Limits...
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: 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.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.

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

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171891&req=5

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171891&req=5

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: 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.Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces.

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