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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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Schwann cell c-Src is activated in response to Lm-1. (a) Transient Src activation in response to Lm: SCs were incubated with 10 μg/ml Lm-1, harvested at the indicated times, lysed, and analyzed for c-Src-PY416 and c-Src. Time course immunoblot and densitometry plot of pSrc/total Src ratio are shown. (b) Fyn activation in response to Lm: lysates from SCs treated as above for 1 h were immunoprecipitated with Fyn-specific antibody followed by immunoblotting with phospho-Src (PY416) antibody that also detects pFyn. (c) Src activation depends on the presence of gal-sulfatide. SCs were treated with Lm-1 as above for 1 h in the presence (+) or absence (−) of 50 U/ml arylsulfatase and analyzed for c-Src phosphorylation. (d) c-Src coimmunoprecipitates with β-DG. SCs untreated or treated with 10 μg/ml Lm-1 for 1 h were extracted with 1% Triton X-100–Tris buffer. Cell lysates were immunoprecipitated with anti–β-DG antibody and the immunoprecipitates were subjected to immunoblot analysis with c-Src-–specific antibody. (e) SCs were untreated or treated with 10 μg/ml Lm-1 for 1 h and immunostained for Lm-γ1, Src, and Src-PY416 (pSrc). Diffusely distributed Src immunofluorescence coalesces into dense plaques that overlap with Lm immunofluorescence after Lm-1 treatment, whereas most of Src-PY416 is associated with the nucleus (arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points). (f) Anti–α-DG antibody IIH6 inhibits Src phosphorylation, whereas anti–β1-integrin (Ha2/5) does not. The bar graph shows the phospho-Src/total Src ratio based on the immunoblot densitometry. (g) Lm-1 fragments E1′ and E3, but not E8 or E3 mutG (which lack a sulfatide-binding sequence), block c-Src phosphorylation. SCs were incubated for 1 h with Lm-1 in the presence of 100 μg/ml BSA, 250 μg/ml E8, 250 μg/ml E1′, 100 μg/ml rE3, or 100 μg/ml rE3 mutant G. Cells were washed, lysed in 1% SDS-Tris buffer, and immunoblotted.
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fig6: Schwann cell c-Src is activated in response to Lm-1. (a) Transient Src activation in response to Lm: SCs were incubated with 10 μg/ml Lm-1, harvested at the indicated times, lysed, and analyzed for c-Src-PY416 and c-Src. Time course immunoblot and densitometry plot of pSrc/total Src ratio are shown. (b) Fyn activation in response to Lm: lysates from SCs treated as above for 1 h were immunoprecipitated with Fyn-specific antibody followed by immunoblotting with phospho-Src (PY416) antibody that also detects pFyn. (c) Src activation depends on the presence of gal-sulfatide. SCs were treated with Lm-1 as above for 1 h in the presence (+) or absence (−) of 50 U/ml arylsulfatase and analyzed for c-Src phosphorylation. (d) c-Src coimmunoprecipitates with β-DG. SCs untreated or treated with 10 μg/ml Lm-1 for 1 h were extracted with 1% Triton X-100–Tris buffer. Cell lysates were immunoprecipitated with anti–β-DG antibody and the immunoprecipitates were subjected to immunoblot analysis with c-Src-–specific antibody. (e) SCs were untreated or treated with 10 μg/ml Lm-1 for 1 h and immunostained for Lm-γ1, Src, and Src-PY416 (pSrc). Diffusely distributed Src immunofluorescence coalesces into dense plaques that overlap with Lm immunofluorescence after Lm-1 treatment, whereas most of Src-PY416 is associated with the nucleus (arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points). (f) Anti–α-DG antibody IIH6 inhibits Src phosphorylation, whereas anti–β1-integrin (Ha2/5) does not. The bar graph shows the phospho-Src/total Src ratio based on the immunoblot densitometry. (g) Lm-1 fragments E1′ and E3, but not E8 or E3 mutG (which lack a sulfatide-binding sequence), block c-Src phosphorylation. SCs were incubated for 1 h with Lm-1 in the presence of 100 μg/ml BSA, 250 μg/ml E8, 250 μg/ml E1′, 100 μg/ml rE3, or 100 μg/ml rE3 mutant G. Cells were washed, lysed in 1% SDS-Tris buffer, and immunoblotted.

Mentions: The possibility that anchorage-dependent BM assembly enabled SC signaling was investigated (Fig. 6). c-Src became tyrosine phosphorylated at its activating residue Y416 (Fig. 6 a), beginning within 15 min of Lm treatment and peaking by 30–60 min. Fyn, another Src family member present in SCs in which the activation-specific antibody shows cross-reactivity, was also activated by Lm treatment (Fig. 6 b). If the SCs were incubated with arylsulfatase, Lm treatment failed to induce Src activation (Fig. 6 c). Immunoprecipitation of SC detergent lysates with β-DG antibody followed by immunoblotting with c-Src–specific antibody revealed that c-Src was associated with the DG-containing complex regardless of whether or not the cells were Lm treated (Fig. 6 d). c-Src underwent a transition from a dispersed pattern to a condensed one, colocalizing with Lm (Fig. 6 e). pY416-Src, on the other hand, was only weakly detected in untreated SCs and strongly detected in Lm-treated SCs. The epitope, although increased throughout the cells, was seen to be predominantly associated with nuclei, and pSrc was not detected in soluble detergent lysates of the cells (Fig. 6 e and not depicted). The data suggest that Lm-activated Src translocates to the nucleus; however, alternative mechanisms cannot be ruled out. Nuclear pSrc was also detected in a patchy distribution of sciatic nerves between P1 and P7 (Fig. 1).


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)

Schwann cell c-Src is activated in response to Lm-1. (a) Transient Src activation in response to Lm: SCs were incubated with 10 μg/ml Lm-1, harvested at the indicated times, lysed, and analyzed for c-Src-PY416 and c-Src. Time course immunoblot and densitometry plot of pSrc/total Src ratio are shown. (b) Fyn activation in response to Lm: lysates from SCs treated as above for 1 h were immunoprecipitated with Fyn-specific antibody followed by immunoblotting with phospho-Src (PY416) antibody that also detects pFyn. (c) Src activation depends on the presence of gal-sulfatide. SCs were treated with Lm-1 as above for 1 h in the presence (+) or absence (−) of 50 U/ml arylsulfatase and analyzed for c-Src phosphorylation. (d) c-Src coimmunoprecipitates with β-DG. SCs untreated or treated with 10 μg/ml Lm-1 for 1 h were extracted with 1% Triton X-100–Tris buffer. Cell lysates were immunoprecipitated with anti–β-DG antibody and the immunoprecipitates were subjected to immunoblot analysis with c-Src-–specific antibody. (e) SCs were untreated or treated with 10 μg/ml Lm-1 for 1 h and immunostained for Lm-γ1, Src, and Src-PY416 (pSrc). Diffusely distributed Src immunofluorescence coalesces into dense plaques that overlap with Lm immunofluorescence after Lm-1 treatment, whereas most of Src-PY416 is associated with the nucleus (arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points). (f) Anti–α-DG antibody IIH6 inhibits Src phosphorylation, whereas anti–β1-integrin (Ha2/5) does not. The bar graph shows the phospho-Src/total Src ratio based on the immunoblot densitometry. (g) Lm-1 fragments E1′ and E3, but not E8 or E3 mutG (which lack a sulfatide-binding sequence), block c-Src phosphorylation. SCs were incubated for 1 h with Lm-1 in the presence of 100 μg/ml BSA, 250 μg/ml E8, 250 μg/ml E1′, 100 μg/ml rE3, or 100 μg/ml rE3 mutant G. Cells were washed, lysed in 1% SDS-Tris buffer, and immunoblotted.
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fig6: Schwann cell c-Src is activated in response to Lm-1. (a) Transient Src activation in response to Lm: SCs were incubated with 10 μg/ml Lm-1, harvested at the indicated times, lysed, and analyzed for c-Src-PY416 and c-Src. Time course immunoblot and densitometry plot of pSrc/total Src ratio are shown. (b) Fyn activation in response to Lm: lysates from SCs treated as above for 1 h were immunoprecipitated with Fyn-specific antibody followed by immunoblotting with phospho-Src (PY416) antibody that also detects pFyn. (c) Src activation depends on the presence of gal-sulfatide. SCs were treated with Lm-1 as above for 1 h in the presence (+) or absence (−) of 50 U/ml arylsulfatase and analyzed for c-Src phosphorylation. (d) c-Src coimmunoprecipitates with β-DG. SCs untreated or treated with 10 μg/ml Lm-1 for 1 h were extracted with 1% Triton X-100–Tris buffer. Cell lysates were immunoprecipitated with anti–β-DG antibody and the immunoprecipitates were subjected to immunoblot analysis with c-Src-–specific antibody. (e) SCs were untreated or treated with 10 μg/ml Lm-1 for 1 h and immunostained for Lm-γ1, Src, and Src-PY416 (pSrc). Diffusely distributed Src immunofluorescence coalesces into dense plaques that overlap with Lm immunofluorescence after Lm-1 treatment, whereas most of Src-PY416 is associated with the nucleus (arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points). (f) Anti–α-DG antibody IIH6 inhibits Src phosphorylation, whereas anti–β1-integrin (Ha2/5) does not. The bar graph shows the phospho-Src/total Src ratio based on the immunoblot densitometry. (g) Lm-1 fragments E1′ and E3, but not E8 or E3 mutG (which lack a sulfatide-binding sequence), block c-Src phosphorylation. SCs were incubated for 1 h with Lm-1 in the presence of 100 μg/ml BSA, 250 μg/ml E8, 250 μg/ml E1′, 100 μg/ml rE3, or 100 μg/ml rE3 mutant G. Cells were washed, lysed in 1% SDS-Tris buffer, and immunoblotted.
Mentions: The possibility that anchorage-dependent BM assembly enabled SC signaling was investigated (Fig. 6). c-Src became tyrosine phosphorylated at its activating residue Y416 (Fig. 6 a), beginning within 15 min of Lm treatment and peaking by 30–60 min. Fyn, another Src family member present in SCs in which the activation-specific antibody shows cross-reactivity, was also activated by Lm treatment (Fig. 6 b). If the SCs were incubated with arylsulfatase, Lm treatment failed to induce Src activation (Fig. 6 c). Immunoprecipitation of SC detergent lysates with β-DG antibody followed by immunoblotting with c-Src–specific antibody revealed that c-Src was associated with the DG-containing complex regardless of whether or not the cells were Lm treated (Fig. 6 d). c-Src underwent a transition from a dispersed pattern to a condensed one, colocalizing with Lm (Fig. 6 e). pY416-Src, on the other hand, was only weakly detected in untreated SCs and strongly detected in Lm-treated SCs. The epitope, although increased throughout the cells, was seen to be predominantly associated with nuclei, and pSrc was not detected in soluble detergent lysates of the cells (Fig. 6 e and not depicted). The data suggest that Lm-activated Src translocates to the nucleus; however, alternative mechanisms cannot be ruled out. Nuclear pSrc was also detected in a patchy distribution of sciatic nerves between P1 and P7 (Fig. 1).

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