Regulation of Neuron Branching by the Interaction of Neuroligin C-Terminus Domain with PIP2

Abstract

Intracellular signaling lipids are key regulators of cell physiology. At neuronal synapses and during neuronal development mounting evidence implicates lipids, such as phosphoinositides, as critical regulators. Hence, regulating their cellular distribution is essential for triggering signals in the relevant location. Here we are discussing how neuroligin (nlg-1) contributes to this signaling with its C-terminal domain leading to a thickening of dendrites and the formation of spiny structure in E18 hippocampal neurons as early as DIV4. We have established that overexpression of nlg-1 in HEK293T cells that naturally lack neuron specific proteins, leads to the formation of branches resembling neurite-like protrusions suggesting that early neuroligin expression could contribute to neurite and dendritic arbor formation. We have demonstrated that the morphological changes were linked to local changes in the plasma membrane lipid composition associated to Pi3K and PKC activity. However none of the proteins known to bind to nlg-1 could explain our results. Guided by computational prediction, we tested the relation between nlg-1 and phosphoinositol lipids. We found an increase in PIP2 in membrane expansions in cell expressing nlg-1 wild type compared to nlg-1 C-terminus deletion mutant. Furthermore only cells expressing nlg-1 were affected by a peptide treatment designed to perturb nlg-1 interaction with PIP2. Finally we showed that Nlg1-C-terminus is targeted to the membrane in a PIP2 dependent fashion supporting the hypothesis that nlg-1 harbor an unconventional PDZ domain that interacts with PIP2 to regulate the number of neurite, as well as neurons' branching pattern.