Abstract
Macrophages perform diverse functions within tissues during immune responses to pathogens and injury, but molecular mechanisms by which physical properties of the tissue regulate macrophage ...behavior are less well understood. Here, we examine the role of the mechanically activated cation channel Piezo1 in macrophage polarization and sensing of microenvironmental stiffness. We show that macrophages lacking Piezo1 exhibit reduced inflammation and enhanced wound healing responses. Additionally, macrophages expressing the transgenic Ca
2+
reporter, Salsa6f, reveal that Ca
2+
influx is dependent on Piezo1, modulated by soluble signals, and enhanced on stiff substrates. Furthermore, stiffness-dependent changes in macrophage function, both in vitro and in response to subcutaneous implantation of biomaterials in vivo, require Piezo1. Finally, we show that positive feedback between Piezo1 and actin drives macrophage activation. Together, our studies reveal that Piezo1 is a mechanosensor of stiffness in macrophages, and that its activity modulates polarization responses.
The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the ...normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.
The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are ...associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca
signaling have been observed in several AD models, we investigated TREM2 regulation of Ca
signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca
signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y
and P2Y
receptors, results in greater release of Ca
from the endoplasmic reticulum stores, which triggers sustained Ca
influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca
probe, Salsa6f, we found that cytosolic Ca
tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca
using a P2Y
receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca
response to purinergic signals, suggesting a window of Ca
signaling for optimal microglial motility.
Key points
•
The endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1), activates Orai1 channels by directly interacting with each Orai1 subunit at the C‐ and N‐termini. Current ...models about the roles of these sites are rooted in notions of modularity, with the C‐terminal site thought to mediate STIM1 binding and the N‐terminal site thought to regulate channel gating.
•
Here we report that the functions of the two sites are not so distinct: the N‐terminal site contributes to the stable association of STIM1 to Orai1, and, conversely, the C‐terminal site regulates channel activation.
•
In addition to channel activation, STIM1 binding also modulates Orai1 channel ion selectivity. The structural requirements for modulation of ion selectivity closely match those seen for gating, suggesting that gating and permeation are closely coupled in Orai1 channels.
•
These results help us understand the molecular requirements of STIM1‐mediated activation of Orai1 channels and regulation of channel ion selectivity.
Ca2+ release‐activated Ca2+ (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+ sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1–Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C‐ and N‐termini. Previous studies have proposed modular functions for these sites, with the C‐terminal site thought to regulate STIM1–Orai1 binding and trapping of Orai1 at the ER–PM junctions, and the N‐terminal site mediating gating. However, here we find that a variety of mutations in the N‐terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N‐ and C‐terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1–SS channels), indicating that loss of gating in these mutants by full‐length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1–SS channels carrying more drastic deletions that removed the STIM1 binding sites (Δ1–85, Δ73–85, or Δ272–279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C‐ and N‐terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1–Orai1 association, Orai1 trapping, and channel activation.
Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato ...optimized for cell tracking while monitoring cytosolic Ca
, and a transgenic Ca
reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca
signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca
signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca
signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca
transients ('sparkles') as cells migrate.
The G-protein-coupled protease-activated receptor 2 (PAR2) plays an important role in the pathogenesis of various inflammatory and auto-immune disorders. In airway epithelial cells (AECs), ...stimulation of PAR2 by allergens and proteases triggers the release of a host of inflammatory mediators to regulate bronchomotor tone and immune cell recruitment. Activation of PAR2 turns on several cell signaling pathways of which the mobilization of cytosolic Ca(2+) is likely a critical but poorly understood event. In this study, we show that Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by stromal interaction molecule 1 and Orai1 are a major route of Ca(2+) entry in primary human AECs and drive the Ca(2+) elevations seen in response to PAR2 activation. Activation of CRAC channels induces the production of several key inflammatory mediators from AECs including thymic stromal lymphopoietin, IL-6, and PGE2, in part through stimulation of gene expression via nuclear factor of activated T cells (NFAT). Furthermore, PAR2 stimulation induces the production of many key inflammatory mediators including PGE2, IL-6, IL-8, and GM-CSF in a CRAC channel-dependent manner. These findings indicate that CRAC channels are the primary mechanism for Ca(2+) influx in AECs and a vital checkpoint for the induction of PAR2-induced proinflammatory cytokines.
Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible ...individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca(2+) is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca(2+) elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca(2+) elevations in AECs through the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca(2+) entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway.
The immune system performs critical functions to defend against invading pathogens and maintain tissue homeostasis. Immune cells reside within or are recruited to a host of mechanically active ...tissues throughout the body and, as a result, are exposed to varying types and degrees of mechanical stimuli. Despite their abundance in such tissues, the role of mechanical stimuli in influencing immune cell function and the molecular mechanisms responsible for mechanics-mediated changes are still poorly understood. The recent emergence of mechanically-gated ion channels, particularly Piezo1, has provided an exciting avenue of research within the fields of mechanobiology and immunology. Numerous studies have identified roles for mechanically-gated ion channels in mechanotransduction within various different cell types, with a few recent studies in immune cells. These initial studies provide strong evidence that mechanically-gated ion channels play pivotal roles in regulating the immune system. In this review, we discuss characteristics of ion channel mediated mechanotransduction, review the current techniques used to quantify and visualize ion channel activity in response to mechanical stimuli, and finally we provide an overview of recent studies examining the role of mechanically-gated ion channels in modulating immune cell function.
Store-operated Ca ²⁺ entry (SOCE) is a universal Ca ²⁺ influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca ²⁺ stores and ...relies on a complex molecular interplay between the plasma membrane (PM) Ca ²⁺ channel ORAI1 and the ER Ca ²⁺ sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER–PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1–ORAI1 interactions. Thus, despite its constitutive localization at ER–PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control ( i ) transition of STIM1 from a quiescent to an active conformational state, ( ii ) cytoplasmic STIM1 oligomerization, and ( iii ) STIM1–ORAI1 binding required for ORAI1 activation.
Significance Stromal interaction molecule (STIM) 1 is an essential activator of the ORAI1 calcium (Ca ²⁺) channel that mediates Ca ²⁺ entry into many cell types. Patients with mutations in STIM1 or ORAI1 genes suffer from a severe immunodeficiency syndrome. Here, we studied a disease-causing mutant of STIM1 that is incapable of activating ORAI1 channels, thereby uncovering key molecular mechanisms of STIM1 function. The autosomal recessive R429C mutation interferes with the structural integrity of a protein–protein interaction domain in STIM1 and impairs the ability of STIM1 to multimerize and bind to ORAI1. Drugs targeting this domain may provide a means to selectively modulate STIM1–ORAI1 interaction and Ca ²⁺ entry as a novel approach to treat autoimmune diseases and other disorders associated with abnormal ORAI1-mediated Ca ²⁺ entry.
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