JCS007351 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. DiI membrane labeling provides no evidence for plasma membrane ruffling adjacent to Kv2.1 surface clusters. HEK cells expressing only GFP-Kv2.1-loopBAD were incubated with DiI for 10 minutes to visualize the plasma membrane itself. (A) GFP-Kv2.1-loopBAD localization along with the DiI signal. (B) The DiI signal only. The perimeters of the Kv2.1-containing clusters are indicated by the white outline.

• Supplemental Figure S2 -

  Fig. S2. Effect of swinholide A treatment on HEK cell morphology. The effects of either 75 or 200 nM swinholide A is illustrated in panels A and B or C and D, respectively. (A and C) Images before swinholide treatment. (B and D) The same cells 40 minutes after Swinholide A addition. These are the same cells as those pictured in Fig. 9.

• Movie 1 -

  Movie 1. FRAP of YFP-Kv1.4-myc within Kv2.1-containing surface clusters. The ROI indicated in Fig. 5C was photobleached as described in Materials and methods and YFP fluorescence recovery then imaged every 1.6 seconds for approximately 2.5 minutes. Note that only 41% of the YFP fluorescence recovered over this time course.

• Movie 2 -

  Movie 2. Trapping of a non-clustered GFP-Kv2.1-loopBAD channel after crossing of the cluster perimeter. The quantum dot bound channel was tracked on the plasma membrane for 27 seconds before it entered an adjacent cluster where it was retained for 110 second before leaving. Cells were imaged every 0.8 seconds for both GFP and 655 quantum dot fluorescence using the DeltaVision RT wide-field microscope system. Note the mobile Qdots inside adjacent GFP-Kv2.1-loopBAD clusters.

• Movie 3 -

  Movie 3. Time course of swinholide-A-induced Kv2.1 declustering. Shown is a time series performed during swinholide A addition. Swinholide A was added to a final concentration of 200 nM at 2 minutes and 49 seconds and the cell then imaged every 5 seconds for almost 25 minutes. Note the increase in background membrane fluorescence as the clusters disappear. In this example, cluster number decreased from 154 to 35, average size of the remaining clusters decreased from 0.81 to 0.18 μm², and the amount of GFP fluorescence that was cluster-associated also decreased markedly.

• Movie 4 -

  Movie 4. Time course of Kv2.1 cluster dynamics under control conditions. Shown is a time-lapse series performed as a DMSO vehicle control for the 200 nM swinholide A treatment. DMSO was added to a final concentration of 0.4% at 1 minute and 29 seconds and the cell then imaged every 5 seconds for 41 minutes. In this example, cluster number changed from 223 to 172, average size increased from 0.8 to 1.4 μm², and the amount of GFP fluorescence that was cluster associated increased by 1.1-fold. These changes reflect the normal cluster dynamics observed over a 40-minute period. In such control cells, the mean changes in cluster number, size, and GFP fluorescence were 0.93±0.37, 1.1±0.33 and 1.05±0.33 fold, respectively. n=8.