title: Measuring localization and diffusion coefficients of basolateral proteins in lateral versus basal membranes using functionalized substrates and kICS analysis.
abstract: Micropatterning enabled semiquantitation of basolateral proteins in lateral and basal membranes of the same cell. Lateral diffusion coefficients of basolateral aquaporin-3 (AQP3-EGFP) and EGFP-AQP4 were extracted from "lateral" and "basal" membranes using identical live-cell imaging and k-space Image Correlation Spectroscopy (kICS). To simultaneously image proteins in "lateral" and "basal" membranes, micropatterning with the extracellular domain of E-cadherin and collagen, to mimic cell-cell and cell-extracellular matrix (ECM) adhesion, respectively, was used. In kidney collecting duct principal cells AQP3 localizes lateral and basal whereas AQP4 localizes mainly basal. On alternating stripes of E-cadherin and collagen, AQP3-EGFP was predominantly localized to "lateral" compared to "basal" membranes, whereas Orange-AQP4 was evenly distributed. Average diffusion coefficients were extracted via kICS analysis of rapid time-lapse sequences of AQP3-EGFP and EGFP-AQP4 on uniform substrates of either E-cadherin or collagen. AQP3-EGFP was measured to 0.022±0.010μm(2)/s on E-cadherin and 0.019±0.004μm(2)/s on collagen, whereas EGFP-AQP4 was measured to 0.044±0.009μm(2)/s on E-cadherin and 0.037±0.009μm(2)/s on collagen, thus, diffusion did not differ between substrates. Cholesterol depletion by methyl-beta-cyclodextrin (MBCD) reduced the AQP3-EGFP diffusion coefficient by 43% from 0.024±0.007μm(2)/s (water) to 0.014±0.003μm(2)/s (MBCD) (p<0.05) on collagen surfaces, and by 41% from 0.023±0.011μm(2)/s (water) to 0.014±0.005μm(2)/s (MBCD) (p<0.05) on E-cadherin surfaces. Thus, protein patterning enables the semiquantitation of protein distribution between the "lateral" and "basal" membranes as well as measurements of lateral diffusion coefficients.
authors.affiliation: Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, C. F. Moellers Allé 3, Aarhus, Denmark.
authors.forename: Saw
authors.initials: S
authors.lastname: Marlar
authors.affiliation: Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, C. F. Moellers Allé 3, Aarhus, Denmark.
authors.forename: Eva C
authors.initials: EC
authors.lastname: Arnspang
authors.affiliation: Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, C. F. Moellers Allé 3, Aarhus, Denmark.
authors.forename: Gitte A
authors.initials: GA
authors.lastname: Pedersen
authors.affiliation: Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, C. F. Moellers Allé 3, Aarhus, Denmark.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Koffman
authors.affiliation: Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, C. F. Moellers Allé 3, Aarhus, Denmark. Electronic address: nejsum@mb.au.dk.
authors.forename: Lene N
authors.initials: LN
authors.lastname: Nejsum
mesh_terms: D000818:Animals; D051400:Aquaporin 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051401:Aquaporin 4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015820:Cadherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003094:Collagen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D051401:Aquaporin 4; D015820:Cadherins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051400:Aquaporin 3; D003094:Collagen
keywords: (4-6) Diffusion coefficient; Aquaporin; MDCK; Micropatterning; kICS: k-space Image Correlation Spectroscopy