Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein

Abstract

Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN-green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.

Figure 1

Identical FN matrices are assembled by CHO cells transfected with native plasma FN (a and d) or with FN–gfp (b, c, e, and f). Cells were cultured 36 h to allow matrix assembly and were fixed with 3.7% formaldehyde in PBS, treated with 0.2% Triton X-100, and then immunostained using polyclonal antibodies for FN (d and e) or gfp (f). (a–c) gfp fluorescence is visualized; (d–f) the rhodamine-labeled second antibody is visualized. (Bar = 50 μm.)

Figure 2

The mobility of the FN matrix network. Selected images show active fibril movement (arrows). Images were originally taken at 5-min intervals after 36 h of culture. White squares in b are magnified in a and c. (Bar = 50 μm.)

Figure 3

The elasticity of FN matrix fibrils demonstrated by exceptionally large movements and breakages. Specific movements are discussed in the text. Images were originally taken at 5-min intervals after 15 h of culture. Arrows indicate fibril ends or predicted cell adhesion sites. (Bar = 20 μm.) A digital movie of this sequence is published as supplemental material on the PNAS web site (www.pnas.org).

Figure 4

The mobility of an FN matrix fibril after laser beam radiation. The spot burned by the laser is indicated by the white circle. (Bar = 20 μm.)

Figure 5

Movement of cells and FN fibrils after EDTA treatment. A cell culture is imaged by Nomarski optics (Left) and gfp fluorescence (Right) before and 20 and 50 min after adding 5 mM EDTA. At 0 min, the fibril indicated by arrows runs near, but not precisely along, some cell edges. At 20 min, the fibril runs closely along the cell edge at the arrowhead, but its other attachments are not clear. At 50 min that cell has rounded; the fibril still contacts the cell at one point, but it maintains a straight path rather than following the rounded edge of the cell. The bottom right end of the fibril has moved about 20 μm from 20 to 50 min, but what it is attached to is not clear. (Bar = 20 μm.)

Figure 6

Schematic diagram of possible mechanisms for stretching and contracting an FN matrix fibril. These diagrams illustrate single FN molecules; it is important to remember that the fluorescent FN fibrils are ten to hundreds of molecules thick, and hundreds of molecules in legnth. (a) In the contracted fibril the molecules are in a compact conformation, with bends between FN-III domains and stabilized by intramolecular bonds. Stretching the fibril breaks these bonds and extends the FN molecules. (b) The fibril could be stretched by unfolding FN-III domains and contracted by refolding the domains. The unfolded domain is 28.5 nm compared with the 3.5-nm length of the folded domain (–15).