Acting as the point of contact for the outside world, the plasma membrane is crucial for cellular signaling events. Proper organization of membrane components is necessary to accomplish this task. Although a number of experiments have demonstrated the compartmentalization of lipids and proteins on the plasma membrane, direct observation of the mechanisms by which the organization occurs has been challenging, in part due to the imaging restrictions of a diffraction-limited system and the dynamic nature of the membrane compartmentalization. Using photoactivated localization microscopy (PALM), a superresolution technique, we have captured the dynamics of compartments formed by the cortical actin cytoskeleton. Live human embryonic kidney (HEK293) cells were imaged with a temporal resolution of 2 s and a spatial resolution of 40 nm. The actin cytoskeleton forms compartments with a mean area of 2.3±0.3 μm2 that are partially outlined by actin bundles. When the PALM images of actin were combined with single particle tracking of membrane proteins, we directly observed the cytoskeleton acting as a barrier to the diffusion of Kv2.1 and Kv1.4, two voltage-gated potassium channels. In addition, we used a novel compartment detection and tracking algorithm to show that Kv2.1 and Kv1.4 channels avoid actin when changing compartments. This work represents the first direct observations of individual membrane protein interactions with barriers formed by the actin cytoskeleton.
