title: Live-cell imaging of aquaporin-4 diffusion and interactions in orthogonal arrays of particles.
abstract: Orthogonal arrays of particles (OAPs) have been visualized for many years by freeze-fracture electron microscopy. Our laboratory discovered that aquaporin-4 (AQP4) is the protein responsible for OAP formation by demonstrating OAPs in AQP4-transfected cells and absence of OAPs in AQP4 knockout mice. We recently developed live-cell, single-molecule imaging methods to study AQP4 diffusion and interactions in OAPs. The methods include single particle tracking of quantum-dot labeled AQP4, and total internal reflection fluorescence microscopy of green fluorescent protein (GFP) and small fluorophore-labeled AQP4. The full-length (M1) form of AQP4 diffuses freely in membranes and does not form OAPs, whereas the shorter (M23) form of AQP4 forms OAPs and is nearly immobile. Analysis of a series of AQP4 truncations, point mutants and chimeras revealed that OAP formation by AQP4-M23 is stabilized by hydrophobic tetramer-tetramer interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from blocking of this interaction by residues just upstream from Met23. These biophysical methods are being extended to identify the cellular site of AQP4 assembly, AQP4 isoform interactions, OAP size and dynamics, and the determinants of regulated OAP assembly.
authors.affiliation: Departments of Medicine and Physiology, University of California, San Francisco, CA 94143-0521, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Crane
authors.forename: M
authors.initials: M
authors.lastname: Tajima
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D051401:Aquaporin 4 / Q000235:genetics / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D006652:Histological Techniques / Q000379:methods; D051379:Mice; D018345:Mice, Knockout; D008853:Microscopy / Q000379:methods; D010539:Permeability
chemical_list: C496031:Aqp4 protein, mouse; D051401:Aquaporin 4
references.citation: Brain. 2008 Apr;131(Pt 4):1087-98
references.pmid: 18267965
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11981-6
references.pmid: 9751776
references.citation: FASEB J. 2008 Mar;22(3):870-9
references.pmid: 17965267
references.citation: Pflugers Arch. 2008 Jul;456(4):693-700
references.pmid: 17968585
references.citation: Proc Natl Acad Sci U S A. 2009 May 5;106(18):7437-42
references.pmid: 19383790
references.citation: Annu Rev Biophys Biomol Struct. 1999;28:319-65
references.pmid: 10410805
references.citation: Nat Med. 2000 Feb;6(2):159-63
references.pmid: 10655103
references.citation: Biophys J. 1997 Aug;73(2):1073-80
references.pmid: 9251823
references.citation: FASEB J. 2008 Sep;22(9):3216-23
references.pmid: 18511552
references.citation: Biochemistry. 1997 Jun 17;36(24):7625-32
references.pmid: 9200715
references.citation: J Cell Sci. 2009 Mar 15;122(Pt 6):813-21
references.pmid: 19240114
references.citation: J Clin Invest. 1997 Sep 1;100(5):957-62
references.pmid: 9276712
references.citation: Nat Methods. 2005 Nov;2(11):825-7
references.pmid: 16278651
references.citation: Am J Physiol Renal Physiol. 2000 Feb;278(2):F310-6
references.pmid: 10662735
references.citation: J Neurosci. 2004 Sep 15;24(37):8049-56
references.pmid: 15371505
references.citation: J Biol Chem. 2007 Jul 27;282(30):21866-72
references.pmid: 17525153
references.citation: Biophys J. 1993 Nov;65(5):2021-40
references.pmid: 8298032
references.citation: Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):14108-13
references.pmid: 11717465
references.citation: J Hirnforsch. 1995;36(2):239-58
references.pmid: 7615928
references.citation: Biochemistry. 2008 Feb 26;47(8):2631-7
references.pmid: 18247481
references.citation: Nature. 2005 Apr 7;434(7034):786-92
references.pmid: 15815633
references.citation: J Biol Chem. 1995 Sep 29;270(39):22907-13
references.pmid: 7559426
references.citation: J Cell Sci. 1995 Sep;108 ( Pt 9):2993-3002
references.pmid: 8537439
references.citation: Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):13052-6
references.pmid: 7528931
references.citation: J Neurochem. 2005 Oct;95(1):254-62
references.pmid: 16181429
references.citation: J Biol Chem. 2008 Aug 29;283(35):23510-3
references.pmid: 18617526
references.citation: Epilepsia. 1984 Apr;25(2):145-51
references.pmid: 6705745
references.citation: Biochim Biophys Acta. 2008 Apr;1778(4):1181-9
references.pmid: 18179769
references.citation: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):2106-11
references.pmid: 12578959
references.citation: J Biol Chem. 2005 Apr 8;280(14):13906-12
references.pmid: 15695511
references.citation: J Cell Sci. 2005 Dec 15;118(Pt 24):5691-8
references.pmid: 16303850
references.citation: J Biol Chem. 1994 Feb 25;269(8):5497-500
references.pmid: 7509789
references.citation: Am J Physiol Renal Physiol. 2004 Sep;287(3):F501-11
references.pmid: 15149973
references.citation: Genomics. 2008 Apr;91(4):367-77
references.pmid: 18255256
references.citation: J Mol Biol. 2006 Jan 27;355(4):628-39
references.pmid: 16325200
references.citation: J Mol Med (Berl). 2008 May;86(5):523-9
references.pmid: 18311471
references.citation: Neuron. 2006 Jul 6;51(1):71-84
references.pmid: 16815333
references.citation: J Biol Chem. 2001 Aug 17;276(33):31233-7
references.pmid: 11406631
references.citation: FASEB J. 2007 Jan;21(1):108-16
references.pmid: 17135365
references.citation: J Biol Chem. 1996 Mar 1;271(9):4577-80
references.pmid: 8617713
references.citation: Biochemistry. 1999 Aug 24;38(34):11156-63
references.pmid: 10460172
references.citation: J Mol Biol. 2009 Jun 19;389(4):694-706
references.pmid: 19406128
references.citation: J Cereb Blood Flow Metab. 2006 Dec;26(12):1527-37
references.pmid: 16552421
references.citation: Acta Neuropathol. 1981;54(3):189-97
references.pmid: 7257728
references.citation: Invest Ophthalmol Vis Sci. 2002 Feb;43(2):573-9
references.pmid: 11818406
references.citation: Biophys J. 2008 Jan 15;94(2):702-13
references.pmid: 17890385
references.citation: Glia. 2006 Apr 15;53(6):631-6
references.pmid: 16470808
references.citation: Neuroscience. 2009 Jul 7;161(3):764-72
references.pmid: 19345723
references.citation: J Neurosci. 1997 Jan 1;17(1):171-80
references.pmid: 8987746
references.citation: Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10908-12
references.pmid: 8855281
references.citation: FASEB J. 2004 Aug;18(11):1291-3
references.pmid: 15208268
references.citation: Mol Biol Cell. 2008 Aug;19(8):3369-78
references.pmid: 18495865
references.citation: J Cell Sci. 1997 Nov;110 ( Pt 22):2855-60
references.pmid: 9427293
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13609-14
references.pmid: 14597700