SARS-CoV-2 infection alkalinizes the ERGIC and lysosomes through the viroporin activity of the viral envelope protein.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.

The lipid transfer proteins Nir2 and Nir3 sustain phosphoinositide signaling and actin dynamics during phagocytosis.

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated on endoplasmic reticulum (ER) cisternae juxtaposed to phagocytic cups when expressed in phagocytic COS-7 cells. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE) and receptor-mediated phagocytosis, stalling particle capture at the cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2 and Nir3 (Nir2/3) double-knockout cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 depletion reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir proteins maintain phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis.

Redox Control of Mitochondrial Calcium Uptake.

Dong et al. (2017) establish how the mitochondrial Ca2+ uniporter (MCU) integrates Ca2+ and oxidative stress signals by identifying a cysteine residue that controls MCU channel activity, a mechanism causing mitochondrial Ca2+ overload and cell death during oxidative stress.

Enforced tethering elongates the cortical endoplasmic reticulum and limits store-operated Ca2+ entry.

Recruitment of STIM proteins to cortical endoplasmic reticulum (cER) domains forming membrane contact sites (MCSs) mediate the store-operated Ca2+ entry (SOCE) pathway essential for human immunity. The cER is dynamically regulated by STIM and tethering proteins during SOCE, but the ultrastructural rearrangement and functional consequences of cER remodeling are unknown. Here, we express natural (E-Syt1 and E-Syt2) and artificial (MAPPER-S and MAPPER-L) protein tethers in HEK-293T cells and correlate the changes in cER length and gap distance, as measured by electron microscopy, with ionic fluxes. We found that native cER cisternae extended during store depletion and remained elongated at a constant ER-plasma membrane (PM) gap distance during subsequent Ca2+ elevations. Tethering proteins enhanced store-dependent cER expansion, anchoring the enlarged cER at tether-specific gap distances of 12-15 nm (E-Syts) and 5-9 nm (MAPPERs). Cells with artificially extended cER had reduced SOCE and reduced agonist-induced Ca2+ release. SOCE remained modulated by calmodulin and exhibited enhanced Ca2+-dependent inhibition. We propose that cER expansion mediated by ER-PM tethering at a close distance negatively regulates SOCE by confining STIM-ORAI complexes to the periphery of enlarged cER sheets, a process that might participate in the termination of store-operated Ca2+ entry.

S-acylation of Ca2+ transport proteins: molecular basis and functional consequences.

Calcium (Ca2+) regulates a multitude of cellular processes during fertilization and throughout adult life by acting as an intracellular messenger to control effector functions in excitable and non-excitable cells. Changes in intracellular Ca2+ levels are driven by the co-ordinated action of Ca2+ channels, pumps, and exchangers, and the resulting signals are shaped and decoded by Ca2+-binding proteins to drive rapid and long-term cellular processes ranging from neurotransmission and cardiac contraction to gene transcription and cell death. S-acylation, a lipid post-translational modification, is emerging as a critical regulator of several important Ca2+-handling proteins. S-acylation is a reversible and dynamic process involving the attachment of long-chain fatty acids (most commonly palmitate) to cysteine residues of target proteins by a family of 23 proteins acyltransferases (zDHHC, or PATs). S-acylation modifies the conformation of proteins and their interactions with membrane lipids, thereby impacting intra- and intermolecular interactions, protein stability, and subcellular localization. Disruptions of S-acylation can alter Ca2+ signalling and have been implicated in the development of pathologies such as heart disease, neurodegenerative disorders, and cancer. Here, we review the recent literature on the S-acylation of Ca2+ transport proteins of organelles and of the plasma membrane and highlight the molecular basis and functional consequence of their S-acylation as well as the therapeutic potential of targeting this regulation for diseases caused by alterations in cellular Ca2+ fluxes.

TMBIM5 is the Ca2+ /H+ antiporter of mammalian mitochondria.

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.

The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex.

The stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor that regulates the activity of Orai plasma membrane Ca2+ channels to mediate the store-operated Ca2+ entry pathway essential for immunity. Uncoordinated 93 homolog B1 (UNC93B1) is a multiple membrane-spanning ER protein that acts as a trafficking chaperone by guiding nucleic-acid sensing toll-like receptors to their respective endosomal signaling compartments. We previously showed that UNC93B1 interacts with STIM1 to promote antigen cross-presentation in dendritic cells, but the STIM1 binding site(s) and activation step(s) impacted by this interaction remained unknown. In this study, we show that UNC93B1 interacts with STIM1 in the ER lumen by binding to residues in close proximity to the transmembrane domain. Cysteine crosslinking in vivo showed that UNC93B1 binding promotes the zipping of transmembrane and proximal cytosolic helices within resting STIM1 dimers, priming STIM1 for translocation. In addition, we show that UNC93B1 deficiency reduces store-operated Ca2+ entry and STIM1-Orai1 interactions and targets STIM1 to lighter ER domains, whereas UNC93B1 expression accelerates the recruitment of STIM1 to cortical ER domains. We conclude that UNC93B1 therefore acts as a trafficking chaperone by maintaining the pool of resting STIM1 proteins in a state primed for activation, enabling their rapid translocation in an extended conformation to cortical ER signaling compartments.

Calcium storage in multivesicular endo-lysosome.

It is now established that endo-lysosomes, also referred to as late endosomes, serve as intracellular calcium store, in addition to the endoplasmic reticulum. While abundant calcium-binding proteins provide the latter compartment with its calcium storage capacity, essentially nothing is known about the mechanism responsible for calcium storage in endo-lysosomes. In this paper, we propose that the structural organization of endo-lysosomal membranes drives the calcium storage capacity of the compartment. Indeed, endo-lysosomes exhibit a characteristic multivesicular ultrastructure, with intralumenal membranes providing a large amount of additional bilayer surface. We used a theoretical approach to investigate the calcium storage capacity of endosomes, using known calcium binding affinities for bilayers and morphological data on endo-lysosome membrane organization. Finally, we tested our predictions experimentally after Sorting Nexin 3 depletion to decrease the intralumenal membrane content. We conclude that the major negatively-charge lipids and proteins of endo-lysosomes serve as calcium-binding molecules in the acidic calcium stores of mammalian cells, while the large surface area of intralumenal membranes provide the necessary storage capacity.

Proteins Interacting with STIM1 and Store-Operated Ca2+ Entry.

The endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) interacts with ORAI Ca2+ channels at the plasma membrane to regulate immune and muscle cell function. The conformational changes underlying STIM1 activation, translocation, and ORAI1 trapping and gating, are stringently regulated by post-translational modifications and accessory proteins. Here, we review the recent progress in the identification and characterization of ER and cytosolic proteins interacting with STIM1 to control its activation and deactivation during store-operated Ca2+ entry (SOCE).

Molecular Mechanisms of Calcium Signaling During Phagocytosis.

Calcium (Ca2+) is a ubiquitous second messenger involved in the regulation of numerous cellular functions including vesicular trafficking, cytoskeletal rearrangements and gene transcription. Both global as well as localized Ca2+ signals occur during phagocytosis, although their functional impact on the phagocytic process has been debated. After nearly 40 years of research, a consensus may now be reached that although not strictly required, Ca2+ signals render phagocytic ingestion and phagosome maturation more efficient, and their manipulation make an attractive avenue for therapeutic interventions. In the last decade many efforts have been made to identify the channels and regulators involved in generating and shaping phagocytic Ca2+ signals. While molecules involved in store-operated calcium entry (SOCE) of the STIM and ORAI family have taken center stage, members of the canonical, melastatin, mucolipin and vanilloid transient receptor potential (TRP), as well as purinergic P2X receptor families are now recognized to play significant roles. In this chapter, we review the recent literature on research that has linked specific Ca2+-permeable channels and regulators to phagocytic function. We highlight the fact that lipid mediators are emerging as important regulators of channel gating and that phagosomal ionic homeostasis and Ca2+ release also play essential parts. We predict that improved methodologies for measuring these factors will be critical for future advances in dissecting the intricate biology of this fascinating immune process.

MitoNEET-dependent formation of intermitochondrial junctions.

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.

L-OPA1 regulates mitoflash biogenesis independently from membrane fusion.

Mitochondrial flashes mediated by optic atrophy 1 (OPA1) fusion protein are bioenergetic responses to stochastic drops in mitochondrial membrane potential (Δψm) whose origin is unclear. Using structurally distinct genetically encoded pH-sensitive probes, we confirm that flashes are matrix alkalinization transients, thereby establishing the pH nature of these events, which we renamed "mitopHlashes". Probes located in cristae or intermembrane space as verified by electron microscopy do not report pH changes during Δψm drops or respiratory chain inhibition. Opa1 ablation does not alter Δψm fluctuations but drastically decreases the efficiency of mitopHlash/Δψm coupling, which is restored by re-expressing fusion-deficient OPA1K301A and preserved in cells lacking the outer-membrane fusion proteins MFN1/2 or the OPA1 proteases OMA1 and YME1L, indicating that mitochondrial membrane fusion and OPA1 proteolytic processing are dispensable. pH/Δψm uncoupling occurs early during staurosporine-induced apoptosis and is mitigated by OPA1 overexpression, suggesting that OPA1 maintains mitopHlash competence during stress conditions. We propose that OPA1 stabilizes respiratory chain supercomplexes in a conformation that enables respiring mitochondria to compensate a drop in Δψm by an explosive matrix pH flash.

The ER phagosome connection in the era of membrane contact sites.

Phagocytosis is an essential mechanism through which innate immune cells ingest foreign material that is either destroyed or used to generate and present antigens and initiate adaptive immune responses. While a role for the ER during phagosome biogenesis has been recognized, whether fusion with ER cisternae or vesicular derivatives occurs has been the source of much contention. Membrane contact sites (MCS) are tight appositions between ER membranes and various organelles that coordinate multiple functions including localized signalling, lipid transfer and trafficking. The discovery that MCS form between the ER and phagosomes now begs the question of whether MCS play a role in connecting the ER to phagosomes under different contexts. In this review, we consider the implications of MCS between the ER and phagosomes during cross-presentation and infection with intracellular pathogens. We also discuss the similarities between these contacts and those between the ER and plasma membrane and acidic organelles such as endosomes and lysosomes. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

The role of STIM proteins in neutrophil functions.

Stromal interaction molecule (STIM) proteins regulate store-operated Ca2+ entry (SOCE) in innate and adaptive immune cells and participate in the Ca2+ signals that control the functions of neutrophils, the first line of host defence against bacterial and fungal infections. Loss-of-function experiments in animal and cellular models indicate that both STIM1 and STIM2 regulate neutrophil functions, but the complexity of the SOCE machinery and the versatility of neutrophils complicate the evaluation of the results. This review aims to summarize the latest progress in the field, with special attention to the details of the experimental designs. Future study design should aim to improve the standardization of experimental procedures and to provide a more holistic understanding of the role of STIM proteins in neutrophils function.

ORAI1 Mutations with Distinct Channel Gating Defects in Tubular Aggregate Myopathy.

Calcium (Ca2+ ) is a physiological key factor, and the precise modulation of free cytosolic Ca2+ levels regulates multiple cellular functions. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ homeostasis, and is mediated by the concerted activity of the Ca2+ sensor STIM1 and the Ca2+ channel ORAI1. Dominant gain-of-function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss-of-function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation-dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non-muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca2+ homeostasis and leading to muscle dysfunction.

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange.

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.

Junctate boosts phagocytosis by recruiting endoplasmic reticulum Ca2+ stores near phagosomes.

Local intracellular Ca(2+) elevations increase the efficiency of phagocytosis, a process that is essential for innate and adaptive immunity. These local Ca(2+) elevations are generated in part by the store-operated Ca(2+) entry (SOCE) sensor STIM1, which recruits endoplasmic reticulum (ER) cisternae to phagosomes and opens phagosomal Ca(2+) channels at ER-phagosome junctions. However, residual ER-phagosome contacts and periphagosomal Ca(2+) hotspots remain in Stim1(-/-) cells. Here, we tested whether junctate (also called ASPH isoform 8), a molecule that targets STIM1 to ER-plasma-membrane contacts upon Ca(2+)-store depletion, cooperates with STIM1 at phagosome junctions. Junctate expression in Stim1(-/-) and Stim1(-/-); Stim2(-/-) phagocytic fibroblasts increased phagocytosis and periphagosomal Ca(2+) elevations, yet with only a minimal impact on global SOCE. These Ca(2+) hotspots were only marginally reduced by the SOCE channel blocker lanthanum chloride (La(3+)) but were abrogated by inositol trisphosphate receptor inhibitors 2-APB and xestospongin-C, revealing that unlike STIM1-mediated hotspots, junctate-mediated Ca(2+) originates predominantly from periphagosomal Ca(2+) stores. Accordingly, junctate accumulates near phagosomes and elongates ER-phagosome junctions in Stim1(-/-) cells. Thus, junctate mediates an alternative mechanism for generating localized Ca(2+) elevations within cells, promoting Ca(2+) release from internal stores recruited to phagosomes, thereby boosting phagocytosis.

STIM1L traps and gates Orai1 channels without remodeling the cortical ER.

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca(2+) entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca(2+) influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca(2+) imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1(-/-)/Stim2(-/-) fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca(2+) elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.

The Role of Mitochondria in the Activation/Maintenance of SOCE: Membrane Contact Sites as Signaling Hubs Sustaining Store-Operated Ca2+ Entry.

Store-operated Ca2+ entry (SOCE) is a cell signaling pathway essential for immune and muscle function controlled by dynamic interactions between Ca2+-sensing STIM proteins on the endoplasmic reticulum (ER) and Ca2+-permeable ORAI channels on the plasma membrane (PM). STIM-ORAI interactions occur at membrane contact sites (MCS), evolutionarily conserved cellular structures characterized by the close apposition (10-20 nm) between the ER and target membranes that facilitate the exchange of lipids by non-vesicular transport mechanisms. STIM-ORAI interactions were considered to be restricted to ER-PM MCS, but recent evidence indicates that productive interactions take place between ER-bound STIM1 and Ca2+ channels located in intracellular organelles. Interactions between the ER and endosomes or lysosomes regulate the lipid homeostasis of these organelles and the propagation of Ca2+ signals initiated by the release of Ca2+ from acidic stores. Intracellular MCS also regulate the efficiency of phagocytosis, a fundamental cellular process essential for immunity and tissue homeostasis, by ensuring the coordinated opening of Ca2+ channels on phagocytic vacuoles and of Ca2+ release channels on juxtaposed ER stores. In this chapter, we review the current knowledge on the molecular composition and architecture of membrane contact sites that sustain Ca2+ signals at the plasma membrane and in intracellular organelles.