the TM4SFs CD151/PETA-3, CD9, and CD81/TAPA-1 are all localized at intercellular contact sites of endothelial cells (Sincock et al., 1997; Yanez-Mo et al., 1998). Experi- ments involving the localization of OSP/claudin-11, OAP-1, and b1 protein complex in TJs are presently ongoing. It has been suggested that members of TM4SF and inte- grins play a role in proliferation, migration, signal trans- duction, cell activation, and tumor invasion (Hemler et al., 1996; Maecker et al., 1997; Sugiura and Berditchevski, 1999). There is some evidence that at least integrins are in- volved in some of these processes in oligodendrocytes (Malek-Hedayat and Rome, 1994; Frost et al., 1999; Blas- chuk et al., 2000). Data presented here suggest that the OAP-1, OSP/claudin-11, and b1 integrin complex is in- volved in regulating proliferation and migration of oligo- dendrocytes. Overexpression of both OSP/claudin-11 and OAP-1 resulted in increased growth in CIMO cells. This finding is consistent with the previously described growth regulatory effects of other TJ-associated proteins (Li and Mrsny, 2000; Ryeom et al., 2000) and the elevated levels of OSP/claudin-11 found in some tumors (Buznikov, A.G., and J.M. Bronstein, unpublished results). Several lines of evidence support an integral role of inte- grins and tetraspanins in cellular migration. Integrins have been shown to regulate the migration of a range of neural cells, including neural crest cells (Bronner-Fraser, 1993), Schwann cells (Milner et al., 1997), oligodendrocyte pre- cursor (Milner et al., 1996), and neuronal precursors within the optic tectum (Galileo et al., 1992). The disrup- tion of the b1 subunit gene by homologous recombination has demonstrated the critical role of b1 integrins in migra- tion (Fassler et al., 1995; Hirsch et al., 1996). Antibodies to TM4SF members CD9, CD53, CD81, CD82, CD151/ PETA-3, and CD81/TAPA-1 alter integrin-mediated cell migration (Domanico et al., 1997; Lagaudriere-Gesbert et al., 1997; Yanez-Mo et al., 1998). Notably, in Schwann cells of the peripheral nervous system, CD9 associates with in- tegrins on the cell surface and an anti-CD9 monoclonal antibody promoted adhesion and proliferation of Schwann cells in vitro (Hadjiargyrou et al., 1996). Given this litera- ture, it was not surprising that antibodies that bind to b1 integrin and OAP-1 proteins caused a profound decrease of oligodendrocyte migration (Fig. 7 A). We did not ex- pect the observed decrease in migration in the presence of anti–OSP/claudin-11 antibody, which was targeted to a pu- tative intracellular COOH termini epitope. To ensure that we were seeing specific binding of this antibody, we per- formed peptide-blocking experiments proving that the an- tibody was recognizing this sequence. It is possible that it is also recognizing another epitope, but this is highly un- likely since immunohistochemical staining using this anti- body is specific and almost identical with that described by Morita et al. (1999) and staining is absent in OSP-null cells. The specificity of the inhibitory response is sup- ported by the fact that an unrelated antibody or inacti- vated antibody had no effect on migration. Thus, the pre- dicted structure of OSP/claudin-11 is wrong or is dynamic or the antibody is gaining access to an intracellular epitope. Given the potential importance of our finding that anti–OSP/claudin-11 antibodies inhibit migration and the unexpected staining of live cells, we felt it was essential to confirm OSP/claudin-11’s role in migration using an- other technique. The alterations in migratory behavior of oligodendrocytes in the OSP-null mouse and the dose dependency of this effect add strong support for our con- clusion. It is possible that these results reflected altered proliferation rates, but we feel this is unlikely. Oligoden- drocyte cell cultures from transgenic animals appeared similar to wild-type cultures, and myelin appears normal in the adult OSP/claudin-11 knockout mouse (Gow et al., 1999). Although quantitation of the number of oligoden- drocytes has not been performed in these mice, it is possi- ble that OSP/OAP-1/integrin–mediated migration is not required for relatively normal myelination to occur during development. Experiments are underway evaluating OSP/ claudin-11’s role in proliferation and migration during re- myelination after injury. The formation of TJs is normally associated with a cellu- lar quiescent nonproliferating and nonmigratory state. Conversely, integrin/TM4SF activation has been positively associated with cell growth and migration, suggesting that the interaction of OSP/claudin-11 with this complex may therefore be a dynamic one. These associations could also be important in cytoskeletal interactions and interactions with different regulatory proteins. Further studies are nec- essary to determine the importance of OSP/claudin-11/ OAP-1/integrin association in oligodendroglial prolifera- tion and migration during myelin formation, maintenance, and repair and to determine if this mechanism can be gen- eralized to other members of the claudin family. We thank Drs. Andrew Charles, Michael Woodruff, and Jim Costantin for helpful discussions. Our thanks to Nilofer Nayama for her excellent tech- nical assistance. This study was supported by grants from the National Institutes of Health to J.M. Bronstein (NS01596) and H.I. Kornblum (NS28383), and from the National Multiple Sclerosis Society to J.M. Bronstein (RG2850B1). Submitted: 24 April 2000 Revised: 27 February 2001 Accepted: 28 February 2001 References Berditchevski, F., M.M. Zutter, and M.E. Hemler. 1996. Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins). Mol. Biol. Cell. 7:193–207. Blaschuk, K.L., E.E. Frost, and C. Ffrench-Constant. 2000. The regulation of proliferation and differentiation in oligodendrocyte progenitor cells by al- phaV integrins. Development. 127:1961–1969. Bronner-Fraser, M. 1993. Segregation of cell lineage in the neural crest. Curr. Opin. Genet. Dev. 3:641–647. Bronstein, J.M., P. Popper, P.E. Micevych, and D.B. Farber. 1996. Isolation and characterization of a novel oligodendrocyte-specific protein. Neurology. 47: 772–778. Bronstein, J.M., P.E. Micevych, and K. Chen. 1997. Oligodendrocyte-specific protein (OSP) is a major component of CNS myelin. J. Neurosci. Res. 50: 713–720. Bronstein, J.M., T.G. Hales, R.F. Tyndale, and A.C. Charles. 1998. A condi- tionally immortalized glial cell line that expresses mature myelin proteins and functional GABA(A) receptors. J. Neurochem. 70:483–491. Bronstein, J.M., K. Chen, S. Tiwari-Woodruff, and H.I. Kornblum. 2000. Devel- opmental expression of OSP/claudin-11. J. Neurosci. Res. 60:284–290. Campagnoni, A.T. 1988. Molecular biology of myelin proteins from the central nervous system. J. Neurochem. 51:1–14. Campagnoni, A.T., and W.B. Macklin. 1988. Cellular and molecular aspects of myelin protein gene expression. Mol. Neurobiol. 2:41–89. Chien, C.T., P.L. Bartel, R. Sternglanz, and S. Fields. 1991. The two-hybrid sys- tem: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA. 88:9578–9582. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Bio- chem. 162:156–159. Citi, S., H. Sabanay, R. Jakes, B. Geiger, and J. Kendrick-Jones. 1988. Cingulin, a new peripheral component of tight junctions. Nature. 19:272–276. Dermietzel, R., A.G. Leibstein, and D. Schunke. 1980. Interlamellar tight junc- The Journal of Cell Biology, Volume 153, 2001 304