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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 FNΔIII1–2. (A) CHOα5 cells were incubated for either 4 or 24 h in the presence of 50 μg/ml FNΔIII1–2. Monoclonal antibody IC3 was used to detect recFN matrix by immunofluorescence. (B) DOC-soluble and -insoluble cell lysates were isolated from CHOα5 cells incubated in the presence of 50 μg/ml FNΔIII1–2 or pFN for 0.5, 4, 7, 16, 24, and 48 h. DOC-soluble and -insoluble material was analyzed as described in Fig. 3. Arrow and bracket indicate locations of high molecular mass multimers. Dash indicates location of 180-kD molecular mass standard. Bar, 10 μm.
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fig5: Assembly of FNΔIII1–2. (A) CHOα5 cells were incubated for either 4 or 24 h in the presence of 50 μg/ml FNΔIII1–2. Monoclonal antibody IC3 was used to detect recFN matrix by immunofluorescence. (B) DOC-soluble and -insoluble cell lysates were isolated from CHOα5 cells incubated in the presence of 50 μg/ml FNΔIII1–2 or pFN for 0.5, 4, 7, 16, 24, and 48 h. DOC-soluble and -insoluble material was analyzed as described in Fig. 3. Arrow and bracket indicate locations of high molecular mass multimers. Dash indicates location of 180-kD molecular mass standard. Bar, 10 μm.

Mentions: Because III1–2 has FN binding activity (Aguirre et al., 1994) and a proteolytic fragment containing III1 plus part of III2 inhibits incorporation of FN into matrix (Chernousov et al., 1991; Morla and Ruoslahti, 1992), we generated FNΔIII1–2 to test whether these two repeats together comprise a matrix regulatory region. Immunofluorescence analysis of FN assembled by CHOα5 cells showed that early during assembly, FNΔIII1–2 formed aggregates on the cell surface (Fig. 5 A) similar to, but much smaller than, those formed by FNΔIII1–7 (Sechler et al., 1996). As assembly progressed, FNΔIII1–2 formed mainly short fibrils between cells although occasional long thin fibrils were also visible. An extensive fibrillar network was never observed even after prolonged incubations. Levels of DOC-insoluble material were significantly reduced, especially at later times of assembly, and no high molecular mass multimers were observed (Fig. 5 B). Incubations with higher concentrations of FNΔIII1–2 did not increase the amount of DOC-insoluble matrix (unpublished data). Unlike FNs lacking the RGD sequence, FNΔIII1–2 was not deficient in binding to cells. In fact, substantially more FNΔIII1–2 than native FN was isolated as cell-associated DOC-soluble material. This indicates that FNΔIII1–2 can efficiently bind to the cell surface to initiate assembly, but is defective in the growth phase when conversion from DOC-soluble to -insoluble matrix occurs.


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 FNΔIII1–2. (A) CHOα5 cells were incubated for either 4 or 24 h in the presence of 50 μg/ml FNΔIII1–2. Monoclonal antibody IC3 was used to detect recFN matrix by immunofluorescence. (B) DOC-soluble and -insoluble cell lysates were isolated from CHOα5 cells incubated in the presence of 50 μg/ml FNΔIII1–2 or pFN for 0.5, 4, 7, 16, 24, and 48 h. DOC-soluble and -insoluble material was analyzed as described in Fig. 3. Arrow and bracket indicate locations of high molecular mass multimers. Dash indicates location of 180-kD molecular mass standard. Bar, 10 μm.
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Related In: Results  -  Collection

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fig5: Assembly of FNΔIII1–2. (A) CHOα5 cells were incubated for either 4 or 24 h in the presence of 50 μg/ml FNΔIII1–2. Monoclonal antibody IC3 was used to detect recFN matrix by immunofluorescence. (B) DOC-soluble and -insoluble cell lysates were isolated from CHOα5 cells incubated in the presence of 50 μg/ml FNΔIII1–2 or pFN for 0.5, 4, 7, 16, 24, and 48 h. DOC-soluble and -insoluble material was analyzed as described in Fig. 3. Arrow and bracket indicate locations of high molecular mass multimers. Dash indicates location of 180-kD molecular mass standard. Bar, 10 μm.
Mentions: Because III1–2 has FN binding activity (Aguirre et al., 1994) and a proteolytic fragment containing III1 plus part of III2 inhibits incorporation of FN into matrix (Chernousov et al., 1991; Morla and Ruoslahti, 1992), we generated FNΔIII1–2 to test whether these two repeats together comprise a matrix regulatory region. Immunofluorescence analysis of FN assembled by CHOα5 cells showed that early during assembly, FNΔIII1–2 formed aggregates on the cell surface (Fig. 5 A) similar to, but much smaller than, those formed by FNΔIII1–7 (Sechler et al., 1996). As assembly progressed, FNΔIII1–2 formed mainly short fibrils between cells although occasional long thin fibrils were also visible. An extensive fibrillar network was never observed even after prolonged incubations. Levels of DOC-insoluble material were significantly reduced, especially at later times of assembly, and no high molecular mass multimers were observed (Fig. 5 B). Incubations with higher concentrations of FNΔIII1–2 did not increase the amount of DOC-insoluble matrix (unpublished data). Unlike FNs lacking the RGD sequence, FNΔIII1–2 was not deficient in binding to cells. In fact, substantially more FNΔIII1–2 than native FN was isolated as cell-associated DOC-soluble material. This indicates that FNΔIII1–2 can efficiently bind to the cell surface to initiate assembly, but is defective in the growth phase when conversion from DOC-soluble to -insoluble matrix occurs.

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