Modulating Liquid-Liquid Phase Separation of Nck Adaptor Protein against Enteropathogenic Escherichia coli Infection.

Signaling proteins often form biomolecular condensates through liquid-liquid phase separation (LLPS) during intracellular signal transduction. Modulating the LLPS property of intracellular protein condensates will redirect intracellular signals and provide a potential way to regulate cellular physiology. Phosphorylation of multiple tyrosine residues of the transmembrane receptor nephrin is known to drive the LLPS of the adaptor protein Nck and neuronal Wiskott-Aldrich Syndrome protein (N-WASP) and form the Nck signaling complex. Phosphorylation of the translocated intimin receptor (Tir) in the host cell may recruit this enteropathogenic Escherichia coli (EPEC) virulence factor to the Nck signaling complex and lead to the entry of EPEC into the intestine cell. In this work, we first identified a phosphotyrosine (pY)-containing peptide 3pY based on the sequence similarity of nephrin and Tir; 3pY promoted the LLPS of Nck and N-WASP, mimicking the role of phosphorylated nephrin. Next, we designed a covalent blocker of Nck, peptide p1 based on the selected pY peptides, which site-selectively reacted with the SH2 domain of Nck (Nck-SH2) at Lys331 through a proximity-induced reaction. The covalent reaction of p1 with Nck blocked the protein binding site of Nck-SH2 and disintegrated the 3pY/Nck/N-WASP condensates. In the presence of membrane-translocating peptide L17E, p1 entered Caco-2 cells in the cytosol, reduced the number of Nck puncta, and rendered Caco-2 cells resistant to EPEC infection. Site-selective covalent blockage of Nck thereby disintegrates intracellular Nck condensates, inhibits actin reorganization, and shuts down the entrance pathway of EPEC. This work showcases the promotion or inhibition of protein phase separation by synthetic peptides and the use of reactive peptides as LLPS disruptors and signal modulators.

Deep tissue multi-photon imaging using adaptive optics with direct focus sensing and shaping.

High-resolution optical imaging deep in tissues is challenging because of optical aberrations and scattering of light caused by the complex structure of living matter. Here we present an adaptive optics three-photon microscope based on analog lock-in phase detection for focus sensing and shaping (ALPHA-FSS). ALPHA-FSS accurately measures and effectively compensates for both aberrations and scattering induced by specimens and recovers subcellular resolution at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 µm below the pia, enabling near-non-invasive high-resolution microscopy in cortex. Functional calcium imaging with high sensitivity and high-precision laser-mediated microsurgery through the intact skull were also demonstrated. Moreover, we achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below the pia within the intact brain.

Study of neurovascular coupling by using mesoscopic and microscopic imaging.

Neuronal activation is often accompanied by the regulation of cerebral hemodynamics via a process known as neurovascular coupling (NVC) which is essential for proper brain function and has been observed to be disrupted in a variety of neuropathologies. A comprehensive understanding of NVC requires imaging capabilities with high spatiotemporal resolution and a field-of-view that spans different orders of magnitude. Here, we present an approach for concurrent multi-contrast mesoscopic and two-photon microscopic imaging of neurovascular dynamics in the cortices of live mice. We investigated the spatiotemporal correlation between sensory-evoked neuronal and vascular responses in the auditory cortices of living mice using four imaging modalities. Our findings unravel drastic differences in the NVC at the regional and microvascular levels and the distinctive effects of different brain states on NVC. We further investigated the brain-state-dependent changes of NVC in large cortical networks and revealed that anesthesia and sedation caused spatiotemporal disruption of NVC.