Live-cell imaging of aquaporin-4 diffusion and interactions in orthogonal arrays of particles

Abstract

Orthogonal arrays of particles (OAPs) have been visualized for many years by freeze-fracture electron microscopy. Our laboratory discovered that aquaporin-4 (AQP4) is the protein responsible for OAP formation by demonstrating OAPs in AQP4-transfected cells and absence of OAPs in AQP4 knockout mice. We recently developed live-cell, single-molecule imaging methods to study AQP4 diffusion and interactions in OAPs. The methods include single particle tracking of quantum-dot labeled AQP4, and total internal reflection fluorescence microscopy of green fluorescent protein (GFP) and small fluorophore-labeled AQP4. The full-length (M1) form of AQP4 diffuses freely in membranes and does not form OAPs, whereas the shorter (M23) form of AQP4 forms OAPs and is nearly immobile. Analysis of a series of AQP4 truncations, point mutants and chimeras revealed that OAP formation by AQP4-M23 is stabilized by hydrophobic tetramer-tetramer interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from blocking of this interaction by residues just upstream from Met23. These biophysical methods are being extended to identify the cellular site of AQP4 assembly, AQP4 isoform interactions, OAP size and dynamics, and the determinants of regulated OAP assembly.

Fig. 1

AQP4 domains and labeling, and static methods to measure OAP formation. (A) AQP4 sequence and topology showing site of Myc or GFP insertion in the second extracellular loop for fluorescence labeling. Black: Met1 and Met23 translation initiation sites; blue: residues where single mutations had no effect on OAP formation or disruption; red: residues where single mutations significantly disrupted OAPs; pink: residues where single mutations mildly disrupted OAPs; yellow: residues where mutation produced loss of plasma membrane expression; green: C-terminal PDZ-binding domains. Horizontal lines indicate tested C-terminal truncation sites. (B) Immunoblots following BN-PAGE of homogenized brain tissues (left) and lysates of COS-7 cells transfected with AQP4-M23 or AQP4-M1 (right). (C) Freeze-fracture electron micrographs of the plasma membrane P-face of COS-7 cells expressing Myc-tagged AQP4-M1 (left) or AQP4-M23 (right).

Fig. 2

Membrane assembly and diffusional mobility of M1 and M23 isoforms of AQP4. (A) Schematic showing the organization of AQP4 tetramers (left) and representative single particle trajectories (right) of quantum dot-labeled AQP4 molecules in the plasma membrane of COS-7 cells expressing AQP4-M1 (top) or AQP4-M23 (bottom). Each grey cylinder represents one AQP4 tetramer. A subset of AQP4 molecules are labeled with quantum dots (red) for single particle tracking. (B) Combined MSD vs. time plots and averaged diffusion coefficients for AQP4-M1 (grey) and AQP4-M23 (black) in COS-7 cells. (C) Cumulative probability distribution of ranges at 1 s (P(range)) for AQP4-M1 (grey) and AQP4-M23 (black), with dashed lines indicating median range.

Fig. 3

Comparison of long and short-range diffusion of full-length AQPs and C-terminal deletion mutants. (A) Representative trajectories superimposed on cellular profiles (white regions) of COS-7 cells expressing AQP1 (left), AQP4-M1 (middle) or AQP4-M23 (right) determined from time-lapse SPT at 1 Hz over 6 min. (B) Combined MSD vs. time plots for AQP1 (grey), AQP4-M1 (black), AQP4-M23 (red), and deletion mutant M23Δ6 (blue). (C) Expansion of Fig. 3B with additional MSD plots for AQP4-M23 after treatment with latrunculin B (green), jasplakinolide (orange), nocodazole (purple) or paraformaldehyde (grey). (D) Average diffusion coefficients of AQP4-M1 (top group), AQP4-M23 (middle group), and AQP1 (bottom group) following indicated treatments or truncations. (E) P(range) for AQP1 (grey), AQP4-M1 (black), AQP4-M23 (red), and deletion mutants M23Δ6 (dark blue) and M1Δ6 (light blue). Data obtained from transiently transfected COS-7 cells. MSD plots (B and C) are results from time-lapse acquisition at 1 Hz over 6 min, while diffusion coefficients and ranges (D and E) were calculated following short-range measurements at 91 Hz over 6 s.

Fig. 4

Mutations in the AQP4 N-terminal domain affect OAP formation. (A) Amino acid sequences leading up to the first transmembrane helix at Gln32 of indicated AQP4 mutants (left), and resulting fraction of each mutant found to reside in OAPs (right). Measurements done by SPT in live, transfected COS-7 cells. (B) P(range) for indicated AQP4 truncation mutants upstream of Met23: M16 (red), M17 (green), M18 (dark blue), M19 (light blue), M20 (orange), M21 (purple). (C) P(range) resulting from polyalanine additions upstream of Met23: MA₂M23 (red), MA₄M23 (green), MA₆M23 (purple), and for alanine point mutations in AQP4-M1: M1-C13A (dark blue), M1-C17A (light blue), M1-C13A/C17A (orange). (D) P(range) for point mutations of hydrophobic N-terminus residues in AQP4-M23: M23-V24A (red), M23-V24Q (orange), M23-A25S (green), M23-A25Q (purple), M23-F26A (dark blue), M23-F26Q (light blue). (E) P(range) for point mutations just downstream of the hydrophobic N-terminus of AQP4-M23: M23-K27A (red), M23-K27P (orange), M23-G28A (green), M23-G28P (blue). (B–E) P(range) for AQP4-M23 (black) and AQP4-M1 (grey) are shown for reference. Dashed line indicates 95th percentile of the range of AQP4-M23.

Fig. 5

Visualization of AQP4 OAPs by total internal reflection fluorescence microscopy. TIRF micrographs showing: (A) Alexa-labeled AQP4-M1.myc (left) and AQP4-M23.myc (right) in transfected primary mouse astrocytes. (B) Alexa-labeled AQP4-M1.myc (left) and AQP4-M23.myc (right) in COS-7 cells. Inset shows an expanded 5×5 μm area. (C) AQP4-M1.GFP (left) and AQP4-M23.GFP (right) in COS-7 cells.