Nanoscale features of fibronectin fibrillogenesis depend on protein-substrate interaction and cytoskeleton structure

Abstract

Cell-reorganized fibronectin layers on polymer films providing a gradation of the binding strength between protein and substrate were analyzed by combined fluorescence and scanning force microscopy. The nanoscale fibronectin patterns exhibited paired parallel fibrils with characteristic spacings of 156, 233, 304, and 373 nm. These spacings depend on the interaction of fibronectin with the substrate: at enhanced fibronectin-substrate anchorage the cells form larger stress fibers, which are assembled by alpha-actinin cross-linked pairs of actin filaments subunits at the focal adhesions. A ubiquitous repeating unit of approximately 71 nm was found within these characteristic distances. We conclude that the dimensions of the actin stress fibers reflect the binding strength of fibronectin to the polymer substrate and act--in turn--as a template for the reorganization of fibronectin into surface-bound nanofibrils with characteristic spacings. This explanation was confirmed by data showing the alpha-actinin/fibronectin colocalization.

FIGURE 1

Dynamic displacement characteristic of FN-TRITC on POMA and PPMA in a solution of 50 μg/ml of human serum albumin over 48 h measured by confocal laser scanning microscopy. The error bars indicate the mean error from three independent experiments with each having 10 measurements per data point.

FIGURE 2

Analysis of TRITC conjugated FN fibrils on PPMA after reorganization by endothelial cells using a coupled fluorescence confocal laser scanning microscopy and SFM. (A) Optical image. Scale bar, 5 μm (B) SFM image of one part of image in Fig. 2 A. The cutout is marked in Fig. 2 A by a square. Scale bar, 5 μm (height scale, 50 nm).

FIGURE 3

SFM height images of FN fibrils on PPMA (A) and POMA (B). The white lines indicate section cuts shown in Fig. 5. Scale bars, 1 μm. Height scale, 70 nm.

FIGURE 4

Graphs of line sections of the SFM topography images in Fig. 3, A (PPMA) and B (POMA), indicating the height of the FN fibrils and the spacing between them.

FIGURE 5

Histogram of typical spacings of paired FN nanofibrils on the two different substrates with 45 measurements for each substrate.

FIGURE 6

Fitting a periodic squared sine function to the histogram data of both substrates of Fig. 6. A repeating unit of 71 nm could be determined.

FIGURE 7

α-Actinin staining of endothelial cells after 50 min of reorganization of preadsorbed TRITC conjugated FN on the two copolymer substrates (A, POMA; B, PPMA). Contours of FN fibrils are drawn over the α-actinin fluorescence intensity image. The asterisks indicate pronounced colocalization or association of FN fibrils and α-actinin clusters. Scale bar, 5 μm.

FIGURE 8

(A) α-Actinin concentration determined by antibody staining intensity measured with confocal laser scanning microscopy. The mean intensity of α-actinin was measured at areas colocalized with FN fibrils (60 clusters) and at areas of α-actinin clusters partly colocalized (associated) with FN fibrils (seven clusters). The mean intensities for the two substrates are statistically different on a significance level of p < 10⁻⁵. (B) Ratio of mean α-actinin concentration colocalized or associated with FN fibrils compared with ratio of mean FN nanofibril spacing on the two different copolymer substrates. Error bars indicate the calculated propagated error of all measurements.

FIGURE 9

Schematic representation of the proposed model for the FN fibrillogenesis explaining the typical spacing of FN nanofibrils and its substrate dependence. Related to the gradated bond strength of FN to the substrate, the tensile force of the cell is adjusted, which acts in the actin stress fibers onto the substrate-bound FN molecules. In this process, the focal adhesion size and the thickness of actin stress fibers is regulated. This provides a distinct thickness of the actin stress fibers due to the α-actinin as the actin cross-linking element, which provides a pattern template for the FN fibril formation along the fibers by the myosin-driven FN stretching and transport. The discrete size of the actin stress fibers depends on their inner structure with a square lattice of α-actinin cross-linked actin filaments. Note that the sketch just illustrates the correlation of FN bond strength, stress fiber size, its inner structure, and fibronectin fibril spacing. Details of protein assembly and the structure of the focal adhesions are not in the scope of the figure.