Marlar, Saw5; Christensen, Eva Arnspang6; Pedersen, Gitte Albinus6; Koffman, Jennifer Skaarup5; Nejsum, Lene Niemann6
1 Department of Molecular Biology and Genetics - Molecular Cell and Developmental Biology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University2 Interdisciplinary Nanoscience Center - INANO-MBG, Gustav Wied 10, Interdisciplinary Nanoscience Center, Science and Technology, Aarhus University3 iNano-School, Science and Technology, Aarhus University4 Department of Clinical Medicine, Health, Aarhus University5 Department of Molecular Biology and Genetics - Molecular Cell and Developmental Biology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University6 Department of Clinical Medicine, Health, Aarhus University
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 localize lateral and basal whereas AQP4 localize 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 ± 0.010 μm2/sec on E-cadherin, and 0.019 ± 0.004 μm2/sec on collagen, whereas EGFP-AQP4 was measured to 0.044 ± 0.009 μm2/sec on E-cadherin and 0.037 ± 0.009 μm2/sec 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 ± 0.007 μm2/sec (water) to 0.014 ± 0.003 μm2/sec (MBCD) (p < 0.05) on collagen surfaces, and by 41% from 0.023 ± 0.011 μm2/sec (water) to 0.014 ± 0.005 μm2/sec (MBCD) (p < 0.05) on E-cadherin surfaces. Thus, protein patterning enables semiquantitation of protein distribution between the “lateral” and “basal” membranes as well as measurements of lateral diffusion coefficients.
B B a - Biomembranes, 2014, Vol 1838, Issue 10, p. 2404-2411