FIG. 1.

Large-scale PRMPs on the surface of living 3T3-L1 adipocytes. (A, C, and D) 3D image stacks of adipocytes transiently expressing EGFP/PLCδ1-PH were acquired with an ultrafast-acquisition microscope (see Materials and Methods), and representative deconvolved images were projected within 3D grids measuring 42 by 43 by 20 μm. These cells are composed of 81, 81, and 30 z sections, respectively, which are stacked successively along the optical z axis. Each z section represents a 0.25-μm slice of cells. The cells are oriented in such a way that the bottom membranes attached to the coverslip are closest to the plane containing the origin (O). All cells were serum starved for >2 h, and the cell in panel C was subsequently insulin simulated for 10 min at 37°C. To facilitate visualization of the PtdIns(4,5)P₂ spatial distribution, the dynamic ranges (i.e., minimum to maximum pixel intensity ranges) of the original 12-bit images are linearly mapped to 8-bit false-color intensity scales. The images are further contrast enhanced between their respective background noise levels and 10 times above those levels (e.g., the false-color intensity scale bar in panel A). Thus, the image in panel A was rescaled at between 10 and 100, and those in panels C and D were rescaled at between 20 and 200. The resulting color images (e.g., panel A) were the first approximations of their 8-bit gray-scale counterparts without contrast enhancement (e.g., inset in panel A). Only the bottom section (i.e., z1 to z30) of the cell in panel D is shown; yellow arrows indicate membrane ruffles, while the red arrow indicates apparently thickened membrane regions. Ripple-like patterns in the 3D images (most apparent in the red regions in panels A and C) are related to the axial-to-lateral magnification ratio used for the ultrafast-acquisition microscope and should be regarded as continuous regions (see Materials and Methods).(B) Full-scale (i.e., 0 to 255) 8-bit 2D intensity plot of the 45th z section of the cell in panel A (yellow outline in panel A). (See movie 1 at http://invitro.umassmed.edu/∼sh/supplements/supplements.html for a complete set of 2D intensity plots.)