Figure 1.
Microdomains of PI 4,5-P2 on the plasma membrane. (A–F) PC12 cell membrane sheets were incubated with 2 μM PLCδ1–PH-GFP fusion protein and 100 μM FM4-64 to image PI 4,5-P2 (A and B) or membrane area (C and D), respectively. (E and F) Membrane sheets were incubated with PLCδ1–PH-GFP fusion protein after incubations with or without 2 mM MgATP, respectively. (G–I) Quantification of membrane PI 4,5-P2. (G) Supported planar bilayers containing indicated mole percentage of PI 4,5-P2 and 0.1 mol% rhodamine-PE were incubated with 2 μM PLCδ1–PH-GFP fusion protein and imaged in red (top) and green (bottom) channels. (H) Fluorescence intensity per pixel was plotted for each supported bilayer to generate a calibration curve. (I) From calibration curves prepared in parallel, pixel intensity on PC12 cell membrane sheets (incubated with or without MgATP) was used to infer PI 4,5-P2 concentrations in microdomains, between microdomains, and averaged across the membrane. Mean ± SEM values are shown for +MgATP (n = 13) and −MgATP (n = 5). (J and K) PI 4,5-P2 microdomains overlap with clusters of CAPS and docked vesicles. (J) PI 4,5-P2 microdomains in PC12 cell membrane sheets were localized by incubation with PLCδ1–PH-GFP and fixed for immunolocalization with CAPS polyclonal or chromogranin B monoclonal antibodies. (K) Triple channel colocalizations were quantified as described in Materials and methods. Mean ± SEM (n = 9) values are shown.