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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.007351

Journal of Cell Science 120, 2413-2423 (2007)
Published by The Company of Biologists 2007
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A cytoskeletal-based perimeter fence selectively corrals a sub-population of cell surface Kv2.1 channels

Michael M. Tamkun1,2,*, Kristen M. S. O'Connell1 and Annah S. Rolig2

1 Department of Biomedical Sciences, Colorado State University, Ft. Collins, CO 80523, USA
2 Department of Biochemistry and Molecular Biology, Colorado State University, Ft. Collins, CO 80523, USA


Figure 1
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  		Fig. 1. GFP-Kv2.1 can show both a clustered and non-clustered distribution in HEK cells. HEK cells transfected with GFP-Kv2.1-loopBAD were imaged to detect GFP fluorescence of the GFP-Kv2.1-loopBAD expression outside the characteristic surface clusters.

 

Figure 2
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  		Fig. 2. Single-particle tracking of wild-type GFP-Kv2.1-loopBAD channels. HEK cells expressing GFP-Kv2.1-loopBAD were enzymatically biotinylated and then tagged at low efficiency with streptavidin-labeled 605 quantum dots as described in the Materials and Methods. The two most common classes of single Qdot tracks observed are indicated. (A) Single-particle track of a channel trapped within a cluster perimeter along with the corresponding MSD analysis of channel diffusion. In this case, a diffusion coefficient of 0.04 µm2/second was calculated from the initial slope. Overall, MSD analysis of Kv2.1 channel movement within clusters yielded a mean diffusion coefficient of 0.03±0.02 µm2/second, n=21. (B) Single-particle track of a channel outside the cluster whose movement was indifferent to the cluster perimeter. Analysis of the corresponding MSD plot for this track yielded a diffusion coefficient of 0.06 µm2/second. Analysis of multiple tracks derived from non-clustered channels yielded a mean diffusion coefficient of 0.06±0.05 µm2/second, n=33. Cells were imaged every 0.8 seconds.

 

Figure 3
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  		Fig. 3. Analysis of GFP-Kv2.1-loopBAD {Delta}C mobility on the cell surface. HEK cells expressing a GFP-Kv2.1-loopBAD channel without the C-terminal 318 amino acids ({Delta}C) were enzymatically biotinylated and tagged with streptavidin-labeled 655 quantum dots as described in the Materials and Methods. (A) Non-clustered cell surface distribution of this mutant channel in three dimensions. (B) The Qdot single-particle track used to generate the MSD plot indicated in C. In this case a diffusion coefficient of 0.10 µm2/second was calculated from the initial slope. Overall the mean diffusion coefficient equalled 0.07±0.04 µm2/second, n=10, for these mutant channels incapable of forming surface clusters. Cells were imaged every 0.55 seconds.

 

Figure 4
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  		Fig. 4. FRAP analysis of Kv2.1 mobility outside the cell surface cluster. (A) Imaging of a HEK cell expressing GFP-Kv2.1 before and after photobleach within the indicated region of interest (yellow circle). Prebleach and postbleach images and the fluorescence recovery at 28.8 seconds postbleach are shown. The perimeters of the clusters observed in the prebleach image are indicated in red. Note that the fluorescence intensity within the cluster perimeter recovers to the prebleach background level over this time interval as illustrated in the lower right panel. (B) Time course of fluorescence recovery within the bleach ROI, illustrating a FRAP time constant of 9.0 seconds in this representative experiment. Using this value, we estimated that non-clustered Kv2.1 has a diffusion coefficient of 0.09 µm2/second. The mean diffusion coefficient calculated for non-clustered channels using this approach was 0.08±0.03 µm2/second, n=10. Cells were imaged every 1.1 seconds.

 

Figure 5
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  		Fig. 5. Detection of Kv1.4 within the Kv2.1 cell surface cluster. FRAP analysis of YFP-Kv1.4-myc mobility. HEK cells expressing both CFP-Kv2.1-HA and YFP-Kv1.4-myc were imaged for each fluorophore. (A) Localization of CFP-Kv2.1-HA. (B) Homogeneous distribution of YFP-Kv1.4-myc. Note that Kv1.4 is present at the same cell surface density on both sides of the Kv2.1 cluster perimeter (outlined in white). (C) Photobleach of YFP-Kv1.4-myc contained within the red ROI. This field of view is identical to that presented in A and B. (D) YFP fluorescence recovery at 146 seconds, illustrating that the mobile YFP-Kv1.4-myc channels have diffused into the CFP-Kv2.1-HA clusters outlined in white. (E) Time course of fluorescence recovery within the bleach ROI, illustrating a FRAP time constant of 42.4 seconds in this representative experiment. Using this value, we calculated that the mobile YFP-Kv1.4-myc channels in this cell have a diffusion coefficient of 0.2 µm2/second. The mean diffusion coefficient for YFP-Kv1.4-myc was 0.28±0.13 µm2/second, n=11. Cells were imaged every 1.6 seconds.

 

Figure 6
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  		Fig. 6. Non-clustered GFP-Kv2.1-loopBAD channels can become trapped after crossing the cluster perimeter. Illustrated is a Qdot655-tagged channel entering a cluster (yellow arrow) and being transiently retained within the cluster perimeter for 112 seconds before escaping. Cluster perimeter crossing events were relatively rare, being observed in only 9 out of more than 80 Qdot tracks analyzed. See supplementary material Movie 2. The Qdot was imaged every 0.8 seconds.

 

Figure 7
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  		Fig. 7. Phalloidin-stained actin filaments direct Kv2.1 surface cluster orientation. HEK cells expressing GFP-Kv2.1-loopBAD were formaldehyde fixed, permeabilized with Triton X-100 and stained with Alexa Fluor 594-labeled phalloidin. (A) GFP-Kv2.1-loopBAD expression pattern. (B) Phalloidin pattern. (C) The overlay of panels A and B.

 

Figure 8
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  		Fig. 8. Kv2.1 surface clusters correlate with an absence of cortical actin. HEK cells were co-transfected with mCherry-Kv2.1-loopBAD and GFP-actin and imaged without fixation. (A) The expression pattern of GFP-actin. (C) Localization of mCherry-Kv2.1-loopBAD overlaid with GFP-actin expression. (B,D) Enlargements of the boxed regions in A and C. The yellow arrow in D indicates that the Kv2.1 cluster perimeter did not always align with the border of the actin-free zone. Nonetheless, Kv2.1 clusters tend to form where actin is reduced.

 

Figure 9
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  		Fig. 9. Concentration-dependent effects of swinholide A on GFP-Kv2.1-HA cluster size. The effect of 75 or 200 nM swinholide A on Kv2.1 cluster size was determined as illustrated in panels A and B, or C and D, respectively. (A,C) Cluster distribution on the bottom of the cell before either 75 or 200 nM swinholide treatment, respectively. (B,D) The cluster pattern 40 minutes later. In this example, 75 nM swinholide reduced the cluster number from 284 to 123 while increasing the average size from 0.34 to 0.50 µm2. There was a 1.2-fold increase in the GFP signal associated with the clusters. With the 200 nm concentration, the cluster number decreased from 485 to 113, with the average cluster area decreasing from 0.58 to 0.27 µm2. The total GFP signal that was cluster associated dropped to 3.5% of its original value.

 

Figure 10
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  		Fig. 10. Proposed model for the Kv2.1-containing cluster perimeter fence showing the clustered Kv2.1 channel trapped in a well within the cortical cytoskeleton. The walls of this well form the functional fence, which retains channels that have sufficient depth owing to their assembly with accessory proteins, perhaps via phosphorylation, at the channel C-terminus. Thus, the channel within the cluster has the same lateral mobility as the non-clustered channel but remains corralled. Other membrane proteins, and non-modified Kv2.1 channels, do not have the depth to be trapped within the well. Cortical actin, illustrated in green, is proposed to play a role in separating and organizing the clusters as opposed to actually being the fence itself. Unknown cytoskeletal elements, illustrated in orange, are proposed to form the actual cortical well. Alternatively, the orange color might represent stable actin filaments that are poorly labeled by GFP-actin but severed by high swinholide A concentrations.

 




© The Company of Biologists Ltd 2007