Deciphering within dendritic spines the nanoscale organizations and dynamics of branched actin network regulators

F-actin network dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of F-actin regulators in spines is unknown. In this study, using single protein tracking and super-resolution imaging (PALM, dSTORM), we show that within spines branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at membrane protrusion tips (Chazeau et al., EMBO J., 2014). This organization is opposite to classical lamellipodial protrusive structures where branched F-actin nucleation and elongation occur at protrusion tips. The initiation of branched F-actin nucleation at the PSD might coordinate synaptic activity and spine structural plasticity. In a second study, in collaboration with the team of Olivier Thoumine (IINS), we revealed that interactions between trans-synaptic N-cadherin adhesions and the actin networks induce the stabilization of dendritic filopodia into mature spines (Chazeau et al., Mol. Biol. Cell, 2014).

Nanoscale segregation of branched F-actin nucleation and elongation factors determines dendritic spine protrusions.
Chazeau A., Mehidi A., Nair D., Gautier J., Leduc C., Chamma I., Kage F., Kechkar A., Thoumine O., Rottner K., Choquet D., Gautreau A., Sibarita J.B., Giannone G.
EMBO J. 2014. 33, 2745-2764.

A mechanical coupling between N-cadherin adhesions and the F-actin flow stabilizes dendritic spines.
Chazeau A, Garcia M, Czöndör K, Perrais P, Tessier B, Giannone G, Thoumine O.
Mol Biol Cell. 2014. 26(5), 859-873.

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Neuroligin1 phosphotyrosine level controls the formation of excitatory and inhibitory synapses

Adhesion between neurexin-1β (Nrx1β) and neuroligin-1 (Nlg1) induces early recruitment of the postsynaptic density protein 95 (PSD-95) scaffold; however, the associated signaling mechanisms are unknown. To dissociate the effects of ligand binding and receptor multimerization, we compared conditions in which Nlg1 in neurons was bound to Nrx1b or non-activating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1b binding stimulates the interaction between Nlg1 and PSD-95. Phosphotyrosine immunoblots and pull-down of gephyrin by Nlg1 peptides in vitro showed that Nlg1can be phosphorylated at a unique tyrosine (Y782), preventing gephyrin binding. Expression of Nlg1 point mutants in neurons indicated that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and accordingly the formation of inhibitory and excitatory synapses. We propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization.

This study was fostered by paradigms coming from the integrin field. Our study demonstrate that like integrins: (1) Neuroligin-1 can be considered as a ligand-activated adhesion molecule; (2) a phosphotyrosine switch controls the competitive binding of scaffolds on the intracellular tail of Neuroligin-1.

Neurexin-1β binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1.
Giannone G., Mondin M., Grillo-Bosch D., Tessier B., Saint-Michel E., Czöndör K., Sainlos M., Choquet D., Thoumine O.
Cell Reports. 2013. 3, 1996-2007.