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TRIO encodes a cytoskeletal regulatory protein with three catalytic domains-two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain-as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders. We provide evidence here that the Trio SRs interact intramolecularly with the GEF1 domain to inhibit its enzymatic activity. We demonstrate that SRs 6-9 decrease GEF1 catalytic activity both in vitro and in cells and show that NDD-associated variants in the SR8 and GEF1 domains relieve this autoinhibitory constraint. Our results from chemical cross-linking and bio-layer interferometry indicate that the SRs primarily contact the pleckstrin homology region of the GEF1 domain, reducing GEF1 binding to the small GTPase Rac1. Together, our findings reveal a key regulatory mechanism that is commonly disrupted in multiple NDDs and may offer a new target for therapeutic intervention for TRIO-associated NDDs.

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 super-resolution 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.

Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 s temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 s temporal resolution. HIDE probes such as DiI-SiR are non-toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super-resolution over biologically relevant timescales.

Background: Repetitive transcranial magnetic stimulation (rTMS) is an effective therapy in post‐stroke motor recovery. However, the underlying mechanisms of rTMS regulates long‐lasting changes with synaptic transmission and glutamate receptors function (including AMPARs or NMDARs) remains unclear. Methods: Mice were received 10‐Hz rTMS treatment once daily on the third day after photothrombotic (PT) stroke for 18 days. Motor behaviors and the Western blot were used to evaluate the therapeutic efficacy of 10‐Hz rTMS in the mice with PT model. Moreover, we used wild‐type (WT) and NEX‐α3−/− mice to further explore the 10‐Hz rTMS effect. Results: We found that 10‐Hz rTMS improved the post‐stroke motor performance in the PT mice. Moreover, the levels of AMPAR, vGlut1, and integrin α3 in the peri‐infarct were significantly increased in the rTMS group. In contrast, 10‐Hz rTMS did not induce these aforementioned effects in NEX‐α3−/− mice. The amplitude of AMPAR‐mediated miniature excitatory postsynaptic currents (EPSCs) and evoked EPSCs was increased in the WT + rTMS group, but did not change in NEX‐α3−/− mice with rTMS. Conclusions: In this study, 10‐Hz rTMS improved the glutamatergic synaptic transmission in the peri‐infract cortex through effects on integrin α3 and AMPARs, which resulted in motor function recovery after stroke. [ABSTRACT FROM AUTHOR]

Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin [alpha]5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site, S651. Active PDE then hydrolyzes antiinflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site of PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in atherosclerosis-prone regions of arteries and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55[alpha] as the factor recruiting PP2A to PDE4D5. The B55[alpha]-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also, unexpectedly, stabilized the PP2A-B55[alpha] complex. The integrin-regulated, proatherosclerotic transcription factor Yap was also dephosphorylated and activated through this pathway. PDE4D5 therefore mediated matrix-specific regulation of endothelial cell phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex that has other targets. We believe these results may have widespread consequences for the control of cell function by integrins., Introduction Endothelial basement membranes in stable vessels, consisting primarily of collagen IV, laminins, and less abundant species, promote vessel maturation and stability (1, 2). By contrast, fibronectin (FN) is present [...]