title: Studying early stages of fibronectin fibrillogenesis in living cells by atomic force microscopy.
abstract: Fibronectin (FN) is an extracellular matrix protein that can be assembled by cells into large fibrillar networks, but the dynamics of FN remodeling and the transition through intermediate fibrillar stages are incompletely understood. Here we used a combination of fluorescence microscopy and time-lapse atomic force microscopy (AFM) to visualize initial stages of FN fibrillogenesis in living fibroblasts at high resolution. Initial FN nanofibrils form within <5 min of cell-matrix contact and subsequently extend at a rate of 0.25 μm/min at sites of cell membrane retraction. FN nanofibrils display a complex linear array of globular features spaced at varying distances, indicating the coexistence of different conformational states within the fibril. In some cases, initial fibrils extended in discrete increments of ∼ 800 nm during a series of cyclical membrane retractions, indicating a stepwise fibrillar extension mechanism. In presence of Mn(2+), a known activator of integrin adhesion to FN, fibrillogenesis was accelerated almost threefold to 0.68 μm/min and fibrillar dimensions were increased, underlining the importance of integrin activation for early FN fibrillogenesis. FN fibrillogenesis visualized by time-lapse AFM thus provides new structural and mechanistic insight into initial steps of cell-driven FN fibrillogenesis.
authors.affiliation: DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany.
authors.forename: Tetyana
authors.initials: T
authors.lastname: Gudzenko
authors.affiliation: DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany clemens.franz@kit.edu).
authors.forename: Clemens M
authors.initials: CM
authors.lastname: Franz
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D059008:Time-Lapse Imaging / Q000379:methods
chemical_list: D005353:Fibronectins; D016023:Integrins