Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking

Jonathan M. Crane, Alfred N. Van Hoek, William Skach, A. S. Verkman

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

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 × 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.

Original languageEnglish (US)
Pages (from-to)3369-3378
Number of pages10
JournalMolecular Biology of the Cell
Volume19
Issue number8
DOIs
StatePublished - Aug 2008

Fingerprint

Aquaporin 4
Quantum Dots
jasplakinolide
Electron Microscopy
Cell Membrane
PDZ Domains
Post-Synaptic Density
Aquaporins
Herpes Zoster
Fluorescence Microscopy
Astrocytes
Actins
Epitopes
Protein Isoforms

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking. / Crane, Jonathan M.; Van Hoek, Alfred N.; Skach, William; Verkman, A. S.

In: Molecular Biology of the Cell, Vol. 19, No. 8, 08.2008, p. 3369-3378.

Research output: Contribution to journalArticle

Crane, Jonathan M. ; Van Hoek, Alfred N. ; Skach, William ; Verkman, A. S. / Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking. In: Molecular Biology of the Cell. 2008 ; Vol. 19, No. 8. pp. 3369-3378.
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