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OSP/claudin-11 forms a complex with a novel member of the tetraspanin super family and beta1 integrin and regulates proliferation and migration of oligodendrocytes.

Tiwari-Woodruff SK, Buznikov AG, Vu TQ, Micevych PE, Chen K, Kornblum HI, Bronstein JM - J. Cell Biol. (2001)

Bottom Line: Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line.Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes.These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of California at Los Angeles School of Medicine, Los Angeles, California 90095, USA.

ABSTRACT
Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths. TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways. We have identified an OSP/claudin-11-associated protein (OAP)1, using a yeast two-hybrid screen. OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes. OAP-1, OSP/claudin-11, and beta1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry. Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line. Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes. These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.

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OAP-1 and OSP/claudin-11 proteins are present on the cell surface. (A) Cell surface proteins of oligodendrocytes were biotinylated using a membrane-impermeant biotin ester. After solubilization and recovery of biotinylated proteins using Strepavidin-agarose, OAP-1 (lane 2) and OSP/claudin-11 (lane 3) proteins expressed at the plasma membrane were identified by SDS-PAGE and immunoblotting. Nonbiotinylated proteins (lane 1) were not observed. (B) Surface biotinylated oligodendrocyte proteins isolated from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) and anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3). Nonbiotinylated protein from wild-type oligodendrocytes was precipitated with anti–OSP/claudin-11 antibody (lane 4). Biotinylated proteins were visualized using Streptavidin-HRP as described in Materials and Methods. (C) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OSP/claudin-11 antibody. (D) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OAP-1 antibody. (E) Immunohistochemical EM of primary oligodendrocytes. OSP/claudin-11 and OAP-1 immunoreactivity was localized to the outer cell membrane (arrows). The asterisks represent the filter matrix the oligodendrocytes were grown on.
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Figure 6: OAP-1 and OSP/claudin-11 proteins are present on the cell surface. (A) Cell surface proteins of oligodendrocytes were biotinylated using a membrane-impermeant biotin ester. After solubilization and recovery of biotinylated proteins using Strepavidin-agarose, OAP-1 (lane 2) and OSP/claudin-11 (lane 3) proteins expressed at the plasma membrane were identified by SDS-PAGE and immunoblotting. Nonbiotinylated proteins (lane 1) were not observed. (B) Surface biotinylated oligodendrocyte proteins isolated from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) and anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3). Nonbiotinylated protein from wild-type oligodendrocytes was precipitated with anti–OSP/claudin-11 antibody (lane 4). Biotinylated proteins were visualized using Streptavidin-HRP as described in Materials and Methods. (C) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OSP/claudin-11 antibody. (D) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OAP-1 antibody. (E) Immunohistochemical EM of primary oligodendrocytes. OSP/claudin-11 and OAP-1 immunoreactivity was localized to the outer cell membrane (arrows). The asterisks represent the filter matrix the oligodendrocytes were grown on.

Mentions: We predicted that both OSP/claudin-11 and OAP-1 are membrane bound based on their high level of hydrophobicity and known subcellular localization of β1 integrin. Consistent with our hypothesis, OAP-1 and OSP/claudin-11 colocalized on the cell surface of nonpermeabilized live oligodendrocytes immunolabeled before fixation (Fig. 5 B). Surface labeling with anti-OSP antibody was absent in OSP- oligodendrocytes (data not shown). Furthermore, surface protein biotinylation of nonpermeabilized oligodendrocytes resulted in biotinylation of OSP/claudin-11 and OAP-1 (Fig. 6A and Fig. B). To determine if OSP/claudin-11 and OAP-1 complex on the surface of cells, oligodendrocytes from wild-type (+/+) and OSP/claudin-11 knockout (−/−) mice were biotinylated and subjected to coimmunoprecipitation. OSP/claudin-11 and OAP-1 protein were detected in OSP/claudin-11 and OAP-1 +/+ immunoprecipitated biotinylated oligodendrocytes (Fig. 6C and Fig. D). Neither OSP/claudin-11 or OAP-1 protein was detected in −/− oligodendrocytes immunoprecipitated with anti–OSP/claudin-11, although OAP-1 was detected in OAP-1 immunoprecipitated −/− oligodendrocytes (Fig. 6C and Fig. D). Surface localization of these proteins was also confirmed using immunohistochemical EM. Immunoreactive protein localized primarily to the outer cell membranes of primary oligodendrocytes and appeared to be more concentrated on their processes (Fig. 6 E).


OSP/claudin-11 forms a complex with a novel member of the tetraspanin super family and beta1 integrin and regulates proliferation and migration of oligodendrocytes.

Tiwari-Woodruff SK, Buznikov AG, Vu TQ, Micevych PE, Chen K, Kornblum HI, Bronstein JM - J. Cell Biol. (2001)

OAP-1 and OSP/claudin-11 proteins are present on the cell surface. (A) Cell surface proteins of oligodendrocytes were biotinylated using a membrane-impermeant biotin ester. After solubilization and recovery of biotinylated proteins using Strepavidin-agarose, OAP-1 (lane 2) and OSP/claudin-11 (lane 3) proteins expressed at the plasma membrane were identified by SDS-PAGE and immunoblotting. Nonbiotinylated proteins (lane 1) were not observed. (B) Surface biotinylated oligodendrocyte proteins isolated from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) and anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3). Nonbiotinylated protein from wild-type oligodendrocytes was precipitated with anti–OSP/claudin-11 antibody (lane 4). Biotinylated proteins were visualized using Streptavidin-HRP as described in Materials and Methods. (C) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OSP/claudin-11 antibody. (D) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OAP-1 antibody. (E) Immunohistochemical EM of primary oligodendrocytes. OSP/claudin-11 and OAP-1 immunoreactivity was localized to the outer cell membrane (arrows). The asterisks represent the filter matrix the oligodendrocytes were grown on.
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Figure 6: OAP-1 and OSP/claudin-11 proteins are present on the cell surface. (A) Cell surface proteins of oligodendrocytes were biotinylated using a membrane-impermeant biotin ester. After solubilization and recovery of biotinylated proteins using Strepavidin-agarose, OAP-1 (lane 2) and OSP/claudin-11 (lane 3) proteins expressed at the plasma membrane were identified by SDS-PAGE and immunoblotting. Nonbiotinylated proteins (lane 1) were not observed. (B) Surface biotinylated oligodendrocyte proteins isolated from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) and anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3). Nonbiotinylated protein from wild-type oligodendrocytes was precipitated with anti–OSP/claudin-11 antibody (lane 4). Biotinylated proteins were visualized using Streptavidin-HRP as described in Materials and Methods. (C) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OSP/claudin-11 antibody. (D) Surface-biotinylated primary oligodendrocyte homogenates from wild-type and OSP/claudin-11 transgenic mice were immunoprecipitated with anti–OAP-1 antibody (+/+, lane 1) or anti–OSP/claudin-11 antibody (+/+, lane 2; −/−, lane 3) and probed with anti–OAP-1 antibody. (E) Immunohistochemical EM of primary oligodendrocytes. OSP/claudin-11 and OAP-1 immunoreactivity was localized to the outer cell membrane (arrows). The asterisks represent the filter matrix the oligodendrocytes were grown on.
Mentions: We predicted that both OSP/claudin-11 and OAP-1 are membrane bound based on their high level of hydrophobicity and known subcellular localization of β1 integrin. Consistent with our hypothesis, OAP-1 and OSP/claudin-11 colocalized on the cell surface of nonpermeabilized live oligodendrocytes immunolabeled before fixation (Fig. 5 B). Surface labeling with anti-OSP antibody was absent in OSP- oligodendrocytes (data not shown). Furthermore, surface protein biotinylation of nonpermeabilized oligodendrocytes resulted in biotinylation of OSP/claudin-11 and OAP-1 (Fig. 6A and Fig. B). To determine if OSP/claudin-11 and OAP-1 complex on the surface of cells, oligodendrocytes from wild-type (+/+) and OSP/claudin-11 knockout (−/−) mice were biotinylated and subjected to coimmunoprecipitation. OSP/claudin-11 and OAP-1 protein were detected in OSP/claudin-11 and OAP-1 +/+ immunoprecipitated biotinylated oligodendrocytes (Fig. 6C and Fig. D). Neither OSP/claudin-11 or OAP-1 protein was detected in −/− oligodendrocytes immunoprecipitated with anti–OSP/claudin-11, although OAP-1 was detected in OAP-1 immunoprecipitated −/− oligodendrocytes (Fig. 6C and Fig. D). Surface localization of these proteins was also confirmed using immunohistochemical EM. Immunoreactive protein localized primarily to the outer cell membranes of primary oligodendrocytes and appeared to be more concentrated on their processes (Fig. 6 E).

Bottom Line: Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line.Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes.These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of California at Los Angeles School of Medicine, Los Angeles, California 90095, USA.

ABSTRACT
Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths. TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways. We have identified an OSP/claudin-11-associated protein (OAP)1, using a yeast two-hybrid screen. OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes. OAP-1, OSP/claudin-11, and beta1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry. Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line. Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes. These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.

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