Figure 4. OAP-1 associates with OSP/claudin-11 and b1 integrin. (A) Mouse brain homogenates were immunoprecipitated with anti-OSP, anti–OAP-1, anti-GFAP, or anti–b1 integrin and then probed with anti–OAP-1 antibody. (B) Primary oligodendrocyte homogenates were immunoprecipitated with anti-OSP, anti– OAP-1, anti-GFAP, or anti–b1 integrin and then probed with anti–OAP-1 antibody. (C) Mouse brain homogenates were im- munoprecipitated with anti-OSP, anti–OAP-1, anti-GFAP, or anti–b1 integrin and then probed with anti–OSP/claudin-11 anti- body. (D) Mouse brain homogenates were immunoprecipitated with anti-OSP, anti-OAP-1, anti-GFAP, or anti–b1 integrin and then probed with anti–b1 integrin antibody. (E) CIMO homoge- nates were immunoprecipitated with anti-OSP, anti–b1 integrin, or anti-GFAP and then probed with anti–OAP-1 antibody. (F) Sil- ver-stained gel of anti–OSP/claudin-11 (lane 1), anti–OAP-1 (lane 2), and anti–b1 integrin (lane 3) immunoprecipitated mouse brain homogenate. Small arrows denote bands that correspond to the molecular masses of OAP-1, OSP/claudin-11, and b1 integrin. The IgG band is visible at z50 kD. complementary experiments indicated that OSP/claudin-11 (Fig. 4 C) and b1 integrin (Fig. 4 D) can be immunoprecipi- tated from mouse brain homogenates with anti–OAP-1. Furthermore, OSP/claudin-11 and OAP-1 were immuno- precipitated with anti–b1 integrin antibody, and b1 integrin was immunoprecipitated with anti–OSP/claudin-11 anti- body (Fig. 4). The identity of b1 integrin on the Western blots was confirmed using both polyclonal and monoclonal antibodies. OSP/claudin-11, OAP-1, and b1 integrin also coimmunoprecipitated in CIMO cell line under permissive and nonpermissive conditions (Fig. 4 E). Neither b1 inte- grin, OAP-1, nor OSP/claudin-11 was precipitated when the primary antibody was omitted or when an unrelated anti- body GFAP was used as the primary antibody. Silver stain- ing of OSP/claudin-11, OAP-1, and b1 integrin immunopre- cipitated mouse brain homogenates showed two to four bands corresponding to the molecular masses of OSP/clau- din-11, OAP-1, and b1 integrin along with the IgG band at z50 kD (Fig. 4 F). These results suggest that OAP-1, OSP/ claudin-11, and b1 integrin form a complex in the brain and oligodendrocyte homogenates. Additional confirmation of an association between OSP/claudin-11, OAP-1, and b1 integrin was obtained us- ing double-labeled immunocytochemistry and confocal microscopy (Fig. 5 A). Controls with GFAP showed no colocalization with any of the experimental proteins (data not shown). A striking colocalization of OSP/claudin-11, OAP-1, and b1 integrin was observed in most oligoden- drocytes (Fig. 5 A, third panel in rows 1 and 2). A few oli- godendrocytes showed only partial colocalization (data not shown), suggesting that complex formation was under dynamic control or that the population of oligodendro- cytes was heterogeneous. OSP/Claudin-11, OAP-1, and b1 Integrin Is a Surface Membrane Protein Complex We predicted that both OSP/claudin-11 and OAP-1 are membrane bound based on their high level of hydropho- bicity and known subcellular localization of b1 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-null oligodendrocytes (data not shown). Further- more, surface protein biotinylation of nonpermeabilized oligodendrocytes resulted in biotinylation of OSP/claudin- 11 and OAP-1 (Fig. 6, A and B). To determine if OSP/ claudin-11 and OAP-1 complex on the surface of cells, oli- godendrocytes from wild-type (1/1) and OSP/claudin-11 knockout (2/2) mice were biotinylated and subjected to coimmunoprecipitation. OSP/claudin-11 and OAP-1 pro- tein were detected in OSP/claudin-11 and OAP-1 1/1 im- munoprecipitated biotinylated oligodendrocytes (Fig. 6, C and D). Neither OSP/claudin-11 or OAP-1 protein was de- tected in 2/2 oligodendrocytes immunoprecipitated with anti–OSP/claudin-11, although OAP-1 was detected in OAP-1 immunoprecipitated 2/2 oligodendrocytes (Fig. 6, C and D). Surface localization of these proteins was also confirmed using immunohistochemical EM. Immunoreac- tive protein localized primarily to the outer cell mem- branes of primary oligodendrocytes and appeared to be more concentrated on their processes (Fig. 6 E). Overexpression of OSP/Claudin-11 and OAP-1 Increases Proliferation in CIMO Cells TM4SFs and TJs have been implicated in the regulation of cellular proliferation. To study the effect of altered OAP-1 and/or OSP/claudin-11 gene expression on growth, CIMO cells were used (Bronstein et al., 1998). Under permissive conditions (338C 1 Ig), CIMO cells grew rapidly and were relatively undifferentiated. When transferred to nonper- missive conditions (378C, lacking Ig), the cells had a more differentiated morphology, grew slowly, and expressed myelin proteins. OSP/claudin-11 mRNA levels were 11- fold higher and OAP-1 levels were 1.5-fold higher in CIMO cells grown under permissive (Fig. 7 A) compared with nonpermissive conditions (Fig. 7 A). Increased mRNA expression could have reflected a response to higher cell proliferative rate or may have helped induce proliferation. To distinguish between these possibilities, CIMO cells were transfected with various constructs to al- ter gene expression, and their effect on proliferation was The Journal of Cell Biology, Volume 153, 2001 300