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Vol. 19, Issue 8, 3369-3378, August 2008

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Aquaporin-4 Dynamics in Orthogonal Arrays in Live Cells Visualized by Quantum Dot Single Particle Tracking

Jonathan M. Crane^*, Alfred N. Van Hoek, William R. Skach, and A. S. Verkman^*

*Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521; Center for Systems Biology, Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital, Boston, MA 02114; and Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239

Submitted March 28, 2008; Revised April 24, 2008; Accepted May 9, 2008
Monitoring Editor: Jennifer Lippincott-Schwartz

Freeze-fracture electron microscopy (FFEM) indicates that aquaporin-4 (AQP4) water channels can assemble in cell plasma membranes in orthogonal arrays of particles (OAPs). We investigated the determinants and dynamics of AQP4 assembly in OAPs by tracking single AQP4 molecules labeled with quantum dots at an engineered external epitope. In several transfected cell types, including primary astrocyte cultures, the long N-terminal "M1" form of AQP4 diffused freely, with diffusion coefficient 5 x 10^–10 cm²/s, covering 5 µm in 5 min. The short N-terminal "M23" form of AQP4, which by FFEM was found to form OAPs, was relatively immobile, moving only 0.4 µm in 5 min. Actin modulation by latrunculin or jasplakinolide did not affect AQP4-M23 diffusion, but deletion of its C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding domain increased its range by approximately twofold over minutes. Biophysical analysis of short-range AQP4-M23 diffusion within OAPs indicated a spring-like potential, with a restoring force of 6.5 pN/µm. These and additional experiments indicated that 1) AQP4-M1 and AQP4-M23 isoforms do not coassociate in OAPs; 2) OAPs can be imaged directly by total internal reflection fluorescence microscopy; and 3) OAPs are relatively fixed, noninterconvertible assemblies that do not require cytoskeletal or PDZ-mediated interactions for formation. Our measurements are the first to visualize OAPs in live cells.

Address correspondence to: Alan S. Verkman (alan.verkman{at}ucsf.edu)

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