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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: 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.

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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: 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