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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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Ultrastructural evidence of cell-surface BM assembly. (top four panels) SCs were prepared for EM untreated (NT), treated with 20 μg/ml Lm-1 for 1 h, treated with Lm-1 in the presence of 50 U/ml arylsulfatase (sulfatase, s'ase), or treated with Lm-1 plus 250 U/ml of bacterial collagenase (col'ase). (bottom three panels) MEFs were treated with 40 μg/ml Lm-1 for 1 h, loaded with 10 μM sulfatide (sulf), and treated with Lm-1 or treated with Lm in the presence of arylsulfatase. Arrows indicate BM and arrowheads delimit plasma membrane. Cross sections through cells that are adhered to plastic with exposed cell surfaces are shown above. (right) Degree of BM coverage is determined from different cross-sectional levels (mean ± SEM; SCs, n = 3–4; fibroblasts, n = 5–7).
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fig4: Ultrastructural evidence of cell-surface BM assembly. (top four panels) SCs were prepared for EM untreated (NT), treated with 20 μg/ml Lm-1 for 1 h, treated with Lm-1 in the presence of 50 U/ml arylsulfatase (sulfatase, s'ase), or treated with Lm-1 plus 250 U/ml of bacterial collagenase (col'ase). (bottom three panels) MEFs were treated with 40 μg/ml Lm-1 for 1 h, loaded with 10 μM sulfatide (sulf), and treated with Lm-1 or treated with Lm in the presence of arylsulfatase. Arrows indicate BM and arrowheads delimit plasma membrane. Cross sections through cells that are adhered to plastic with exposed cell surfaces are shown above. (right) Degree of BM coverage is determined from different cross-sectional levels (mean ± SEM; SCs, n = 3–4; fibroblasts, n = 5–7).

Mentions: SCs and sulfatide-treated MEFs that were maintained in confluent cultures and incubated with Lm-1 were found to achieve maximal Lm surface immunostaining with 20–40 μg/ml of protein, extending over almost the entire surface. The SCs and MEFs treated under these conditions were examined by transmission EM (Fig. 4). After incubation with Lm-1, nearly the entire SC surface was covered by a continuous BM deposit (lamina densa overlying a lamina lucida) that was absent in untreated cells or in cells treated with arylsulfatase. Sulfatide loading of the arylsulfatase-treated SCs restored the linear ECM deposit. The deposit was dependent primarily on Lm deposition rather than type IV collagen and was shown by incubating the SCs in the presence of Lm-1 and bacterial collagenase (which eliminated detectable type IV collagen immunostaining). MEFs had almost no extracellular deposits either without or with Lm-1 incubation. However, if cells were first loaded with sulfatide, and then treated with Lm-1, a continuous BM was noted in all sections examined. Treatment of the MEFs with arylsulfatase after sulfatide loading prevented the appearance of an ECM deposit on the exposed fibroblast plasma membrane.


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)

Ultrastructural evidence of cell-surface BM assembly. (top four panels) SCs were prepared for EM untreated (NT), treated with 20 μg/ml Lm-1 for 1 h, treated with Lm-1 in the presence of 50 U/ml arylsulfatase (sulfatase, s'ase), or treated with Lm-1 plus 250 U/ml of bacterial collagenase (col'ase). (bottom three panels) MEFs were treated with 40 μg/ml Lm-1 for 1 h, loaded with 10 μM sulfatide (sulf), and treated with Lm-1 or treated with Lm in the presence of arylsulfatase. Arrows indicate BM and arrowheads delimit plasma membrane. Cross sections through cells that are adhered to plastic with exposed cell surfaces are shown above. (right) Degree of BM coverage is determined from different cross-sectional levels (mean ± SEM; SCs, n = 3–4; fibroblasts, n = 5–7).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171891&req=5

fig4: Ultrastructural evidence of cell-surface BM assembly. (top four panels) SCs were prepared for EM untreated (NT), treated with 20 μg/ml Lm-1 for 1 h, treated with Lm-1 in the presence of 50 U/ml arylsulfatase (sulfatase, s'ase), or treated with Lm-1 plus 250 U/ml of bacterial collagenase (col'ase). (bottom three panels) MEFs were treated with 40 μg/ml Lm-1 for 1 h, loaded with 10 μM sulfatide (sulf), and treated with Lm-1 or treated with Lm in the presence of arylsulfatase. Arrows indicate BM and arrowheads delimit plasma membrane. Cross sections through cells that are adhered to plastic with exposed cell surfaces are shown above. (right) Degree of BM coverage is determined from different cross-sectional levels (mean ± SEM; SCs, n = 3–4; fibroblasts, n = 5–7).
Mentions: SCs and sulfatide-treated MEFs that were maintained in confluent cultures and incubated with Lm-1 were found to achieve maximal Lm surface immunostaining with 20–40 μg/ml of protein, extending over almost the entire surface. The SCs and MEFs treated under these conditions were examined by transmission EM (Fig. 4). After incubation with Lm-1, nearly the entire SC surface was covered by a continuous BM deposit (lamina densa overlying a lamina lucida) that was absent in untreated cells or in cells treated with arylsulfatase. Sulfatide loading of the arylsulfatase-treated SCs restored the linear ECM deposit. The deposit was dependent primarily on Lm deposition rather than type IV collagen and was shown by incubating the SCs in the presence of Lm-1 and bacterial collagenase (which eliminated detectable type IV collagen immunostaining). MEFs had almost no extracellular deposits either without or with Lm-1 incubation. However, if cells were first loaded with sulfatide, and then treated with Lm-1, a continuous BM was noted in all sections examined. Treatment of the MEFs with arylsulfatase after sulfatide loading prevented the appearance of an ECM deposit on the exposed fibroblast plasma membrane.

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