Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation  

Abstract

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-b-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.

Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization

“…Evidence suggests that the behavior of individual molecules is of particular importance because it can be correlated to the environmental conditions (12). It has been reported that AMT1;3 is widely distributed in the epidermal cells of Arabidopsis roots and makes an important contribution to the absolute ammonium uptake capacity (2,3).…”

“…Single-particle fluorescence imaging was performed on an inverted microscope (IX71; Olympus) equipped with a total internal reflective-fluorescence illuminator and a 100× oil-immersion objective (Olympus; NA 1.45) as described (12). The gain of our EM CCD camera was set at 300 throughout all singleparticle imaging experiments, and the setting was in the linear dynamic range of the EM CCD camera.…”

A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development

“…This technique is particularly suitable for the investigation of endocytosis and exocytosis, owing to its capacity to provide high signal-to-noise imaging of fluorescently labeled puncta located near the cover slip (Simons and Gerl, 2010). When the incident angle of the laser was adjusted, this technique can be successfully extended to the quantitative analysis of membrane-associated events within intact plant cells Konopka et al, 2008;Bednarek, 2008a, 2008b;Li et al, 2011;Wan et al, 2011). In our study, we used VA-TIRFM to clearly document the budding into the cell of GFP-Flot1 punctuate structures.…”

Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development

“…Co-immunoprecipitation and fluorescence cross-correlation spectroscopy (FCCS) analyses showed that AP2 σ interacts with clathrin light chain (CLC) in vivo. To investigate the molecular dynamics at high spatiotemporal resolution on the plasma membrane, we made used of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) combined with a single-particle tracking (SPT) assay (Li et al, 2011;Wan et al, 2011) to reveal that AP2 σ displays similar dynamic behavior to CLC and accumulates at the endocytic clathrin-coated pits (CCPs). These findings provide new insight into the machinery and function of clathrin-mediated endocytosis in plant cells.…”