In situ identification of cellular drug targets in mammalian tissue.

The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.

Xiphoid nucleus of the midline thalamus controls cold-induced food seeking.

Maintaining body temperature is calorically expensive for endothermic animals1. Mammals eat more in the cold to compensate for energy expenditure2, but the neural mechanism underlying this coupling is not well understood. Through behavioural and metabolic analyses, we found that mice dynamically switch between energy-conservation and food-seeking states in the cold, the latter of which are primarily driven by energy expenditure rather than the sensation of cold. To identify the neural mechanisms underlying cold-induced food seeking, we used whole-brain c-Fos mapping and found that the xiphoid (Xi), a small nucleus in the midline thalamus, was selectively activated by prolonged cold associated with elevated energy expenditure but not with acute cold exposure. In vivo calcium imaging showed that Xi activity correlates with food-seeking episodes under cold conditions. Using activity-dependent viral strategies, we found that optogenetic and chemogenetic stimulation of cold-activated Xi neurons selectively recapitulated food seeking under cold conditions whereas their inhibition suppressed it. Mechanistically, Xi encodes a context-dependent valence switch that promotes food-seeking behaviours under cold but not warm conditions. Furthermore, these behaviours are mediated by a Xi-to-nucleus accumbens projection. Our results establish Xi as a key region in the control of cold-induced feeding, which is an important mechanism in the maintenance of energy homeostasis in endothermic animals.

HYBRiD: hydrogel-reinforced DISCO for clearing mammalian bodies.

The recent development of solvent- and polymer-based brain-clearing techniques has advanced our ability to visualize the mammalian nervous system in three dimensions. However, it remains challenging to image the mammalian body en bloc. Here we developed HYBRiD (hydrogel-based reinforcement of three-dimensional imaging solvent-cleared organs (DISCO)), by recombining components of organic- and polymer-based clearing pipelines. We achieved high transparency and protein retention, as well as compatibility with direct fluorescent imaging and immunostaining in cleared mammalian bodies. Using parvalbumin- and somatostatin-Cre models, we demonstrated the utility of HYBRiD for whole-body imaging of genetically encoded fluorescent reporters without antibody enhancement of signals in newborn and juvenile mice. Using K18-hACE2 transgenic mice, HYBRiD enabled perfusion-free clearing and visualization of SARS-CoV-2 infection in a whole mouse chest, revealing macroscopic and microscopic features of viral pathology in the same sample. HYBRiD offers a simple and universal solution to visualize large heterogeneous body parts or entire animals for basic and translational research.

Phosphorylation of pyruvate dehydrogenase inversely associates with neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Xiphoid nucleus of the midline thalamus controls cold-induced food seeking.

Maintaining body temperature is calorically expensive for endothermic animals. Mammals eat more in the cold to compensate for energy expenditure, but the neural mechanism underlying this coupling is not well understood. Through behavioral and metabolic analyses, we found that mice dynamically switch between energy conservation and food-seeking states in the cold, the latter of which is primarily driven by energy expenditure rather than the sensation of cold. To identify the neural mechanisms underlying cold-induced food seeking, we use whole-brain cFos mapping and found that the xiphoid (Xi), a small nucleus in the midline thalamus, was selectively activated by prolonged cold associated with elevated energy expenditure but not with acute cold exposure. In vivo calcium imaging showed that Xi activity correlates with food-seeking episodes in cold conditions. Using activity-dependent viral strategies, we found that optogenetic and chemogenetic stimulation of cold-activated Xi neurons recapitulated cold-induced feeding, whereas their inhibition suppressed it. Mechanistically, Xi encodes a context-dependent valence switch promoting food-seeking behaviors in cold but not warm conditions. Furthermore, these behaviors are mediated by a Xi to nucleus accumbens projection. Our results establish Xi as a key region for controlling cold-induced feeding, an important mechanism for maintaining energy homeostasis in endothermic animals.

Phosphorylation of pyruvate dehydrogenase marks the inhibition of in vivo neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as c- fos has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity (i.e., inhibition). Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected neuronal inhibition across the brain induced by a wide range of factors including general anesthesia, sensory experiences, and natural behaviors. Thus, as an in vivo marker for neuronal inhibition, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Pathogens manipulate host autophagy through injected effector proteins.

Macroautophagy/autophagy plays a dual role in many physiological processes of multicellular eukaryotes. In plants, autophagy can be used by both host and pathogen for a beneficiary infection outcome. Plants employ a two-tier innate immune system to defend against invading pathogens. Cell surface localized pattern recognition receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and launch pattern-triggered immunity (PTI) to provide broad-spectrum resistance. Pathogens inject a battery of effector proteins into their hosts to counter PTI and compromise the primary immune response. Hosts induce a second layer of defense called effector-triggered immunity (ETI) to counter the effects of these effectors. In addition to ETI and PTI, autophagy is emerging as a central cellular process modulated by both host and pathogens toward their respective advantage. Pathogens lacking the ability to inject effectors are compromised in virulence. However, molecular targets and biochemical characterization of most of these effector proteins remain elusive. In a recent paper we presented a systematic analysis of interaction between autophagy proteins of Arabidopsis thaliana with effectors from bacterial, fungal, oomycete and nematode pathogens. Abbreviations: ATG, autophagy related; BiFC, bimolecular fluorescence complementation; ETI, effector-triggered immunity; PAMPs, pathogen-associated molecular patterns; PTI, pattern-triggered immunity.

Phytopathogen Effectors Use Multiple Mechanisms to Manipulate Plant Autophagy.

Autophagy is a central part of immunity and hence is a key target of pathogens. However, the precise molecular mechanisms by which plant pathogens manipulate autophagy remain elusive. We identify a network of 88 interactions between 184 effectors from bacterial, fungal, oomycete, and nematode pathogens with 25 Arabidopsis autophagy (ATG) proteins. Notably, Pseudomonas syringae pv tomato (Pto) bacterial effectors HrpZ1, HopF3, and AvrPtoB employ distinct molecular strategies to modulate autophagy. Calcium-dependent HrpZ1 oligomerization targets ATG4b-mediated cleavage of ATG8 to enhance autophagy, while HopF3 also targets ATG8 but suppresses autophagy, with both effectors promoting infection. AvrPtoB affects ATG1 kinase phosphorylation and enhances bacterial virulence. Since pathogens inject limited numbers of effectors into hosts, our findings establish autophagy as a key target during infection. Additionally, as autophagy is enhanced and inhibited by these effectors, autophagy likely has different functions throughout infection and, thus, must be temporally and precisely regulated for successful infection.

Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD.

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection. The plant NADPH oxidase RBOHD is a primary player in ROS production during innate immunity. However, how RBOHD is negatively regulated remains elusive. Here we show that RBOHD is regulated by C-terminal phosphorylation and ubiquitination. Genetic and biochemical analyses reveal that the PBL13 receptor-like cytoplasmic kinase phosphorylates RBOHD's C-terminus and two phosphorylated residues (S862 and T912) affect RBOHD activity and stability, respectively. Using protein array technology, we identified an E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. Mimicking phosphorylation of RBOHD (T912D) results in enhanced ubiquitination and decreased protein abundance. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Thus, our study reveals an intricate post-translational network that negatively regulates the abundance of a conserved NADPH oxidase.

TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity.

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play a critical role in defence against pathogens in plants and animals. However, we know very little about NLR-interacting proteins and the mechanisms that regulate NLR levels. Here, we used proximity labeling (PL) to identify the proteome proximal to N, which is an NLR that confers resistance to Tobacco mosaic virus (TMV). Evaluation of different PL methods indicated that TurboID-based PL provides more efficient levels of biotinylation than BioID and BioID2 in plants. TurboID-based PL of N followed by quantitative proteomic analysis and genetic screening revealed multiple regulators of N-mediated immunity. Interestingly, a putative E3 ubiquitin ligase, UBR7, directly interacts with the TIR domain of N. UBR7 downregulation leads to an increased amount of N protein and enhanced TMV resistance. TMV-p50 effector disrupts the N-UBR7 interaction and relieves negative regulation of N. These findings demonstrate the utility of TurboID-based PL in plants and the N-interacting proteins we identified enhance our understanding of the mechanisms underlying NLR regulation.

The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity.

Plants employ cell-surface pattern recognition receptors (PRRs) to detect pathogens. Although phytohormones produced during PRR signaling play an essential role in innate immunity, a direct link between PRR activation and hormone regulation is unknown. EFR is a PRR that recognizes bacterial EF-Tu and activates immune signaling. Here we report that EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1. The BIK1 structure revealed that the EFR-phosphorylated sites reside on a uniquely extended loop away from the BIK1 kinase core domain. Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections. In addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation. These findings demonstrate the mechanistic basis of signal transduction from PRR to phytohormones, mediated through a PRR-BIK1-WRKY axis.

The MAP4 Kinase SIK1 Ensures Robust Extracellular ROS Burst and Antibacterial Immunity in Plants.

Microbial patterns are recognized by cell-surface receptors to initiate pattern-triggered immunity (PTI) in plants. Receptor-like cytoplasmic kinases (RLCKs), such as BIK1, and calcium-dependent protein kinases (CPKs) are engaged during PTI to activate the NADPH oxidase RBOHD for reactive oxygen species (ROS) production. It is unknown whether protein kinases besides CPKs and RLCKs participate in RBOHD regulation. We screened mutants in all ten Arabidopsis MAP4 kinases (MAP4Ks) and identified the conserved MAP4K SIK1 as a positive regulator of PTI. sik1 mutants were compromised in their ability to elicit the ROS burst in response to microbial features and exhibited compromised PTI to bacterial infection. SIK1 directly interacts with, phosphorylates, and stabilizes BIK1 in a kinase activity-dependent manner. Furthermore, SIK1 directly interacts with and phosphorylates RBOHD upon flagellin perception. Thus, SIK1 positively regulates immunity by stabilizing BIK1 and activating RBOHD to promote the extracellular ROS burst.

Arabidopsis receptor-like cytoplasmic kinase BIK1: purification, crystallization and X-ray diffraction analysis.

Receptor-like cytoplasmic kinases (RLCKs) in Arabidopsis play a central role in the integration of signaling input from various growth and immune signaling pathways. BOTRYTIS-INDUCED KINASE 1 (BIK1), belonging to the RLCK family, is an important player in defense against bacterial and fungal pathogens and in ethylene and brassinosteroid hormone signaling. In this study, the purification and crystallization of a first member of the class VI family of RLCK proteins, BIK1, are reported. BIK1 was crystallized using the microbatch-under-oil method. X-ray diffraction data were collected to 2.35 Šresolution. The crystals belonged to the monoclinic space group C2, with two monomers per asymmetric unit.