Studying the roles of different proteins and the mechanisms involved in synaptogenesis is hindered by the complexity and heterogeneity of synapse types, and by the spatial and temporal unpredictability of spontaneous synapse formation. Here we demonstrate a robust and high-content method to induce selectively presynaptic or postsynaptic structures at controlled locations. Neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with various synaptogenic adhesion molecules. When plated on neurexin-1b-coated micropatterns, neurons expressing neuroligin-1 exhibit specific dendritic organization and selective recruitment of the postsynaptic scaffolding molecule PSD-95. Furthermore, functional AMPA receptors are trapped at neurexin-1b dots, as revealed by live-imaging experiments. In contrast, neurons plated on SynCAM1-coated substrates exhibit strongly patterned axons and selectively assemble functional presynapses. N-cadherin coating, however, is not able to elicit synapses, indicating the specificity of our system. This method opens the way to both fundamental and therapeutic studies of various synaptic systems.

Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short-and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.growth cone | actin flow | N-cadherin adhesion | micropatterned substrates | single-molecule tracking G rowth cones are motile structures at the extremity of axons responsible for path finding and neurite extension during nervous system development and repair. Growth cones translate extracellular signals into directional migration through a coordinated regulation of cytoskeleton, adhesion, and membrane processes (1). At the cytoskeletal level, motility is generated by polarized actin treadmilling, which, together with myosin contraction, generates a continuous retrograde actin flow from the periphery to the base of growth cones (2-7). At the membrane level, adhesion proteins form dynamic bonds with immobilized extracellular ligands, allowing step-by-step locomotion (8).The molecular clutch model postulates that the mechanical coupling between ligand-bound transmembrane adhesion receptors and the actin flow allows traction forces to be transmitted to the substrate, resulting in local diminution of the retrograde flow and forward progression (9-11). Optical tweezers and flexible substrate experiments using microspheres coated with adhesion molecules revealed clutch-like mechanisms for integrins (12, 13), Ig cell adhesion molecules (14, 15), and cadherins (16,17). However, the mechanism of clutch engagement at the individual molecular level remains elusive. For integrin-based adhesion, single-molecule tracking experiments suggested that talin and vinculin could switch between a state bound to flowing actin and a state bound to immobilized integrins (18, 19). For cadherin-based adhesion,...

identified in the gene encoding desmin leading to rare diseases belonging to MyoFibrillar Myopathy group (MFM). These pathologies are mainly characterized by aggregates formation in muscle tissue, associated with misorganizations of the contractile apparatus. Moreover patients progressively develop muscle weakness. Currently, pathophysiology and molecular defects of MFMs remain largely unknown, and no treatments are available. In this context, the aim of our study is to clarify whether desmin mutations implicated in MFMs plays a role at early stage of expression, by impairing the properties of pre-muscular cells, the myoblasts. First we have studied the formation of desmin aggregates in living myoblasts over-expressing for 24h wild-type (WT) or different mutant desmins. We show that each mutant has a specific impact on the desmin network organization. Second we have performed mechanical measurements on C2C12 cells, focusing on the E413K mutant, which induces a large desmin network disorganization associated with important aggregate formation: we have compared the mechanical properties of WT-cells, C2C12 over-expressing desmin-WT-GFP and C2C12 overexpressing mutated desmin E413K-GFP. Visco-elastic properties of cells have been evaluated by using two custom-made set-ups, optical tweezers and a single-cell rheometer: we show that the 3 cells types share the same viscoelastic behaviour. Finally, we have investigated the impact of mutated desmin on the contractility of myoblasts, and we demonstrate that E413K-mutation significantly decreases cell contraction abilities specifically for cells with desmin aggregates, while aggregates of WT-desmin do no induce the same effect.