Mitochondria-ER contact sites expand during mitosis.

Mitochondria-ER contact sites (MERCS) are involved in energy homeostasis, redox and Ca2+ signaling, and inflammation. MERCS are heavily studied; however, little is known about their regulation during mitosis. Here, we show that MERCS expand during mitosis in three cell types using various approaches, including transmission electron microscopy, serial EM coupled to 3D reconstruction, and a split GFP MERCS marker. We further show enhanced Ca2+ transfer between the ER and mitochondria using either direct Ca2+ measurements or by quantifying the activity of Ca2+-dependent mitochondrial dehydrogenases. Collectively, our results support a lengthening of MERCS in mitosis that is associated with improved Ca2+ coupling between the two organelles. This augmented Ca2+ coupling could be important to support the increased energy needs of the cell during mitosis.

SOCE as a regulator of neuronal activity.

Store operated Ca2+ entry (SOCE) is a ubiquitous signalling module with established roles in the immune system, secretion and muscle development. Recent evidence supports a complex role for SOCE in the nervous system. In this review we present an update of the current knowledge on SOCE function in the brain with a focus on its role as a regulator of brain activity and excitability.

STAT1- and NFAT-independent amplification of purinoceptor function integrates cellular senescence with interleukin-6 production in preadipocytes.

BACKGROUND AND PURPOSE: Senescent preadipocytes promote adipose tissue dysfunction by secreting pro-inflammatory factors, although little is known about the mechanisms regulating their production. We investigated if up-regulated purinoceptor function sensitizes senescent preadipocytes to cognate agonists and how such sensitization regulates inflammation. EXPERIMENTAL APPROACH: Etoposide was used to trigger senescence in 3T3-L1 preadipocytes. CRISPR/Cas9 technology or pharmacology allowed studies of transcription factor function. Fura-2 imaging was used for calcium measurements. Interleukin-6 levels were quantified using quantitative PCR and ELISA. Specific agonists and antagonists supported studies of purinoceptor coupling to interleukin-6 production. Experiments in MS1 VEGF angiosarcoma cells and adipose tissue samples from obese mice complemented preadipocyte experiments. KEY RESULTS: DNA damage-induced senescence up-regulated purinoceptor expression levels in preadipocytes and MS1 VEGF angiosarcoma cells. ATP-evoked Ca2+ release was potentiated in senescent preadipocytes. ATP enhanced interleukin-6 production, an effect mimicked by ADP but not UTP, in a calcium-independent manner. Senescence-associated up-regulation and activation of the adenosine A3 receptor also enhanced interleukin-6 production. However, nucleotide hydrolysis was not essential because exposure to ATPγS also enhanced interleukin-6 secretion. Pharmacological experiments suggested coupling of P2X ion channels and P2Y12 -P2Y13 receptors to downstream interleukin-6 production. Interleukin-6 signalling exacerbated inflammation during senescence and compromised adipogenesis. CONCLUSIONS AND IMPLICATIONS: We report a previously uncharacterized link between cellular senescence and purinergic signalling in preadipocytes and endothelial cancer cells, raising the possibility that up-regulated purinoceptors play key modulatory roles in senescence-associated conditions like obesity and cancer. There is potential for exploitation of specific purinoceptor antagonists as therapeutics in inflammatory disorders.

Chronic reduction of store operated Ca2+ entry is viable therapeutically but is associated with cardiovascular complications.

Loss of function mutations in store-operated Ca2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1) hypomorph mouse model was generated with significant reduction in Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.

Native SOCE complexes: Small but mighty?

Our current understanding of the molecular mechanisms underlying activation of store-operated Ca2+ entry (SOCE) relies in large part on studies that modulate the expression of STIM1 and Orai1. Shen et al. present the first detailed study to address the dynamics and stoichiometry of endogenous STIM1 and Orai1. They argue for an active SOCE cluster centered around a single Orai1 channel per punctum linked to 12 STIM1 dimers, which could have significant implications on SOCE-dependent Ca2+ signaling.

SIRT1 promotes lipid metabolism and mitochondrial biogenesis in adipocytes and coordinates adipogenesis by targeting key enzymatic pathways.

The NAD+-dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD+ boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD+ booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD+ metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.

Correction: Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

[This corrects the article DOI: 10.1371/journal.pbio.3000901.].

The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis.

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260-275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

Multifunctional rhodamine B appended ROMP derived fluorescent probe detects Al3+ and selectively labels lysosomes in live cells.

There a few reports of rhodamine-based fluorescent sensors for selective detection of only Al3+, due to the challenge of identifying a suitable ligand for binding Al3+ ion. The use of fluorophore moieties appended to a polymer backbone for sensing applications is far from mature. Here, we report a new fluorescent probe/monomer 4 and its ROMP derived polymer P for specific detection of Al3+ ions. Both monomer 4 and its polymer P exhibit high selectivity toward only Al3+ with no interference from other metal ions, having a limit detection of 0.5 and 2.1 µM, respectively. The reversible recognition of monomer 4 and P for Al3+ was also proved in presence of Na2EDTA by both UV-Vis and fluorometric titration. The experimental data indicates the behavior of 4 and P toward Al3+ is pH independent in medium conditions. In addition, the switch-on luminescence response of 4 at acidic pH (0 < 5.0), allowed us to specifically stain lysosomes (pH ~ 4.5-5.0) in live cells.

Membrane progesterone receptor induces meiosis in Xenopus oocytes through endocytosis into signaling endosomes and interaction with APPL1 and Akt2.

The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRß is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRß signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRß into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.

L-type Ca2+ channel blockers promote vascular remodeling through activation of STIM proteins.

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.

The CCT chaperonin is a novel regulator of Ca2+ signaling through modulation of Orai1 trafficking.

Store-operated Ca2+ entry (SOCE) encodes a range of cellular responses downstream of Ca2+ influx through the SOCE channel Orai1. Orai1 recycles at the plasma membrane (PM), with ~40% of the total Orai1 pool residing at the PM at steady state. The mechanisms regulating Orai1 recycling remain poorly understood. We map the domains in Orai1 that are required for its trafficking to and recycling at the PM. We further identify, using biochemical and proteomic approaches, the CCT [chaperonin-containing TCP-1 (T-complex protein 1)] chaperonin complex as a novel regulator of Orai1 recycling by primarily regulating Orai1 endocytosis. We show that Orai1 interacts with CCT through its intracellular loop and that inhibition of CCT-Orai1 interaction increases Orai1 PM residence. This increased residence is functionally significant as it results in prolonged Ca2+ signaling, early formation of STIM1-Orai1 puncta, and more rapid activation of NFAT (nuclear factor of activated T cells) downstream of SOCE. Therefore, the CCT chaperonin is a novel regulator of Orai1 trafficking and, as such, a modulator of Ca2+ signaling and effector activation kinetics.

Spatially restricted subcellular Ca2+ signaling downstream of store-operated calcium entry encoded by a cortical tunneling mechanism.

Agonist-dependent Ca2+ mobilization results in Ca2+ store depletion and Store-Operated Calcium Entry (SOCE), which is spatially restricted to microdomains defined by cortical ER - plasma membrane contact sites (MCS). However, some Ca2+-dependent effectors that localize away from SOCE microdomains, are activated downstream of SOCE by mechanisms that remain obscure. One mechanism proposed initially in acinar cells and termed Ca2+ tunneling, mediates the uptake of Ca2+ flowing through SOCE into the ER followed by release at distal sites through IP3 receptors. Here we show that Ca2+ tunneling encodes exquisite specificity downstream of SOCE signal by dissecting the sensitivity and dependence of multiple effectors in HeLa cells. While mitochondria readily perceive Ca2+ release when stores are full, SOCE shows little effect in raising mitochondrial Ca2+, and Ca2+-tunneling is completely inefficient. In contrast, gKCa displays a similar sensitivity to Ca2+ release and tunneling, while the activation of NFAT1 is selectively responsive to SOCE and not to Ca2+ release. These results show that in contrast to the previously described long-range Ca2+ tunneling, in non-specialized HeLa cells this mechanism mediates spatially restricted Ca2+ rise within the cortical region of the cell to activate a specific subset of effectors.

The VLDL receptor regulates membrane progesterone receptor trafficking and non-genomic signaling.

Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRß (PAQR8) partner that is required for mPRß plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper.

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

BACKGROUND: Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. METHODS: We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4+ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. RESULTS: After 8 days in culture, 94% ± 6% of the NKX2-5GFP+ cells were beating when hESCs embryonic bodies were plated on E4+ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP+ cardiomyocytes were close to the E4+ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. CONCLUSIONS: The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony.

Ca2+ tunnelling through the ER lumen as a mechanism for delivering Ca2+ entering via store-operated Ca2+ channels to specific target sites.

Ca2+ signalling is perhaps the most universal and versatile mechanism regulating a wide range of cellular processes. Because of the many different calcium-binding proteins distributed throughout cells, signalling precision requires localized rises in the cytosolic Ca2+ concentration. In electrically non-excitable cells, for example epithelial cells, this is achieved by primary release of Ca2+ from the endoplasmic reticulum via Ca2+ release channels placed close to the physiological target. Because any rise in the cytosolic Ca2+ concentration activates Ca2+ extrusion, and in order for cells not to run out of Ca2+ , there is a need for compensatory Ca2+ uptake from the extracellular fluid. This Ca2+ uptake occurs through a process known as store-operated Ca2+ entry. Ideally Ca2+ entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca2+ tunnelling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca2+ entering via store-operated Ca2+ channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept.

Store-Operated Ca2+ Entry in Oocytes Modulate the Dynamics of IP3 -Dependent Ca2+ Release From Oscillatory to Tonic.

Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store-operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low-amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3 R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3 Rs at distant sites, a process we refer to as "Ca2+ teleporting." Therefore, sustained SOCE activation in the presence of an IP3 -dependent "leak" pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 232: 1095-1103, 2017. © 2016 Wiley Periodicals, Inc.

Xenopus Oocyte As a Model System to Study Store-Operated Ca(2+) Entry (SOCE).

Store-operated Ca(2+) entry (SOCE) is a ubiquitous Ca(2+) influx pathway at the cell membrane that is regulated by Ca(2+) content in intracellular stores. SOCE is important for a multitude of physiological processes, including muscle development, T-cell activation, and fertilization. Therefore, understanding the molecular regulation of SOCE is imperative. SOCE activation requires conformational and spatial changes in proteins located in both the endoplasmic reticulum and plasma membrane. This leads to the generation of an ionic current of very small amplitude. Both biochemical and electrophysiological parameters of SOCE can be difficult to record in small mammalian cells. In this protocol we present the different methodologies that enable the study of SOCE in a unique model system, the frog oocyte, which provides several advantages and have contributed significantly to our understanding of SOCE regulation.

The Ca2+-activated Cl- channel Ano1 controls microvilli length and membrane surface area in the oocyte.

Ca(2+)-activated Cl(-) channels (CaCCs) play important physiological functions in epithelia and other tissues. In frog oocytes the CaCC Ano1 regulates resting membrane potential and the block to polyspermy. Here, we show that Ano1 expression increases the oocyte surface, revealing a novel function for Ano1 in regulating cell morphology. Confocal imaging shows that Ano1 increases microvilli length, which requires ERM-protein-dependent linkage to the cytoskeleton. A dominant-negative form of the ERM protein moesin precludes the Ano1-dependent increase in membrane area. Furthermore, both full-length and the truncated dominant-negative forms of moesin co-localize with Ano1 to the microvilli, and the two proteins co-immunoprecipitate. The Ano1-moesin interaction limits Ano1 lateral membrane mobility and contributes to microvilli scaffolding, therefore stabilizing larger membrane structures. Collectively, these results reveal a newly identified role for Ano1 in shaping the plasma membrane during oogenesis, with broad implications for the regulation of microvilli in epithelia.

The inhibitory input to mouse cerebellar Purkinje cells is reciprocally modulated by Bergmann glial P2Y1 and AMPA receptor signaling.

Synaptic transmission has been shown to be modulated by glial functions, but the modes of specific glial action may vary in different neural circuits. We have tested the hypothesis, if Bergmann GLIA (BG) are involved in shaping neuronal communication in the mouse cerebellar cortex, using acutely isolated cerebellar slices of wild-type (WT) and of glia-specific receptor knockout mice. Activation of P2Y1 receptors by ADP (100 µM) or glutamatergic receptors by AMPA (0.3 µM) resulted in a robust, reversible and repeatable rise of evoked inhibitory input in Purkinje cells by 80% and 150%, respectively. The ADP-induced response was suppressed by prior application of AMPA, and the AMPA-induced response was suppressed by prior application of ADP. Genetic deletion or pharmacological blockade of either receptor restored the response to the other receptor agonist. Both ADP and AMPA responses were sensitive to Rose Bengal, which blocks vesicular glutamate uptake, and to the NMDA receptor antagonist D-AP5. Our results provide strong evidence that activation of both ADP and AMPA receptors, located on BGs, results in the release of glutamate, which in turn activates inhibitory interneurons via NMDA-type glutamate receptors. This infers that BG cells, by means of metabotropic signaling via their AMPA and P2Y1 receptors, which mutually suppress each other, would interdependently contribute to the fine-tuning of Purkinje cell activity in the cerebellar cortex. GLIA 2016. GLIA 2016;64:1265-1280.