CFTR Clustering and Tethering in Ceramide-Platforms in Response to Post-Infection PKC Stimulation

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Cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel located on the apical surface of epithelia, functions in fluid secretion and is essential for mucociliary clearance of bacteria from the lung. The loss of CFTR function leads to recurrent bacterial infections and inflammation, however the precise role of CFTR in host-pathogen interactions is not well understood. Here, we examine the effects of Pseudomonas aeruginosa(Pa) and the soluble bacterial effectors flagellin and LPS on the distribution and dynamics of CFTR on the plasma membrane of human bronchial epithelial primary cells. Both image correlation spectroscopy (ICS) and its derivative the k-space correlation spectroscopy (kICS) were used to quantitatively measure CFTR aggregation level and dynamical tethering in lipid rafts before and during Pa infection. Before infection, 30% of CFTR was found to partition into rafts and undergo slow transport dynamics (D=8x10−3m2/s). Acute exposure to Pa or its effectors stimulated the aggregation of lipid rafts into m-size platforms. This significantly increased the fraction of the confined CFTR (>50%), reduced its transport (D=2x10−3m2/s), and increased CFTR aggregation level (3-fold) due to its entrapment into these platforms. Intact lipid rafts, protein kinase C (PKC) activation and the increase in ceramide production by acid sphingomyelinase (aSMase) were essential for platforms formation, and for CFTR tethering and clustering post-infection. PKC activation alone (independent of infection) induced platform formation and increased CFTR stability and aggregation, revealing a central role for PKC in modulating the formation of ceramide-rich platforms from subresolution lipid rafts upon infection, an action mediated via aSMase activation. The formation of ceramide-rich platforms may be involved in bacterial internalization by host epithelial cells as suggested previously, or may stimulate CFTR function by bringing it into close proximity with receptors and their associated signaling molecules.