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. INTRODUCTIONFibronectin (FN) is a large dimeric glycoprotein and an abundant component of the extracellular matrix (ECM) in different tissues, where it mediates integrin-dependent cell attachment and matrix cross-linking (Schwarzbauer and DeSimone, 2011). FN also plays an indispensable role during development, wound healing, and matrix repair (Grinnell, 1984a). A hallmark of FN is the cell-mediated reorganization of FN dimers into fibrils, which activates a range of its biological functions (Mao and Schwarzbauer, 2005;Singh et al., 2010). Fibrillar FN networks stably anchor cells to the matrix environment, but FN fibrillogenesis also often coincides with large morphogenetic changes during embryonic development in conjunction with cell movement. For instance, FN fibrils can guide cell migration during development (Winklbauer and Keller, 1996;Marsden and DeSimone, 2001;Davidson et al., 2004) and may immobilize growth factor gradients (Nagel et al., 2004) and contribute to tissue patterning (Sakai et al., 2003;Larsen et al., 2006).Despite the well-established physiological importance of fibrillar FN, the molecular mechanisms leading to FN assembly and the ultrastructure of the resulting FN fibrils are incompletely understood. Both plasma and cellular FN, two closely related isoforms, are initially secreted in a compact, inactive conformation. The conversion of globular FN molecules into the extended, active conformation is driven by cellular contraction forces transmitted by integrin receptors at focal adhesion sites (Dzamba et al., 1994;Christopher et al., 1997). Extension of the FN molecule exposes different FN-FN binding sites (Singh et al., 2010), allowing FN molecules to interact lateral...

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

“…The authors respectively recorded correlative topography/fluorescence images of fixed cells which were incubated on fluorescently-labelled fibronectin for 4 and 16 h (Figure 7), and thereby acquired the structure information of fibronectin. Based on the fibronectin structure, they monitored the early fibrillogenesis dynamics at high resolution by time-lapse AFM, and they also found that Mn 2+ enhanced the early fibrillogenesis [57]. Oreopoulos et al reported the coupling of AFM and polarized TIRFM technique, which can correlate the topographic features and the information (e.g., molecular orientation, conformation and rotational mobility) provided by polarized TIRFM [58,59].…”

“…( E ) Three-dimensional topography intuitively showing the FN fibril structure. Reproduced from [57] with permission.…”

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review

“…The prerequisite of AFM measurements is immobilizing cells onto a substrate. Adherent cells can naturally grow and spread on the substrate [28], while suspended cells can be immobilized onto the substrate via poly-L-lysine electrostatic adsorption [29] or microfabricated devices (e.g., porous polymer membrane [30], microwell [31] and micropillar [32]). For mechanical measurements, the piezoelectric ceramic tube drives the AFM tip (as shown in Fig.…”

Controlling Fibronectin Fibrillogenesis Using Visible Light

“…However, studies using conventional light microscopy techniques are unsuitable to resolve the earliest steps of fibrillogenesis occurring at the nanoscale. Atomic force microscopy (AFM)-based imaging has been used to visualize molecular-scale structural changes of FN fibrils extended through cellular (Gudzenko and Franz, 2015) or external forces (Szymanski et al, 2017). Nevertheless, AFM live-cell imaging still suffers from limited scan speeds and therefore cannot adequately time-resolve the earliest steps of FN fibrillogenesis, which occur on the scale of seconds to minutes.…”