Clathrin-Mediated Endocytosis Introduces a Nonergodic Diffusion Process in the Plasma Membrane

Abstract

Tracking individual potassium channels in the plasma membrane reveals complex dynamics involving anomalous diffusion. Theoretical models show that anomalous subdiffusion can be caused by several different processes. In particular, transient binding events, modeled by a continuous time random walk (CTRW), may not only induce anomalous subdiffusion but also weak ergodicity breaking, that is, the ensemble and time averages do not coincide. We studied the physical mechanism underlying Kv2.1 and Kv1.4 potassium channel anomalous dynamics by performing time series analysis of extensive single molecule tracking in the membrane of live mammalian cells. We find ample evidence showing that the ensemble and temporal distributions are different. Our data reveal that two anomalous subdiffusion processes simultaneously coexist and only one of them is ergodic. Weak ergodicity breaking is found to be maintained by immobilization events as long as 60 seconds. In the presence of either actin or chlathrin inhibitors, ergodicity is recovered. In order to elucidate the effects of clathrin endocytosis on Kv2.1 trafficking and diffusion, we have performed simultaneous total internal reflection fluorescence imaging of quantum-dot-tagged Kv2.1 and RFP-tagged clathrin. Our results show that Kv2.1 channels are frequently recycled from and to the plasma membrane. Retrieval of Kv2.1 from the membrane is found to be attained via a clathrin-mediated endocytic pathway. These endocytic/insertion processes are needed to maintain a nonergodic CTRW. Furthermore, the Kv2.1 stalling events colocalize with clathrin clusters. These results suggest that abortive endocytic events are responsible for the observed channel immobilization in the plasma membrane.