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Specific function of the multimeric fibronectin formed in vitro as revealed by the interaction with HT-1080 cells
Sixth International Conference on the Molecular Biology and Pathology of Matrix
Specific Function of the Multimeric Fibronectin Formed in vitro as Revealed by the Interaction with HT-1080 Cells. Keiko Sakai, Hitomi Fujisaki, Eijiro Adachi*, Shunji Hattori, and Toshihiko Hayashit Nippi Biomatrix Res. Lab., Tokyo, Japan, *Kitasato Univ. Sch. of Med., Dept. Anat. and Cell Biol., Sagamihara, Kanagawa, Japan; tDept. Life Sci. Grad. Sch. of Arts & Sci., Univ. Tokyo, Tokyo, Japan Purified plasma fibronectin multimerized upon incubation with a SH reagent (Sakai, K. et al., J. Biochem. 115, 415-421, 1994). The multimeric fibronectin (mFN) has a fibrillar structure with about 100 pm in length in the immunofluorescent microscopic image and with a rather Uniform diameter at the immunoelectron microscopic level. The mFN was compared with the dimeric fibronectin (dFN) as cell culture substrate in the interaction with fibrosarcoma HT-1080, which cannot synthesize fibronectin. At a low protein concentration (0.2 I~g/ml), 3-fold larger number of HT-1080 cells attached to mFN than to dFN. Cell spreading started at an earlier timing on mFN, suggesting a stronger affinity. The direct immunostaining pattern of vinculin of the cells on mFN showed patched plaquelike appearances, suggesting that adhesion focal contacts containing vinculin were more readily formed. The dFN caused the vinculin distribution to be diffuse. The 1131integrin was localized on the cells cultured on either fibronectin form. In contrast, (z3 integrin staining pattern was distinct on mFN, but the staining of the cells on dFN was obscured. Fibronectin and actin filamenst showed a colocalization by double-fluorescent staining in the course of cell spreading on mFN. it appears that the direct vicinity of the cell enriched fibronectin, while the region just away from the vicinity lost the staining, as if mFN was removed by the cell and transferred to the cell vicinity. Such transfer of supramolecular aggregates by the cell was reported for prelabeled type I collagen fibrils and fibroblasts (Yamato et al., J. Biochem., 117, 940-946, 1995).
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Separation of the Subtypes of Type V Collagen Molecules, [(zl(V)]=(x2(V) and (xl(V)u2(V)(z3(V), by Chain CompositionDependent Affinity for Heparin. K. Mizuno, and T. Hayashi Department of Life Sciences, Graduate School of Arts & Sciences, University of Tokyo, Tokyo, Japan The heparin affinities of heat-treated type V collagen 0~ chains and the triple-helical molecules were evaluated by the NaCI concentration required for eluting bound polypeptide(s) from heparin column. Among the three kinds of (x chain, c~l(V) chain showed by far the highest affinity for heparin. The type V collagen molecules in triple-helical conformation with a lower affinity showed apparent chain composition of e~I(V):(~2(V):(x3(V) with 1:1:1 ratio, while the molecules with a higher affinity showed the chain composition of c¢I(V):(x2(V) with 2:1. it appeared that the differential heparin affinity of the two fractions, each of which could be taken to comprise one subtype of the type V collagen with the chain composition in a molecule, was due to the number of (xl(V) chain in a triple-helical molecule. The affinity of the subtype of [(zl(V)]2a2(V) was higher than that of separated (xl(V) chain, while that of e~l (V)(72(V)(x3(V) was lower than separated ~.1 (V) chain. A possible explanation for the affinity difference among the type V collagen molecules and the separated (x chains is discussed on the assumption that heparin affinity of type V collagen molecules is governed by the number of c~l(V) chain contained in a molecule. The chain shows a high affinity for heparin as a separated chain, but which might less strongly bind to heparin in the triple-helical structure by conformational restraint. The subtype with two c~l(V) chains was suggested to have roughly two-fold affinity than that with one c~l(V) chain.
Specific Function of the Multimeric Fibronectin Formed in vitro as Revealed by the Interaction with HT-1080 Cells. Keiko Sakai, Hitomi Fujisaki, Eijiro Adachi*, Shunji Hattori, and Toshihiko Hayashit Nippi Biomatrix Res. Lab., Tokyo, Japan, *Kitasato Univ. Sch. of Med., Dept. Anat. and Cell Biol., Sagamihara, Kanagawa, Japan; tDept. Life Sci. Grad. Sch. of Arts & Sci., Univ. Tokyo, Tokyo, Japan Purified plasma fibronectin multimerized upon incubation with a SH reagent (Sakai, K. et al., J. Biochem. 115, 415-421, 1994). The multimeric fibronectin (mFN) has a fibrillar structure with about 100 pm in length in the immunofluorescent microscopic image and with a rather Uniform diameter at the immunoelectron microscopic level. The mFN was compared with the dimeric fibronectin (dFN) as cell culture substrate in the interaction with fibrosarcoma HT-1080, which cannot synthesize fibronectin. At a low protein concentration (0.2 I~g/ml), 3-fold larger number of HT-1080 cells attached to mFN than to dFN. Cell spreading started at an earlier timing on mFN, suggesting a stronger affinity. The direct immunostaining pattern of vinculin of the cells on mFN showed patched plaquelike appearances, suggesting that adhesion focal contacts containing vinculin were more readily formed. The dFN caused the vinculin distribution to be diffuse. The 1131integrin was localized on the cells cultured on either fibronectin form. In contrast, (z3 integrin staining pattern was distinct on mFN, but the staining of the cells on dFN was obscured. Fibronectin and actin filamenst showed a colocalization by double-fluorescent staining in the course of cell spreading on mFN. it appears that the direct vicinity of the cell enriched fibronectin, while the region just away from the vicinity lost the staining, as if mFN was removed by the cell and transferred to the cell vicinity. Such transfer of supramolecular aggregates by the cell was reported for prelabeled type I collagen fibrils and fibroblasts (Yamato et al., J. Biochem., 117, 940-946, 1995).
157
Separation of the Subtypes of Type V Collagen Molecules, [(zl(V)]=(x2(V) and (xl(V)u2(V)(z3(V), by Chain CompositionDependent Affinity for Heparin. K. Mizuno, and T. Hayashi Department of Life Sciences, Graduate School of Arts & Sciences, University of Tokyo, Tokyo, Japan The heparin affinities of heat-treated type V collagen 0~ chains and the triple-helical molecules were evaluated by the NaCI concentration required for eluting bound polypeptide(s) from heparin column. Among the three kinds of (x chain, c~l(V) chain showed by far the highest affinity for heparin. The type V collagen molecules in triple-helical conformation with a lower affinity showed apparent chain composition of e~I(V):(~2(V):(x3(V) with 1:1:1 ratio, while the molecules with a higher affinity showed the chain composition of c¢I(V):(x2(V) with 2:1. it appeared that the differential heparin affinity of the two fractions, each of which could be taken to comprise one subtype of the type V collagen with the chain composition in a molecule, was due to the number of (xl(V) chain in a triple-helical molecule. The affinity of the subtype of [(zl(V)]2a2(V) was higher than that of separated (xl(V) chain, while that of e~l (V)(72(V)(x3(V) was lower than separated ~.1 (V) chain. A possible explanation for the affinity difference among the type V collagen molecules and the separated (x chains is discussed on the assumption that heparin affinity of type V collagen molecules is governed by the number of c~l(V) chain contained in a molecule. The chain shows a high affinity for heparin as a separated chain, but which might less strongly bind to heparin in the triple-helical structure by conformational restraint. The subtype with two c~l(V) chains was suggested to have roughly two-fold affinity than that with one c~l(V) chain.