Membrane Lateral Diffusion and Capture of CFTR within Transient Confinement Zones

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) channel interacts with scaffolding and other proteins that are expected to restrict its lateral movement, yet previous studies have reported predominantly free diffusion. We examined the lateral mobility of CFTR channels on live baby hamster kidney cells using three complementary methods. Channels bearing an extracellular biotinylation target sequence were labeled with streptavidin conjugated with fluorescent dyes (Alexa Fluor 488 or 568) or quantum dots (qDot605). Fluorescence recovery after photobleaching and image correlation spectroscopy of the dye-labeled channels revealed a significant immobile population ( approximately 50%), which was confirmed by direct single particle tracking (SPT) of qDot605-labeled CFTR. Adding 10 histidine residues at the C-terminus of CFTR to mask the postsynaptic density 95, Discs large, ZO-1 (PDZ) binding motif abolished its association with EBP50/NHERF1, reduced the immobile fraction, and increased mobility. Other interactions that are not normally detected on this timescale became apparent when binding of PDZ domain proteins was disrupted. SPT revealed that CFTR(His-10) channels diffuse randomly, become immobilized for periods lasting up to 1 min, and in some instances are recaptured at the same location. The impact of transient confinement on the measured diffusion using the three fluorescence techniques were assessed using computer simulations of the biological experiments. Finally, the impact of endosomal CFTR on mobility measurements was assessed by fluorescence correlation spectroscopy. These results reveal unexpected features of CFTR dynamics which may influence its ion channel activity.

Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling

“…The central finding is that expression of defective CFTR, i.e., mutated DF508CFTR or decreased CFTR expression, increases sphingolipid synthesis. Increased sphingolipid synthesis results in significantly increased mass of sphingomyelin, sphingosine, and sphinganine, sphingolipids that affect membrane properties and function as parent compounds of signaling lipids (3,4,13). The data suggest that the effects of CFTR on sphingolipid metabolism are specific and independent of expression in human lung epithelial model cell lines.…”

“…Possibly, expression of defective CFTR, previously reported to coincide with characteristics of a destabilized membrane, triggers the synthesis of a class of lipids that promote membrane stabilization (2). If membrane stability were indeed affected by expression of defective CFTR, then an increase in sphingolipid and sphingomyelin synthesis and mass would be a compensatory mechanism directed to increase membrane stability or embedment of CFTR into the sphingolipid and cholesterol-rich membrane regions it has been associated with (3,4). According to this model, increased sphingolipid synthesis would reflect a state in which significantly decreased CFTR expression, as in 16HBE14o (2) antisense cells, would reflect a state that makes it necessary to maximally increase membrane stability.…”

“…CFTR is expressed in the apical membrane of epithelial cells and is connected through filamin with the actin cytoskeleton (2). Several studies suggested that CFTR localizes within cholesterol-and sphingomyelin-rich membrane regions (3,4). It is well accepted that reduced airway surface liquid volume is the initiating event in CF airways disease pathogenesis; however, how defective CFTR causes the complex phenotype associated with CF that includes an increased inflammatory state, dyslipidemia and osteoporosis, has not been fully understood (5).…”

Imaging CFTR in its native environment

“…In a first approach, we combined immunofluorescence using quantum dot (Qdot)-labeled antibodies with AFM. Qdots are crystalline fluorophores with an extreme photostability [4,63,37,36,106], appearing as bright spots and as distinct fluorescence events on the membranes (Fig. 3a and b).…”

Fine Tuning of CFTR Traffic and Function by PDZ Scaffolding Proteins

“…CFTR binding to this station is PDZ-dependent and results in increased stability at the plasma membrane. Indeed, a CFTR mutant truncated for its C-terminal PDZ interacting motif is highly mobile at the plasma membrane, whereas intact CFTR exhibits a greater immobile fraction or a more confined diffusion (Bates et al, 2006;Haggie et al, 2004;. Moreover, C-terminal truncation of CFTR alters its endocytic/recycling dynamics.…”