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A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly.

Sechler JL, Rao H, Cumiskey AM, Vega-Colón I, Smith MS, Murata T, Schwarzbauer JE - J. Cell Biol. (2001)

Bottom Line: In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded.Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site.Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

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RecFN assembly in the presence of human FN matrix. CHOα5 cells were incubated with 50 μg/ml human pFN for 8 h. Medium was then removed, cell layers were washed to remove unbound FN, and cells were fed with fresh medium containing 25 μg/ml full-length recFN (A), FNΔIII1–2 (B), or FNΔIII2–5 (C). After incubation for an additional 16 h, cells were fixed and recFN fibrils detected with an anti–rat FN-specific monoclonal antibody. Bar, 20 μm.
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fig6: RecFN assembly in the presence of human FN matrix. CHOα5 cells were incubated with 50 μg/ml human pFN for 8 h. Medium was then removed, cell layers were washed to remove unbound FN, and cells were fed with fresh medium containing 25 μg/ml full-length recFN (A), FNΔIII1–2 (B), or FNΔIII2–5 (C). After incubation for an additional 16 h, cells were fixed and recFN fibrils detected with an anti–rat FN-specific monoclonal antibody. Bar, 20 μm.

Mentions: FN lacking the RGD sequence is unable to initiate matrix assembly but can be incorporated once assembly has been primed by native FN (Sechler et al., 1996). However, preinitiation by native FN would not be expected to rescue FNΔIII1–2 assembly, as this protein shows a defect in fibril growth. In fact, neither FNΔIII1–2 nor FNΔIII2–5 was able to efficiently incorporate into a preformed human FN matrix. A very few short fibrils were formed by FNΔIII1–2 (Fig. 6 B) and FNΔIII2–5 fibrils were found in small patches distributed unevenly throughout the matrix (Fig. 6 C). In contrast, an extensive fibrillar network was assembled by native FN (Fig. 6 A). In all cases, a fibrillar human plasma FN (pFN) matrix could be readily detected with a human FN-specific monoclonal antibody (unpublished data). Thus, the presence of repeat III2 is required for the continued growth of FN fibrils.


A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly.

Sechler JL, Rao H, Cumiskey AM, Vega-Colón I, Smith MS, Murata T, Schwarzbauer JE - J. Cell Biol. (2001)

RecFN assembly in the presence of human FN matrix. CHOα5 cells were incubated with 50 μg/ml human pFN for 8 h. Medium was then removed, cell layers were washed to remove unbound FN, and cells were fed with fresh medium containing 25 μg/ml full-length recFN (A), FNΔIII1–2 (B), or FNΔIII2–5 (C). After incubation for an additional 16 h, cells were fixed and recFN fibrils detected with an anti–rat FN-specific monoclonal antibody. Bar, 20 μm.
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Related In: Results  -  Collection

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fig6: RecFN assembly in the presence of human FN matrix. CHOα5 cells were incubated with 50 μg/ml human pFN for 8 h. Medium was then removed, cell layers were washed to remove unbound FN, and cells were fed with fresh medium containing 25 μg/ml full-length recFN (A), FNΔIII1–2 (B), or FNΔIII2–5 (C). After incubation for an additional 16 h, cells were fixed and recFN fibrils detected with an anti–rat FN-specific monoclonal antibody. Bar, 20 μm.
Mentions: FN lacking the RGD sequence is unable to initiate matrix assembly but can be incorporated once assembly has been primed by native FN (Sechler et al., 1996). However, preinitiation by native FN would not be expected to rescue FNΔIII1–2 assembly, as this protein shows a defect in fibril growth. In fact, neither FNΔIII1–2 nor FNΔIII2–5 was able to efficiently incorporate into a preformed human FN matrix. A very few short fibrils were formed by FNΔIII1–2 (Fig. 6 B) and FNΔIII2–5 fibrils were found in small patches distributed unevenly throughout the matrix (Fig. 6 C). In contrast, an extensive fibrillar network was assembled by native FN (Fig. 6 A). In all cases, a fibrillar human plasma FN (pFN) matrix could be readily detected with a human FN-specific monoclonal antibody (unpublished data). Thus, the presence of repeat III2 is required for the continued growth of FN fibrils.

Bottom Line: In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded.Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site.Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.

Show MeSH