Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging

We investigated the molecular determinants of aquaporin-4 (AQP4) assembly in orthogonal arrays of particles (OAPs) by visualizing fluorescently labeled AQP4 mutants in cell membranes using quantum-dot single-particle tracking and total internal reflection fluorescence microscopy. The full-length `lo...

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Published inJournal of cell science Vol. 122; no. 6; pp. 813 - 821
Main Authors Crane, Jonathan M, Verkman, Alan S
Format Journal Article
LanguageEnglish
Published England The Company of Biologists Limited 15.03.2009
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Summary:We investigated the molecular determinants of aquaporin-4 (AQP4) assembly in orthogonal arrays of particles (OAPs) by visualizing fluorescently labeled AQP4 mutants in cell membranes using quantum-dot single-particle tracking and total internal reflection fluorescence microscopy. The full-length `long' (M1) form of AQP4 diffused freely in membranes and did not form OAPs, whereas the `short' (M23) form of AQP4 formed OAPs and was nearly immobile. Analysis of AQP4 deletion mutants revealed progressive disruption of OAPs by the addition of three to seven residues at the AQP4-M23 N-terminus, with polyalanines as effective as native AQP4 fragments. OAPs disappeared upon downstream deletions of AQP4-M23, which, from analysis of point mutants, involves N-terminus interactions of residues Val24, Ala25 and Phe26. OAP formation was also prevented by introducing proline residues at sites just downstream from the hydrophobic N-terminus of AQP4-M23. AQP1, an AQP4 homolog that does not form OAPs, was induced to form OAPs upon replacement of its N-terminal domain with that of AQP4-M23. Our results indicate that OAP formation by AQP4-M23 is stabilized by hydrophobic intermolecular interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from non-selective blocking of this interaction by seven residues just upstream from Met23.
Bibliography:Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/6/813/DC1
Author for correspondence (e-mail: Alan.Verkman@ucsf.edu)
This work was supported by NIH grants DK35124, EB00415, HL73856, HL59198, EY13574 and DK72517, and Research Development Program and Drug Discovery grants from the Cystic Fibrosis Foundation. J.M.C. was supported in part by NIH NRSA award GM808512. Deposited in PMC for release after 12 months.
ISSN:0021-9533
1477-9137
DOI:10.1242/jcs.042341