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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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Assembly of FNΔIII2–5. (A) Native pFN and FNΔIII2–5 were added to CHOα5 cells at a concentration of 50 μg/ml and cultured for the indicated times. Fibrils were visualized by indirect immunofluorescence as in Fig. 2. (B) DOC-insoluble material isolated at the indicated times was analyzed under reducing conditions by immunoblotting with IC3 monoclonal antibody. Bar, 10 μm.
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fig4: Assembly of FNΔIII2–5. (A) Native pFN and FNΔIII2–5 were added to CHOα5 cells at a concentration of 50 μg/ml and cultured for the indicated times. Fibrils were visualized by indirect immunofluorescence as in Fig. 2. (B) DOC-insoluble material isolated at the indicated times was analyzed under reducing conditions by immunoblotting with IC3 monoclonal antibody. Bar, 10 μm.

Mentions: In contrast to the normal matrix formed with FNΔIII1, deletion of III2–5 decreased matrix accumulation. Some fibril assembly was initiated, but the amount of FNΔIII2–5 matrix assembled by CHOα5 cells was less than that of native FN, and the distribution of fibrils was more sparse than for native FN (Fig. 4 A). Furthermore, DOC-insoluble matrix was at least threefold less for FNΔIII2–5 than for native FN (Fig. 4 B). Limited conversion of DOC-soluble FNΔIII2–5 into DOC-insoluble matrix suggests that this recFN is impaired in its ability to participate in fibril growth.


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)

Assembly of FNΔIII2–5. (A) Native pFN and FNΔIII2–5 were added to CHOα5 cells at a concentration of 50 μg/ml and cultured for the indicated times. Fibrils were visualized by indirect immunofluorescence as in Fig. 2. (B) DOC-insoluble material isolated at the indicated times was analyzed under reducing conditions by immunoblotting with IC3 monoclonal antibody. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196193&req=5

fig4: Assembly of FNΔIII2–5. (A) Native pFN and FNΔIII2–5 were added to CHOα5 cells at a concentration of 50 μg/ml and cultured for the indicated times. Fibrils were visualized by indirect immunofluorescence as in Fig. 2. (B) DOC-insoluble material isolated at the indicated times was analyzed under reducing conditions by immunoblotting with IC3 monoclonal antibody. Bar, 10 μm.
Mentions: In contrast to the normal matrix formed with FNΔIII1, deletion of III2–5 decreased matrix accumulation. Some fibril assembly was initiated, but the amount of FNΔIII2–5 matrix assembled by CHOα5 cells was less than that of native FN, and the distribution of fibrils was more sparse than for native FN (Fig. 4 A). Furthermore, DOC-insoluble matrix was at least threefold less for FNΔIII2–5 than for native FN (Fig. 4 B). Limited conversion of DOC-soluble FNΔIII2–5 into DOC-insoluble matrix suggests that this recFN is impaired in its ability to participate in fibril growth.

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