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First published online 24 February 2009
doi: 10.1242/jcs.042341

Journal of Cell Science 122, 813-821 (2009)
Published by The Company of Biologists 2009
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Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging

Jonathan M. Crane and Alan S. Verkman*

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA


Figure 1
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  		Fig. 1. 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 Qdot-labeled AQP4 molecules in the plasma membranes 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) AQP4 sequence and topology. Black indicates Met1 and Met23 translation initiation sites; orange, engineered Myc site; 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; and green, C-terminal PDZ-binding domains. Horizontal lines indicate sites of C-terminal truncations. (C) Combined MSD vs time plots and averaged diffusion coefficients for AQP4-M1 (grey) and AQP4-M23 (black) in COS-7 cells. (D) Cumulative probability distribution of ranges at 1 second [P(range)] for AQP4-M1 (grey) and AQP4-M23 (black), with dashed lines indicating median range. (E) TIRF micrographs of Alexa Flour 555-labeled AQP4-M1 and AQP4-M23 in COS-7 cells. Inset shows expanded 4x4 µm area. Scale bar: 10 µm.

 

Figure 2
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  		Fig. 2. N-terminal deletion mutants of AQP4 show altered ability to form OAPs. (A) P(range) for indicated AQP4 truncation mutants upstream of Met23: M16 (red), M17 (green), M18 (dark blue), M19 (light blue), M20 (orange), M21 (purple). (B) P(range) for AQP4 truncations downstream from Met23: M24 (red), M25 (green), M26 (blue), M27 (orange). (C) P(range) for alanine mutant M23-R108A (blue) and C-terminal deletion mutants: M23{Delta}6 (red), M23{Delta}71 (green), M1{Delta}6 (purple), M1{Delta}71 (orange). P(range) for AQP4-M23 (black) and AQP4-M1 (grey) are shown in A-C for reference. Dashed line indicates 95th percentile of the range of AQP4-M23. (D) Estimated percentage of indicated AQP4 mutants in OAPs based on the 95th percentile of range for AQP4-M23. (E) TIRF micrographs of COS-7 cells transfected with AQP4-M1, AQP4-M23, and N-terminal deletion mutants M16, M17 and M20. Each image is a 5x5 µm square. Scale bar: 2 µm. (F) BN-PAGE (top) and SDS-PAGE (bottom) immunoblots of cell lysates from COS-7 cells transfected with AQP4-M23, AQP4-M1 and N-terminal deletion mutants M16, M17 and M20. Molecular size markers are indicated to the right.

 

Figure 3
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  		Fig. 3. Disruption of OAPs by residues upstream of Met23 is not sequence specific. (A) P(range) for alanine mutations in AQP4-M1: M1-C13A/C17A (red), M1-C17A (green), M1-S18A (dark blue), M1-R19A (light blue), M1-E20A (orange), AQP4-M23 (black) and AQP4-M1 (grey). (B) Estimated percentage of AQP4-M1 alanine mutants in OAPs. (C) N-terminal sequences of selected AQP4-M23 mutants containing polypeptide additions upstream of Met23. (D) P(range) resulting from polypeptide additions upstream of Met23: MA2M23 (red), MA4M23 (green), MA6M23 (dark blue), MQ6M23 (purple), MVAFKGVM23 (orange), M16 (light blue), AQP4-M23 (black) and AQP4-M1 (grey). (E) Estimated percentage of indicated polypeptide addition mutants of AQP4-M23 in OAPs.

 

Figure 4
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  		Fig. 4. N-terminus residues just downstream of Met23 are responsible for AQP4-M23 OAP formation. (A) Kyte-Doolittle hydropathy plot of the first 25 residues of AQP4-M23 using a 5-residue running sum. The dashed line indicates the start of the first transmembrane helix according to the published crystal structure (Hiroaki et al., 2006Go). * indicates aromatic residues. (B) P(range) for alanine mutations in AQP4-M23: M23-V24A (red), M23-F26A (green), M23-V24A/F26A (orange), M23-K27A (dark blue), M23-G28A (pink), M23-V29A (light blue), M23-W30A (purple), M23-T31A (yellow), M23-Q32A (dark green). (C) P(range) for glutamine point mutations of hydrophobic and aromatic residues: M23-V24Q (red), M23-A25Q (green), M23-F26Q (dark blue), M23-V24Q/A25Q/F26Q (orange), M23-W30Q (purple) and M23-A33Q (light blue). (D) Estimated percentage of indicated AQP4-M23 point mutants in OAPs. (E) P(range) following mutations of AQP4-M23 at Ala25: M23-A25L (red), M23-A25S (blue), M23-A25Q (green). (F) P(range) following mutations of AQP4-M23 at Phe26: M23-F26L (red), M23-F26A (green), M23-F26Y (purple), M23-F26Q (dark blue), M23-F26E (light blue), M23-F26K (orange). (G) P(range) following proline mutations in AQP4-M23: M23-K27P (green), M23-K27A (blue), M23-G28P (red), M23-G28A (pink), with estimated percentage of proline mutants in OAPs given below. P(range) for AQP4-M23 (black) and AQP4-M1 (grey) are shown in B, C and E-G for reference.

 

Figure 5
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  		Fig. 5. AQP1 forms OAPs after replacement of its N-terminal domain with that of AQP4-M23. (A) Sequence alignment of the first 20 residues of AQP4-M23 and the first 18 residues of AQP1. Red and yellow highlight identical and conserved residues, respectively. Blue and yellow rectangles represent the beginning of transmembrane domains of AQP4 and AQP1, respectively, according to published structures. (B) Crystal structure of AQP1 (de Groot et al., 2001Go) (yellow) shown with the addition of nine N-terminal residues from AQP4-M23 (blue), forming AQP1ch1. Below is a schematic of various AQP4-AQP1 chimeras, showing the relative contributions from AQP4 (blue) and AQP1 (yellow), with numbers indicating the initial and final residues taken from each protein, and chimera names listed to the right. (C) P(range) of AQP4-AQP1 chimeras: AQP1ch1 (red), AQP1ch2 (green), AQP1ch3 (dark blue), AQP1ch4 (light blue), ch1-F26Q (orange), AQP4-M23 (black) and AQP1 (grey). (D) Estimated percentage of indicated AQPs and chimeras in OAPs.

 

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