Supplementary Material for: The Type I Inositol 1,4,5-Trisphosphate Receptor Interacts with Protein 4.1N to Mediate Neurite Formation through Intracellular Ca<sup>2+</sup> Waves

2011-03-10T00:00:00Z (GMT) by Fiedler M.J. Nathanson M.H.
Ca<sup>2+</sup> waves are an important mechanism for encoding Ca<sup>2+</sup> signaling information, but the molecular basis for wave formation and how this regulates neuronal function is not entirely understood. Using nerve growth factor-differentiated PC12 cells as a model system, we investigated the interaction between the type I inositol 1,4,5-trisphosphate receptor (IP3R1) and the cytoskeletal linker, protein 4.1N, to examine the relationship between Ca<sup>2+</sup> wave formation and neurite development. This was examined using RNAi and overexpressed dominant negative binding regions of each protein. Confocal microscopy was used to monitor neurite formation and Ca<sup>2+</sup> waves. Knockdown of IP3R1 or 4.1N attenuated neurite formation, as did binding regions of IP3R1 and 4.1N, which colocalized with endogenous 4.1N and IP3R1, respectively. Upon stimulation with the IP3-producing agonist carbachol, both RNAi and dominant negative molecules shifted signaling events from waves to homogeneous patterns of Ca<sup>2+</sup> release. These findings provide evidence that IP3R1 localization, via protein 4.1N, is necessary for Ca<sup>2+</sup> wave formation, which in turn mediates neurite formation.