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Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.

Li S, Liquari P, McKee KK, Harrison D, Patel R, Lee S, Yurchenco PD - J. Cell Biol. (2005)

Bottom Line: This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

ABSTRACT
Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

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Lm 1–sulfatide associations on cultured SCs. (a) SCs that adhered to plastic were incubated with 10 μg/ml Lm-1 added to medium. Lm-α1 (red; DAPI, blue) initially accumulated in diffusely dispersed aggregates and then condensed into a compact, sheetlike pattern. Dotted lines indicate outer cell borders. (b) Nidogen-1 (red) and type IV collagen (red) were detected in a colocalizing pattern when treated with Lm (green). (c) Gal-sulfatide (nonpermeabilized cells, Cy3, red), which was initially diffuse, condensed and colocalized with Lm-α1 (FITC, green). (d and e) Lm-1 did not accumulate (d) on SCs when incubated for 1 h (10 μg/ml) in the presence of malarial CS protein (150 μg/ml), whereas it accumulated (e) on SCs treated with a mixture (h/hp) of heparitinase (0.1 U/ml) and heparinase (1 U/ml). (f) Sulfatide and Lm-α1 were largely undetectable if 50 U/ml arylsulfatase was added to SC medium. However, if cells were washed and incubated with 10 μM gal-sulfatide/BSA (for 30 min) followed by 10 μg/ml Lm-1, sulfatide and Lm immunofluorescence were restored. (g) Cell gal-sulfatide is bound to Lm-1. SCs were labeled with 5 μM BODIPY-gal-sulfatide; incubated with Lm-1, BSA, or nonpolymerizing AEBSF-Lm-1 (10 μg/ml) for 1 h; lysed with 1% Triton X-100; extracted; centrifuged to remove insoluble material; and were immunoprecipitated with anti-E1′ Lm antibody. The precipitate was solubilized with 1% SDS and BODIPY fluorescence was determined (excitation, 490 nm; emission, 513 nm). (h) Lm–LG domain and polymerization are required for BM. SCs were incubated for 1 h with 10 μg/ml of recombinant Lm-1 (rLm-1) or 10 μg/ml Lm-1 lacking G-domain (rLm-1ΔG) and immunostained for Lm-γ1. SCs were also incubated with either native Lm-1 (Lm-1, 10 μg/ml), alone, or in the presence of competing recombinant E3 (rE3 WT; 100 μg/ml); with E3 mutant G (rE3 Mut G; 100 μg/ml) to block LG-modular interactions; or with fragment E1′ to block Lm polymerization (AEBSF-E1′ is control).
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fig2: Lm 1–sulfatide associations on cultured SCs. (a) SCs that adhered to plastic were incubated with 10 μg/ml Lm-1 added to medium. Lm-α1 (red; DAPI, blue) initially accumulated in diffusely dispersed aggregates and then condensed into a compact, sheetlike pattern. Dotted lines indicate outer cell borders. (b) Nidogen-1 (red) and type IV collagen (red) were detected in a colocalizing pattern when treated with Lm (green). (c) Gal-sulfatide (nonpermeabilized cells, Cy3, red), which was initially diffuse, condensed and colocalized with Lm-α1 (FITC, green). (d and e) Lm-1 did not accumulate (d) on SCs when incubated for 1 h (10 μg/ml) in the presence of malarial CS protein (150 μg/ml), whereas it accumulated (e) on SCs treated with a mixture (h/hp) of heparitinase (0.1 U/ml) and heparinase (1 U/ml). (f) Sulfatide and Lm-α1 were largely undetectable if 50 U/ml arylsulfatase was added to SC medium. However, if cells were washed and incubated with 10 μM gal-sulfatide/BSA (for 30 min) followed by 10 μg/ml Lm-1, sulfatide and Lm immunofluorescence were restored. (g) Cell gal-sulfatide is bound to Lm-1. SCs were labeled with 5 μM BODIPY-gal-sulfatide; incubated with Lm-1, BSA, or nonpolymerizing AEBSF-Lm-1 (10 μg/ml) for 1 h; lysed with 1% Triton X-100; extracted; centrifuged to remove insoluble material; and were immunoprecipitated with anti-E1′ Lm antibody. The precipitate was solubilized with 1% SDS and BODIPY fluorescence was determined (excitation, 490 nm; emission, 513 nm). (h) Lm–LG domain and polymerization are required for BM. SCs were incubated for 1 h with 10 μg/ml of recombinant Lm-1 (rLm-1) or 10 μg/ml Lm-1 lacking G-domain (rLm-1ΔG) and immunostained for Lm-γ1. SCs were also incubated with either native Lm-1 (Lm-1, 10 μg/ml), alone, or in the presence of competing recombinant E3 (rE3 WT; 100 μg/ml); with E3 mutant G (rE3 Mut G; 100 μg/ml) to block LG-modular interactions; or with fragment E1′ to block Lm polymerization (AEBSF-E1′ is control).

Mentions: Cultured rat sciatic nerve SCs did not possess detectable Lm-γ1 or α2 epitopes on their exposed surfaces or, as determined in immunoblots in cell extracts and conditioned medium (unpublished data), evidence that the cells had lost the ability to synthesize relevant Lms. However, these cells assemble a BM when incubated with exogenous Lm-1 or -2 (Tsiper and Yurchenco, 2002). When subconfluent cells (passage 25–32) were treated with 10 μg/ml Lm-1, Lm epitope, which was initially distributed diffusely (Fig. 2 a), condensed within 30 min into large confluent regions in which the aggregate edges were retracted from one or more of the outer cell borders, allowing for the assessment of colocalization with other components. A similar time course was observed with recombinant Lm-2 (unpublished data). The Lm pattern colocalized with endogenous nidogen-1 and type IV collagen (Fig. 2 b). Accumulation of the latter component was not appreciated previously (Tsiper and Yurchenco, 2002), likely because a short accumulation time in medium (1 h) during culturing was used in that study to permit detection of collagen as now seen with overnight accumulation.


Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.

Li S, Liquari P, McKee KK, Harrison D, Patel R, Lee S, Yurchenco PD - J. Cell Biol. (2005)

Lm 1–sulfatide associations on cultured SCs. (a) SCs that adhered to plastic were incubated with 10 μg/ml Lm-1 added to medium. Lm-α1 (red; DAPI, blue) initially accumulated in diffusely dispersed aggregates and then condensed into a compact, sheetlike pattern. Dotted lines indicate outer cell borders. (b) Nidogen-1 (red) and type IV collagen (red) were detected in a colocalizing pattern when treated with Lm (green). (c) Gal-sulfatide (nonpermeabilized cells, Cy3, red), which was initially diffuse, condensed and colocalized with Lm-α1 (FITC, green). (d and e) Lm-1 did not accumulate (d) on SCs when incubated for 1 h (10 μg/ml) in the presence of malarial CS protein (150 μg/ml), whereas it accumulated (e) on SCs treated with a mixture (h/hp) of heparitinase (0.1 U/ml) and heparinase (1 U/ml). (f) Sulfatide and Lm-α1 were largely undetectable if 50 U/ml arylsulfatase was added to SC medium. However, if cells were washed and incubated with 10 μM gal-sulfatide/BSA (for 30 min) followed by 10 μg/ml Lm-1, sulfatide and Lm immunofluorescence were restored. (g) Cell gal-sulfatide is bound to Lm-1. SCs were labeled with 5 μM BODIPY-gal-sulfatide; incubated with Lm-1, BSA, or nonpolymerizing AEBSF-Lm-1 (10 μg/ml) for 1 h; lysed with 1% Triton X-100; extracted; centrifuged to remove insoluble material; and were immunoprecipitated with anti-E1′ Lm antibody. The precipitate was solubilized with 1% SDS and BODIPY fluorescence was determined (excitation, 490 nm; emission, 513 nm). (h) Lm–LG domain and polymerization are required for BM. SCs were incubated for 1 h with 10 μg/ml of recombinant Lm-1 (rLm-1) or 10 μg/ml Lm-1 lacking G-domain (rLm-1ΔG) and immunostained for Lm-γ1. SCs were also incubated with either native Lm-1 (Lm-1, 10 μg/ml), alone, or in the presence of competing recombinant E3 (rE3 WT; 100 μg/ml); with E3 mutant G (rE3 Mut G; 100 μg/ml) to block LG-modular interactions; or with fragment E1′ to block Lm polymerization (AEBSF-E1′ is control).
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Related In: Results  -  Collection

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fig2: Lm 1–sulfatide associations on cultured SCs. (a) SCs that adhered to plastic were incubated with 10 μg/ml Lm-1 added to medium. Lm-α1 (red; DAPI, blue) initially accumulated in diffusely dispersed aggregates and then condensed into a compact, sheetlike pattern. Dotted lines indicate outer cell borders. (b) Nidogen-1 (red) and type IV collagen (red) were detected in a colocalizing pattern when treated with Lm (green). (c) Gal-sulfatide (nonpermeabilized cells, Cy3, red), which was initially diffuse, condensed and colocalized with Lm-α1 (FITC, green). (d and e) Lm-1 did not accumulate (d) on SCs when incubated for 1 h (10 μg/ml) in the presence of malarial CS protein (150 μg/ml), whereas it accumulated (e) on SCs treated with a mixture (h/hp) of heparitinase (0.1 U/ml) and heparinase (1 U/ml). (f) Sulfatide and Lm-α1 were largely undetectable if 50 U/ml arylsulfatase was added to SC medium. However, if cells were washed and incubated with 10 μM gal-sulfatide/BSA (for 30 min) followed by 10 μg/ml Lm-1, sulfatide and Lm immunofluorescence were restored. (g) Cell gal-sulfatide is bound to Lm-1. SCs were labeled with 5 μM BODIPY-gal-sulfatide; incubated with Lm-1, BSA, or nonpolymerizing AEBSF-Lm-1 (10 μg/ml) for 1 h; lysed with 1% Triton X-100; extracted; centrifuged to remove insoluble material; and were immunoprecipitated with anti-E1′ Lm antibody. The precipitate was solubilized with 1% SDS and BODIPY fluorescence was determined (excitation, 490 nm; emission, 513 nm). (h) Lm–LG domain and polymerization are required for BM. SCs were incubated for 1 h with 10 μg/ml of recombinant Lm-1 (rLm-1) or 10 μg/ml Lm-1 lacking G-domain (rLm-1ΔG) and immunostained for Lm-γ1. SCs were also incubated with either native Lm-1 (Lm-1, 10 μg/ml), alone, or in the presence of competing recombinant E3 (rE3 WT; 100 μg/ml); with E3 mutant G (rE3 Mut G; 100 μg/ml) to block LG-modular interactions; or with fragment E1′ to block Lm polymerization (AEBSF-E1′ is control).
Mentions: Cultured rat sciatic nerve SCs did not possess detectable Lm-γ1 or α2 epitopes on their exposed surfaces or, as determined in immunoblots in cell extracts and conditioned medium (unpublished data), evidence that the cells had lost the ability to synthesize relevant Lms. However, these cells assemble a BM when incubated with exogenous Lm-1 or -2 (Tsiper and Yurchenco, 2002). When subconfluent cells (passage 25–32) were treated with 10 μg/ml Lm-1, Lm epitope, which was initially distributed diffusely (Fig. 2 a), condensed within 30 min into large confluent regions in which the aggregate edges were retracted from one or more of the outer cell borders, allowing for the assessment of colocalization with other components. A similar time course was observed with recombinant Lm-2 (unpublished data). The Lm pattern colocalized with endogenous nidogen-1 and type IV collagen (Fig. 2 b). Accumulation of the latter component was not appreciated previously (Tsiper and Yurchenco, 2002), likely because a short accumulation time in medium (1 h) during culturing was used in that study to permit detection of collagen as now seen with overnight accumulation.

Bottom Line: This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

ABSTRACT
Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.

Show MeSH
Related in: MedlinePlus