STING orchestrates the neuronal inflammatory stress response in multiple sclerosis.

Inflammation-induced neurodegeneration is a defining feature of multiple sclerosis (MS), yet the underlying mechanisms remain unclear. By dissecting the neuronal inflammatory stress response, we discovered that neurons in MS and its mouse model induce the stimulator of interferon genes (STING). However, activation of neuronal STING requires its detachment from the stromal interaction molecule 1 (STIM1), a process triggered by glutamate excitotoxicity. This detachment initiates non-canonical STING signaling, which leads to autophagic degradation of glutathione peroxidase 4 (GPX4), essential for neuronal redox homeostasis and thereby inducing ferroptosis. Both genetic and pharmacological interventions that target STING in neurons protect against inflammation-induced neurodegeneration. Our findings position STING as a central regulator of the detrimental neuronal inflammatory stress response, integrating inflammation with glutamate signaling to cause neuronal cell death, and present it as a tractable target for treating neurodegeneration in MS.

Sensory circuitry controls cytosolic calcium-mediated phytochrome B phototransduction.

Although Ca2+ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca2+ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca2+ increases via phyB, which are sensed by Ca2+-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca2+ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyBS80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyBS80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca2+ increases to specific responses in sensory stimulus processing.

Structural Mechanism of EMRE-Dependent Gating of the Human Mitochondrial Calcium Uniporter.

Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.

trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages.

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo.

Circadian Rhythms and Astrocytes: The Good, the Bad, and the Ugly.

This review explores the interface between circadian timekeeping and the regulation of brain function by astrocytes. Although astrocytes regulate neuronal activity across many time domains, their cell-autonomous circadian clocks exert a particular role in controlling longer-term oscillations of brain function: the maintenance of sleep states and the circadian ordering of sleep and wakefulness. This is most evident in the central circadian pacemaker, the suprachiasmatic nucleus, where the molecular clock of astrocytes suffices to drive daily cycles of neuronal activity and behavior. In Alzheimer's disease, sleep impairments accompany cognitive decline. In mouse models of the disease, circadian disturbances accelerate astroglial activation and other brain pathologies, suggesting that daily functions in astrocytes protect neuronal homeostasis. In brain cancer, treatment in the morning has been associated with prolonged survival, and gliomas have daily rhythms in gene expression and drug sensitivity. Thus, circadian time is fast becoming critical to elucidating reciprocal astrocytic-neuronal interactions in health and disease.

Chronic calcium signaling in IgE+ B cells limits plasma cell differentiation and survival.

In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. To better understand IgE-BCR-mediated control of IgE responses, we developed whole-genome CRISPR screening that enabled comparison of IgE+ and IgG1+ B cell requirements for proliferation, survival, and differentiation into PCs. IgE+ PCs exhibited dependency on the PI3K-mTOR axis that increased protein amounts of the transcription factor IRF4. In contrast, loss of components of the calcium-calcineurin-NFAT pathway promoted IgE+ PC differentiation. Mice bearing a B cell-specific deletion of calcineurin B1 exhibited increased production of IgE+ PCs. Mechanistically, sustained elevation of intracellular calcium in IgE+ PCs downstream of the IgE-BCR promoted BCL2L11-dependent apoptosis. Thus, chronic calcium signaling downstream of the IgE-BCR controls the self-limiting character of IgE responses and may be relevant to the accumulation of IgE-producing cells in allergic disease.

BCL6 controls contact-dependent help delivery during follicular T-B cell interactions.

BCL6 is required for development of follicular T helper (Tfh) cells to support germinal center (GC) formation. However, it is not clear what unique functions programmed by BCL6 can explain its absolute essentiality in T cells for GC formation. We found that ablation of one Bcl6 allele did not appreciably alter early T cell activation and follicular localization but inhibited GC formation and Tfh cell maintenance. BCL6 impinged on Tfh calcium signaling and also controlled Tfh entanglement with and CD40L delivery to B cells. Amounts of BCL6 protein and nominal frequencies of Tfh cells markedly changed within hours after strengths of T-B cell interactions were altered in vivo, while CD40L overexpression rectified both defective GC formation and Tfh cell maintenance because of the BCL6 haploinsufficiency. Our results reveal BCL6 functions in Tfh cells that are essential for GC formation and suggest that BCL6 helps maintain Tfh cell phenotypes in a T cell non-autonomous manner.

Mechanisms and significance of tissue-specific MICU regulation of the mitochondrial calcium uniporter complex.

Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.

Mechanisms and physiological implications of cooperative gating of clustered ion channels.

Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.

Calcium-release channels: structure and function of IP3 receptors and ryanodine receptors.

Ca2+-release channels are giant membrane proteins that control the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. The two members, ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate receptors (IP3Rs), are evolutionarily related and are both activated by cytosolic Ca2+. They share a common architecture, but RyRs have evolved additional modules in the cytosolic region. Their massive size allows for the regulation by tens of proteins and small molecules, which can affect the opening and closing of the channels. In addition to Ca2+, other major triggers include IP3 for the IP3Rs and depolarization of the plasma membrane for a particular RyR subtype expressed in skeletal muscle. Their size has made them popular targets for study via electron microscopic methods, with current structures culminating near 3 Å. The available structures have provided many new mechanistic insights into the binding of auxiliary proteins and small molecules, how these can regulate channel opening, and the mechanisms of disease-associated mutations. They also help scrutinize previously proposed binding sites, as some of these are now incompatible with the structures. Many questions remain around the structural effects of posttranslational modifications, additional binding partners, and the higher order complexes these channels can make in situ. This review summarizes our current knowledge about the structures of Ca2+-release channels and how this informs on their function.

The roles of calcium and ATP in the physiology and pathology of the exocrine pancreas.

This review deals with the roles of calcium ions and ATP in the control of the normal functions of the different cell types in the exocrine pancreas as well as the roles of these molecules in the pathophysiology of acute pancreatitis. Repetitive rises in the local cytosolic calcium ion concentration in the apical part of the acinar cells not only activate exocytosis but also, via an increase in the intramitochondrial calcium ion concentration, stimulate the ATP formation that is needed to fuel the energy-requiring secretion process. However, intracellular calcium overload, resulting in a global sustained elevation of the cytosolic calcium ion concentration, has the opposite effect of decreasing mitochondrial ATP production, and this initiates processes that lead to necrosis. In the last few years it has become possible to image calcium signaling events simultaneously in acinar, stellate, and immune cells in intact lobules of the exocrine pancreas. This has disclosed processes by which these cells interact with each other, particularly in relation to the initiation and development of acute pancreatitis. By unraveling the molecular mechanisms underlying this disease, several promising therapeutic intervention sites have been identified. This provides hope that we may soon be able to effectively treat this often fatal disease.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Ca2+-Stimulated AMPK-Dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection.

Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.

Luminal transport through intact endoplasmic reticulum limits the magnitude of localized Ca2+ signals.

The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca[Formula: see text] signaling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca[Formula: see text] releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca[Formula: see text] release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca[Formula: see text] delivery system.

Formation of recurring transient Ca2+-based intercellular communities during Drosophila hematopoiesis.

Tissue development occurs through a complex interplay between many individual cells. Yet, the fundamental question of how collective tissue behavior emerges from heterogeneous and noisy information processing and transfer at the single-cell level remains unknown. Here, we reveal that tissue scale signaling regulation can arise from local gap-junction mediated cell-cell signaling through the spatiotemporal establishment of an intermediate-scale of transient multicellular communication communities over the course of tissue development. We demonstrated this intermediate scale of emergent signaling using Ca2+ signaling in the intact, ex vivo cultured, live developing Drosophila hematopoietic organ, the lymph gland. Recurrent activation of these transient signaling communities defined self-organized signaling "hotspots" that gradually formed over the course of larva development. These hotspots receive and transmit information to facilitate repetitive interactions with nonhotspot neighbors. Overall, this work bridges the scales between single-cell and emergent group behavior providing key mechanistic insight into how cells establish tissue-scale communication networks.

NAADP-binding proteins find their identity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from endosomes and lysosomes by activating ion channels called two-pore channels (TPCs). However, no NAADP-binding site has been identified on TPCs. Rather, NAADP activates TPCs indirectly by engaging NAADP-binding proteins (NAADP-BPs) that form part of the TPC complex. After a decade of searching, two different NAADP-BPs were recently identified: Jupiter microtubule associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12). These discoveries bridge the gap between NAADP generation and NAADP activation of TPCs, providing new opportunity to understand and manipulate the NAADP-signaling pathway. The unmasking of these NAADP-BPs will catalyze future studies to define the molecular choreography of NAADP action.

How do plants feel the heat and survive?

Climate change is increasingly affecting the quality of life of organisms on Earth. More frequent, extreme, and lengthy heat waves are contributing to the sixth mass extinction of complex life forms in the Earth's history. From an anthropocentric point of view, global warming is a major threat to human health because it also compromises crop yields and food security. Thus, achieving agricultural productivity under climate change calls for closer examination of the molecular mechanisms of heat-stress resistance in model and crop plants. This requires a better understanding of the mechanisms by which plant cells can sense rising temperatures and establish effective molecular defenses, such as molecular chaperones and thermoprotective metabolites, as reviewed here, to survive extreme diurnal variations in temperature and seasonal heat waves.

Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation-Contraction Complex.

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca2+-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.

TR(i)P Goes On: Auxiliary TRP Channel Subunits?

Transient receptor potential (TRP) cation channels are a diverse family of channels whose members play prominent roles as cellular sensors and effectors. The important role of TRP channels (and mechanosensitive piezo channels) in the complex interaction of our senses with the environment was underlined by the award of the Nobel Prize in Physiology or Medicine to 2 pioneers in this field, David Julius and Ardem Patapoutian. There are many competent and comprehensive reviews on many aspects of the TRP channels, and there is no intention to expand on them. Rather, after an introduction to the nomenclature, the molecular architecture of native TRP channel/protein complexes in vivo will be summarized using TRP channels of the canonical transient receptor potential subfamily as an example. This molecular architecture provides the basis for the signatures of native canonical transient receptor potential currents and their control by endogenous modulators and potential drugs.