Role of the Planar Cell Polarity (PCP) signaling in the dynamic organization of synapses and the integration of synaptic information: from basic mechanisms to patho-physiological consequences – NanoPlanSYN

Our goal is to decipher the spatiotemporal mechanisms controlled by the planar cell polarity (PCP) signaling in spine during specification and morpho-fonctional plasticity that depend on the dynamics of the actin cytoskeleton and trans-synaptic adhesions. Because PCP components sculpt cell morphology by inducing polarized changes in the cytoskeleton, they are strong candidates to regulate F-actin networks in dendritic spines. Our specific goal is to decrypt and monitor at the nanoscale level the organization and dynamics of PCP main players, Vangl2, along with its main interactors, Scribble1 and Prickle2 and to study the interplay between these PCP proteins, actin regulators and adhesion complexes in normal and pathological conditions.
PCP is well known for participating in intercellular and intracellular transmission of information, and notably for integrating local and long-range directionality cues to organize cellular polarity at the tissue level. In the brain, growing evidences suggest that PCP signaling is fundamental for neuronal development, including neuronal migration, neuronal polarity, axonal guidance, but also dendrite morphogenesis and synaptogenesis. Recent work from our lab (Montcouquiol/Sans) reveals specific behavioral consequences to brain-specific disruptions of PCP signaling in mice (Moreau et al, J. Neurosci. 2010; Hilal et al, in revision; Ezan et al., or Robert et al., in preparation). Mechanistically, we know little about Vangl2-dependent signaling. Because of the well-known asymmetrical clustering of PCP molecules in subcellular compartments or membrane regions that seem required for PCP signaling in different systems, we hypothesize that PCP protein nanoscale organization within the spine could similarly control cytoskeleton and/or adhesion dynamics. To address this, we plan to study the coupling or interplay between PCP signaling and the assembly of actin cytoskeleton macromolecular complexes and its connection to cadherin-adhesion molecules using biochemistry, biophysics and 3D super-resolution optical microscopy techniques.
Here, we will use well-established and new molecular, cellular and genetic tools to study PCP signaling (Sans lab) combined with single-molecule-based localization microscopy (SMLM) to study actin regulators and adhesion in spine (Giannone lab; Rossier et al., Nat Cell Biol 2012, Chazeau et al, EMBO J. 2014). A strong innovative aspect of this proposal is to develop and apply quantitative 3D SMLM using recent cutting-edge technological developments achieved in Sibarita’s lab such as single objective selective plane illumination microscopy (soSPIM) (Galland et al, Nat Methods 2015) and tessellation-based data analysis (Levet et al, Nat Methods 2015). From a methodological standpoint, soSPIM and SMLM will bring in-depth nanoscopy capabilities of thick biological samples. Combined with two-photon glutamate uncaging, it will be possible to achieve super-resolution imaging of brain slices from wild-type and PCP cKO animals before and after synaptic structural plasticity. The tessellation-based SMLM analysis software will be used to quantify the molecular organization and dynamics of various dendritic spines proteins at the single-molecule level. We will take a bottom-up approach, from simpler in vitro systems, such as dissociated hippocampal neurons, to more complex and challenging systems, such as live brain slices.
This project will provide novel conceptual insights into the nanoscale segregation of PCP proteins in spines and how individual PCP proteins control the actin cytoskeleton to build functional synapses. This project will offer a multilevel and interdisciplinary approach to understand the consequences of mutations of PCP signaling at the nanoscale, cellular and physiological levels, on the establishment, maturation and function of the basic functional unit of neuronal integration in the complex circuit of the hippocampus: the dendritic spine.