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