Winklbauer R and Stoltz C (1995), Fibronectin fibril growth in the extracellular ...

XB-ART-19920

J Cell Sci 1995 Apr 01;108 ( Pt 4):1575-86.

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Fibronectin fibril growth in the extracellular matrix of the Xenopus embryo.

Winklbauer R , Stoltz C .

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We show that the mechanism of fibronectin fibril formation on the blastocoel roof of the Xenopus embryo is comparable to that in other systems. Fibril assembly is inhibited by RGD peptide, by an amino-terminal fragment of fibronectin, and by cytochalasin B. When added exogenously, intact fibronectin, but not a 110 kDa cell binding fragment of fibronectin, is incorporated into fibrils. Thus, the blastocoel roof of Xenopus represents a valid model system for the study of fibronectin fibril formation in situ. Moreover, we show that fibril formation can be induced experimentally in this system. Examination of fibril elongation by double-labelling experiments reveals that individual, unbranched fibronectin fibrils grow only at one end, i.e. in a unipolar fashion. The rate of elongation is 4.7 microns/min. Most fibrils grow only for a short time, and the increase in total fibril length per cell is driven by the repeated initiation of new fibrils. Assembly of fibronectin into fibrils precedes cross-linking of fibronectin into multimers in this system.

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Species referenced: Xenopus laevis
Genes referenced: fn1

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	Fig. 1. Diagram of a FN monomer outlining the locations of sites involved in matrix assembly, and of FN fragments relevant to this work. The three repeating units of FN are depicted as follows: type I repeats, hatched; type II repeats, lightly dotted; type III repeats, open boxes. Differentially spliced type III repeats E III A and E III B (heavily dotted) and the variably spliced region (III CS) are indicated. Heparin-, fibrinand collagen-binding domains are outlined, and the RGD cell binding site in the 10th type III repeat is indicated.
	Fig. 2. FN fibril formation on the blastocoel roof of the Xenopus embryo. Blastocoel roof from stage 9 (a), stage 10 (b), stage 10+ (c) and stage 11 (d) embryos was stained with antibody to Xenopus plasma FN and FITC-conjugated secondary antibody, and mounted whole.
	Fig. 3. Characterization of FN fibril formation on the X e n o p u s blastocoel roof. GRGDSP peptide (4 mg/ml) (a), but not GRGESP control peptide (b) inhibits fibril formation. A 110 kDa fluorescein-labelled cell binding fragment is not incorporated into FN fibrils (c), but intact labelled FN is (d). FN fibril formation is strongly inhibited by a 30 kDa fragment from the amino terminus of FN (e). When the blastocoel roof of stage 10 embryos is used to condition the substratum in the presence of 20 mg/ml of cytochalasin B for 2 hours, a punctate, diffuse immunoflu o r e s c e n c e staining pattern is observed (f). Double-labelling of a blastocoel roof whole-mount for FN, with FN antibody and FITC-conjugated secondary antibody (g), and F-actin, with rhodamine-phalloidin (h), reveals partial overlap of extracellular FN fibrils and intracellular F-actin bundles. The high background fluorescence in blastocoel roof whole-mounts seems to be due to the presence of labelled F-actin well below the cell surface. Intense rhodamine staining of cell boundaries breaks through to the fluorescein image (arrows), but can be easily distinguished from FN fib r i l s . Arrowheads indicate identical positions in g and h. All micrographs are at the same magnification. Bar, 20 mm.
	Fig. 4. Visualization of FN fibril elongation by double-labelling. (a) Schematic representation of double-labelling technique. Existing fibrils are labelled with FN antibody (1). Then further fibril growth occurs in the absence of antibody (2). Newly added fibril segments are decorated with Fab fragments of the same IgG antibody preparation (3). Whole IgG molecules and Fab fragments are visualized by staining with rhodamine-conjugated antibodies specific for the Fc part (R) and fluorescein-conjugated antibodies directed against the Fab part (F), respectively (4). (b,b’) Double-labelling of growing FN fibrils. Fibrils were decorated with IgG at stage 10+, and with Fab fragment 2 hours later. (b) Rhodamine fluorescence, marking FN fibrils present at the beginning of the experiment. (b’) Same field as in (b), fluorescein label, visualizing both preexisting and newly formed fibrils. Fibrils that appear during the experiment are labelled with fluorescein only (arrow). To a preexisting fibril (ends marked with arrowheads in (b) and in (b’)) new fibril length has been added at one end. (c,c’) Further example, as in (b,b’). (d,d’) Modification of the double-labelling method. Existing fibrils (d) were labelled by the incorporation of fluorescein-FN. After further incubation (30 minutes) in the absence of labelled FN, blastocoel roofs were stained with FN antibody and rhodamine-conjugated secondary antibody (d’). Newly added fibrils are labelled with rhodamine only. Arrows and arrowheads as in (b,c). (e,e’) Combination of double-labelling with substratum conditioning. Preexisting fibrils were labelled with antibody and blastocoel roofs were then pressed against the bottom of the dish for 20 minutes. Antibody-decorated fibrils, and also fibril segments forming anew during conditioning, are transferred to the plastic substratum. The transferred fibril network was incubated with Fab fragments of the same antibody, and stained with secondary antibodies ((e), rhodamine; (e’), fluorescein) as described above. Arrowheads as in b-d; arrow, interrupted new fibril. Most fibrils, although short, did not elongate during the experiment. Bar, 10 mm.
	Fig. 5. FN fibril formation in the presence of antibody. Continuous incubation from stage 9 to stage 10+ in 25 mg/ml of IgG against Xenopus plasma FN does not inhibit fibril formation on the blastocoel roof (a), as compared to buffer-incubated controls (b). No fibrils were present at the beginning of the experiment in this batch of embryos (c). Bar, 10 mm.
	Fig. 6. Fibril branching. Standard double-staining experiments (Fig. 4a) were performed. Fibril growth after labelling of preexisting fibrils was for 6 minutes (a,a’) or 2 minutes (b,b’). Rhodamine (a,b) labels FN fibrils present at the beginning of the experiment, fluorescein (a’,b’) labels both preexisting and newly formed fibrils. (a,a’) Lateral addition of new fibril segment (arrowheads) to older fibril. (b,b’) Lateral addition of preexisting fibril (arrow) to newly formed fibril. Bar, 5 mm.
	Fig. 7. Quantitation of FN fibril elongation. (a) Maximum fibril length as a function of elongation time. Double-staining experiments (DS) were performed as described (Fig. 4a). The average length of the three largest newly formed fibrils for each time point is indicated, and the range between the longest and the shortest of these fibrils is represented as a vertical bar. In a second approach, stage 9 blastocoel roofs were used to condition substratum for varying times (CS). For each time point, the 3 longest fibrils were selected, and treated as above. The increase in maximum fibril length during the first 4 minutes (DS) or 3 minutes (CS), respectively, is 4.7 mm/min in both cases. (b) Frequency distribution of fibril lengths. The lengths of newly added segments of elongating fibrils as seen in double-labelling experiments were measured as described above. Fibril elongation was allowed for 2 minutes (top; 57 cases of fibril elongation evaluated) or for 8 minutes, respectively (bottom; 78 cases), and fibril lengths were classed according to 2 mm steps.
	Fig. 8. FN fibril growth on conditioned substratum. Plastic substratum was conditioned with stage 9 blastocoel roofs for 0.75 minute (a), 3.25 minutes (b), 8 minutes (c) and 18 minutes (d), and stained with FN antibody. Isolated patches of fibrils corresponding in size to single cells were photographed. Long fibrils sometimes appear to be interrupted (arrowheads). Bar, 10 mm.
	Fig. 9. Quantitation of FN fibril growth on conditioned substratum. Substratum was conditioned with stage 9 blastocoel roofs for varying times. For isolated patches of fibrils corresponding to single cells, total fibril length, i.e. the sum of the lengths of all individual fibrils, and fibril number were determined. Dots were not counted as fibrils. For each time point, those three patches with the largest total fibril length, or the highest number of fibrils, were selected, and averages were calculated as for Fig. 7a. Vertical bars represent the range between the highest and the lowest value, as in Fig. 7a, not standard deviations. The average increase in total fibril length per cell (L) during the first 5 minutes is 18 mm/min, the average increase in fibril number (n) is 10 fibrils/min. The growth of single fibrils (Fig. 7a) is shown for comparison (l, broken line).
	Fig. 10. Solubility of FN fibrils in urea. (a,b) Fibril formation was induced in stage 9 blastocoel roofs and allowed to proceed for 10 minutes. Deposited fibrils were incubated for 30 minutes in MBS (a; control) or 3 M urea in MBS (b). After extraction with urea, only faint traces of FN fibrils are left (arrowheads). (c) About 2 hours after the onset of FN fibril formation, at stage 10 , fibrils were transfered to plastic and incubated in 3 M urea in MBS for 30 minutes. Arrowheads, traces of extracted FN fibrils. Arrow, FN fibril resistant to extraction by urea.