title: Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling.
abstract: We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1-6 capped actin filaments efficiently, whereas FliI GLD 2-6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1-6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2-6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2-6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1-6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts.
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada.
authors: Pamma D
authors: PD
authors: Arora
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada.
authors: Yongqiang
authors: Y
authors: Wang
authors: Department of Biochemistry, Albert Einstein College of Medicine, New York, NY 10461.
authors: Anne
authors: A
authors: Bresnick
authors: Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104.
authors: Paul A
authors: PA
authors: Janmey
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada christopher.mcculloch@utoronto.ca.
authors: Christopher A
authors: CA
authors: McCulloch
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D022081:Cell Surface Extensions / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins; D018995:Myosin Heavy Chains; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism*; D011485:Protein Binding; D015534:Trans-Activators
chemical_list: D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C000609263:FlII protein, mouse; D008840:Microfilament Proteins; C525088:Myh9 protein, mouse; D015534:Trans-Activators; D003094:Collagen; D024507:Nonmuscle Myosin Type IIA; D018995:Myosin Heavy Chains