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 mem- brane-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 (1/1, lane 1) and anti–OSP/claudin-11 antibody (1/1, lane 2; 2/2, 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 (1/1, lane 1) or anti–OSP/claudin-11 antibody (1/1, lane 2; 2/2, 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 ( 1/1, lane 1) or anti–OSP/claudin-11 antibody (1/1, lane 2; 2/2, 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. under both growth permissive and nonpermissive condi- tions (Fig. 7 C). OAP-1–OSP/Claudin-11–b1 Integrin Complex Regulates Oligodendrocyte Migration Cell proliferation, differentiation, and migration are at least partially mediated by the interaction between cell membrane proteins and extracellular matrix (ECM). Inte- grins are a key family of ECM receptors, and avb1 integrin has been shown to play a role in oligodendrocyte migration (Milner et al., 1996). Since OSP/claudin-11 and OAP-1 form a complex with b1 integrins, it is likely that they are involved in cellular interactions with ECM. The possible involvement of OSP/claudin-11 and OAP-1 in oligoden- drocyte migration was tested using an established in vitro assay used previously to demonstrate that integrins are in- volved in oligodendrocyte migration (Varani et al., 1978; Milner et al., 1996). Primary oligodendrocytes were plated in an agarose drop in the presence (positive control) or ab- sence (negative control) of fibronectin. Maximal migration in positive controls was achieved in 4 d, with 58 6 12 cells per well appearing in the migratory zone. Migration of oli- godendrocytes was inhibited significantly in the presence of anti–OSP/claudin-11, anti–OAP-1, and anti–b1 integrin antibodies by 95, 76, and 88% of the control, respectively (Fig. 8 A). The inhibitory effect of the antibodies on migra- tion was specific, since inactive anti–OAP-1 (boiled for 10 min) and anti-GFAP (unrelated) antibody had no effect. Reduced migration also did not reflect decreased viability since .95% of all cells under all conditions remained via- ble as determined by trypan blue staining. We were surprised by the significant inhibition of migra- tion in the presence of anti–OSP/claudin-11 antibody, since the recognizable epitope is predicted to be intracellular. To confirm that the anti–OSP/claudin-11 antibody was indeed recognizing protein epitopes on live oligodendrocytes, cul- tures were incubated with anti–OSP/claudin-11 antibodies and with antibody that had been preincubated with OSP/ claudin-11 peptide (Fig. 5, B and C). We observed specific labeling of live cells, although not as pronounced as with permeabilized oligodendrocytes (data not shown). This staining was markedly attenuated by preincubation with peptide and was absent in OSP-null oligodendrocytes, demonstrating specificity of the antibody labeling. Migration assays using oligodendrocytes isolated from OSP/claudin-11–null mice add further support to OSP/ claudin-11’s role in migration. Oligodendrocytes com- pletely lacking OSP/claudin-11 had markedly reduced ability to migrate compared with wild-type cells, whereas migration assays using heterozygote cells resulted in inter- mediate values (Fig. 8 B). Interestingly, only fibronectin- dependent migration was affected by alterations in OSP/ claudin-11 gene dosage. Discussion Although several proteins have been localized to TJs, none have been shown to associate directly with OSP/claudin-11. Reported here is the identification of an OSP/claudin-11– interacting protein, OAP-1. It is evident from sequence analysis that OAP-1 is a member of the TM4SF. OAP-1, like other members of TM4SF, has (a) four highly con- served hydrophobic transmembrane domains, (b) charged residues in or near the transmembrane domains, and (c) conserved cysteine residues in the second transmembrane domain (Maecker et al., 1997). Tspan-1–Tspan-6 are also new members of the TM4SF family, which were identified by Todd et al. (1998) by searching the dbEST database. OAP-1 shares highest homology with human Tspan-3 and is likely the mouse homologue of human Tspan-3. Like hu- man Tspan-3 mRNA expression, murine OAP-1 showed widespread tissue expression. Within the CNS, OAP-1 lo- calized to oligodendrocytes, neurons, and astrocytes. Members of TM4SF have been implicated in several ba- sic biological processes (Hemler et al., 1996; Maecker et al., The Journal of Cell Biology, Volume 153, 2001 302