Localization of Ganglioside 9-O-Acetyl GD3 in Point Contacts of Neuronal Growth Cones Erika M. A. Negreiros, Ana C. M. Lea ˜ o, Marcelo F. Santiago, Rosalia Mendez-Otero Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, RJ, Brazil Received 12 December 2002; accepted 7 March 2003 ABSTRACT: Gangliosides are a large group of sia- lylated glycosphingolipids widely expressed in mamma- lian tissues. We have shown previously that the expres- sion of 9-O-acetyl GD3 is highly correlated with periods of neurite outgrowth in the developing nervous system, and that the advance of dorsal root ganglia growth cones on laminin was halted in presence of an antibody specific for 9-O-acetyl GD3. In this work, we examined by im- munocytochemistry and confocal microscopy whether this ganglioside is localized in point contacts in neuronal growth cones. We identified point contacts by immuno- reactions with proteins, such as vinculin and 1 integrin, known to be associated with these structures in growth cones. Our observations indicate that 9-O-acetyl GD3 is specifically associated with vinculin and 1 integrin in point contacts of growth cones, suggesting a possible role for this particular ganglioside in the modulation of these contacts during neurite outgrowth. © 2003 Wiley Periodi- cals, Inc. J Neurobiol 57: 31–37, 2003 Keywords: gangliosides; growth cone; point contacts; dorsal root ganglia; integrin INTRODUCTION Cell movement requires that several events occur in the appropriate site and sequence. During movement, the leading edge of the cell forms adhesion structures with the substrate and these structures contain inte- grins. In non-neuronal cells as the adhesions coalesce, they become sites of attachment for stress fibers on the inside of the cell and are known as focal adhesions (for review see Yamada and Geiger, 1997). In neuro- nal cells, during axonal elongation or regeneration on laminin substrate, the growth cone forms transient adhesive integrin-mediated contacts, called “point contacts” (Arregui et al., 1994; Gomez et al., 1996; Renaudin et al., 1999). Point contacts are small inte- grin-containing adhesion sites that differ in size and distribution from the focal contact type of adhesions, and it has been suggested that these focal contacts represent sites of strong anchorage to the extracellular matrix and may reduce the rate of cell migration (Luna and Hitt, 1992). Point contacts, on the other hand, are abundant in highly motile cells (Bershadsky et al., 1985; Nermut et al., 1991), and it has been suggested that they represent an adhesion structure specialized for transient attachment during motility, which can be dynamically and rapidly regulated in response to environmental influences (Arregui et al., 1994; Renaudin et al., 1999). It has also been shown that point contacts in growth cones from rat dorsal root ganglia (DRG) are mature adhesion sites defined by the presence of both 1 integrin and vinculin, and that they are associated with signaling proteins. Renaudin et al., (1999) were also able to show that whereas 1 integrin clusters are located on both the upper and lower plasma membranes, only those asso- Correspondence to: R. Mendez-Otero (rmotero@biof.ufrj.br or rmotero@abc.org.br). Contract grant sponsor: PRONEX (R.M.-O.). Contract grant sponsor: CNPq (R.M.-O.). Contract grant sponsor: FAPERJ (R.M.-O.). Contract grant sponsor: Ministry of Science and Technology (MCT). © 2003 Wiley Periodicals, Inc. DOI 10.1002/neu.10248 31