Live-cell imaging of aquaporin-4 supramolecular assembly and diffusion

Abstract

Aquaporin-4 (AQP4) is a water channel expressed in astrocytes throughout the central nervous system, as well as in epithelial cells in various peripheral organs. AQP4 is involved in brain water balance, neuroexcitation, astrocyte migration, and neuroinflammation and is the target of pathogenic autoantibodies in neuromyelitis optica. Two AQP4 isoforms produced by alternative splicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular aggregates called orthogonal arrays of particles (OAPs). OAPs have been studied morphologically, by freeze-fracture electron microscopy, and biochemically, by native gel electrophoresis. We have applied single-molecule and high-resolution fluorescence microscopy methods to visualize AQP4 and OAPs in live cells. Quantum dot single particle tracking of fluorescently labeled AQP4 has quantified AQP4 diffusion in membranes, and has elucidated the molecular determinants and regulation of OAP formation. The composition, structure, and kinetics of OAPs containing fluorescent protein-AQP4 chimeras have been studied utilizing total internal reflection fluorescence microscopy, single-molecule photobleaching, and super-resolution imaging methods. The biophysical data afforded by live-cell imaging of AQP4 and OAPs has provided new insights in the roles of AQP4 in organ physiology and neurological disease.

Figure 17.1

Approaches to visualize AQP4 and OAPs. (A) Freeze-fracture electron micrographs of the plasma membrane P-face of COS-7 cells expressing the M1 and M23 isoforms of AQP4. (B) AQP4 immunoblot following Blue-native gel electrophoresis of cell lysates from AQP4-expressing COS-7 cells. (C) Total internal reflection fluorescence micrographs of GFP-AQP4 chimeras. (D) (left) Schematic showing the organization of AQP4 tetramers (left) and examples of single particle trajectories of Qdot-labeled AQP4 molecules in the plasma membrane of AQP4-expressing COS-7 cells. Each cylinder represents one AQP4 tetramer in which a subset of AQP4 molecules are labeled with quantum dots (red) for single particle tracking. (Center) Combined mean squared displacement (MSD) versus time plots and averaged diffusion coefficients for AQP4-M1 (gray) and AQP4-M23 (black) in COS-7 cells. (Right) Cumulative probability distribution of range at 1 s (P(range)) deduced from SPT measurements, with dashed lines indicating median range. Adapted from Crane et al. (2008) and Tajima et al. (2010).

Figure 17.2

Quantum dot single particle tracking reveals determinants of AQP4 OAP assembly. (A) AQP4 sequence and topology showing site of GFP or epitope (myc) insertion in the second extracellular loop. Black: Met-1 and Met-23 translation initiation sites; blue: residues where mutations did not affect OAP assembly; red: mutations disrupt OAPs; pink: mutations mildly disrupt OAPs; yellow: mutations reduce plasma membrane expression; green: C-terminal PDZ-binding domains. (B) P(range) for indicated AQP4 truncation mutants. (C.) OAP modulation by coexpression of M1-AQP4 and M23-AQP4. (Left) P(range) for cells transfected with M23 only (black) or M1 only (gray), or cotransfected with M23 (red) and M1 (green) at M23-to-M1 ratios of 1:1. (Right) P(range) comparing M1/M23 cotransfection (solid) versus “separate” (dashed), computed by summing P(range) curves for separate transfections. (D) TIRFM of Alexa-labeled AQP4 in cells expressing M23-F26Q or M23-G28P and fixed at 4 or 37 °C. Adapted from Crane and Verkman (2009a, 2009b) and Crane et al. (2009).

Figure 17.3

OAP dynamics and structure revealed by TIRFM of GFP-AQP4 chimeras. (A) TIRFM image (left) showing distinct fluorescent spots in cells expressing M23-AQP4, corresponding to OAPs, with deduced single OAP trajectories over 3 h shown at the right. (Bottom) High magnification of boxed region showing spontaneous OAP disruption events; trajectories of original OAP (black) and daughter OAPs (red, green, yellow, blue) shown at the right. (B) U87MG cells were transfected with GFP-M23 and GFP-M1 AQP4 at indicated ratios. Representative TIRF micrographs show fluorescent spots (top). Deduced number histograms of single-spot fluorescence (background-subtracted, area-integrated intensities), proportional to OAP size, shown at the bottom. Unity represents the intensity of monomeric GFP. (C) U87MG cells were transfected with GFP-M23 and (untagged) M1 AQP4 at a ratio of 20:1. TIRFM of two large AQP4 aggregates (left), showing relative concentration of fluorescence at the periphery. Line profiles (dashed white lines at the left) shown at the right. Adapted from Tajima et al. (2010) and Jin et al. (2011).

Figure 17.4

Photobleaching and super-resolution imaging of AQP4 OAPs. (A) Single molecule step-photobleaching and intensity analysis shows AQP4 heterotetrameric association. (Top) Serial TIRFM images of recombinant monomeric GFP-labeled AQP4-M1 showing multistep loss of fluorescence. (Bottom, left) Single-spot (background-subtracted) integrated fluorescence intensity histograms for GFP-labeled AQP4-M1 without or with excess unlabeled AQP4-M1. (Right) Single-spot intensities as a function of time during continuous illumination, showing single versus multistep photobleaching. (B) Diffusion of GFP-labeled M1 and M23 AQP4 at the plasma membrane in live cells. Arrowheads indicate bleached area. Adapted from Tajima et al. (2010). (C) Super-resolution image of AQP4 OAPs. PALM image of AQP4-M23 chimera containing PA-GFP at its C-terminus. Inset: TIRFM (non-super-resolution) of area in white box.