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Vol. 17, Issue 12, 4937-4945, December 2006

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Tracking of Quantum Dot-labeled CFTR Shows Near Immobilization by C-Terminal PDZ Interactions

Peter M. Haggie^*, Jung Kyung Kim^*, Gergely L. Lukacs, and A. S. Verkman^*

*Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, CA 94143-0521; and Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada

Submitted August 3, 2006; Accepted September 6, 2006
Monitoring Editor: Vivek Malhotra

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100–200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-08-0670) on September 20, 2006.

Address correspondence to: Alan S. Verkman (alan.verkman{at}ucsf.edu)

Abbreviations used: CFTR, cystic fibrosis transmembrane conductance regulator; EBP50, ezrin-radixin-moesin (ERM) binding phosphoprotein of 50 kDa; PDZ, postsynaptic density protein 95/discs large/zonula occludens-1 (PSD95/Dlg/ZO-1); Qdots, quantum dots; SPT, single particle tracking.

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