Figure 7. Overexpression of OSP/claudin-11 and OAP-1 in- creases proliferation in CIMO cells. (A) Northern blot of CIMO cells grown at 378C (lanes 1 and 3) and 338C (lanes 2 and 4) probed with a 32P-labeled 2-kb cDNA of OSP/claudin-11 (lanes 1 and 2) and 1.9-kB cDNA of OAP-1 (lanes 3 and 4). Both OSP/ claudin-11 and OAP-1 expression is increased at 338C during proliferation. (B) Western blot of CIMO cells transfected with control vector (lanes 1 and 3) and OSP (lanes 2 and 4) probed with anti-OSP antibody at 338C (lanes 1 and 2) and 378C (lanes 3 and 4). (Bottom panel) Western blot of CIMO cells transfected with OAP-1 (lane 1) and control vector (lane 2) at 378C probed with anti–OAP-1 antibody confirms increased expression in these stable transfectents. (C) [3H]Thymidine incorporation into DNA of CIMO cells transfected with control vectors (pBabe and pcDNA) and OSP/claudin-11, antisense OSP/claudin-11, PLP, MBP, and OAP-1 constructs were observed under nonpermissive (378C, minus Ig) and permissive conditions (338C, plus Ig). OSP/ claudin-11–transfected cells had a significant effect on proliferation at 338C, whereas OAP-1 had a more generalized effect at both 338C and 378C. Control CIMO cells incorporated 4,000 6 800 cpm/well/ 12 h, similar to the control vector transfected CIMO cells (aver- age 6 SEM, n 5 24–32; 500 cells/well, each well counted in tripli- cates). OSP/claudin-11 versus pBabe vector at 378C (***P , 0.0005), and OAP-1 versus pcDNA vector at 338C and 378C (**P , 0.005) (Student’s t test). 1997; Sugiura and Berditchevski, 1999), and it has been proposed that they exert their function by promoting as- sembly of signaling complexes. TM4SF proteins associate with other TM4SF members, for example, CD4, CD8, PETA-3, NAG-2, and CR2/CD19 (Imai and Yoshie, 1993; Tachibana et al., 1997; Yanez-Mo et al., 1998), and with in- tegrins (Berditchevski et al., 1996; Hadjiargyrou et al., 1996; Maecker et al., 1997; Yanez-Mo et al., 1998; Yauch et al., 1998). The discovery that OSP/claudin-11 associated with a member of the TM4SF led us to investigate whether OAP-1 and OSP/claudin-11 formed a complex that in- cludes integrins. Oligodendrocyte precursors in vitro ex- press the integrins a6b1, avb1, and avb3 subunits (Milner and Ffrench-Constant, 1994; Shaw et al., 1996). Because Figure 8. Oligodendrocyte migration in the presence of OSP/ claudin-11, OAP-1, and b1 integrin antibodies and in OSP/ claudin-11–deficient cells. (A) Cells were migrated out of agarose drops in medium: 2control, minus fibronectin; 1control, plus fibronectin; anti–b1 integrin antibody; anti–OSP/claudin-11 anti- body; anti–OAP-1 antibody; boiled inactive anti–OAP-1 antibody, I anti–OAP-1; anti-GFAP antibody; GRGDSP peptide; and GRGESP peptide. Except for the 2control, all other conditions contained fibronectin. The total number of cells migrating out of the agar drop was counted after 4 d. Statistical values represent each condition versus positive control: **P , 0.0005; *P , 0.005 (Student’s t test). (B) Migration assays were performed using oligodendrocytes isolated from wild-type (1/1), heterozygous OSP (1/2), and homozygous knockout OSP (2/2) mice, and cells were allowed to migrate in the presence (black bars) and absence (stippled bars) of fibronectin. There was marked attenu- ation of fibronectin-dependent migration in OSP/claudin-11– deficient cells. Values are expressed as percentage of wild-type. Heterozygous (1/2) versus wild-type (*P , 0.05) and homozygous recessive (2/2) versus wild-type (**P , 0.005) (Student’s t test). many tetraspanins interact with b1 integrin subunits, we tested the presence and association of b1 integrin subunit with OSP/claudin-11 and OAP-1. The association of these three proteins was first suggested by coimmunoprecipita- tion in mouse brain homogenates and in primary oligoden- drocytes homogenates under relatively stringent condi- tions. Double immunocytochemistry showed colocalization of OSP/claudin-11, OAP-1, and b1 integrin in the cell body and processes of most oligodendrocytes. Surface biotinyla- tion and immunoelectron microscopy demonstrated the presence of OSP/claudin-11 and OAP-1 on the oligoden- drocyte membrane surface, but it is still not clear whether OSP/claudin-11 complexes with OAP-1 and b1 integrin at TJs. This would not be unprecedented, since some mem- bers of the TM4SF have been found at cell-to-cell contacts where they are also associated with integrins. For example, Tiwari-Woodruff et al. OSP/Claudin-11, b1 Integrin, and OAP-1 Form a Complex 303