Figure 2.

Ankyrin-binding activity of the cytoplasmic domain of neurofascin is required for neurofascin-directed cell segregation and aggregation. Native neurofascin, or neurofascin lacking either the entire cytoplasmic domain or FIGQY residues within the cytoplasmic domain (Fig. 1A) were expressed in neuroblastoma cells and evaluated for ankyrin-binding activity (A), ability to direct cell segregation (B–D), and cell aggregation activity (E). (A) HA epitope-tagged neurofascin constructs were immunoprecipitated from crude cell lysates, resolved by SDS/PAGE, and analyzed by immunoblotting with a brain ankyrin-specific polyclonal antibody (Methods). In a separate experiment, cells expressing either native neurofascin or FIGQY→F mutant of neurofascin were treated with NGF (100 ng/ml) as described in Fig. 4, followed by the same immunoprecipitation and immunoblotting protocol. Cell segregation (B–D) was observed by the same method as in Fig. 1. DiA-labeled cells expressing neurofascin with a truncated cytoplasmic domain (B), cells expressing neurofascin missing FIGQY (C), or cells expressing native neurofascin (D) were mixed with unlabeled cells that were untransfected and express no neurofascin. (E) Cell aggregation was measured as a function of time.