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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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Inhibition of Src kinase reduces the Lm-dependent protection from apoptosis. SCs and MEFs grew as spherical clusters in suspension. (a) SCs were cultured in suspension in the absence (−) or presence (+) of Lm-1 (10 μg/ml) plus DG-blocking antibody IIH6 (10 μg/ml), β1-integrin–blocking antibody Ha2/5, or control IgM for 6 h. Cell lysates were subjected to immunoblot analysis for c-Src-Py416 (pSrc) and total c-Src (Src). (b) SCs were grown in suspension in the absence (NT), presence of Lm-1 (10 μg/ml), or Lm-1 + Src kinase inhibitor PP2 (2 μM) in serum-free medium for 24 h, cryosectioned, and stained for Lm-γ1, activated caspase-3, and DAPI (blue). The Lm-treated cell clusters developed linear, pericellular deposits of Lm. Caspase-3 fluorescence that was detected in untreated cells was almost completely absent in the Lm-treated cells. Src inhibition eliminated the Lm-dependent survival effect. (c) Relative quantitation of SC apoptosis. The ratio of cleaved caspase-3 immunostain summed intensities, divided by the DAPI-stained nuclear areas, was plotted (mean ± SEM; n = 10 fields). (d) Lm matrix assembly protects MEFs from apoptosis. Cells untreated or treated with gal-sulfatide were cultured in suspension and incubated for 24 h with 20 μg/ml Lm-1 in serum-free DME. Cell clusters were immunostained for Lm and cleaved caspase-3. Lm-1 accumulated in a linear pattern, mostly on the surface of sulfatide-loaded MEF clusters with the cells showing little apoptosis. In untreated MEFs, Lm-1 did not accumulate in the cell clusters with the cells undergoing substantial apoptosis.
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fig8: Inhibition of Src kinase reduces the Lm-dependent protection from apoptosis. SCs and MEFs grew as spherical clusters in suspension. (a) SCs were cultured in suspension in the absence (−) or presence (+) of Lm-1 (10 μg/ml) plus DG-blocking antibody IIH6 (10 μg/ml), β1-integrin–blocking antibody Ha2/5, or control IgM for 6 h. Cell lysates were subjected to immunoblot analysis for c-Src-Py416 (pSrc) and total c-Src (Src). (b) SCs were grown in suspension in the absence (NT), presence of Lm-1 (10 μg/ml), or Lm-1 + Src kinase inhibitor PP2 (2 μM) in serum-free medium for 24 h, cryosectioned, and stained for Lm-γ1, activated caspase-3, and DAPI (blue). The Lm-treated cell clusters developed linear, pericellular deposits of Lm. Caspase-3 fluorescence that was detected in untreated cells was almost completely absent in the Lm-treated cells. Src inhibition eliminated the Lm-dependent survival effect. (c) Relative quantitation of SC apoptosis. The ratio of cleaved caspase-3 immunostain summed intensities, divided by the DAPI-stained nuclear areas, was plotted (mean ± SEM; n = 10 fields). (d) Lm matrix assembly protects MEFs from apoptosis. Cells untreated or treated with gal-sulfatide were cultured in suspension and incubated for 24 h with 20 μg/ml Lm-1 in serum-free DME. Cell clusters were immunostained for Lm and cleaved caspase-3. Lm-1 accumulated in a linear pattern, mostly on the surface of sulfatide-loaded MEF clusters with the cells showing little apoptosis. In untreated MEFs, Lm-1 did not accumulate in the cell clusters with the cells undergoing substantial apoptosis.

Mentions: To determine whether cell survival was affected by Lm assembly, SCs and MEFs were incubated in suspension in the absence of serum in order to eliminate the possibility that the plastic substrate and serum component were alternative substrate and soluble inhibitors of apoptosis (Fig. 8). Both cells cultured in this manner formed cell aggregates in spherical clusters with scattered single cells. When the suspended SCs were treated with Lm-1 in the presence or absence of receptor-blocking antibodies, c-Src phosphorylation was induced by Lm and was selectively blocked with the DG-specific reagent (Fig. 8 a), which provided evidence that the Lm-initiated process occurred in suspended SCs as well as in adherent SCs in a DG-dependent, but integrin-independent, fashion (Fig. 8 a). SC clusters and sulfatide-loaded fibroblasts were treated with the same concentration of Lm-1 for 24 h. Sections of cell aggregates were immunostained for Lm and activated caspase-3, which is a marker for apoptosis (Fig. 8, b–d). SC and MEF cell clusters that were not treated with Lm-1 developed irregular borders and were stained with antibody specific for activated caspase-3. In those SC and MEF cell clusters treated with Lm-1, the cell edges were smooth, and prominent pericellular and peri-spherule Lm immunostaining was noted, and was accompanied by almost no detectable activated caspase-3. The SC clusters were also treated with Lm-1 and PP2 or SU6656: apoptosis was observed in the presence of Lm-1 at levels approaching those of cells incubated without Lm, implicating Src family kinases as mediators of Lm-dependent cell survival.


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)

Inhibition of Src kinase reduces the Lm-dependent protection from apoptosis. SCs and MEFs grew as spherical clusters in suspension. (a) SCs were cultured in suspension in the absence (−) or presence (+) of Lm-1 (10 μg/ml) plus DG-blocking antibody IIH6 (10 μg/ml), β1-integrin–blocking antibody Ha2/5, or control IgM for 6 h. Cell lysates were subjected to immunoblot analysis for c-Src-Py416 (pSrc) and total c-Src (Src). (b) SCs were grown in suspension in the absence (NT), presence of Lm-1 (10 μg/ml), or Lm-1 + Src kinase inhibitor PP2 (2 μM) in serum-free medium for 24 h, cryosectioned, and stained for Lm-γ1, activated caspase-3, and DAPI (blue). The Lm-treated cell clusters developed linear, pericellular deposits of Lm. Caspase-3 fluorescence that was detected in untreated cells was almost completely absent in the Lm-treated cells. Src inhibition eliminated the Lm-dependent survival effect. (c) Relative quantitation of SC apoptosis. The ratio of cleaved caspase-3 immunostain summed intensities, divided by the DAPI-stained nuclear areas, was plotted (mean ± SEM; n = 10 fields). (d) Lm matrix assembly protects MEFs from apoptosis. Cells untreated or treated with gal-sulfatide were cultured in suspension and incubated for 24 h with 20 μg/ml Lm-1 in serum-free DME. Cell clusters were immunostained for Lm and cleaved caspase-3. Lm-1 accumulated in a linear pattern, mostly on the surface of sulfatide-loaded MEF clusters with the cells showing little apoptosis. In untreated MEFs, Lm-1 did not accumulate in the cell clusters with the cells undergoing substantial apoptosis.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171891&req=5

fig8: Inhibition of Src kinase reduces the Lm-dependent protection from apoptosis. SCs and MEFs grew as spherical clusters in suspension. (a) SCs were cultured in suspension in the absence (−) or presence (+) of Lm-1 (10 μg/ml) plus DG-blocking antibody IIH6 (10 μg/ml), β1-integrin–blocking antibody Ha2/5, or control IgM for 6 h. Cell lysates were subjected to immunoblot analysis for c-Src-Py416 (pSrc) and total c-Src (Src). (b) SCs were grown in suspension in the absence (NT), presence of Lm-1 (10 μg/ml), or Lm-1 + Src kinase inhibitor PP2 (2 μM) in serum-free medium for 24 h, cryosectioned, and stained for Lm-γ1, activated caspase-3, and DAPI (blue). The Lm-treated cell clusters developed linear, pericellular deposits of Lm. Caspase-3 fluorescence that was detected in untreated cells was almost completely absent in the Lm-treated cells. Src inhibition eliminated the Lm-dependent survival effect. (c) Relative quantitation of SC apoptosis. The ratio of cleaved caspase-3 immunostain summed intensities, divided by the DAPI-stained nuclear areas, was plotted (mean ± SEM; n = 10 fields). (d) Lm matrix assembly protects MEFs from apoptosis. Cells untreated or treated with gal-sulfatide were cultured in suspension and incubated for 24 h with 20 μg/ml Lm-1 in serum-free DME. Cell clusters were immunostained for Lm and cleaved caspase-3. Lm-1 accumulated in a linear pattern, mostly on the surface of sulfatide-loaded MEF clusters with the cells showing little apoptosis. In untreated MEFs, Lm-1 did not accumulate in the cell clusters with the cells undergoing substantial apoptosis.
Mentions: To determine whether cell survival was affected by Lm assembly, SCs and MEFs were incubated in suspension in the absence of serum in order to eliminate the possibility that the plastic substrate and serum component were alternative substrate and soluble inhibitors of apoptosis (Fig. 8). Both cells cultured in this manner formed cell aggregates in spherical clusters with scattered single cells. When the suspended SCs were treated with Lm-1 in the presence or absence of receptor-blocking antibodies, c-Src phosphorylation was induced by Lm and was selectively blocked with the DG-specific reagent (Fig. 8 a), which provided evidence that the Lm-initiated process occurred in suspended SCs as well as in adherent SCs in a DG-dependent, but integrin-independent, fashion (Fig. 8 a). SC clusters and sulfatide-loaded fibroblasts were treated with the same concentration of Lm-1 for 24 h. Sections of cell aggregates were immunostained for Lm and activated caspase-3, which is a marker for apoptosis (Fig. 8, b–d). SC and MEF cell clusters that were not treated with Lm-1 developed irregular borders and were stained with antibody specific for activated caspase-3. In those SC and MEF cell clusters treated with Lm-1, the cell edges were smooth, and prominent pericellular and peri-spherule Lm immunostaining was noted, and was accompanied by almost no detectable activated caspase-3. The SC clusters were also treated with Lm-1 and PP2 or SU6656: apoptosis was observed in the presence of Lm-1 at levels approaching those of cells incubated without Lm, implicating Src family kinases as mediators of Lm-dependent cell survival.

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