Down syndrome is molecularly characterized by a trisomy of chromosome 21, which results phenotypically in malformations of the neuroanatomy and malfunctions in neurochemistry. The founding member of the Down Syndrome Cell Adhesion Molecule (Dscam) family was isolated through a direct screen of a region of chromosome 21. Recently, a new member of the Dscam family, Dsam-1, was identified in Drosophila and statistically exists in over 30,000 isoforms due to alternative splicing of four variable exons. It was shown to be necessary for proper development of the Drosophila central nervous system,and was identified as an axon guidance receptor. Following the discovery of Dscam-1, three new members of the family have been identified in Drosophila. Since the Dscam molecules belong to the immunoglobulin (Ig) superfamily of cell adhesion molecules, they have the potential to act as homo/heterophilic receptors. To identify the molecular function of Dscam-1 and its paralogues, stable cell lines will be constructed expressing one of the four family members. These cell lines will be tested for homo/heterophilic cell adhesion using a standard cell adhesion assay. Briefly, cells will be incubated in suspension either with cells expressing the same Dscam receptor or a different one. The cells will be differentially labeled with lipophilic dyes and checked for aggregation using confocal microscopy. Dscam receptors may also interact in cis (on the same cell surface). This will be tested through transient transfection of one receptor into a stable line expressing another. Co-immunoprecipitation experiments will be done to identify interactions between the two receptors. All possible combinations will be tested. These experiments will begin to elucidate the specificity of the Dscam receptors for each other and their role in axon guidance.
| Wojtowicz, Woj M; Flanagan, John J; Millard, S Sean et al. (2004) Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding. Cell 118:619-33 |
