title: Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology.
abstract: The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption.
authors: Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy; Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari, Italy.
authors: Maria
authors: M
authors: Lasalvia
authors: Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy.
authors: Stefano
authors: S
authors: Castellani
authors: Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy.
authors: Palma
authors: P
authors: D'Antonio
authors: Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy; Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari, Italy.
authors: Giuseppe
authors: G
authors: Perna
authors: Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy.
authors: Annalucia
authors: A
authors: Carbone
authors: Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy.
authors: Anna Laura
authors: AL
authors: Colia
authors: Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy.
authors: Angela Bruna
authors: AB
authors: Maffione
authors: Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy; Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari, Italy.
authors: Vito
authors: V
authors: Capozzi
authors: Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy. Electronic address: massimo.conese@unifg.it.
authors: Massimo
authors: M
authors: Conese
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D001826:Body Fluids / Q000378:metabolism; D001980:Bronchi / Q000473:pathology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D048430:Cell Shape; D003550:Cystic Fibrosis / Q000473:pathology*; D055119:Elastic Modulus; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*
keywords: Airway epithelial cells; Atomic force microscopy; Cystic fibrosis; Elastic module; Fluid absorption; Roughness