Biology subjects

Johnson, M. T.

Publications and source records attributed to Johnson, M. T..

4 recordsLinked to original sources

bioRxiv2022

Orai3 and Orai1 are essential for CRAC channel function and metabolic reprogramming in B cells

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in expression of Orai isoforms in response to B cell activation. We show that Orai3 and Orai1 are essential components of native CRAC channels in B cells and are critical for primary B cell proliferation and survival. The combined loss of Orai1 and Orai3 strongly impairs SOCE, nuclear factor for activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of B cells in response to antigenic stimulation. Our results clarify the molecular composition and cellular functions of SOCE in B lymphocytes.

immunology↗

bioRxiv2021

Peripheral Coupling Sites Formed by STIM1 Govern the Contractility of Vascular Smooth Muscle Cells

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs), although the mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal-interaction molecule 1), a single transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased and SR-dependent Ca2+ signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.

physiology↗

bioRxiv2021

Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

cell biology↗

bioRxiv2020

Colorectal adenocarcinomas downregulate the mitochondrial Na+/Ca2+ exchanger NCLX to drive metastatic spread

Despite the established role of mitochondria in tumorigenesis, the molecular mechanisms by which mitochondrial Ca2+ (mtCa2+) signaling regulates tumor growth and metastasis remain unknown. The crucial role of mtCa2+ in tumorigenesis is highlighted by the altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer cells. Here, we demonstrate that expression of the mitochondrial Na+/Ca2+ exchanger NCLX (SLC8B1) is decreased in colorectal tumors and is associated with advanced-stage disease in patients. We reveal that downregulation of NCLX leads to mtCa2+ overload, mitochondrial depolarization, mitophagy, and reduced tumor size. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, the expression of epithelial-to-mesenchymal transition (EMT), hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to an increase in mitochondrial reactive oxygen species (mtROS) which activates HIF1 signaling supporting the metastatic behavior of tumor cells lacking NCLX. Our results reveal that loss of NCLX expression is a novel driver of metastatic progression, indicating that control of mtCa2+ levels is a novel therapeutic approach in metastatic colorectal cancer. HighlightsO_LIThe expression of NCLX is decreased in colorectal tumors and is associated with advanced-stage disease in patients. C_LIO_LINCLX plays a dichotomous role in colorectal tumor growth and metastasis. C_LIO_LINCLX downregulation causes mitophagy and reduced colorectal cancer tumor growth. C_LIO_LINCLX downregulation induces stemness, chemoresistance and metastasis through mtCa2+/ROS/HIF1 signaling axis. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/083071v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@30360dorg.highwire.dtl.DTLVardef@12ca72borg.highwire.dtl.DTLVardef@9328ddorg.highwire.dtl.DTLVardef@15ffcdb_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceMitochondrial Ca2+ (mtCa2+) homeostasis is essential for cellular metabolism and growth and plays a critical role in cancer progression. mtCa2+ uptake is mediated by an inner membrane protein complex containing the mitochondrial Ca2+ uniporter (MCU). mtCa2+ uptake by the MCU is followed by a [~]100-fold slower mtCa2+ extrusion mediated by the inner mitochondrial membrane ion transporter, the mitochondrial Na+/Ca2+ exchanger NCLX. Because NCLX is a slower transporter than the MCU, it is a crucial rate-limiting factor of mtCa2+ homeostasis that cannot easily be compensated by another Ca2+ transport mechanism. This represents the first study investigating the role of NCLX in tumorigenesis and metastasis. We demonstrate for the first time that colorectal cancers exhibit loss of NCLX expression and that this is associated with advanced-stage disease. Intriguingly, decreased NCLX function has a dichotomous role in colorectal cancer. Thus, we reveal that NCLX loss leads to reduced primary tumor growth and overall tumor burden in vivo. Yet, the consequential increases in mtCa2+ elicit pro-survival, hypoxic and gene transcription pathways that enhance metastatic progression. This dichotomy is a well-established feature of chemoresistant and recurrent tumor cells including cancer stem cells. Moreover, the downstream changes elicited by NCLX loss are reminiscent of mesenchymal colorectal cancer subtypes that display poor patient survival. Our data indicate that the demonstrated changes to the mtCa2+/mtROS/HIF1 signaling axis elicited through the loss of NCLX are a key adaptation and driver of metastatic colorectal cancer.

cancer biology↗