SUMMARY Although growing body of evidence supports that Wnt-Frizzled signaling controls axon guidance from vertebrates to worms, whether and how this is mediated by planar cell polarity (PCP) signaling remains elusive. We show here that the core PCP components are required for Wnt5a-stimulated outgrowth and anterior-posterior guidance of commissural axons. Dishevelled1 can inhibit PCP signaling by increasing hyperphosphorylation of Frizzled3 and preventing its internalization. Vangl2 antagonizes that by reducing Frizzled3 phosphorylation and promotes internalization. In commissural axon growth cones, Vangl2 is predominantly localized on the plasma membrane and is highly enriched on the tips of the filopodia as well as in patches of membrane where new filopodia emerge. Taken together, we propose that the antagonistic functions of Vangl2 and Dvl1 (over Frizzled3 hyperphosphorylation and endocytosis) allows sharpening of PCP signaling locally on the tips of the filopodia for sensing directional cues, Wnts, eventually causing turning of growth cones.

How growth cones detect small concentration differences of guidance cues for correct steering remains a long-standing puzzle. Commissural axons engage planar cell polarity (PCP) signaling components to turn anteriorly in a Wnt gradient after midline crossing. We found here that Frizzled3, a Wnt receptor, undergoes endocytosis via filopodia tips. Wnt5a increases Frizzled3 endocytosis, which correlates with filopodia elongation. We discovered an unexpected antagonism between Dishevelleds, which may function as a signal amplification mechanism in filopodia where PCP signaling is activated: Dishevelled2 blocks Dishevelled1-induced Frizzled3 hyperphosphorylation and membrane accumulation. A key component of apical-basal polarity (A-BP) signaling, aPKC, also inhibits Dishevelled1-induced Frizzled3 hyperphosphorylation. Celsr3, another PCP component, is required in commissural neurons for anterior turning. Frizzled3 hyperphosphorylation is increased in Celsr3 mutant mice, where PCP signaling is impaired, suggesting Frizzled3 hyperphosphorylation does correlate with loss of PCP signaling in vivo. Furthermore, we found that the small GTPase, Arf6, which is required for Frizzled3 endocytosis, is essential for Wnt-promoted outgrowth, highlighting the importance of Frizzled3 recycling in PCP signaling in growth cone guidance. In a Wnt5a gradient, more Frizzled3 endocytosis and activation of atypical protein kinase C was observed on the side of growth cones facing higher Wnt5a concentration, suggesting that spatially controlled Frizzled3 endocytosis is part of the key mechanism for growth cone steering.

The signaling mechanisms that choreograph the assembly of the highly asymmetric pre-and postsynaptic structures are still poorly defined. Using synaptosome fractionation, immunostaining, and coimmunoprecipitation, we found that Celsr3 and Vangl2, core components of the planar cell polarity (PCP) pathway, are localized at developing glutamatergic synapses and interact with key synaptic proteins. Pyramidal neurons from the hippocampus of Celsr3 knockout mice exhibit loss of ∼50% of glutamatergic synapses, but not inhibitory synapses, in culture. Wnts are known regulators of synapse formation, and our data reveal that Wnt5a inhibits glutamatergic synapses formed via Celsr3. To avoid affecting earlier developmental processes, such as axon guidance, we conditionally knocked out Celsr3 in the hippocampus 1 week after birth. The CA1 neurons that lost Celsr3 also showed a loss of ∼50% of glutamatergic synapses in vivo without affecting the inhibitory synapses assessed by miniature excitatory postsynaptic current (mEPSC) and electron microscopy. These animals displayed deficits in hippocampus-dependent behaviors in adulthood, including spatial learning and memory and fear conditioning. In contrast to Celsr3 conditional knockouts, we found that the conditional knockout of Vangl2 in the hippocampus 1 week after birth led to a large increase in synaptic density, as evaluated by mEPSC frequency and spine density. PCP signaling is mediated by multiple core components with antagonizing functions. Our results document the opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.Celsr3 | Vangl2 | glutamatergic | synapse formation G lutamatergic synapses, the predominant excitatory synapses in the brain, are asymmetric cell-cell junctions formed from distinct pre-and postsynaptic components involving highly organized complexes of hundreds of proteins across the 20-nm synaptic cleft (1, 2). The signaling pathway that directly assembles these asymmetric protein complexes has not been well understood. Understanding mechanisms of glutamatergic synapse formation will provide important insights into the function and plasticity as well as dysfunction of glutamatergic synapses, which underlie numerous nervous system disorders.Many epithelial tissues show planar cell polarity, the global asymmetry of cellular and tissue morphology and/or structure along the tissue plane (3, 4). The conserved core planar cell polarity (PCP) components, Frizzled, Dishevelled, Diego, Prickle, Vang(l), and Flamingo (Fmi)/Celsr, form asymmetric complexes at the cadherin-mediated adherens junctions that connect neighboring epithelial cells (3, 4). Recent studies suggest that mutations of some components of the PCP signaling pathway, Celsr3/Fmi and Vangl2, affect GABAergic circuit development in zebrafish retina, GABAergic motoneuron synapse development in Caenorhabditis elegans, and hippocampal/cortical glutamatergic and GABAergic synapse formation (5-11). PCP components are critical regulators of neuronal migration and axon guidance, which take p...

Super-resolution imaging methods such as structured illumination microscopy and others have offered various compromises between resolution, imaging speed, and biocompatibility. Here we experimentally demonstrate a physical mechanism for super-resolution that offers advantages over existing technologies. Using finely structured, resonant, and controllable near-field excitation from localized surface plasmons in a planar nanoantenna array, we achieve wide-field surface imaging with resolution down to 75 nm while maintaining reasonable speed and compatibility with biological specimens.