Controlling Fibronectin Fibrillogenesis Using Visible Light

Abstract

We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.

Native extracellular matrix probes to target patient- and tissue-specific cell–microenvironment interactions by force spectroscopy

“…2E–G). The UV laser of the LMD apparatus could induce thermal damage and autofluorescence, 44 and fibronectin inactivation 45 around the edge of the ablated region; nevertheless, the central area used to produce the probes and interacting with the cells is not affected by these phenomena.…”

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

“…[157] OxEA requires DL-lactate as a substrate and supports 1-2% of the steady-state molecular oxygen concentration without significantly changing the solution pH, as well as the intracellular functions allowing live-cell imaging. [158] As a result, OxEA permits efficient photoswitching of both cyanine and rhodamine fluorophores under the same buffer compositions, enabling simultaneous multicolor GSDIM imaging with Alexa488, Alexa555, and Alexa647 (Figure 2J,K). [71] Alternative photostabilizing buffers include the sulfite buffer and the heavy water (D 2 O) instead of normal water (H 2 O).…”

Native extracellular matrix probes to target patient- and tissue- specific cell-microenvironment interactions by force spectroscopy

“…These results are in line with the results obtained by quantitative staining of major ECM proteins such as collagen, laminin, and fibronectin on different cut pieces of ECM, demonstrating that the composition of the matrix is not affected by the cut (Figure 3). The UV laser of the LMD apparatus could induce thermal damage and autofluorescence [57], and also fibronectin inactivation [58] around the edge of the ablated region; nevertheless, the central area used for the production of the probes and interacting with the cells is not affected by these phenomena. Stress tests were performed to assess the firm attachment of the matrix to the cantilever and the force spectroscopy functionality after repeated use.…”