Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.

Astrocyte-secreted IL-33 mediates homeostatic synaptic plasticity in the adult hippocampus.

Hippocampal synaptic plasticity is important for learning and memory formation. Homeostatic synaptic plasticity is a specific form of synaptic plasticity that is induced upon prolonged changes in neuronal activity to maintain network homeostasis. While astrocytes are important regulators of synaptic transmission and plasticity, it is largely unclear how they interact with neurons to regulate synaptic plasticity at the circuit level. Here, we show that neuronal activity blockade selectively increases the expression and secretion of IL-33 (interleukin-33) by astrocytes in the hippocampal cornu ammonis 1 (CA1) subregion. This IL-33 stimulates an increase in excitatory synapses and neurotransmission through the activation of neuronal IL-33 receptor complex and synaptic recruitment of the scaffold protein PSD-95. We found that acute administration of tetrodotoxin in hippocampal slices or inhibition of hippocampal CA1 excitatory neurons by optogenetic manipulation increases IL-33 expression in CA1 astrocytes. Furthermore, IL-33 administration in vivo promotes the formation of functional excitatory synapses in hippocampal CA1 neurons, whereas conditional knockout of IL-33 in CA1 astrocytes decreases the number of excitatory synapses therein. Importantly, blockade of IL-33 and its receptor signaling in vivo by intracerebroventricular administration of its decoy receptor inhibits homeostatic synaptic plasticity in CA1 pyramidal neurons and impairs spatial memory formation in mice. These results collectively reveal an important role of astrocytic IL-33 in mediating the negative-feedback signaling mechanism in homeostatic synaptic plasticity, providing insights into how astrocytes maintain hippocampal network homeostasis.

Phase-Separated Multienzyme Compartmentalization for Terpene Biosynthesis in a Prokaryote.

Liquid-liquid phase separation (LLPS) forms biomolecular condensates or coacervates in cells. Metabolic enzymes can form phase-separated subcellular compartments that enrich enzymes, cofactors, and substrates. Herein, we report the construction of synthetic multienzyme condensates that catalyze the biosynthesis of a terpene, α-farnesene, in the prokaryote E. coli. RGGRGG derived from LAF-1 was used as the scaffold protein to form the condensates by LLPS. Multienzyme condensates were then formed by assembling two enzymes Idi and IspA through an RIAD/RIDD interaction. Multienzyme condensates constructed inside E. coli cells compartmentalized the cytosolic space into regions of high and low enzyme density and led to a significant enhancement of α-farnesene production. This work demonstrates LLPS-driven compartmentalization of the cytosolic space of prokaryotic cells, condensation of a biosynthetic pathway, and enhancement of the biosynthesis of α-farnesene.

Synthetic Multienzyme Complexes Assembled on Virus-like Particles for Cascade Biosynthesis In Cellulo.

Multienzyme complexes, or metabolons, are natural assemblies or clusters of sequential enzymes in biosynthesis. Spatial proximity of the enzyme active sites results in a substrate channeling effect, streamlines the cascade reaction, and increases the overall efficiency of the metabolic pathway. Engineers have constructed synthetic multienzyme complexes to acquire better control of the metabolic flux and a higher titer of the target product. As most of these complexes are assembled through orthogonal interactions or bioconjugation reactions, the number of enzymes to be assembled is limited by the number of orthogonal interaction or reaction pairs. Here, we utilized the Tobacco mosaic virus (TMV) virus-like particle (VLP) as protein scaffold and orthogonal reactive protein pairs (SpyCatcher/SpyTag and SnoopCatcher/SnoopTag) as linker modules to assemble three terpene biosynthetic enzymes in Escherichia coli. The enzyme assembly switched on the production of amorpha-4,11-diene, whereas the product was undetectable in all the controls without assembly. This work demonstrates a facile strategy for constructing scaffolded catalytic nanomachineries to biosynthesize valuable metabolites in bacterial cells, and a unique assembly induced the switch-on mechanism in biosynthesis for the first time.

Phase-Separated Multienzyme Biosynthesis.

Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, for example, the purinosome, which forms subcellular macrobodies responsible for de novo purine biosynthesis. Here, we construct synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. A synthetic protein phase separation system using component proteins from postsynaptic density in neuronal synapses, GKAP, Shank, and Homer provides the scaffold for assembly. Three sets of guest proteins: a pair of fluorescent proteins (CFP and YFP), three sequential enzymes in menaquinone biosynthesis pathway (MenF, MenD, and MenH), and two enzymes in terpene biosynthesis pathway (Idi and IspA) are assembled via peptide-peptide interactions in the condensate. First, we discover that coassembly of CFP and YFP exhibited a broad distribution of the FRET signal within the condensate. Second, a spontaneous enrichment of the rate-limiting enzyme MenD in the condensate is sufficient to increase the 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate production rate by 70%. Third, coassembly of both Idi and IspA in the protein condensate increases the farnesyl pyrophosphate production rate by more than 50%. Altogether, we show here that phase separation significantly accelerates the efficiency of multienzyme biocatalysis.

Quantitative Imaging of Lipid Synthesis and Lipolysis Dynamics in Caenorhabditis elegans by Stimulated Raman Scattering Microscopy.

Quantitative methods to precisely measure cellular states in vivo have become increasingly important and desirable in modern biology. Recently, stimulated Raman scattering (SRS) microscopy has emerged as a powerful tool to visualize small biological molecules tagged with alkyne (C≡C) or carbon-deuterium (C-D) bonds in the cell-silent region. In this study, we developed a technique based on SRS microscopy of vibrational tags for quantitative imaging of lipid synthesis and lipolysis in live animals. The technique aims to overcome the major limitations of conventional fluorescent staining and lipid extraction methods that do not provide the capability of in vivo quantitative analysis. Specifically, we used three bioorthogonal lipid molecules (the alkyne-tagged fatty acid 17-ODYA, deuterium-labeled saturated fatty acid PA-D31, and unsaturated fatty acid OA-D34) to investigate the metabolic dynamics of lipid droplets (LDs) in live Caenorhabditis elegans ( C. elegans). Using a hyperspectral SRS (hsSRS) microscope and subtraction method, the interfering non-Raman background was eliminated to improve the accuracy of lipid quantification. A linear relationship between SRS signals and fatty acid molar concentrations was accurately established. With this quantitative analysis tool, we imaged and determined the changes in concentration of the three fatty acids in LDs of fed or starved adult C. elegans. Using the hsSRS imaging mode, we also observed the desaturation of fatty acids in adult C. elegans via spectral analysis on the SRS signals from LDs. The results demonstrated the unique capability of hsSRS microscopy in quantitative analysis of lipid metabolism in vivo.

Mitochondrial Imaging with Combined Fluorescence and Stimulated Raman Scattering Microscopy Using a Probe of the Aggregation-Induced Emission Characteristic.

In vivo quantitative measurement of biodistribution plays a critical role in the drug/probe development and diagnosis/treatment process monitoring. In this work, we report a probe, named AIE-SRS-Mito, for imaging mitochondria in live cells via fluorescence (FL) and stimulated Raman scattering (SRS) imaging. The probe features an aggregation-induced emission (AIE) characteristic and possesses an enhanced alkyne Raman peak at 2223 cm-1. The dual-mode imaging of AIE-SRS-Mito for selective mitochondrion-targeting was examined on a homemade FL-SRS microscope system. The detection limit of the probe in the SRS imaging was estimated to be 8.5 µM. Due to the linear concentration dependence of SRS and inertness of the alkyne Raman signal to environmental changes, the intracellular distribution of the probe was studied, showing a local concentration of >2.0 mM in the mitochondria matrix, which was >100-fold higher than the incubation concentration. To the best of our knowledge, this is the first time that the local concentration of AIE molecules inside cells has been measured noninvasively and directly. Also, the nonquenching effect of such AIE molecules in cell imaging has been verified by the positive correlation of FL and SRS signals. Our work will encourage the utilization of SRS microscopy for quantitative characterization of FL probes or other nonfluorescent compounds in living biological systems and the development of FL-SRS dual-mode probes for specific biotargets.

Ultrafast Delivery of Aggregation-Induced Emission Nanoparticles and Pure Organic Phosphorescent Nanocrystals by Saponin Encapsulation.

Saponins are a class of naturally occurring bioactive and biocompatible amphiphilic glycosides produced by plants. Some saponins, such as α-hederin, exhibit unique cell membrane interactions. At concentrations above their critical micelle concentration, they will interact and aggregate with membrane cholesterol to form transient pores in the cell membrane. In this project, we utilized the unique permeabilization and amphiphilic properties of saponins for the intracellular delivery of deep-red-emitting aggregation-induced emission nanoparticles (AIE NPs) and pure organic room-temperature phosphorescent nanocrystals (NCs). We found this method to be biocompatible, inexpensive, ultrafast, and applicable to deliver a wide variety of AIE NPs and NCs into cancer cells.

Microsecond protein folding events revealed by time-resolved fluorescence resonance energy transfer in a microfluidic mixer.

We demonstrate the combination of the time-resolved fluorescence resonance energy transfer (tr-FRET) measurement and the ultrarapid hydrodynamic focusing microfluidic mixer. The combined technique is capable of probing the intermolecular distance change with temporal resolution at microsecond level and structural resolution at Angstrom level, and the use of two-photon excitation enables a broader exploration of FRET with spectrum from near-ultraviolet to visible wavelength. As a proof of principle, we used the coupled microfluidic laminar flow and time-resolved two-photon excitation microscopy to investigate the early folding states of Cytochrome c (cyt c) by monitoring the distance between the tryptophan (Trp-59)-heme donor-acceptor (D-A) pair. The transformation of folding states of cyt c in the early 500 µs of refolding was revealed on the microsecond time scale. For the first time, we clearly resolved the early transient state of cyt c, which is populated within the dead time of the mixer (<10 µs) and has a characteristic Trp-59-heme distance of ∼31 Å. We believe this tool can find more applications in studying the early stages of biological processes with FRET as the probe.

Mapping live cell viscosity with an aggregation-induced emission fluorogen by means of two-photon fluorescence lifetime imaging.

Intracellular viscosity is a crucial parameter that indicates the functioning of cells. In this work, we demonstrate the utility of TPE-Cy, a cell-permeable dye with aggregation-induced emission (AIE) property, in mapping the viscosity inside live cells. Owing to the AIE characteristics, both the fluorescence intensity and lifetime of this dye are increased along with an increase in viscosity. Fluorescence lifetime imaging of live cells stained with TPE-Cy reveals that the lifetime in lipid droplets is much shorter than that from the general cytoplasmic region. The loose packing of the lipids in a lipid droplet results in low viscosity and thus shorter lifetime of TPE-Cy in this region. It demonstrates that the AIE dye could provide good resolution in intracellular viscosity sensing. This is also the first work in which AIE molecules are applied in fluorescence lifetime imaging and intracellular viscosity sensing.

In Vivo nonlinear optical imaging of immune responses: tissue injury and infection.

In this study, we demonstrate a noninvasive imaging approach based on multimodal nonlinear optical microscopy to in vivo image the responses of immune cells (neutrophils) to the tissue injury and bacterial infection in a zebrafish model. Specifically, the second harmonic generation from myosin thick filaments in sarcomere enabled a clear visualization of the muscle injury and infection. Two-photon excited fluorescence was used to track the behavior of the neutrophils that were transgenically labeled by red fluorescent protein. The corresponding reduced nicotinamide adenine dinucleotide (NADH) two-photon excited fluorescence images revealed a detailed morphological transformation process of individual neutrophils during muscle tissue injury and bacterial infection. The analysis of time-resolved NADH signals from the neutrophils provided important biological insights of the cellular energy metabolism during the immune responses. We found a significant increase of free/protein-bound NADH ratios in activated neutrophils in bacterial-infected tissue. In this study, we also discovered that, under 720 nm excitation, two wild-type strains (DH5? and BL21) of bacteria Escherichia coli emitted distinct endogenous fluorescence of double-peak at ?450 and ?520 nm, respectively. We demonstrated that the double-peak fluorescence signal could be used to differentiate the E. coli from surrounding tissues of dominant NADH signals, and to achieve label-free tracking of E. coli bacteria in vivo.

col1a2+ fibroblasts/muscle progenitors finetune xanthophore countershading by differentially expressing csf1a/1b in embryonic zebrafish.

Animals evolve diverse pigment patterns to adapt to the natural environment. Countershading, characterized by a dark-colored dorsum and a light-colored ventrum, is one of the most prevalent pigment patterns observed in vertebrates. In this study, we reveal a mechanism regulating xanthophore countershading in zebrafish embryos. We found that csf1a and csf1b mutants altered xanthophore countershading differently: csf1a mutants lack ventral xanthophores, while csf1b mutants have reduced dorsal xanthophores. Further study revealed that csf1a is expressed throughout the trunk, whereas csf1b is expressed dorsally. Ectopic expression of csf1a or csf1b in neurons attracted xanthophores into the spinal cord. Blocking csf1 signaling by csf1ra mutants disrupts spinal cord distribution and normal xanthophores countershading. Single-cell RNA sequencing identified two col1a2+ populations: csf1ahighcsf1bhigh muscle progenitors and csf1ahighcsf1blow fibroblast progenitors. Ablation of col1a2+ fibroblast and muscle progenitors abolished xanthophore patterns. Our study suggests that fibroblast and muscle progenitors differentially express csf1a and csf1b to modulate xanthophore patterning, providing insights into the mechanism of countershading.

Intravital microscopy of satellite cell dynamics and their interaction with myeloid cells during skeletal muscle regeneration.

Skeletal muscle regeneration requires the highly coordinated cooperation of muscle satellite cells (MuSCs) with other cellular components. Upon injury, myeloid cells populate the wound site, concomitant with MuSC activation. However, detailed analysis of MuSC-myeloid cell interaction is hindered by the lack of suitable live animal imaging technology. Here, we developed a dual-laser multimodal nonlinear optical microscope platform to study the dynamics of MuSCs and their interaction with nonmyogenic cells during muscle regeneration. Using three-dimensional time-lapse imaging on live reporter mice and taking advantages of the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH), we studied the spatiotemporal interaction between nonmyogenic cells and muscle stem/progenitor cells during MuSC activation and proliferation. We discovered that their cell-cell contact was transient in nature. Moreover, MuSCs could activate with notably reduced infiltration of neutrophils and macrophages, and their proliferation, although dependent on macrophages, did not require constant contact with them. These findings provide a fresh perspective on myeloid cells' role during muscle regeneration.

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.

Interhemispheric cortical long-term potentiation in the auditory cortex requires heterosynaptic activation of entorhinal projection.

Long-term potentiation (LTP), which underlies learning and memory, can be induced by high-frequency electrical stimulation (HFS or HFES) and is thought to occur at the synapses of efferent projection. Here, the contralateral connectivity in mice auditory cortex was investigated to reveal the fundamental corticocortical connection properties. After HFES, plasticity was not observed at the terminal synapses at the recording site. The optogenetic HFS at the recording site of the interhemispheric cortical projections could not induce LTP, but HFES at the recording site could induce the interhemispheric cortical LTP. Our subsequent results uncovered that it is the cholecystokinin (CCK) released from the entorhino-neocortical pathway induced by HEFS that modulates the neuroplasticity of the afferent projections, including interhemispheric auditory cortical afferents. Our study illustrates a heterosynaptic mechanism as the basis for cortical plasticity. This regulation might contribute new spots for the understanding and treatment of neurological disorders.

Cell reprogramming in a predictable manner on the superhydrophobic microwell array chip.

Reprogramming of somatic cells into the pluripotent state is stochastic and inefficient using the conventional culture plates. Novel micro-culture systems employing precisely controlled biophysical cues can improve the reprogramming efficiencies dramatically. Here we perform iPSC induction on our previously developed superhydrophobic microwell array chip (SMAR-chip) where cells undergo distinctive morphology change, switching from 2D monolayers to 3D clumps, and develop into bona fide colonies in more than 90% of the microwells. The PDMS substrate, together with the microwell structure and the superhydrophobic layer constitute a well-controlled microenvironment favorable for the morphogenesis and pluripotency induction. Investigation of the molecular roadmap demonstrates that the SMAR-chip promotes the transition from the initiation phase to the maturation phase and overcomes the roadblocks for reprogramming. In addition, the SMAR-chip also promotes the reprogramming of human cells, opening our method for translational applications. In summary, our study provides a novel platform for efficient cell reprogramming and emphasizes the advantages of employing the insoluble microenvironmental cues for the precise control of cell fate conversion.

Two-photon excitation chlorophyll fluorescence lifetime imaging: a rapid and noninvasive method for in vivo assessment of cadmium toxicity in a marine diatom Thalassiosira weissflogii.

We demonstrate that a two-photon excitation fluorescence lifetime imaging technology can rapidly and noninvasively assess the cadmium (Cd)-induced toxic effects in a marine diatom Thalassiosira weissflogii. The chlorophyll, an intrinsic fluorophore, was used as a contrast agent for imaging of cellular structures and for assessment of cell toxicity. The assessment is based on an imaging-guided statistical analysis of chlorophyll fluorescence decay. This novel label-free imaging method is physically based and free of tedious preparation and preprocessing of algal samples. We first studied the chlorophyll fluorescence quenching induced by the infrared two-photon excitation laser and found that the quenching effects on the assessment of Cd toxicity could be well controlled and calibrated. In the toxicity study, chlorophyll fluorescence lifetime images were collected from the diatom samples after exposure to different concentrations of Cd. The alteration of chloroplast structure at higher Cd concentration was clearly identified. The decay of chlorophyll fluorescence extracted from recorded pixels of high signal-to-noise ratio in the fluorescence lifetime image was analyzed. The increase of average chlorophyll fluorescence lifetime following Cd treatment was observed, indicating the Cd inhibition effect on the electron transport chain in photosynthesis system. The findings of this study show that the temporal characteristics of chlorophyll fluorescence can potentially be utilized as a biomarker for indicating Cd toxicity noninvasively in algal cells.

Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

Long-term in vivo imaging of mouse spinal cord through an optically cleared intervertebral window.

The spinal cord accounts for the main communication pathway between the brain and the peripheral nervous system. Spinal cord injury is a devastating and largely irreversible neurological trauma, and can result in lifelong disability and paralysis with no available cure. In vivo spinal cord imaging in mouse models without introducing immunological artifacts is critical to understand spinal cord pathology and discover effective treatments. We developed a minimally invasive intervertebral window by retaining the ligamentum flavum to protect the underlying spinal cord. By introducing an optical clearing method, we achieve repeated two-photon fluorescence and stimulated Raman scattering imaging at subcellular resolution with up to 15 imaging sessions over 6-167 days and observe no inflammatory response. Using this optically cleared intervertebral window, we study neuron-glia dynamics following laser axotomy and observe strengthened contact of microglia with the nodes of Ranvier during axonal degeneration. By enabling long-term, repetitive, stable, high-resolution and inflammation-free imaging of mouse spinal cord, our method provides a reliable platform in the research aiming at interpretation of spinal cord physiology and pathology.

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.