ZBTB21 suppresses CRE-mediated transcription to impair synaptic function in Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder and a major cause of intellectual disability. The genetic etiology of DS is the extra copy of chromosome 21 (HSA21)-encoded genes; however, the contribution of specific HSA21 genes to DS pathogenesis remains largely unknown. Here, we identified ZBTB21, an HSA21-encoded zinc-finger protein, as a transcriptional repressor in the regulation of synaptic function. We found that normalization of the Zbtb21 gene copy number in DS mice corrected deficits in cognitive performance, synaptic function, and gene expression. Moreover, we demonstrated that ZBTB21 binds to canonical cAMP-response element (CRE) DNA and that its binding to CRE could be competitive with CRE-binding factors such as CREB. ZBTB21 represses CRE-dependent gene expression and results in the negative regulation of synaptic plasticity, learning and memory. Together, our results identify ZBTB21 as a CRE-binding protein and repressor in cAMP-dependent gene regulation, contributing to cognitive defects in DS.

Signal-sustained imaging of mitophagy with an Enzyme-Activatable Metabolic Lipid-Labeling Probe.

Imaging of mitophagy is of significance as aberrant mitophagy is engaged in multiple diseases. Mitophagy has been imaged with synthetic or biotic pH sensors by reporting pH acidification en route delivery into lysosomes. To circumvent uncertainty of acidity-dependent signals, we herein report an enzyme-activatable probe covalently attached on mitochondrial inner membrane (ECAM) for signal-persist mitophagy imaging. ECAM is operated via ΔΨm-driven accumulation of Mito-proGreen in mitochondria and covalent linking of the trapped probe with azidophospholipids metabolically incorporated into the mitochondrial inner membrane. Upon mitophagy, ECAM is delivered into lysosomes and hydrolyzed by LNPEP/leucyl aminopeptidase, yielding turn-on green fluorescence that is immune to lysosomal acidity changes and stably retained in fixed cells. With ECAM, phorbol-12-myristate-13-acetate (PMA) was identified as a highly potent inducer of mitophagy. Overcoming signal susceptibility of pH probes and liability of ΔΨm probes to dissipation from stressed mitochondria, ECAM offers an attractive tool to study mitophagy and mitophagy-inducing therapeutic agents.

AMPK targets PDZD8 to trigger carbon source shift from glucose to glutamine.

The shift of carbon utilization from primarily glucose to other nutrients is a fundamental metabolic adaptation to cope with decreased blood glucose levels and the consequent decline in glucose oxidation. AMP-activated protein kinase (AMPK) plays crucial roles in this metabolic adaptation. However, the underlying mechanism is not fully understood. Here, we show that PDZ domain containing 8 (PDZD8), which we identify as a new substrate of AMPK activated in low glucose, is required for the low glucose-promoted glutaminolysis. AMPK phosphorylates PDZD8 at threonine 527 (T527) and promotes the interaction of PDZD8 with and activation of glutaminase 1 (GLS1), a rate-limiting enzyme of glutaminolysis. In vivo, the AMPK-PDZD8-GLS1 axis is required for the enhancement of glutaminolysis as tested in the skeletal muscle tissues, which occurs earlier than the increase in fatty acid utilization during fasting. The enhanced glutaminolysis is also observed in macrophages in low glucose or under acute lipopolysaccharide (LPS) treatment. Consistent with a requirement of heightened glutaminolysis, the PDZD8-T527A mutation dampens the secretion of pro-inflammatory cytokines in macrophages in mice treated with LPS. Together, we have revealed an AMPK-PDZD8-GLS1 axis that promotes glutaminolysis ahead of increased fatty acid utilization under glucose shortage.

Phosphorylation of caspase-8 by RSKs via organ-constrained effects controls the sensitivity to TNF-induced death.

Caspase-8 (Casp8) serves as an initiator of apoptosis or a suppressor of necroptosis in context-dependent manner. Members of the p90 RSK family can phosphorylate caspase-8 at threonine-265 (T265), which can inactivate caspase-8 for bypassing caspase-8-mediated blockade of necroptosis and can also decrease caspase-8 level by promoting its degradation. Mutating T265 in caspase-8 to alanine (A) in mice blocked TNF-induced necroptotic cecum damage but resulted in unexpectedly massive injury in the small intestine. Here, we show RSK1, RSK2, and RSK3 redundantly function in caspase-8 phosphorylation, and the duodenum is the most severely affected part of the small intestine when T265 phosphorylation of caspase-8 was prevented. Eliminating caspase-8 phosphorylation resulted in a duodenum-specific increase in basal caspase-8 protein level, which shall be responsible for the increased sensitivity to TNF-induced damage. Apoptosis of intestinal epithelial cells (IECs) was predominant in the duodenum of TNF-treated Rsk1-/-Rsk2-/-Rsk3-/- and Casp8T265A/T265A mice, though necroptosis was also observed. The heightened duodenal injury amplified systemic inflammatory responses, as evidenced by the contribution of hematopoietic cells to the sensitization of TNF-induced animal death. Further analysis revealed that hematopoietic and non-hematopoietic cells contributed differentially to cytokine production in response to the increased cell death. Collectively, RSKs emerges as a previously overlooked regulator that, via tissue/organ-constrained inactivating caspase-8 and/or downregulating caspase-8 protein level, controls the sensitivity to TNF-induced organ injury and animal death.

Cell biology is .

50 years ago, cell biology was a nascent field. Today, it is a vast discipline whose principles and tools are also applied to other disciplines; vice versa, cell biologists are inspired by other fields. So, the question begs: what is cell biology? The answers are as diverse as the people who define it.

Anthrax lethal toxin and tumor necrosis factor-α synergize on intestinal epithelia to induce mouse death.

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish.

CRISPR-mediated knock-in (KI) technology opens a new era of fluorescent-protein labeling in zebrafish, a preferred model organism for in vivo imaging. We described here an optimized zebrafish gene-tagging strategy, which enables easy and high-efficiency KI, ensures high odds of obtaining seamless KI germlines and is suitable for wide applications. Plasmid donors for 3'-labeling were optimized by shortening the microhomologous arms and by reducing the number and reversing the sequence of the consensus Cas9/sgRNA binding sites. To allow for scar-less KI across the genome, linearized dsDNA donors with 5'-chemical modifications were generated and successfully incorporated into our method. To refine the germline screen workflow and expedite the screen process, we combined fluorescence enrichment and caudal-fin junction-PCR. Furthermore, to trace proteins expressed at a low abundance, we developed a fluorescent signal amplifier using the transcriptional activation strategy. Together, our strategies enable efficient gene-tagging and sensitive expression detection for almost every gene in zebrafish.

Protocol for reconstitution of oligomeric assembly of NAIP5-NLRC4 inflammasome in vitro.

Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2023).1.

Fluorescence-On Imaging of Reticulophagy Enabled by an Acidity-Reporting Solvatochromic Probe.

Aberrant autophagy of the endoplasmic reticulum (reticulophagy) is engaged in diverse pathological disorders. Herein, we reported sensitive imaging of reticulophagy with ER-Green-proRed, a diad combining a solvatochromic entity of trifluoromethylated naphthalimide for long-term ER tracking by green fluorescence and an entity of rhodamine-lactam fluorogenic to lysosomal acidity. Stringently accumulated in the ER to give green fluorescence, ER-Green-proRed exhibits robust red fluorescence upon codelivery with the ER subdomain into lysosomes. The relevance of turn-on red fluorescence to reticulophagy was validated by reticulophagy modulated by starvation, reticulophagic receptors, and autophagy inhibition. This imaging method was successfully employed to discern reticulophagy induced by various pharmacological agents. These results show the potential of ER-targeted pH probes, as exemplified by ER-Green-proRed, to image reticulophagy and to identify reticulophagy inducers.

Dear-DIAXMBD: Deep Autoencoder Enables Deconvolution of Data-Independent Acquisition Proteomics.

Data-independent acquisition (DIA) technology for protein identification from mass spectrometry and related algorithms is developing rapidly. The spectrum-centric analysis of DIA data without the use of spectra library from data-dependent acquisition data represents a promising direction. In this paper, we proposed an untargeted analysis method, Dear-DIAXMBD, for direct analysis of DIA data. Dear-DIAXMBD first integrates the deep variational autoencoder and triplet loss to learn the representations of the extracted fragment ion chromatograms, then uses the k-means clustering algorithm to aggregate fragments with similar representations into the same classes, and finally establishes the inverted index tables to determine the precursors of fragment clusters between precursors and peptides and between fragments and peptides. We show that Dear-DIAXMBD performs superiorly with the highly complicated DIA data of different species obtained by different instrument platforms. Dear-DIAXMBD is publicly available at https://github.com/jianweishuai/Dear-DIA-XMBD.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

Sensitive imaging of Endoplasmic reticulum (ER) autophagy with an acidity-reporting ER-Tracker.

Macroautophagic/autophagic turnover of endoplasmic reticulum (reticulophagy) is critical for cell health. Herein we reported a sensitive fluorescence-on imaging of reticulophagy using a small molecule probe (ER-proRed) comprised of green-emissive fluorinated rhodol for ER targeting and nonfluorescent rhodamine-lactam prone to lysosome-triggered red fluorescence. Partitioned in ER to exhibit green fluorescence, ER-proRed gives intense red fluorescence upon co-delivery with ER into acidic lysosomes. Serving as the signal of reticulophagy, the turning on of red fluorescence enables discernment of reticulophagy induced by starvation, varied levels of reticulophagic receptors, and chemical agents such as etoposide and sodium butyrate. These results show ER probes optically activatable in lysosomes, such as ER-proRed, offer a sensitive and simplified tool for studying reticulophagy in biology and diseases.Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CQ, chloroquine diphosphate; ER, endoplasmic reticulum; FHR, fluorinated hydrophobic rhodol; GFP, green fluorescent protein; Reticulophagy, selective autophagy of ER; RFP, red fluorescent protein; ROX, X-rhodamine; UPR, unfolded protein response.

A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation.

Plasma cells (PC) are antibody-secreting cells and terminal effectors in humoral responses. PCs differentiate directly from activated B cells in response to T cell-independent (TI) antigens or from germinal center B (GCB) cells in T cell-dependent (TD) antigen-induced humoral responses, both of which pathways are essentially regulated by the transcription factor BLIMP1. The p38 mitogen-activated protein kinase isoforms have already been implicated in B cell development, but the precise role of p38α in B cell differentiation is still largely unknown. Here we show that PC differentiation and antibody responses are severely impaired in mice with B cell-specific deletion of p38α, while B cell development and the GCB cell response are spared. By utilizing a Blimp1 reporter mouse model, we show that p38α-deficiency results in decreased BLIMP1 expression. p38α-driven BLIMP1 up-regulation is required for both TI and TD PCs differentiation. By combining CRISPR/Cas9 screening and other approaches, we identify TCF3, TCF4 and IRF4 as downstream effectors of p38α to control PC differentiation via Blimp1 transcription. This study thus identifies an important signalling pathway underpinning PC differentiation upstream of BLIMP1, and points to a highly specialized and non-redundant role for p38α among p38 isoforms.

Protocol to estimate cell type proportions from bulk RNA-seq using DAISM-DNNXMBD.

Computational protocols for cell type deconvolution from bulk RNA-seq data have been used to understand cellular heterogeneity in disease-related samples, but their performance can be impacted by batch effect among datasets. Here, we present a DAISM-DNN protocol to achieve robust cell type proportion estimation on the target dataset. We describe the preparation of calibrated samples from human blood samples. We then detail steps to train a dataset-specific deep neural network (DNN) model and cell type proportion estimation using the trained model. For complete details on the use and execution of this protocol, please refer to Lin et al. (2022).

Immunostaining of phospho-RIPK3 in L929 cells, murine yolk sacs, ceca, and small intestines.

Though phospho-receptor-interacting protein 3 (RIP3 or RIPK3) antibodies are used in western blot, immunostaining of murine phospho-RIPK3 is challenging. Here, we verify and describe a detailed protocol for immunofluorescent detection of phospho-RIPK3 in L929 cells and mouse yolk sacs. We also describe in detail the model construction methods, sample preparation steps, and staining procedures for immunohistochemical labeling of RIPK3 activation in mouse ceca and small intestines by utilizing a specific commercially available antibody. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021) and Wang et al. (2020).

Biocapture-Directed Chemical Labeling for Discerning Stressed States of Organelles.

Lysosomal rupture engaged in diverse diseases remains poorly discerned from lysosomal membrane permeabilization (LMP). We herein reported biocapture-directed chemical labeling (BCCL) for the discern of lysosomal rupture by tracking the release of optically labeled cathepsins from damaged lysosomes into the cytosol. BCCL entails covalent anchoring of an azide-tagged suicide substrate (Epo-LeuTyrAz) to the enzyme active site and bioorthogonal ligation of the introduced azide with DBCORC, a ratiometric sensor featuring an acidity-reporting red emissive X-rhodamine-lactam (ROX), blue emissive coumarin (CM) inert to pH, and DBCO reactive to azide. Aided with fluorescein isocyanate-labeled sialic acid (FITC-Sia), a probe remained in pH-elevated lysosomes but dissipated from LMP+ lysosomes, BCCL enables optical discern of four states of lysosomes: ruptured lysosomes (blue in cytosol), LMP+ lysosomes (blue in lysosomes), pH-elevated lysosomes (blue and green in lysosomes), and physiological lysosomes (blue, green and red in lysosomes). This approach could find applicability to study lysosome rupture over LMP in diseases and to evaluate lysosome rupture-inducing drugs.