Measuring localization and diffusion coefficients of basolateral proteins in lateral versus basal membranes using functionalized substrates and kICS analysis

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 ± 0.010 μm ²/s on E-cadherin and 0.019 ± 0.004 μm ²/s on collagen, whereas EGFP-AQP4 was measured to 0.044 ± 0.009 μm ²/s on E-cadherin and 0.037 ± 0.009 μm 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 ± 0.007 μm ²/s (water) to 0.014 ± 0.003 μm ²/s (MBCD) (p < 0.05) on collagen surfaces, and by 41% from 0.023 ± 0.011 μm ²/s (water) to 0.014 ± 0.005 μm 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.