Size of cell-surface Kv2.1 domains is governed by growth fluctuations

Kv2.1 channel counting In order to obtain the density of Kv2.1 channels inside clusters, we measured both the cluster area and the number of channels. The intensity of an individual GFP was measured from the bleaching steps of single fluorophores (1). Individual channels located away from clusters were manually identified and they were then tracked with a custom-written center-of-mass algorithm. A 10x10 region of interest (ROI) was centered around the selected channels and the image was thresholded to reduce bias due to background fluorescence (2). The XY centroid of the thresholded image was obtained and the ROI was moved to the new location to account for channel motion. The center of mass in the next frame was then found and the algorithm was looped until the particle was lost. Particles are lost by the tracking algorithm either because they are photobleached or because they collide with a Kv2.1 cluster or with another individual channel. The tracking algorithm was implemented in LabView. The photobleaching of an individual fluorophore occurs in a discrete manner and a step can be observed for each bleaching molecule. Because Kv2.1 is tetrameric, each channel is labeled with four GFP fluorophores and several steps are seen before the channel trajectory is lost. In order t