins; D029681:Arabidopsis Proteins; D002966:Clathrin; D011993:Recombinant Fusion Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C491680:plasma membrane intrinsic protein 2 Arabidopsis; D014867:Water; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride