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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 binding to sulfatide recruits DG and utrophin in SCs. (a) SCs, untreated (NT), treated with 10 μg/ml Lm-1 (for 1 h), treated with 50 U/ml arylsulfatase followed by Lm-1, or treated with arylsulfatase and then loaded with sulfatide followed by Lm-1, were immunostained for the indicated components (dots indicate cell borders). α-DG and utrophin condensation (and Lm colocalization) were prevented if the cells were treated with arylsulfatase. (b) Lm-1–DG association. SCs untreated (−) or treated (+) with 10 μg/ml Lm-1 (for 1 h) were washed and extracted with 1% Triton X-100. The cell lysates were immunoprecipitated (IP) with anti–β-DG antibody and probed for Lm-α1 chain. The input cell lysates were also immunoblotted (IB) with anti–β-DG antibody. Exogenous Lm was detected in the precipitates when added to the medium. (c and d) Recruitment of utrophin to DG. The immunoprecipitates formed with anti–β-DG antibody were subjected to immunoblotting with utrophin-specific antibody (c). Utrophin recruitment was blocked by antibody IIH6 (d).
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fig5: Lm-1 binding to sulfatide recruits DG and utrophin in SCs. (a) SCs, untreated (NT), treated with 10 μg/ml Lm-1 (for 1 h), treated with 50 U/ml arylsulfatase followed by Lm-1, or treated with arylsulfatase and then loaded with sulfatide followed by Lm-1, were immunostained for the indicated components (dots indicate cell borders). α-DG and utrophin condensation (and Lm colocalization) were prevented if the cells were treated with arylsulfatase. (b) Lm-1–DG association. SCs untreated (−) or treated (+) with 10 μg/ml Lm-1 (for 1 h) were washed and extracted with 1% Triton X-100. The cell lysates were immunoprecipitated (IP) with anti–β-DG antibody and probed for Lm-α1 chain. The input cell lysates were also immunoblotted (IB) with anti–β-DG antibody. Exogenous Lm was detected in the precipitates when added to the medium. (c and d) Recruitment of utrophin to DG. The immunoprecipitates formed with anti–β-DG antibody were subjected to immunoblotting with utrophin-specific antibody (c). Utrophin recruitment was blocked by antibody IIH6 (d).

Mentions: The topographical associations of Lm-1 with DG and utrophin were examined in adherent SCs (Fig. 5). Lm-1 treatment (10 μg/ml for 1 h) induced condensation of previously diffusely distributed α-DG and utrophin, confirming earlier observations (Tsiper and Yurchenco, 2002). However, if the cells were also treated with arylsulfatase, their condensation was not observed (Fig. 5 a). Although α-DG is known to bind to Lms (Ervasti and Campbell, 1993), such complexes have not been shown to occur during BM assembly. To evaluate this, SCs were incubated with Lm-1 under the above conditions, detergent extracted, immunoprecipitated with β-DG antibody, and immunoblotted with Lm-α1 antibody (Fig. 5 b). Lm was detected in the Lm-1–treated cell fraction without changing total amount of DG. β-DG–containing immunoprecipitates of SC detergent lysates were also examined for the presence of utrophin in immunoblots (Fig. 5, c and d). Utrophin was seen in the DG complex only from lysates extracted from cells treated with Lm-1 and was prevented with DG-blocking antibody, whereas the total amount of cellular utrophin and β-DG remained constant; i.e., utrophin was recruited to a sulfatide-associated Lm–DG complex by Lm interaction with DG.


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 binding to sulfatide recruits DG and utrophin in SCs. (a) SCs, untreated (NT), treated with 10 μg/ml Lm-1 (for 1 h), treated with 50 U/ml arylsulfatase followed by Lm-1, or treated with arylsulfatase and then loaded with sulfatide followed by Lm-1, were immunostained for the indicated components (dots indicate cell borders). α-DG and utrophin condensation (and Lm colocalization) were prevented if the cells were treated with arylsulfatase. (b) Lm-1–DG association. SCs untreated (−) or treated (+) with 10 μg/ml Lm-1 (for 1 h) were washed and extracted with 1% Triton X-100. The cell lysates were immunoprecipitated (IP) with anti–β-DG antibody and probed for Lm-α1 chain. The input cell lysates were also immunoblotted (IB) with anti–β-DG antibody. Exogenous Lm was detected in the precipitates when added to the medium. (c and d) Recruitment of utrophin to DG. The immunoprecipitates formed with anti–β-DG antibody were subjected to immunoblotting with utrophin-specific antibody (c). Utrophin recruitment was blocked by antibody IIH6 (d).
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Related In: Results  -  Collection

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fig5: Lm-1 binding to sulfatide recruits DG and utrophin in SCs. (a) SCs, untreated (NT), treated with 10 μg/ml Lm-1 (for 1 h), treated with 50 U/ml arylsulfatase followed by Lm-1, or treated with arylsulfatase and then loaded with sulfatide followed by Lm-1, were immunostained for the indicated components (dots indicate cell borders). α-DG and utrophin condensation (and Lm colocalization) were prevented if the cells were treated with arylsulfatase. (b) Lm-1–DG association. SCs untreated (−) or treated (+) with 10 μg/ml Lm-1 (for 1 h) were washed and extracted with 1% Triton X-100. The cell lysates were immunoprecipitated (IP) with anti–β-DG antibody and probed for Lm-α1 chain. The input cell lysates were also immunoblotted (IB) with anti–β-DG antibody. Exogenous Lm was detected in the precipitates when added to the medium. (c and d) Recruitment of utrophin to DG. The immunoprecipitates formed with anti–β-DG antibody were subjected to immunoblotting with utrophin-specific antibody (c). Utrophin recruitment was blocked by antibody IIH6 (d).
Mentions: The topographical associations of Lm-1 with DG and utrophin were examined in adherent SCs (Fig. 5). Lm-1 treatment (10 μg/ml for 1 h) induced condensation of previously diffusely distributed α-DG and utrophin, confirming earlier observations (Tsiper and Yurchenco, 2002). However, if the cells were also treated with arylsulfatase, their condensation was not observed (Fig. 5 a). Although α-DG is known to bind to Lms (Ervasti and Campbell, 1993), such complexes have not been shown to occur during BM assembly. To evaluate this, SCs were incubated with Lm-1 under the above conditions, detergent extracted, immunoprecipitated with β-DG antibody, and immunoblotted with Lm-α1 antibody (Fig. 5 b). Lm was detected in the Lm-1–treated cell fraction without changing total amount of DG. β-DG–containing immunoprecipitates of SC detergent lysates were also examined for the presence of utrophin in immunoblots (Fig. 5, c and d). Utrophin was seen in the DG complex only from lysates extracted from cells treated with Lm-1 and was prevented with DG-blocking antibody, whereas the total amount of cellular utrophin and β-DG remained constant; i.e., utrophin was recruited to a sulfatide-associated Lm–DG complex by Lm interaction with DG.

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