Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.
Highlights
Recycling is a dynamic trafficking event during which proteins are transported from intracellular compartments and inserted into the plasma membrane
The major new observation in this study is that myosin Vb regulates CFTR-mediated ClϪ secretion across human airway epithelial cells by facilitating the apical membrane recycling of WT-CFTR and ⌬F508-CFTR
Subsequent studies confirmed the role of myosin Vb in plasma membrane recycling in Madin-Darby canine kidney and HeLa cells (66) and demonstrated that, together with Rab11a, myosin Vb facilitates the recycling of the M4 muscarinic receptor in neuronotypic PC12 cells (23) and of the CXCR2 chemokine receptor in leukemia 2H3 cells (24) and the canalicular formation of bile in hepatic WIF-B9 cells (26)
Summary
Recycling is a dynamic trafficking event during which proteins are transported from intracellular compartments and inserted into the plasma membrane. A recent study demonstrated that the recycling of CFTR in human airway epithelial cells occurs in the Rab11a recycling compartment (21). Studies using dominant-negative fragments of myosin Vb have demonstrated that, together with Rab11a, myosin Vb facilitates protein recycling in several polarized and nonpolarized cell models (23–26). Nothing is known about the role of myosin Vb in CFTR trafficking in human airway epithelial cells. The objective of this study was to test the hypothesis that endogenous myosin Vb facilitates CFTR recycling in Rab11a-specific apical recycling compartments in polarized human airway epithelial cells. We report that myosin Vb is required for the recycling of wild-type (WT) CFTR and ⌬F508CFTR to the apical membrane of polarized human airway epithelial cells in a Rab11a-dependent fashion
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