JCS02348 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. GFP-Kv2.1 exhibits limited mobility after photobleach. Images from a FRAP series in a GFP-Kv2.1HA expressing HEK cell.  Panel A illustrates the single cluster selected for bleach.  ROI 2 (red circle) was bleached (panel B) using the 405-nm line of an Olympus FV1000 confocal in tornado scan mode and recovery monitored every 10 s for 250 scans.  To quantitate recovery, a freehand ROI was drawn around the putative membrane compartment and fluorescence intensity over this area plotted.  Recovery within the ROI occurs on a timescale inconsistent with diffusion.  The yellow arrow points to a GFP-Kv2.1 cluster that appears to approach (panel C) and then insert into the membrane (panel D).

• Supplemental Figure 2 -

  Fig. S2. Analysis of GFP-Kv1.4 recovery following photobleach. Panels A-D show prebleach, bleach, and recovery at the indicated times, respectively. ROI 2 (red circle) represents the photobleach region while the gray ROI 3 is the membrane compartment displayed in the XY-Intensity plots within each panel. Unlike Kv2.1, recovery of YFP fluorescence occurs via lateral diffusion from membrane adjacent to the bleach region. 

• Supplemental Figure 3 -

  Fig. S3. Photoactivation occurs through the entire thickness of the cell.  (A) Photoactivated GFP in a fixed HEK cell.  Soluble mRFP was coexpressed to highlight the cell volume (red) and a 30-pixel radius ROI photoactivated by the 405-nm laser for 1-s at 10% attenuation through a 60X, 1.4 NA oil immersion objective that was focused on a middle plane of the cell.  The images along the side are X-Z and Y-Z images of the cell volume, illustrating that at these settings, photoactivation occurred though the entire volume of the cell.  The ROI size, placement and laser settings used were chosen because they give optimal activation of PA-GFP tagged channels. (B) To determine the three dimensional volume of the photoactivation region, the 3-D package included in the FV1000 software was used to generate a reconstruction of the entire cell post-activation.  As expected for a collimated laser with a Gaussian profile, the resulting excitation point spread function results in an ellipsoidal activation volume whose widest radius is at the focal plane.