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: 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

β1-integrin association with Lm and FAK phosphorylation. SCs or ES cell–derived fibroblasts were cultured as aggregates in poly-HEMA–coated dishes for 24 h in the presence or absence of 10 μg/ml Lm-1. (a) Cryosections of SC aggregates were immunostained for Lm-α1 and β1-integrin. The integrin colocalized with Lm at cell edges. Arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points. (b) Lm-1 induces FAK phosphorylation in suspended SCs. SC aggregates were maintained overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. The aggregates were washed, lysed in 1% Triton X-100–Tris buffer, immunoprecipitated with anti-FAK antibody (IP: FAK), and immunoblotted with antiphosphotyrosine (Py) antibody. The immunoblot of total FAK was used as the control. (c) Lm-1 induction of FAK phosphorylation in fibroblasts is dependent on β1-integrin. GD25 ES cell–derived fibroblasts and GD25 cells transduced with a β1-integrin expression vector (control) were loaded with sulfatide and grown in suspension overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. Cell extracts were analyzed for tyrosine phosphorylation of FAK and total FAK as described above. Lm-1 treatment increased FAK phosphorylation level in the control β1AGD25 cells but not the β1-integrin–deficient GD25 fibroblasts.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171891&req=5

fig9: β1-integrin association with Lm and FAK phosphorylation. SCs or ES cell–derived fibroblasts were cultured as aggregates in poly-HEMA–coated dishes for 24 h in the presence or absence of 10 μg/ml Lm-1. (a) Cryosections of SC aggregates were immunostained for Lm-α1 and β1-integrin. The integrin colocalized with Lm at cell edges. Arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points. (b) Lm-1 induces FAK phosphorylation in suspended SCs. SC aggregates were maintained overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. The aggregates were washed, lysed in 1% Triton X-100–Tris buffer, immunoprecipitated with anti-FAK antibody (IP: FAK), and immunoblotted with antiphosphotyrosine (Py) antibody. The immunoblot of total FAK was used as the control. (c) Lm-1 induction of FAK phosphorylation in fibroblasts is dependent on β1-integrin. GD25 ES cell–derived fibroblasts and GD25 cells transduced with a β1-integrin expression vector (control) were loaded with sulfatide and grown in suspension overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. Cell extracts were analyzed for tyrosine phosphorylation of FAK and total FAK as described above. Lm-1 treatment increased FAK phosphorylation level in the control β1AGD25 cells but not the β1-integrin–deficient GD25 fibroblasts.

Mentions: In our analysis of Lm on adherent SCs (Tsiper and Yurchenco, 2002; this study), we did not observe a colocalization of Lms with β1-integrin and found that BM assembly could occur in the absence of β1-integrin. Although this was useful in ruling out a role for integrin in BM assembly, in agreement with observations made in embryoid bodies and supported by genetic studies (Li et al., 2003), the lack of integrin colocalization was nonetheless puzzling, as this was known to occur in developing peripheral nerves (Previtali et al., 2003). Because β1-integrin staining was largely confined to the basal adherent side of the SCs, we reasoned that adhesion to the plastic substrate may have recruited most of the integrin, leaving little to interact with the upper exposed cell surface. We therefore evaluated SCs grown in suspension culture. When Lm-1 was added to the medium, it accumulated between the cells of the cluster (Fig. 9 a) and colocalized with β1-integrin. Furthermore, tyrosine phosphorylation of FAK greatly increased after Lm treatment overnight under suspension conditions (Fig. 9 b). Substrate-adherent cells, on the other hand, revealed constitutively high levels of pFAK, likely obscuring a Lm response (unpublished data). To further examine the relationship between β1-integrin and FAK phosphorylation, fibroblasts isolated from mouse ES cells that were for β1-integrin (DG25) were compared with ones that had been transfected with a construct that allowed the cells to express β1-integrin (β1AGD25). These cells were first sulfatide loaded, and then grown in suspension. Both in the absence or presence of exogenous 10 μg/ml Lm-1, the fibroblasts formed spherical aggregates. After incubation for 18 h, the cells were harvested, extracted with detergent, precipitated with FAK antibody, and evaluated for tyrosine phosphorylation in immunoblots (Fig. 9 c). Low levels of pFAK were detected in the untreated controls. Treatment of integrin-expressing cells with Lm resulted in about a threefold increase of phosphorylation without a significant change in total FAK. In contrast, treatment of the β1-integrin– cells with Lm caused no detectable increase in pFAK.


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)

β1-integrin association with Lm and FAK phosphorylation. SCs or ES cell–derived fibroblasts were cultured as aggregates in poly-HEMA–coated dishes for 24 h in the presence or absence of 10 μg/ml Lm-1. (a) Cryosections of SC aggregates were immunostained for Lm-α1 and β1-integrin. The integrin colocalized with Lm at cell edges. Arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points. (b) Lm-1 induces FAK phosphorylation in suspended SCs. SC aggregates were maintained overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. The aggregates were washed, lysed in 1% Triton X-100–Tris buffer, immunoprecipitated with anti-FAK antibody (IP: FAK), and immunoblotted with antiphosphotyrosine (Py) antibody. The immunoblot of total FAK was used as the control. (c) Lm-1 induction of FAK phosphorylation in fibroblasts is dependent on β1-integrin. GD25 ES cell–derived fibroblasts and GD25 cells transduced with a β1-integrin expression vector (control) were loaded with sulfatide and grown in suspension overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. Cell extracts were analyzed for tyrosine phosphorylation of FAK and total FAK as described above. Lm-1 treatment increased FAK phosphorylation level in the control β1AGD25 cells but not the β1-integrin–deficient GD25 fibroblasts.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: β1-integrin association with Lm and FAK phosphorylation. SCs or ES cell–derived fibroblasts were cultured as aggregates in poly-HEMA–coated dishes for 24 h in the presence or absence of 10 μg/ml Lm-1. (a) Cryosections of SC aggregates were immunostained for Lm-α1 and β1-integrin. The integrin colocalized with Lm at cell edges. Arrowheads indicate colocalizations of antibody immunofluorescence between paired panels, establishing the relationship at various points. (b) Lm-1 induces FAK phosphorylation in suspended SCs. SC aggregates were maintained overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. The aggregates were washed, lysed in 1% Triton X-100–Tris buffer, immunoprecipitated with anti-FAK antibody (IP: FAK), and immunoblotted with antiphosphotyrosine (Py) antibody. The immunoblot of total FAK was used as the control. (c) Lm-1 induction of FAK phosphorylation in fibroblasts is dependent on β1-integrin. GD25 ES cell–derived fibroblasts and GD25 cells transduced with a β1-integrin expression vector (control) were loaded with sulfatide and grown in suspension overnight in the absence (−) or presence (+) of 10 μg/ml Lm-1. Cell extracts were analyzed for tyrosine phosphorylation of FAK and total FAK as described above. Lm-1 treatment increased FAK phosphorylation level in the control β1AGD25 cells but not the β1-integrin–deficient GD25 fibroblasts.
Mentions: In our analysis of Lm on adherent SCs (Tsiper and Yurchenco, 2002; this study), we did not observe a colocalization of Lms with β1-integrin and found that BM assembly could occur in the absence of β1-integrin. Although this was useful in ruling out a role for integrin in BM assembly, in agreement with observations made in embryoid bodies and supported by genetic studies (Li et al., 2003), the lack of integrin colocalization was nonetheless puzzling, as this was known to occur in developing peripheral nerves (Previtali et al., 2003). Because β1-integrin staining was largely confined to the basal adherent side of the SCs, we reasoned that adhesion to the plastic substrate may have recruited most of the integrin, leaving little to interact with the upper exposed cell surface. We therefore evaluated SCs grown in suspension culture. When Lm-1 was added to the medium, it accumulated between the cells of the cluster (Fig. 9 a) and colocalized with β1-integrin. Furthermore, tyrosine phosphorylation of FAK greatly increased after Lm treatment overnight under suspension conditions (Fig. 9 b). Substrate-adherent cells, on the other hand, revealed constitutively high levels of pFAK, likely obscuring a Lm response (unpublished data). To further examine the relationship between β1-integrin and FAK phosphorylation, fibroblasts isolated from mouse ES cells that were for β1-integrin (DG25) were compared with ones that had been transfected with a construct that allowed the cells to express β1-integrin (β1AGD25). These cells were first sulfatide loaded, and then grown in suspension. Both in the absence or presence of exogenous 10 μg/ml Lm-1, the fibroblasts formed spherical aggregates. After incubation for 18 h, the cells were harvested, extracted with detergent, precipitated with FAK antibody, and evaluated for tyrosine phosphorylation in immunoblots (Fig. 9 c). Low levels of pFAK were detected in the untreated controls. Treatment of integrin-expressing cells with Lm resulted in about a threefold increase of phosphorylation without a significant change in total FAK. In contrast, treatment of the β1-integrin– cells with Lm caused no detectable increase in pFAK.

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