Tracking of quantum dot-labeled CFTR shows near immobilization by C-terminal PDZ interactions.
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.
Haggie
Peter M
PM
Department of Medicine and Physiology, University of California, San Francisco, San Francisco, CA 94143-0521, USA.
Kim
Jung Kyung
JK
Lukacs
Gergely L
GL
Verkman
A S
AS
D000199:Actins / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D004058:Diffusion; D015870:Gene Expression; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D045663:Quantum Dots; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013997:Time Factors
D000199:Actins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator