SUMMARY Heterozygous coding mutations in TRIO are associated with neurodevelopmental disorders, including autism, schizophrenia, bipolar disorder, and epilepsy, and impair TRIO’s biochemical activities. To model mutant alleles, we ablated one or both Trio alleles from excitatory neurons in the cortex and hippocampus of mice. Trio haploinsufficiency increases anxiety and impairs social preference and motor coordination. Trio loss reduces forebrain size and dendritic arborization but increases dendritic spine densities. Cortical synapses in Trio haploinsufficient mice are small, exhibit pre– and postsynaptic deficits, and cannot undergo long–term potentiation. Similar phenotypes are observed in Trio knockout mice. Overall, Trio haploinsufficiency causes severe disease–relevant deficits in behavior and neuronal structure and function. Interestingly, phosphodiesterase 4A5 (PDE4A5) levels are reduced and protein kinase A (PKA) signaling is increased when TRIO levels are reduced. Elevation of PDE4A5 and drug–based attenuation of PKA signaling rescue Trio haploinsufficiency–related dendritic spine defects, suggesting an avenue for therapeutic intervention for TRIO –related neurodevelopmental disorders.

In BriefNoonan syndrome (NS) is caused by hyperactive SHP2 and is associated with cognitive deficits. Levy et al. find that NMDA receptor (NMDAR)-mediated currents are disrupted in NS and identify GluN2B Y1252 as a neural substrate of SHP2. Y1252 in turn binds the actin regulator Nck2 to control spine actin and regulate NMDAR currents.

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect cell lines by superresolution for over an order of magnitude longer than with tagged proteins. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.

Synapses are enriched in the cytoskeletal protein actin, which determines the shape of the pre-and postsynaptic compartments, organizes the neurotransmitter release machinery, and provides a framework for trafficking of components. In the postsynaptic compartment, interactions with actin or its associated proteins are also critical for the localization and activity of synaptic neurotransmitter receptors and ion channels. Actin binding proteins, including spectrin and α-actinin, serve as molecular linkages between the actin cytoskeleton and a diverse collection of receptors, including the NMDA receptor (NMDAR) and voltage-gated Na + channels. The

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore and a bio-orthogonal labeling strategy that enables both super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize organelle pairs (ER + mitochondria, ER + plasma membrane) in hard-to-transfect cell lines at super-resolution for up to 7 minutes. The extended time domain possible using these new tools reveal novel dynamic nanoscale targeting between organelles. Super-resolution microscopy ('nanoscopy') can visualize cellular components with resolutionsas high as ~10 nanometers, far below the Abbe diffraction limit 1 . When combined with multicolor labeling strategies, nanoscopy can reveal key features of organelle structure and interactions that remain opaque when visualized using diffraction-limited approaches 2, 3 .However, visualizing organelle dynamics at super-resolution remains challenging because even the most photostable fluorophores bleach within tens to hundreds of seconds under conditions required for STED or SMS nanoscopy 4 .Recently, we reported a set of high-density environmentally sensitive (HIDE) probes that support long time-lapse, single-color nanoscopy of organelles including the ER, mitochondria, Golgi apparatus, and plasma membrane (PM) 5-7 . HIDE probes consist of an organelle-specific lipid or lipid-like small molecule with a reactive trans-cyclooctene (TCO) moiety and a silicon rhodamine-tetrazine (SiR-Tz) 8 reaction partner. These two components undergo a rapid, in situ tetrazine ligation reaction 9, 10 that localizes the SiR dye at high density within the organelle membrane. In this environment, HIDE probes support the acquisition of continuous SMS and