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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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Sulfatide loading renders fibroblasts competent for BM assembly. (a) Mouse embryonic fibroblasts were incubated for 1 h with 10 μg/ml Lm-1, treated with 10 μM sulfatide/BSA (1:1 molar ratio), and incubated with 10 μg/ml Lm-1, or (b) treated with sulfatide/BSA in the presence of 50 U/ml arylsulfatase, followed by incubation with Lm-1. Lm, type IV collagen, and nidogen were detected in a colocalized pattern on fibroblast surfaces only if they were first loaded with sulfatide but not treated with arylsulfatase. The dotted line indicates outer cell borders. (c) Adherent fibroblasts were treated with BSA coupled to GM1-ganglioside, gal-sulfatide, glc-sulfatide, or cholesterol-3-sulfate and treated for 1 h with exogenous Lm-1 (10 μg/ml) followed by Lm-α1 immunostaining. Only the sulfatides enabled Lm accumulation.
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fig3: Sulfatide loading renders fibroblasts competent for BM assembly. (a) Mouse embryonic fibroblasts were incubated for 1 h with 10 μg/ml Lm-1, treated with 10 μM sulfatide/BSA (1:1 molar ratio), and incubated with 10 μg/ml Lm-1, or (b) treated with sulfatide/BSA in the presence of 50 U/ml arylsulfatase, followed by incubation with Lm-1. Lm, type IV collagen, and nidogen were detected in a colocalized pattern on fibroblast surfaces only if they were first loaded with sulfatide but not treated with arylsulfatase. The dotted line indicates outer cell borders. (c) Adherent fibroblasts were treated with BSA coupled to GM1-ganglioside, gal-sulfatide, glc-sulfatide, or cholesterol-3-sulfate and treated for 1 h with exogenous Lm-1 (10 μg/ml) followed by Lm-α1 immunostaining. Only the sulfatides enabled Lm accumulation.

Mentions: Fibroblasts produce several BM macromolecules but typically do not assemble BM on their cell surfaces, contributing their molecules instead to adjacent BMs (Cornbrooks et al., 1983; Marinkovich et al., 1993). We reasoned that fibroblasts may lack a molecule that anchors Lms to their surface. Mouse embryonic lung fibroblasts (MEFs) did not express detectable γ1-Lm but secreted type IV collagen and nidogen-1 into the culture medium (determined by antibody immunofluorescence of detergent-permeabilized and nonpermeabilized cells with BM-specific antibodies and conditioned medium immunoblots). These cells were intercalated with sulfatides and evaluated for their ability to assemble a BM (Fig. 3). After incubation of untreated MEFs with Lm-1, little Lm or other BM component epitopes were detected on cell surfaces. However, if the MEFs were first loaded with gal-sulfatide, then the added Lm-1 accumulated on their surfaces (Fig. 3 a). Nidogen-1 and type IV collagen epitopes were also now detected on the exposed fibroblast surfaces. If the gal-sulfatide–treated MEFs were subsequently incubated with arylsulfatase and then incubated with Lm-1, Lm, then cell-surface nidogen and type IV collagen were not detected (Fig. 3 b).


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)

Sulfatide loading renders fibroblasts competent for BM assembly. (a) Mouse embryonic fibroblasts were incubated for 1 h with 10 μg/ml Lm-1, treated with 10 μM sulfatide/BSA (1:1 molar ratio), and incubated with 10 μg/ml Lm-1, or (b) treated with sulfatide/BSA in the presence of 50 U/ml arylsulfatase, followed by incubation with Lm-1. Lm, type IV collagen, and nidogen were detected in a colocalized pattern on fibroblast surfaces only if they were first loaded with sulfatide but not treated with arylsulfatase. The dotted line indicates outer cell borders. (c) Adherent fibroblasts were treated with BSA coupled to GM1-ganglioside, gal-sulfatide, glc-sulfatide, or cholesterol-3-sulfate and treated for 1 h with exogenous Lm-1 (10 μg/ml) followed by Lm-α1 immunostaining. Only the sulfatides enabled Lm accumulation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171891&req=5

fig3: Sulfatide loading renders fibroblasts competent for BM assembly. (a) Mouse embryonic fibroblasts were incubated for 1 h with 10 μg/ml Lm-1, treated with 10 μM sulfatide/BSA (1:1 molar ratio), and incubated with 10 μg/ml Lm-1, or (b) treated with sulfatide/BSA in the presence of 50 U/ml arylsulfatase, followed by incubation with Lm-1. Lm, type IV collagen, and nidogen were detected in a colocalized pattern on fibroblast surfaces only if they were first loaded with sulfatide but not treated with arylsulfatase. The dotted line indicates outer cell borders. (c) Adherent fibroblasts were treated with BSA coupled to GM1-ganglioside, gal-sulfatide, glc-sulfatide, or cholesterol-3-sulfate and treated for 1 h with exogenous Lm-1 (10 μg/ml) followed by Lm-α1 immunostaining. Only the sulfatides enabled Lm accumulation.
Mentions: Fibroblasts produce several BM macromolecules but typically do not assemble BM on their cell surfaces, contributing their molecules instead to adjacent BMs (Cornbrooks et al., 1983; Marinkovich et al., 1993). We reasoned that fibroblasts may lack a molecule that anchors Lms to their surface. Mouse embryonic lung fibroblasts (MEFs) did not express detectable γ1-Lm but secreted type IV collagen and nidogen-1 into the culture medium (determined by antibody immunofluorescence of detergent-permeabilized and nonpermeabilized cells with BM-specific antibodies and conditioned medium immunoblots). These cells were intercalated with sulfatides and evaluated for their ability to assemble a BM (Fig. 3). After incubation of untreated MEFs with Lm-1, little Lm or other BM component epitopes were detected on cell surfaces. However, if the MEFs were first loaded with gal-sulfatide, then the added Lm-1 accumulated on their surfaces (Fig. 3 a). Nidogen-1 and type IV collagen epitopes were also now detected on the exposed fibroblast surfaces. If the gal-sulfatide–treated MEFs were subsequently incubated with arylsulfatase and then incubated with Lm-1, Lm, then cell-surface nidogen and type IV collagen were not detected (Fig. 3 b).

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