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.

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.

Publisher Correction: The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells.

In the version of this article initially published, the institution name for affiliation 3 (Maryland Anderson Cancer Center) was incorrect. The correct institution is MD Anderson Cancer Center. The error has been corrected in the HTML and PDF versions of the article.

Corrigendum: The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells.

This corrects the article DOI: 10.1038/ni.3748.

The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells.

An imbalance in the lineages of immunosuppressive regulatory T cells (Treg cells) and the inflammatory TH17 subset of helper T cells leads to the development of autoimmune and/or inflammatory disease. Here we found that TAZ, a coactivator of TEAD transcription factors of Hippo signaling, was expressed under TH17 cell-inducing conditions and was required for TH17 differentiation and TH17 cell-mediated inflammatory diseases. TAZ was a critical co-activator of the TH17-defining transcription factor RORγt. In addition, TAZ attenuated Treg cell development by decreasing acetylation of the Treg cell master regulator Foxp3 mediated by the histone acetyltransferase Tip60, which targeted Foxp3 for proteasomal degradation. In contrast, under Treg cell-skewing conditions, TEAD1 expression and sequestration of TAZ from the transcription factors RORγt and Foxp3 promoted Treg cell differentiation. Furthermore, deficiency in TAZ or overexpression of TEAD1 induced Treg cell differentiation, whereas expression of a transgene encoding TAZ or activation of TAZ directed TH17 cell differentiation. Our results demonstrate a pivotal role for TAZ in regulating the differentiation of Treg cells and TH17 cells.

Bacterial Endotoxin Activates the Coagulation Cascade through Gasdermin D-Dependent Phosphatidylserine Exposure.

Excessive activation of the coagulation system leads to life-threatening disseminated intravascular coagulation (DIC). Here, we examined the mechanisms underlying the activation of coagulation by lipopolysaccharide (LPS), the major cell-wall component of Gram-negative bacteria. We found that caspase-11, a cytosolic LPS receptor, activated the coagulation cascade. Caspase-11 enhanced the activation of tissue factor (TF), an initiator of coagulation, through triggering the formation of gasdermin D (GSDMD) pores and subsequent phosphatidylserine exposure, in a manner independent of cell death. GSDMD pores mediated calcium influx, which induced phosphatidylserine exposure through transmembrane protein 16F, a calcium-dependent phospholipid scramblase. Deletion of Casp11, ablation of Gsdmd, or neutralization of phosphatidylserine or TF prevented LPS-induced DIC. In septic patients, plasma concentrations of interleukin (IL)-1α and IL-1ß, biomarkers of GSDMD activation, correlated with phosphatidylserine exposure in peripheral leukocytes and DIC scores. Our findings mechanistically link immune recognition of LPS to coagulation, with implications for the treatment of DIC.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

TCR-induced sumoylation of the kinase PKC-θ controls T cell synapse organization and T cell activation.

Sumoylation regulates many cellular processes, but its role in signaling via the T cell antigen receptor (TCR) remains unknown. We found that the kinase PKC-θ was sumoylated upon costimulation with antigen or via the TCR plus the coreceptor CD28, with Lys325 and Lys506 being the main sumoylation sites. We identified the SUMO E3 ligase PIASxß as a ligase for PKC-θ. Analysis of primary mouse and human T cells revealed that sumoylation of PKC-θ was essential for T cell activation. Desumoylation did not affect the catalytic activity of PKC-θ but inhibited the association of CD28 with PKC-θ and filamin A and impaired the assembly of a mature immunological synapse and central co-accumulation of PKC-θ and CD28. Our findings demonstrate that sumoylation controls TCR-proximal signaling and that sumoylation of PKC-θ is essential for the formation of a mature immunological synapse and T cell activation.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity.

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway.

The NLRP3 inflammasome functions as a crucial component of the innate immune system in recognizing viral infection, but the mechanism by which viruses activate this inflammasome remains unclear. Here we found that inhibition of the serine-threonine kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA virus-induced activation of the NLRP3 inflammasome. Infection with an RNA virus initiated assembly of the RIP1-RIP3 complex, which promoted activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove the NLRP3 inflammasome independently of MLKL, an essential downstream effector of RIP1-RIP3-dependent necrosis. Together our results reveal a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus-induced activation of the NLRP3 inflammasome and establish a direct link between inflammation and cell-death signaling pathways.

Gut stem cell necroptosis by genome instability triggers bowel inflammation.

The aetiology of inflammatory bowel disease (IBD) is a multifactorial interplay between heredity and environment1,2. Here we report that deficiency in SETDB1, a histone methyltransferase that mediates the trimethylation of histone H3 at lysine 9, participates in the pathogenesis of IBD. We found that levels of SETDB1 are decreased in patients with IBD, and that mice with reduced SETDB1 in intestinal stem cells developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability3, and the loss of SETDB1 in intestinal stem cells released repression of endogenous retroviruses (retrovirus-like elements with long repeats that, in humans, comprise approximately 8% of the genome). Excessive viral mimicry generated by motivated endogenous retroviruses triggered Z-DNA-binding protein 1 (ZBP1)-dependent necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted bowel inflammation. Genome instability, reactive endogenous retroviruses, upregulation of ZBP1 and necroptosis were all seen in patients with IBD. Pharmaceutical inhibition of RIP3 showed a curative effect in SETDB1-deficient mice, which suggests that targeting necroptosis of intestinal stem cells may represent an approach for the treatment of severe IBD.

A unique death pathway keeps RIPK1 D325A mutant mice in check at embryonic day 10.5.

Tumor necrosis factor receptor-1 (TNFR1) signaling, apart from its pleiotropic functions in inflammation, plays a role in embryogenesis as deficiency of varieties of its downstream molecules leads to embryonic lethality in mice. Caspase-8 noncleavable receptor interacting serine/threonine kinase 1 (RIPK1) mutations occur naturally in humans, and the corresponding D325A mutation in murine RIPK1 leads to death at early midgestation. It is known that both the demise of Ripk1D325A/D325A embryos and the death of Casp8-/- mice are initiated by TNFR1, but they are mediated by apoptosis and necroptosis, respectively. Here, we show that the defects in Ripk1D325A/D325A embryos occur at embryonic day 10.5 (E10.5), earlier than that caused by Casp8 knockout. By analyzing a series of genetically mutated mice, we elucidated a mechanism that leads to the lethality of Ripk1D325A/D325A embryos and compared it with that underlies Casp8 deletion-mediated lethality. We revealed that the apoptosis in Ripk1D325A/D325A embryos requires a scaffold function of RIPK3 and enzymatically active caspase-8. Unexpectedly, caspase-1 and caspase-11 are downstream of activated caspase-8, and concurrent depletion of Casp1 and Casp11 postpones the E10.5 lethality to embryonic day 13.5 (E13.5). Moreover, caspase-3 is an executioner of apoptosis at E10.5 in Ripk1D325A/D325A mice as its deletion extends life of Ripk1D325A/D325A mice to embryonic day 11.5 (E11.5). Hence, an unexpected death pathway of TNFR1 controls RIPK1 D325A mutation-induced lethality at E10.5.

Gasdermin E mediates photoreceptor damage by all-trans-retinal in the mouse retina.

The breakdown of all-trans-retinal (atRAL) clearance is closely associated with photoreceptor cell death in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its mechanisms remain elusive. Here, we demonstrate that activation of gasdermin E (GSDME) but not gasdermin D promotes atRAL-induced photoreceptor damage by activating pyroptosis and aggravating apoptosis through a mitochondria-mediated caspase-3-dependent signaling pathway. Activation of c-Jun N-terminal kinase was identified as one of the major causes of mitochondrial membrane rupture in atRAL-loaded photoreceptor cells, resulting in the release of cytochrome c from mitochondria to the cytosol, where it stimulated caspase-3 activation required for cleavage of GSDME. Aggregation of the N-terminal fragment of GSDME in the mitochondria revealed that GSDME was likely to penetrate mitochondrial membranes in photoreceptor cells after atRAL exposure. ABC (subfamily A, member 4) and all-trans-retinol dehydrogenase 8 are two key proteins responsible for clearing atRAL in the retina. Abca4-/-Rdh8-/- mice exhibit serious defects in atRAL clearance upon light exposure and serve as an acute model for dry AMD and STGD1. We found that N-terminal fragment of GSDME was distinctly localized in the photoreceptor outer nuclear layer of light-exposed Abca4-/-Rdh8-/- mice. Of note, degeneration and caspase-3 activation in photoreceptors were significantly alleviated in Abca4-/-Rdh8-/-Gsdme-/- mice after exposure to light. The results of this study indicate that GSDME is a common causative factor of photoreceptor pyroptosis and apoptosis arising from atRAL overload, suggesting that repressing GSDME may represent a potential treatment of photoreceptor atrophy in dry AMD and STGD1.

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.

Programmed necrosis: backup to and competitor with apoptosis in the immune system.

Programmed cell death is essential for the development and maintenance of the immune system and its responses to exogenous and endogenous stimuli. Studies have demonstrated that in addition to caspase-dependent apoptosis, necrosis dependent on the kinases RIP1 and RIP3 (also called necroptosis) is a major programmed cell-death pathway in development and immunity. These two programmed cell-death pathways may suppress each other, and necroptosis also serves as an alternative when caspase-dependent apoptosis is inhibited or absent. Here we summarize recent advancements that have identified the molecular mechanisms that underlie necroptosis and explore the mechanisms that regulate the interplay between apoptosis and necroptosis.

RNA N6-Methyladenosine Methyltransferase METTL3 Facilitates Colorectal Cancer by Activating the m6A-GLUT1-mTORC1 Axis and Is a Therapeutic Target.

BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) modification has recently emerged as a new regulatory mechanism in cancer progression. We aimed to explore the role of the m6A regulatory enzyme METTL3 in colorectal cancer (CRC) pathogenesis and its potential as a therapeutic target. METHODS: The expression and clinical implication of METTL3 were investigated in multiple human CRC cohorts. The underlying mechanisms of METTL3 in CRC were investigated by integrative m6A sequencing, RNA sequencing, and ribosome profiling analyses. The efficacy of targeting METTL3 in CRC treatment was elucidated in CRC cell lines, patient-derived CRC organoids, and Mettl3-knockout mouse models. RESULTS: Using targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 dropout screening, we identified METTL3 as the top essential m6A regulatory enzyme in CRC. METTL3 was overexpressed in 62.2% (79/127) and 88.0% (44/50) of primary CRCs from 2 independent cohorts. High METTL3 expression predicted poor survival in patients with CRC (n = 374, P < .01). Functionally, silencing METTL3 suppressed tumorigenesis in CRC cells, human-derived primary CRC organoids, and Mettl3-knockout mouse models. We discovered the novel functional m6A methyltransferase domain of METTL3 in CRC cells by domain-focused CRISPR screening and mutagenesis assays. Mechanistically, METTL3 directly induced the m6A-GLUT1-mTORC1 axis as identified by integrated m6A sequencing, RNA sequencing, ribosome sequencing, and functional validation. METTL3 induced GLUT1 translation in an m6A-dependent manner, which subsequently promoted glucose uptake and lactate production, leading to the activation of mTORC1 signaling and CRC development. Furthermore, inhibition of mTORC1 potentiated the anticancer effect of METTL3 silencing in CRC patient-derived organoids and METTL3 transgenic mouse models. CONCLUSIONS: METTL3 promotes CRC by activating the m6A-GLUT1-mTORC1 axis. METTL3 is a promising therapeutic target for the treatment of CRC.

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.

The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1.

Negative regulation of immunoreceptor signaling is required for preventing hyperimmune activation and maintaining immune homeostasis. The roles of p38IP in immunoreceptor signaling remain unclear. Here, we show that p38IP suppresses T-cell receptor (TCR)/LPS-activated NF-κB and p38 by targeting TAK1 kinase and that p38IP protein levels are downregulated in human PBMCs from rheumatoid arthritis (RA) patients, inversely correlating with the enhanced activity of NF-κB and p38. Mechanistically, p38IP interacts with TAK1 to disassemble the TAK1-TAB (TAK1-binding protein) complex. p38IP overexpression decreases TCR-induced binding of K63-linked polyubiquitin (polyUb) chains to TAK1 but increases that to TAB2, and p38IP knockdown shows the opposite effects, indicating unanchored K63-linked polyUb chain transfer from TAB2 to TAK1. p38IP dynamically interacts with TAK1 upon stimulation, because of the polyUb chain transfer and the higher binding affinity of TAK1 and p38IP for polyUb-bound TAB2 and TAK1, respectively. Moreover, p38IP scaffolds the deubiquitinase USP4 to deubiquitinate TAK1 once TAK1 is activated. These findings reveal a novel role and the mechanisms of p38IP in controlling TCR/LPS signaling and suggest that p38IP might participate in RA pathogenesis.

Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane.

Classical chemical probes are prone to dissipation from stressed organelles, as evidenced by the incapability of mitochondrial dyes to image mitophagy linked to multiple diseases. We herein reported mitophagy imaging via covalent anchoring of a lysosomal probe to the mitochondrial inner membrane (CALM). Utilizing DBCORC-TPP, an azide-conjugatable probe with acidity-triggered fluorescence, CALM is operated via ΔΨm-promoted probe accumulation in mitochondria and thereby bioorthogonal ligation of the trapped probe with azido-choline (Azcholine) metabolically installed on the mitochondrial membrane. Overcoming the limitation of synthetic probes to dissipate from stressed organelles, CALM enables signal-on fluorescence imaging of mitophagy induced by starvation and is further employed to reveal mitophagy in ferroptosis. These results suggest the potential of CALM as a new tool to study mitophagy.