The copper(II) complex of the tricationic dye with negligible fluorescence quantum yield works as an SHG-only dye. The free-base tricationic dye has been demonstrated for two-photon fluorescence and SHG-based multimodal imaging. This study demonstrates the importance of a balance between the hydrophobicity and hydrophilicity of amphiphilic dyes for effective plasma membrane localization.Second harmonic generation (SHG)-based probes are useful for nonlinear optical imaging of biological structures, such as the plasma membrane. Several amphiphilic porphyrin-based dyes with high SHG coefficients have been synthesized with different hydrophilic head groups, and their cellular targeting has been studied. The probes with cationic head groups localize better at the plasma membrane than the neutral probes with zwitterionic or non-charged ethylene glycol-based head groups. Porphyrin dyes with only dications as hydrophilic head groups localize inside HEK293T cells to give SHG, whereas tricationic dyes localize robustly at the plasma membrane of cells, including neurons,   •Amphiphilic porphyrin dyes with different hydrophilic head groups are synthesized•Necessity of balance between hydrophobicity and hydrophilicity for membrane dyes•Far-red to NIR absorbing dyes for multimodal and SHG-only imaging are presented•SHG from a dye labeling the intracellular organelles of live cells is shown Chemistry; Organic Synthesis; Imaging Methods in Chemistry; Nonlinear Optics Based on the results from cellular studies of dyes with the different hydrophilic head groups, we designed and synthesized a new tricationic donor-acceptor-based porphyrin dye that localizes effectively in the plasma membrane of cells to give bright SHG signals at low laser powers (≤5 mW). We demonstrated the SHG and TPEF-based multimodal imaging of the tricationic dye with commercial cellular organelle trackers in the conventional green and red light regions, which are frequently used in fluorescence microscopy. The porphyrin-based dyes emit at wavelengths greater than 630 nm, and they do not give any background signal in the conventional green and red regions. To quench its fluorescence, we synthesized the copper(II) complex of the tricationic dye and demonstrated its plasma membrane localization in HEK293T cells. Here we present the synthesis of six new amphiphilic porphyrin dyes and investigate their use in multiphoton imaging of live cells along with other porphyrin dyes  and second harmonic generation (SHG) offer various advantages over linear optical microscopy, such as deep light penetration, less photodamage, and reduced background signal . Both TP ex vivo . TPEF ca ex vivo . SHG is  ex vivo . For mem ex vivo . Despite ex vivo . One of  ex vivo  has beenn CHCl3) , and exhn CHCl3) . We haven FM4-64 . One of n FM4-64 . Dicatioand JR-3 , have beand JR-3 . Such a rin dyes .Figure 1JR-2 and JR-3 as previously reported -substituted aryl groups attached at the meso positions of the porphyrins. The complete procedures for the synthesis of JF-1 and JF-2 are given in the AK-1 and the neutral dyes IG-1 and JW-1 were synthesized from porphyrins 1 and 2, respectively ; however, if the alkylation is performed in other solvents such as dimethylformamide (DMF), decomposition predominates. To the best of our knowledge, this is the first example of an isolated linear tricationic porphyrin-based amphiphilic dye. AK-1.Cu was synthesized by treating AK-1 with copper(II) acetate. Neutral amphiphilic dye IG-1 was synthesized by in situ removal of the trihexylsilyl group of 2 using tetrabutylammonium fluoride and Sonogashira coupling with 4 followed by removal of zinc with TFA (JW-1 was prepared similarly using the hexaethylene glycol (HEG)-substituted iodoisophthalic acid instead of 4. JW-1 and IG-1 dyes were functionalized with isophthalic derivatives substituted with four HEG and twelve TEG groups, respectively, instead of the pyridinium-based electron-acceptor group as the hydrophilic moiety. The dyes do not require pyridinium-based electron-acceptor groups because it has been previously shown that an acceptor group does not substantially contribute toward the nonlinear optical properties of free-base donor-acceptor-substituted porphyrin dyes  light absorbing and emitting amphiphilic porphyrin dyes functionalized with different hydrophilic head groups  and S1, reported . Dyes JRthe dyes . Like JRectively ; howeverectively . Since thilicity . JF-1 anectively . While swith TFA . Porphyrrin dyes . MultiplAK-1, AK-1.Cu, JR-2, JR-3, JF-1, JF-2, JW-1, and IG-1, have similar absorption spectra  at 20°C in Hank’s balanced salt solution (HBSS) buffer. The incubated cells were imaged under the microscope at 870 nm using up to 5 mW laser power .JF-1 and JR-2 localize at the plasma membrane of HEK293T cells  leads to aggregation and does not improve the brightness of SHG. We postulate that the reason for low SHG signal could be dual: (1) the uptake of the dye in the plasma membrane of the cells is limited by the TEG-substituted aryl groups located at the meso positions of porphyrin, resulting in overall reduced fluorescence and SHG signals or (2) the TDM of the dye is not well aligned perpendicular to the plane of the membrane  in acute mouse brain slices  and red (570–625 nm) regions. This is because AK-1 emits fluorescence at wavelengths greater than 630 nm  that is suitable for multimodal imaging  and nickel(II) cations are known to quench the fluorescence of porphyrins without generating singlet oxygen and, hence, phototoxicity  complex of AK-1  complex of toxicity . Previoutoxicity . Howevertoxicity . As expe3T cells . We also of AK-1  to give JR-3 and JF-2, both entered the cells without any plasma membrane localization  complexes of AK-1 and JF-1 are the second examples of SHG-based dyes reported so far that give negligible TPEF and the first for porphyrin-based dyes. Although the aqueous compatible neutral porphyrin-based dyes JF-1 and IG-1 do not generate SHG in live cells, they are potential candidates for photodynamic therapy (PDT) because free-base porphyrins are known to generate singlet oxygen for PDT (JR-2, stains intracellular organelles to give to SHG signals. Here, we present several highly SHG-efficient probes that localize reliably in cellular membranes to give SHG at low laser powers and that are suitable for deep imaging and TPEF/SHG-based multimodal imaging.We have synthesized a library of far-red to NIR light absorbing and emitting donor-acceptor-based porphyrin dyes with different live cell localization properties depending on the type of hydrophilic head groups. Of cationic, zwitterionic, and non-charged hydrophilic head groups, we found that the cationic porphyrin dyes have the highest affinity toward the plasma membrane. Although fluorescence generally gives brighter images than SHG, the porphyrin dyes reported here generate comparable or better SHG images. The tricationic dye  for PDT . Apart fAll methods can be found in the accompanying


The transient receptor potential cation channel subfamily M member 4 (TRPM4) channel influences calcium homeostasis during many physiological activities such as insulin secretion, immune response, respiratory reaction, and cerebral vasoconstriction. This calcium-activated, monovalent, selective cation channel also plays a key role in cardiovascular pathophysiology; for example, a mutation in the TRPM4 channel leads to cardiac conduction disease. Recently, it has been suggested that the TRPM4 channel is also involved in the development of cardiac ischemia-reperfusion injury, which causes myocardial infarction. In the present review, we discuss the physiological function of the TRPM4 channel, and assess its role in cardiovascular pathophysiology. TRPM4, which is located on human chromosome 19 [+ and K+ across the plasma membrane into the cytoplasm [2+-permeable cation channels, TRPM4 and TRPM5 are permeable only to monovalent cations, and not to Ca2+ or Mg2+. The Ca2+ impermeability of the TRPM4 channel plays a role in the accumulation of intracellular Ca2+, which leads to the depolarization of the plasma membrane [2+, TRPM4 is a Ca2+- and voltage-activated channel, and its activation is regulated via a variety of methods. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an effective modulator of TRPM4 as the channel can be rapidly desensitized to intracellular Ca2+ [The transient receptor potential (TRP) melastatin-like subfamily member 4 (TRPM4) is a 1214-amino-acid-long transmembrane protein encoded by ytoplasm . In contmembrane ,4,5. Altlar Ca2+ ,7. In adlar Ca2+ ,9,10,11,2 and COOH terminal regions of TRPM4 contain binding sites that are related to the channel’s activation [Like other TRP channels, TRPM4 also comprises six transmembrane domains. The NHtivation ,10. TRPMtivation ,13,14,15tivation ,22,23,24Drosophila spp. in 1975, and over 50 family members have been characterized to date [2+ is involved in many important cellular response mechanisms as a major intracellular messenger [2+ concentration are closely related to the physiological and pathological mechanisms in important tissue systems; for example, alteration in calcium concentration in culture medium affects epidermal cell proliferation and differentiation [TRP cation channels were first reported in  to date . In gene to date ,26. The  to date ,15,26,27essenger ,29. The ntiation .2+ is beneficial to bone health and in maintaining the endocrine balance [2+ overload has been regarded as key in a series of cardiovascular risks such as coronary artery diseases, arrhythmia, and cardiac failure; and clinically, calcium antagonists have been used effectively in treating angina, hypertension, and supraventricular tachyarrhythmias, which illustrates that maintenance of Ca2+ homeostasis confers cardioprotective benefits [2+ and play a unique role as cell sensors [2+, such as contraction, proliferation, and apoptosis [2+ and Ca2+ in epithelial tissues [It is widely acknowledged that the dynamic stability of extracellular and intracellular Ca balance ,32,33. Ibenefits ,38,39,40 sensors . They arpoptosis ,43,44. Ipoptosis ,46,47,48 tissues .The multiple functions played by the TRP channels and their activated multimodality characteristics imply that correct channel gating or infiltration may facilitate the study of complex pathophysiological mechanisms ,14,16,502+ [+ > K+ > Cs+ > Li+ [2+ permeability, which induces intracellular Ca2+ accumulation and overload to cause depolarization of the cell membrane, which further leads to cell damage or death [2+ is concerned.In contrast to other TRP family members, TRPM4 can be activated by an intracellular accumulation of Ca2+ ,51. In as+ > Li+ . Howeveror death . Therefo2+-permeability, except for TRPM4 and TRPM5. It has been described that there is 50% amino acid sequence homology between the two channels [2+, which is quite similar to TRPM4, also being involved in numerous physiological and pathological mechanisms by modulating Ca2+ homeostasis. For example, according to Banik et al., TRPM4 and TRPM5 are both essential in transduction of taste stimuli [Additionally, TRPM is a multifunctional group of TRP channels. As well as other TRPs, the majority of TRPM members are cation channels with Cachannels ,4. The a stimuli . Additio stimuli ,54,55,56In contrast to the similarities described above, there are several differences between TRPM4 and TRPM5. For example, the tissue distributions of TRPM4 and TRPM5 are widely divergent. In research by Fonfria and colleagues, compared to TRPM4 expression, TRPM5 expression was observed only in a limited number of tissues including the intestine, pancreas, prostate, kidney, and pituitary. On the other hand, TRPM4 is highly expressed in the heart, lung, brain, bone, and stomach . TRPM4 i2 and COOH terminal regions influence the tetrameric composition of TRPM4, and two ATP-binding cassette transporter-like motifs at the N-terminal nucleotide-binding domain are involved in the inhibition of TRPM4 activity [As a member of the TRP channel family, TRPM4 contains various transmembrane and cytosolic domains that form a three-decker structure ,61. Theractivity .2-binding site, and a coiled-coil domain, all of which participate in the modulation of TRPM4 function [Moreover, there are several PKC phosphorylation sites, five CaM-binding sites, four Walker B motifs (which are putative ATP-binding sites), a putative PIPfunction ,10,60.2+- and voltage-activated channel, there are diverse ways in which TRPM4 is activated, and we will discuss the mechanisms underlying TRPM4 activation in terms of the following modulators: PIP2, ATP, PKC-dependent phosphorylation, and CaM.As a Ca2 is a minor phospholipid component of the cell membrane, where it is a substrate for a number of important signaling proteins [2 regulates all types of ion channels and transporters, including the voltage-gated K+ and Ca2+ channels [2 is a powerful enhancer of TRPM4, which may lead to a desensitization effect on TRPM4 activity through PLC-mediated PIP2 decomposition [2 in the plasma membrane due to Ca2+-mediated PLC activation, TRPM4 gradually becomes insensitive to Ca2+, which leads to a move toward the state of negative potential [2+-desensitized condition, TRPM4 shows voltage-dependent gating with increased open-state probability at a depolarizing membrane potential [l-lysine, which is a type of PIP2 scavenger, can trigger a sharp desensitization [PIPproteins ,64. As achannels ,66. In 2position . With thotential . Under aotential ,60. It htization .2 results in a rapid attenuation of the TRPM4 current, in accordance with Nilius et al., which has been detected by adding PIP2 or inhibiting PIP2 extracellularly of HEK293 cells, both of which can reverse the depletion of the TRPM4 current [2 [Previous studies have additionally shown that loss of PIP current . Further current , is alsorrent [2 ,9,68.2 is a critical auxiliary factor in the activation of TRPM4. PIP2 cannot activate TRPM4 on its own, but can rectify desensitization, increase the sensitivity of TRPM4 to Ca2+, and restrict the voltage dependence of TRPM4 [2-binding sites have been discovered at the C-terminal of TRPM4, which implies that the alteration of these binding sites may affect the sensitivity of TRPM4 to PIP2 and Ca2+ [In summary, PIPof TRPM4 ,10,69,70and Ca2+ .2+ sensitivity of TRPM4 can be modulated by ATP, PKC phosphorylation, and binding of CaM to the C-terminus [2+ sensitivity of TRPM4 after desensitization has been clarified previously [2+, TRPM4 can revert from desensitization when the cytoplasmic side of the membrane is exposed to Mg2+-chelated ATP [2+ sensitivity [Owing to the structure of TRPM4, Caterminus ,9,10,11.eviously . In the ated ATP ,9. Howevated ATP ,72,73. Isitivity .2+ sensitivity of TRPM4. A PKC activator, phorbol 12-myristate 13-acetate (PMA), can reduce the Half maximal effective concentration (EC50) level of Ca2+ in TRPM4 from 15 to 4 μM [2+ sensitivity of TRPM4 and resist its desensitization. This effect of PMA disappears when both the readily-phosphorylated amino acid residues, S1145A and S1152A, at the C-terminus of TRPM4 carry mutations [PKC-mediated TRPM4 phosphorylation can increase the Ca to 4 μM ,74, whicutations .2+ sensitivity of endogenous TRPM4. Mutations in the C-terminal binding site of CaM can significantly reduce TRPM4 current amplitude and promote faster current decay. This suggests that the C-terminal binding site of CaM is vital for the Ca2+ sensitivity of TRPM4 [A previous study by Nilius et al. found that dominant-negative mutants of CaM reduce the activation of TRPM4. CaM partially counters the reduction of Caof TRPM4 ,75.The following sections discuss why it is important to understand TRPM4’s role in diseases by exploring the activated mechanism of the channel.Flufenamic acid is one of the inhibitors of TRPM4 and widely used in research . Origina+ channels, which are vital therapeutic targets of type II diabetes [Glibenclamide is another TRPM4 inhibitor, which can block TRPM4-like currents in sinoatrial node cells completely at a concentration of 100 μM . The inhdiabetes . Comparediabetes ,80.2+ overload [2+ signaling) is able to disrupt intracellular Na+ and Ca2+ concentration, which produces a sudden accumulation of intracellular Ca2+, especially in the case of TRPC3 [Arrhythmia is a group of conditions with abnormal frequency of cardiac pulsation and/or rhythm, caused in the origin of heart activity and/or conduction disturbances, such as atrial sinus node dysregulation, excitement outside the sinoatrial node, agitation, slow conduction, and blocked or abnormal channel conduction ,82,83,84overload ,87,88,89overload ,90,91,92of TRPC3 ,94,95.TRPM4 mutation was detected in a patient with hereditary heart disease: the glutamate at position 7 was replaced with lysine, which induced progressive cardiac bundle-branch block [TRPM4 mutation to the pathophysiology of human cardiovascular disease [It is clear that TRPM4 is highly expressed in atrial cardiomyocytes ,77. Beinch block . This is disease ,96. The  disease ,17,18,97+ channel takes place, considering that TRPM4 switches on the depolarization of the cell membrane potential, which subsequently injures the cardiac conduction system [TRPM4 mutations are associated with isolated cardiac conduction disease, right bundle-branch block, tachycardia, and Brugada syndrome [However, the link between the function of TRPM4 and cardiac conduction block remains unclear. Perhaps the dysfunction of the voltage-dependent Nan system ,98,99. Rsyndrome ,19,20,21+ and Ca2+ is involved in the regulation of the cardiac conduction system and is closely linked to the development of arrhythmia. As a result, TRPM4, which is an important Ca2+ regulator, may also be involved in cardiac conduction system diseases.In summary, the fluctuation of NaCardiac hypertrophy is one of powerful adaptive mechanisms of the cardiovascular system, which is mainly manifested by an increase in cell volume and weight ,101,102.2+ levels in the cardiomyocytes of humans with myocardial hypertrophy were sustained at high levels [2, both of which function as secondary messengers mediating the release of Ca2+ from the smooth endoplasmic reticulum and sarcoplasmic reticulum [2+ plays a significant role in inducing cardiac hypertrophy. Therefore, if the mechanisms that maintain intracellular Ca2+ homeostasis are perturbed, the evolution of the hypertrophic alteration at the stage of compensation and decompensation in the myocardium is affected. Thus, calcium-regulated ion channels act as typical mediators.Calcium is indispensable in the excitation–contraction coupling and heart contractility under normal physiological conditions ,88. In th levels , which meticulum ,109,110.eticulum ,113,114.TRP channels are important targets in myocardial remodeling because they are capable of transmitting long-term calcium signals ,116,117.Although a significant level of TRPM4 current exists in SHRs, it is quite challenging to find it in WKY rats even by facilitating the TRPM4 current with application of PKC. TRPM4 properties can be monitored by the Patch-Clamp measurement to illustrate the existence of a nonselective cation channel in ventricular cardiomyocytes isolated from SHRs. Thus, TRPM4 is crucial during the process of ventricular remodeling ,120.TRPM4 for establishing a cardiac-specific TRPM4-knockout mouse line (TRPM4cKO). They intended to compare cardiac hypertrophic changes between TRPM4cKO mice and wild-type mice  under AII stimulation. Their results showed significantly higher cardiac hypertrophy at the morphological level in TRPM4cKO mice compared with the wild-type mice, including increased heart size and weight. In addition, high levels of hypertrophy-related genes such as atrial natriuretic peptide (ANP), α-actin, and Rcan1 were detected in the TRPM4cKO mice.TRPM4 is a negative regulator of cardiac hypertrophy induced by angiotensin II (AII) stimulation . In this2+ concentration in isolated ventricular cardiomyocytes of TRPM4cKO mice after AII stimulation reconfirmed that TRPM4 is a key regulator of intracellular Ca2+. There was also a significant rise in CN phosphatase activity in the hearts of TRPM4cKO mice following AII treatment. In these mice, Rcan1 mRNA, which is a reporter of the calcineurin–NFAT activation typical in cardiac hypertrophy, is overexpressed. Additionally, store-operated calcium entry (SOCE), which is helpful in promoting intracellular Ca2+ collection from the extracellular environment, has been regarded as having an important role in mediating cardiac hypertrophy [−/− myocytes [2+ influx through SOCE to induce pathological hypertrophy [TRPM4 deficiency increases the hypertrophic response to chronic AII in cultured neonatal myocytes. Increased intracellular Caertrophy ,117,121.myocytes . It meanertrophy ,117,121 +/+ mice, enhanced cardiac function in response to endurance training was observed. In contrast, a high number of apoptotic DNA fragments are detectable in TRPM4−/− mice after endurance training. This illustrates that TRPM4 can obstruct the progress from adaptive hypertrophy to irreversible cardiac remodeling. In the pathological cardiac hypertrophy, Ca2+ entry via SOCs causes activation of the calcineurin–NFAT pathway. The TRPM4 channel inhibits the SOCs and thus prevents pathological remodeling [TRPM4 is important for the beneficial cardiac remodeling induced by endurance training, and the role of TRPM4 in physiological and pathological cardiac hypertrophy has also been studied . In TRPMmodeling .The link between TRPM4 and arrhythmia, wherein 9-phenanthrol can inhibit TRPM4 and can shorten the duration of action potentials (APs), has been confirmed previously, which implies that TRPM4 can delay AP repolarization . It has Thus, TRPM4 is an essential regulator of the hypertrophic alteration and is likely to supply a novel direction for the treatment of cardiovascular diseases caused by cardiac hypertrophy ,122.Ischemic heart diseases (IHDs) have become the most frequently reported threat to human health ,126. Myo2+, especially in the mitochondria. This promotes the depletion of ATP with increased Ca2+, leading to cell death. With the return of blood flow, even if it is conducive to the recovery of ATP, a large burst of Ca2+ and reactive oxygen species in the mitochondria occurs at the onset of reperfusion, which opens the mitochondrial permeability transition pore and causes further myocardial damage. Thus, reduced ATP and disrupted calcium metabolism eventually lead to myocardial IRI. It has also been identified that Ca2+ increase and ATP depletion are involved in the activation of TRPM4, and a link between TRPM4 and myocardial IRI has been reported [The mechanism underlying myocardial ischemia-reperfusion injury (IRI) is complex ,131,132.reported ,134.Inhibition of TRPM4 with 9-phenanthrol (9-Phe) has been shown to confer cardioprotective effects against IRI in the rat heart . In the 2O2) exposure, and the other being hypoxia/reoxygenation (H/R) induction. Moreover, the viability of cardiomyocytes with the TRPM4 knocked down through small interfering RNA (siRNA) transfection was significantly higher than that of the nontransfection group following ROS damage and H/R induction. It can also be suggested that inhibition of TRPM4 protects cardiomyocytes against IRI both in vivo and in vitro, and 9-Phe treatment confers remarkable cardioprotection against oxidative stress injury. Therefore, inhibiting TRPM4 might be promising in treating myocardial IRI for IHD patients [The relationship between TRPM4 and myocardial IRI has been explored more deeply in vitro as well . In rat patients .2+ dynamic equilibrium, and the mechanism of intracellular Ca2+ overload-induced IRI has already been elucidated [2+ during myocardial IRI is being explored.TRPM4 is closely related to intracellular Caucidated ,122,135.Endothelial injury is the risk factor and may cause a series of cardiovascular system disorders ,137. Due2O2), which results in endothelial cell apoptosis. However, this could be reversed through the inhibition of TRPM4 activity with 9-Phe [In a lipopolysaccharide-treated group, more than 40% of the endothelial cells were apoptotic, whereas the apoptosis in the TRPM4-inhihition group reduced significantly . Lipopolth 9-Phe . Thus, ith 9-Phe ,134,144.+ and Cl− is able to cause enlargement of osmotic pressure, which drives the influx of H2O, inducing cell swelling or rupture [It is clear that under the influence of various vascular injury factors, the upregulation of TRPM4 in vascular endothelial cells produces complete or maximal depolarization, resulting in the continuous influx of sodium ions ,138,139. rupture .Overall, TRPM4 is an important nonselective cation channel involved in cellular calcium regulation. A majority of cardiovascular diseases are closely related to calcium homeostasis. As the research on TRPM4 progresses, more functions will be recognized and intrinsic links between TRPM4 and the development of cardiovascular diseases may be discovered.


On the other hand, aberrant Ca2+-signaling and loss of [Ca2+]i homeostasis contributes to tumor initiation proliferation, angiogenesis, and other key processes that support tumor progression in several different cancers. Currently, chemically and functionally distinct drugs are used as chemotherapeutic agents in the treatment and management of cancer among which certain anti-cancer drugs reportedly suppress pro-survival signals and activate pro-apoptotic signaling through modulation of Ca2+-signaling-dependent mechanisms. Most importantly, the modulation of [Ca2+]i levels via the endoplasmic reticulum-mitochondrial axis and corresponding action of channels and pumps within the plasma membrane play an important role in the survival and death of cancer cells. The endoplasmic reticulum-mitochondrial axis is of prime importance when considering Ca2+-signaling-dependent anti-cancer drug targets. This review discusses how calcium signaling is targeted by anti-cancer drugs and highlights the role of calcium signaling in epigenetic modification and the Warburg effect in tumorigenesis. Calcium (Ca Binding of Ca2+ with CBP regulates signal transduction and gene expression [2+ is sequestered. Nuclear Ca2+ has specific biological functions involved in gene expression and regulation [2+]i-regulation and its role in cell proliferation is extensively reviewed by Resende and colleagues [2+] is dependent on changes in [Ca2+]i [The concentration of Caivation) . [Ca2+]i cytosol  i ,19.2+]i-signaling is initiated by the entry of Ca2+ from an extracellular pool or by releasing Ca2+ from ER stores or mitochondria. This increases [Ca2+]i from 100 nM (at rest) to approximately 1000 nM generating an “ON” signal for multiple processes. As a prolonged increase in [Ca2+]i may be harmful, the [Ca2+]i signals are spatially and temporally regulated [2+/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C) decode the Ca2+ signals to various cellular processes [2+]i. [Ca2+]i-signaling is involved in both proliferation and apoptosis. Ca2+-oscillations stimulate cell proliferation via Ca2+ sensitive transcription factor (NFAT) and conversely, an increase in [Ca2+]i for a longer duration activates apoptosis i-signaling are associated with various cancers and is also implicated in therapy resistance i is a versatile second messenger in both proliferation and cell death. [Ca2+]i-signaling involves the participation of various proteins combined differently depending upon the type of cellular process initiated , and the ER and mitochondrial axis in proliferation. [Ca2+]o) modulates various cellular processes via calcium channels and extracellular calcium-sensing G-protein coupled receptors, which include calcium-sensing receptor (CaSR) and GPRC6a [2+]o as a key regulator of proliferation in chicken fibroblast [2+]o. Similar observations were made in mouse 3T3 cells, with cell proliferation being dependent on [Ca2+]o, while a calcium driven mechanism initiated DNA synthesis and cell cycle progression that ultimately resulted in cell division [2+]o and its role in proliferation is reviewed in detail by Borowiec [2+]o potentially exerts biological actions via sensor proteins on the plasma membrane. CaSR senses [Ca2+]o and thus triggers the influx of Ca2+ through specific channels and regulates Ca2+ absorption and homeostasis in various organs. A reduction of Ca2+ influx by blocking calcium channels at the plasma membrane (PM) or reduction of [Ca2+]o attenuates cell proliferation i measurements showed that an androgen stimulated Ca2+ influx was sensitive to nifedipine, indicating that L-type calcium channels were responsible for the androgen-stimulated Ca2+-influx in LNCaP cells. Interestingly, blocking of L-type channels resulted in decreased cell growth i-regulation, and impaired PMCA function results in Ca2+ overload and cell death. Glycolytic inhibition and subsequent ATP depletion impairs PMCA function and induces cells to undergo apoptosis i -signals as the Ca2+ influx is triggered by a calcium release from the ER which is mediated through stimulation of surface receptors i homeostasis is observed in normal human bronchial epithelial (NHBE) cells vs. different lung cancers. Lung cancer cells showed lower ER-Ca2+-content which correlated with reduced expression of SERCA2b and increased expression of IP3R and calreticulin  [SERCA is a calcium pump located in endoplasmic reticulum which maintains a low calcium level and thus enables signaling of various physiological processes . Inhibitpathways . A closeer cells . A signi the ER) .2+]i related to IP3 mediated Ca2+-release and are important determinants of [Ca2+]i-signaling, thus controlling survival, autophagy, apoptosis, metastasis, and invasiveness i. There is a calcium crosstalk in the domain between ER and mitochondria known as mitochondria-associated membrane (MAM). Importantly, tumor cells modify their MAM which leads to alterations of tumor homeostasis and consequently to promotion of migration, invasiveness, metastasis, resistance to apoptosis, and induction of EMT [2+]i homeostasis also may be influenced by Bcl-2 proteins. Overall, members of the Bcl-2 family play an important role in cell death and survival via modulation of Ca2+-transport at the ER, plasma membrane, or mitochondria i rise elevates VDAC1 expression and positively modulate apoptosis i which is related to activation of apoptosis. The release of Ca2+ from ER store as well as influx of Ca2+ through the SOC channels influence apoptosis. As reported by Wertz and Dixit, the release of Ca2+ from the intracellular store alone activated apoptosis via caspase 3/7 in LNCaP prostate cancer cells i mediated induction of cell death.Anti-cancer drugs have multiple modes of action and, based on their mechanism of action, can interfere at the DNA level, act as antimetabolites, inhibit enzyme synthesis, and inhibit microtubule function. In the past decade, much attention has been drawn to calcium signaling. Altered calcium signaling is crucial for the development and progression of cancer through the induction of proliferation, invasion, and metastasis . Figure 2+]o influences the mechanism of action of paclitaxel in inducing apoptosis in breast cancer cells. Apoptosis induced by low dose paclitaxel is independent of [Ca2+]o whereas for high dose paclitaxel, normal [Ca2+]o is critical for inducing apoptosis in MDA-MB-468 TNBC cells, which is mediated via capacitative Ca2+-entry [2+]o reduced anti-cancer action of tamoxifen (TM) on HepG2, human hepatoblastoma cells. To understand the route of Ca2+ entry, Ca2+ channel blockers nifedipine and verapamil were used, but neither of them reversed the effect of TM, while flufenamic effectively reversed the effect of TM. A non-selective cation channel (NSCC) blocker caused significant inhibition of TM-induced apoptosis, indicating NSCC as the main route of Ca2+-influx [2+]o improved the cytotoxic action of CDDP and topotecan [[Ca2+-entry . Similar+-influx . Modifyiopotecan . cis-diamminedichloridoplatinum) is used in treatment of solid tumors and modulates [Ca2+]i. It blocks voltage gated channels at higher concentrations [2+ ]i followed by an increased expression of VDAC1 and oligomerization of VDAC1-triggered cytochrome c release and concomitant apoptosis [2+-dependent mechanism. A study in HeLa S3 cells revealed a rise in [Ca2+]i following CDDP treatment, which correlated with the activation of calpain, an important molecule in the induction of apoptosis [2+]i in MCF-7 breast cancer cells  (PtAcD) is selectively toxic on the breast cancer cell line and less toxic on normal breast cells due to differentially activated JNK and p38 i. Treatment with 1 µM As2O3 in SH-SY5Y NB cells elevated [Ca2+]i by ER-Ca2+ store depletion [2+]i, transient calcium elevation, and spikes. Mechanistic studies have revealed increased H2O2 level, Bax expression, growth arrest at the G1 phase of the cell cycle, and inhibition of vascular endothelial growth factor (VEGF) upon As2O3 treatment o concentration dependently suppressed the cytotoxic activity of doxorubicin in MCF-7 breast cancer cell line. High [Ca2+]o (100 and 140 mM CaCl2) almost completely rescued the cells treated with 0.2 µg/mL of doxorubicin (LD50). Mechanism revealed that Ca2+ activated V-ATPase (Vacuolar-type H+-ATPase), reduced the bioavailability of drug by acidifying the intracellular organelles and sequestering doxorubicin into subcellular compartments [2+]o confers insensitivity to doxorubicin treatment. In another study, silencing of PMCA2 calcium channel increases the sensitivity of MDA-MB-231 to doxorubicin treatment. Doxorubicin and simvastatin treatment in breast cancer cells caused a persistent release of Ca2+, resulting in activation of proapoptotic BIM pathway and subsequent overload of mitochondrial Ca2+ triggering apoptosis. In addition, both drugs significantly reduced the activation of pro survival pathway ERK1/2 signaling, which is calcium-dependent in doxorubicin treatment o or mitochondrial calcium but dependent on IP3R mediated calcium influx from ER. The molecular basis of paclitaxel induced cytosolic Ca2+-oscillation is due to the enhanced binding of protein neuronal Ca2+-sensor 1 (NCS-1) to IP3R [2+]o, Ca2+ entered from the extracellular pool following low dose paclitaxel (10−7 M) treatment and was independent of ER store, but high dose paclitaxel (10−6 M) induced Ca2+-influx extracellularly and a gradual depletion of ER store occurred, culminating in apoptosis. The mechanism of paclitaxel-mediated apoptosis is dependent on [Ca2+]o and dosage of the drug i homeostasis in B lymphocytes, while [Ca2+]i has a complex role in the induction of apoptosis by glucocorticoids. Release of Ca2+ from the stores initiates apoptosis in GC-treated lymphocytes. Here, Ca2+-dependent proteases such as calpain were activated, and blocking of calpain activation prevented GC-induced cell death [2+]i attenuated dexamethasone sensitivity in acute lymphoblastic leukemia (ALL) cells. Dexamethasone increased [Ca2+]i mainly by SOC-operated calcium entry i i-signaling in re-activating tumor suppressor genes via CamK. Reactivation of silenced TSG by epigenetic drug azatidine revealed a novel mechanism, which traced back to Ca2+-dependent activation of CamK and subsequent release of MeCP2 methyl-binding protein from promotion of the silenced genes i level and results in chemoresistance and suppressed apoptosis in human colorectal cancer (CRC) cells [2+]i homeostasis i appointed a positive feedback loop that caused the decrease in tonic inhibition of caspases. CDDP in combination with DCA exhibited synergy [2+]i overload inducing cell death i regulating proteins i rise i-signaling studies. A study in HeLa cells  indicated that combined treatment with (BAPTA-AM) or 2-APB attenuated CDDP-mediated [Ca2+]i rise and apoptosis [2+]i rise. As in the case of dexamethasone, BAPTA-AM synergistically enhanced the cytotoxicity of dexamethasone in ALL cell lines [2+ ]i rise and decrease in cell death [2+]i-dependent apoptotic mechanism in PC3 human prostate cancer cells exposed to thapsigargin (1–10 µM). Here, the rise in [Ca2+]i was attributed to both Ca2+-influx from extracellular environment and depletion of ER stores i rise . 1,2-Bispoptosis . The aboll lines . On the ll death . Huang eR stores . CombinaR stores . In MCF-R stores . Calciumg tumors . Similarg tumors .Collectively, the combination of calcium channel modulators with anti-cancer drugs can have a beneficial effect with some combination of drugs.2+]i-signaling has substantial effects on proliferation and apoptosis. Prolonged increase in [Ca2+]i triggers apoptotic cascade and is the principle behind the action of many anti-cancer drugs. In the context of cancer, many drugs such as CDDP, topotecan, and As2O3 induce apoptosis depending on extra- and intracellular calcium. The modification of either [Ca2+]o or [Ca2+]i by calcium chelating agents can significantly augment the action of anti-cancer drugs such as doxorubicin and dexamethasone. This observation is relevant in a clinical context as caution should be exercised when using calcium-altering drugs and diets. Calcium signaling plays a dual role in cell survival and death. ER–mitochondrial Ca2+ fluxes are crucial in steps  leading to cancer growth, and it is the most targeted site for many of the anti-cancer drugs (2+-flux, SOC entry, channels, and pumps on the PM coordinately control the fate of the cell. Hence, components of the calcium tool kit are potential targets of anti-cancer drugs. Nuclear calcium controls gene regulation, but its dependence on cytosolic calcium is a subject of debate. Hence, a drug which modulates [Ca2+]i may or may not affect nuclear calcium, but there is no adequate information on this. Cancer research reveals that i level, which could lead to [Ca2+]i overload and promote apoptosis, is a new challenge for oncological research. Targeting epigenetic pathways is a newer approach in cancer chemotherapy, and using the calcium signaling pathway to reactivate silenced genes was recently introduced. In this regard, certain calcium-modulating agents have been proven to epigenetically increase the protein phosphorylation included in apoptosis/cell-cycle signaling, which is repressed in several cancers such as in the liver, pancreatic, breast, or prostate.Dysregulated expression of many calcium regulation proteins is associated with migration, proliferation, apoptosis, angiogenesis, and invasion in cancer. However, it is beyond the scope of this review to discuss all the calcium signaling components dysregulated in various cancers. A growing body of evidence emphasizes targeting altered calcium homeostasis in cancer as a potential tool in cancer chemotherapy by modulating either the expression of calcium regulating proteins or their function. CDDP and topotecan are drugs identified to modulate the expression of IPAn increasing number of studies have identified and listed various proteins in the calcium signaling tool kit which are altered in various cancers . Studies


American Journal of Tropical Medicine and Hygiene. Seven articles offer recommendations on how to tailor mentoring to the context and culture of LMICs, and provide guidance on how to implement mentoring programs. This introductory article provides both a prelude and executive summary to the seven articles, describing the motivation, cultural context and relevant background, and presenting key findings, conclusions, and recommendations.Mentoring is a proven path to scientific progress, but it is not a common practice in low- and middle-income countries (LMICs). Existing mentoring approaches and guidelines are geared toward high-income country settings, without considering in detail the differences in resources, culture, and structure of research systems of LMICs. To address this gap, we conducted five Mentoring-the-Mentor workshops in Africa, South America, and Asia, which aimed at strengthening the capacity for evidence-based, LMIC-specific institutional mentoring programs globally. The outcomes of the workshops and two follow-up working meetings are presented in this special edition of the  Mentorship is recognized as a deeply rooted tradition in academics2 and a proven path to the development of future generations of scientists. Historically, investing in the success of others has been an expected responsibility, although even accomplished scientists in high-income countries were typically not formally trained as mentors. Mentorship was performed somewhat intuitively and unofficially, with occasional reports of conflicts of interest and negative outcomes.3 Mentorship frameworks, tools, and programs have only emerged in the last decades.5 Regardless of potential imperfections in its implementation, mentorship can result in deep, continued friendships that evolve and mature over time. In fact, some scientists consider their relationships with mentees their most enduring contributions.Mentorship is the professional relationship by which the mentor, “an experienced and highly regarded, emphatic person,” guides a more junior colleague, the mentee, in developing and reassessing his/her ideas, learning and development,6 However, formal mentoring remains an infrequent and largely unsupported practice in LMIC institutions conducting global health research7 and perhaps this has historically inhibited the growth of their scientists and research  Global Infectious Diseases programs, Global Health Program for Fellows and Scholars,23 and Medical Education Partnership Initiative.24 Other recent efforts include the Wellcome Trust’s African Institutions Initiative25 and Brazil’s Science without Borders,26 in addition to activities supported by the U.S. Centers for Disease Control and Prevention, and the U.S. Agency for International Development. None of these investments, however, has specifically addressed the need for local mentoring models or approaches in LMICs. Cole describes a few examples of mentoring experiences and programs in high-income and LMICs, highlighting multiple research gaps and the need for greater evaluation and systematic assessment.27Historically, LMICs have been the focus of substantial research capacity-building investments, ranging from the Rockefeller Foundation's International Clinical Epidemiology NetworkTo help address these needs, the FIC Global Health Program for Fellows and Scholar consortia members partnered to offer five regional “Mentoring the Mentors in Global Health Research” workshops at LMIC institutions. Each 2-day workshop was led by experienced faculty from each region in collaboration with senior U.S. faculty affiliated with the FIC-supported consortia and provided participants with the definitions, methods, and tools to become effective mentors in LMIC settings. Workshops included between 19 and 37 mid- and senior-level scientists primarily from universities and other global health research institutions. The first two workshops were conducted back-to-back in May 2013 in Lima, Peru, for South American training sites and in June 2013 in Mombasa, Kenya, for East African sites. The third workshop was held in November 2014 in Bangalore, India, for the South Asian sites and the fourth workshop took place in Johannesburg, South Africa, in March 2016 for southern African sites. A fifth workshop was conducted for West and Central Africa in Accra, Ghana, in May 2018. All five workshops received positive reviews addressing a much-needed training gap.American Journal of Tropical Medicine and Hygiene (AJTMH) series of articles is to help herald in a new era of increased mentoring in LMICs that leads to advancement of global health research and practice around the world.Given the success of the first four workshops, in 2017 we held a senior-level technical advisory meeting on global health mentoring at LMIC institutions. Leaders from the first four workshops, along with the principal investigators of the six FIC Global Health Program for Fellows and Scholars consortia and staff from FIC gathered to discuss and plan how to best support the development, sustainability, and productivity of strong mentorship programs in global health in LMIC institutions. The goal for this This special issue addresses the challenge of implementing mentoring programs in LMICs and provides guidance on how to adapt mentoring practices used in high-income countries to the settings and cultural practices of research and academic institutions in resource-limited settings. These publications are directed at scientists, institutional leaders, administrators, and trainees in LMIC institutions in collaboration with partners in high-income countries interested in expanding mentorship at LMIC institutions to advance global health research. The articles can also serve as a reference guide for LMIC institutions to develop strategies, approaches, and programs to support mentorship across institutional units, including departments, schools, and colleges, and to select priority mentoring practices for implementation supported by a strong, LMIC-specific evidence base.The recommendations presented in the articles follow four principles highlighting the philosophy of this special issue. First, LMIC scientists led content development as first or senior authors, for an intended audience of LMIC scientists and their high-income country partners in mentoring efforts, as well as LMIC institutions in resource-limited settings. Second, all content was developed exclusively to guide the implementation of mentoring programs and activities in LMICs and to address LMIC-specific gaps. Third, embracing diversity is paramount, as culture, settings, resources, and opportunities for mentoring differ substantially across LMICs. A one-size-fits-all approach is neither viable nor desirable, and implementation will demand customization to each institution, program, or group interested in advancing mentoring. Fourth, a rigorous evidence- and best practices–based approach to mentoring in LMICs is critically needed, and these articles are only the first step in the process. Further refinement of the proposed recommendations is warranted.28 propose a conceptual framework of mentoring tailored to LMIC settings and David Hamer and Laetitia Rispel et al.29 identify critical, technical, and cultural competencies required for mentoring of LMIC scientists. Monica Gandhi, Craig Cohen, and others compare the barriers and facilitators of mentoring identified during the LMIC mentorship workshops sponsored by the FIC Global Health training programs that started this initiative. The evolution of mentorship efforts after the workshops is presented via case studies by Emilia Noormahomed, Craig Cohen et al.,30 describing existing gap and mentoring needs in various institutions in Africa, Asia, and South America. A framework for the evaluation of mentoring programs, including domains and metrics, is proposed by Benjamin Chi, Tony Raj, and others,31 whereas common issues related to ethics and research integrity in LMICs are examined by Elizabeth Bukusi, Joseph Zunt, and others32 via four selected case study scenarios. The collective knowledge of available mentoring toolkits relevant for LMICs and related gaps is discussed in a scoping review conducted by Bhakti Hansoti and others.33 All authors have attempted to link the contents of each of these articles, recognizing that over time, mentoring practice and implementation in LMICs will lead to more cohesive and updated versions of these tools.This overview piece serves both as a prelude to the seven detailed articles, describing the overall motivation and questions and issues each paper addresses, as well as an executive summary, presenting the key findings, conclusions, and recommendations for next steps. The seven other articles in this special issue address specific aspects of the introduction and strengthening of mentorship practices in LMICs. Shailendra Prasad and othersThe advancement of global health research demands sustained career development opportunities for LMIC scientists that can only be attained via the implementation and dissemination of culturally compatible mentoring practices. Institutional resources and local academic and cultural factors should guide the phased implementation of tailored mentoring activities and programs for each setting, with planned, periodic evaluation of progress. Low- and middle-income country institutions also need to support existing mentors and train additional ones, while mentees can contribute to prevent overburdening the few trained mentors available, by playing an active role in the operational efforts of mentoring programs via progressing and peer mentoring. We hope this special issue will become part of the foundation of LMIC-specific mentoring approaches around the globe.


The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5–6 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca 2+ levels to maintain cellular Ca2+ balance. Here the authors find that the STIM1 luminal domain monomer has multiple Ca2+ - binding sites which set the threshold for physiological activation of STIM1 in cells.Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca Each STIM1 monomer consists of an ER-luminal domain specialized for Ca2+ sensing in the range ~100–400 μM, a single transmembrane helix, and a cytoplasmic domain capable of regulated interaction with plasma membrane ORAI Ca2+ channels at ER-plasma membrane contacts , an ER-membrane protein, is a pivotal regulator of cellular Ca2+ signalling focused on the single predicted EF-hand in the STIM1 luminal domain. Mutations that compromised Ca2+ binding to the EF-hand led to constitutive STIM1 activation, evidenced by the relocalization of STIM1 to ER-plasma membrane junctions in resting cells and by constitutive Ca2+ influx7. An NMR structure of the luminal region STIM1(58-201) with Ca2+ bound revealed a compact domain consisting of paired EF-hands—one canonical, the second noncanonical and unable to bind Ca2+— closely interacting with a sterile α motif (SAM) domain8. STIM1(58-201) is termed the ‘EFSAM’ domain on this basis. Two other elements with clearly defined functions, both located in the STIM1 cytoplasmic region, are the STIM1 ORAI activating region  or CRAC activation domain  that interacts with and gates the ORAI channel12; and the C-terminal polybasic tail (residues 671-685) that targets active STIM1 to ER-plasma membrane junctions14.Early mechanistic investigations into how STIM1 controls Ca2+ in ER stores. Inactive STIM1 in cells is a dimer held together by SOAR/CAD–SOAR/CAD interactions19. The SOAR/CAD domain is folded back against CC1 in a way that precludes its effective interaction with plasma membrane ORAI channels20. STIM1 activation is triggered when Ca2+ dissociates from STIM1 ER-luminal domains, the EFSAM domains rearrange or dimerize, and the STIM1 cytoplasmic domains undergo a conformational change that releases the SOAR/CAD domains and the polybasic tails27. Biochemical and biophysical studies have established that STIM communicates the store-depletion signal to the cytoplasm via STIM–STIM association in the transmembrane and predicted coiled coil 1 (CC1) regions28. These intradimer interactions stabilize a coiled coil and bury the CC1 surface that would otherwise retain SOAR/CAD near the ER in resting cells. Active STIM1 relocalizes to ER-plasma membrane junctions, and its extended cytoplasmic domain bridges the ~ 15 nm ER-plasma membrane distance, and recruits and gates ORAI channels3.The STIM1 cytoplasmic domain undergoes an inactive > active transition in which availability of the SOAR/CAD domain and the polybasic tail is regulated by the level of free Ca2+ has come from studies of the isolated recombinant STIM1 luminal domain. The triggering event has been thought to be Ca2+ dissociation from canonical EF-hand sites, leading in turn to a major loss of EFSAM secondary structure and dimerization of EFSAM domains22, and apposition of TM-CC1 regions28. Here we find that Ca2+ sensing entails loss of Ca2+ from both the EF-hand and other sites in EFSAM; that the additional binding sites interact with the EF-hand site to set the Ca2+ sensitivity of STIM1; and that luminal domain unfolding is not a prerequisite for the EFSAM-EFSAM dimerization that drives STIM1 activation.The accepted description of how STIM1 senses reduced ER Ca2+ response. In cells, the EFSAM domain is linked via the STIM1 transmembrane helix and CC1 region to dimeric SOAR/CAD, in both the STIM1 inactive and active conformations . The most plausible explanation is that Ca2+ binding to the EF-hand in the full EFSAM domain is stabilized allosterically by the interaction of the EF-hand portion with the SAM domain, much as Ca2+ binding to calmodulin can be stabilized by interaction with target proteins or peptides33. The value measured in the EFSAM-GrpE FRET assay also differs from the measured IC50 for inhibition by Ca2+ of STIM1 activation in cells  and from that determined in our Ca2+ titration of STIM1(A230C) crosslinking28. The higher IC50 characteristic of full-length STIM1 is most likely to reflect an energetic cost exacted by the coupling between Ca2+ binding to the STIM1 EFSAM domain and the concerted conformational change of full-length STIM1 from the active to the inactive form.Notably, in both cases, the midpoint of the transition to lower FRET falls at ~1–10 μM Ca2+ store depletion9 and measurement of the Ca2+ concentration dependence of STIM activation35, were conducted at room temperature. We have carried out this initial FRET experiment and other experiments in this paper under comparable conditions. In a series of parallel experiments, we have seen no qualitative difference in STIM1 relocalization in cells responding to store depletion at room temperature and at 37 C.The key experiments defining STIM1 function in cells, including the observation of STIM–STIM FRET upon Ca3). To examine the contribution of EFSAM rearrangements to STIM–STIM FRET in cells, we engineered EFSAM-TM-CC1-GrpE constructs extending to residue 343 of CC1, fused to GrpE in place of SOAR/CAD, and labelled at the N terminus with either CFP or YFP . Second, the sites are Ca2+-specific, since similar Ca2+ binding is observed in the presence of 2 mM Mg2+ .Isothermal titration calorimetry (ITC) measures the heat released or absorbed in a chemical reaction following sequential injections of one of the reactants, and thus permits quantitative studies of ligand binding. To assess Carol Fig. . The plo mM Fig. , legend. mM Fig. , it is l2+-binding site per monomer, but in fact EFSAM(D76A)-GrpE exhibited no Ca2+ binding detectable by ITC .In simpler cases, ITC data can be analyzed to parse ligand binding to individual sites. Here, given the interdependence of binding at the EF-hand and the other Ca2+-binding sites in the STIM1 luminal domain by an independent technique , will not appreciably alter free Ca2+. A Ca2+-binding competitor protein, in this case EFSAM, that is present at a fixed concentration will bind a fraction of added Ca2+ at each step in the titration and shift the titration curve. D4 surveys Ca2+ concentrations in the range from tens to hundreds of μM (ref. 36), and was chosen as the fluorescent Ca2+ sensor based on the ITC measurements of Fig. We sought to confirm the existence of multiple Caque Fig. . We chosent Fig.  is that 2+ to the D4 sensor was fitted with Kd 200 μM -GrpE at 35 μM caused no shift in the titration curve, indicating no detectable binding of Ca2+ to the D76A protein  takes place in the range from 50–400 μM Ca2+, suggesting that the sites are physiologically relevant for ER-luminal Ca2+ binding.In a control titration including 35 μM GrpE, binding of Ca μM Fig. , in lineein Fig. . Inclusia2+ Fig. . The res2+ ligands nor in close contact with other parts of the protein. We replaced aspartate with asparagine (D > N) and glutamate with glutamine (E > Q)— conservative and sterically similar replacements that neutralize the negative charge— in three spatially separate groupings of these EFSAM acidic residues  spectroscopy, which reports on secondary structure and, in particular, on α-helix content. The α-helix content of both EFSAM-2NQ-GrpE and wild-type EFSAM-GrpE in the presence of Caain Fig. 8, and ina2+ Fig. , in line 2+ Fig. , not the∘C and 65 ∘C .To verify that we could observe a loss of EFSAM secondary structure by CD if it occurred, we followed the thermal denaturation of EFSAM-GrpE. EFSAM-GrpE showed a clear decrease in α-helical secondary structure between 25 2+ binding and thus provides an independent way to examine the effect of Ca2+ dissociation on STIM1 protein structure. The far-UV CD spectrum of EFSAM(D76A)-GrpE was comparable to the spectrum of the wild-type protein in the absence of Ca2+, reflecting a largely α-helical structure, and was unchanged by addition of Ca2+  is a sensitive way to probe local structure and dynamics of the protein backbone. A standard protocol is to expose the protein briefly to D2+ and in the absence of Ca2+, and assessed deuterium incorporation  was similar to exchange in wild-type EFSAM in the absence of Ca2+, and, conversely, exchange in EFSAM-2NQ largely matched exchange of wild-type EFSAM in the presence of 30 μM Ca2+  and EFSAM-2NQ buttressed the evidence that there are two structured states of EFSAM. H-D exchange in these proteins was almost Ca2+–low Ca2+ conformational change.Measurements with intrinsic and extrinsic fluorescent probes  responded to depletion of ER Ca2+ stores by forming STIM1 puncta . We incubated the membranes in buffer with 0.5 mM EGTA or 2 mM Ca2+, probed exposure of the individual engineered cysteine residues by reaction with biotin-maleimide, then captured biotinylated proteins on streptavidin resin, followed by SDS-polyacrylamide gel electrophoresis and western blotting for STIM1. The residues tested showed similar low accessibility in the presence of Ca2+ for full-length STIM1 and for EFSAM-GrpE , a protein with N/Q substitutions only at E94, D100, E111, D112, E118, and D119. STIM1-2NQ(6–11) was partially constitutively activated, as evidenced by the presence of some puncta already in resting cells, and by the prominent appearance of puncta upon brief exposure to Ca2+-free solution  showed constitutive Ca2+ entry through ORAI channels , but the heat released upon Ca2+ binding to EFSAM-2NQ(6–11) was reduced compared to that with wild-type EFSAM  titration curve in the ITC experiments even though protein concentration in Fig. 28. In ER membranes with ‘wildtype’ STIM1(A230C), the Ca2+ concentration dependence of crosslinking28 mirrors the concentration dependence of STIM1 activation in cells35, whereas the permanently active STIM1(D76A) protein crosslinks regardless of ambient Ca2+ concentration28. Tellingly, in this assay, the Ca2+ dependence of the conformational change of STIM1(A230C)-2NQ(6–11) in isolated ER membranes was shifted rightward into or beyond the range of resting ER Ca2+ concentrations  replacements both alter STIM1 Ca2+ binding at EFSAM surface sites and shift the Ca2+ concentration dependence of STIM1 activation in vitro and in cells.In vitro experiments established the basis of the partial constitutive activation. ITC still reported ~5–6 CaSAM Fig. , indicatons Fig. . As in t2+ dissociation from EFSAM-GrpE or from full-length STIM1 controls a switch between two structured conformations of the EFSAM domain; that Ca2+ at physiological concentrations occupies multiple sites in the EFSAM domain, not solely the EF-hand site; and that the additional EFSAM Ca2+-binding sites are required together with the EF-hand site to control physiological activation of STIM1 in cells. We show further that Ca2+ binding at the EF-hand and Ca2+ binding at the EFSAM surface sites are energetically coupled, as evidenced by the fact that binding to the EF-hand enables or stabilizes binding to the other sites  would be accounted for by the altered binding at those sites without invoking the coupling to the EF-hand site. Measurements of Ca2+ binding to full-length STIM1 in cells or in ER membranes will be necessary to distinguish between these scenarios. Such measurements could also address the possibility, admissible under either scenario, that Ca2+ binding at some of the EFSAM surface sites is further stabilized as part of an EFSAM-protein or EFSAM-lipid interface.A second more radical interpretation posits that CarpE Fig. , but tha2+ sensing has been built on studies of the isolated recombinant EFSAM domain24. Isolated EFSAM differs from EFSAM-GrpE in two key respects: It has a propensity to unfold at low Ca2+ concentrations, and it exhibits negligible Ca2+ binding at 10 μM Ca2+, whether binding is measured directly as 45Ca2+ binding or indirectly as conformational stabilization. The unfolding simply reflects poor structural stability of the isolated EFSAM domain in the absence of Ca2+. The inability of Ca2+ to bind and stabilize isolated EFSAM at concentrations that elicit a conformational change of EFSAM-GrpE could have same root cause—unfolding of isolated recombinant EFSAM is energetically favored at low Ca2+ concentrations, and thus EF-hand Ca2+ binding is opposed by the unfavorable free energy change of refolding the protein.The prevailing model for STIM1 Ca2+ binding to isolated EFSAM found a single 45Ca2+-binding site per monomer21. Furukawa et al. subsequently reported that an isolated EFSAM domain, purified and refolded under different conditions, exhibited a secondary-structure change that was cooperative with respect to Ca2+ concentration, with Hill coefficient 4.7 (ref. 24). This finding might have led to a re-evaluation of the number of Ca2+-binding sites in EFSAM, but Furukawa et al. instead attempted to explain the cooperativity on the basis of a single Ca2+-binding site per monomer, and did not determine the number of binding sites experimentally. It seems more likely in light of our current data that the Furukawa EFSAM preparation had multiple Ca2+-binding sites, and that the structural change reflected cooperative binding at the EF-hand and surface Ca2+-binding sites. The stoichiometry of physiological Ca2+ binding is central to understanding STIM1 Ca2+ sensing, because the overall process of Ca2+ dissociation, STIM1 oligomerization, and STIM1 redistribution in cells is a steep function of ER Ca2+ concentration, with Hill coefficient in the range 4–8 (refs. 35). Based on the premise that there is only a single Ca2+-binding site per monomer, STIM1 oligomerization has been thought to play a dominant role in this cooperativity. Our results indicate that it is time to reconsider this interpretation.Early quantitative studies of Ca2+ binding to EFSAM-GrpE and the associated protein conformational changes offers a fresh perspective on physiological Ca2+ sensing by STIM1. The next obvious steps are to define the additional Ca2+-binding sites structurally, and to investigate the extent to which Ca2+ dissociation from the multiple sites contributes to the cooperativity of STIM1 activation. Perhaps most intriguingly, the finding that EFSAM does not unfold upon dissociation of Ca2+ will spur renewed efforts to define the structure of the EFSAM–EFSAM dimer that triggers STIM1 activation.Our analysis of CaThermus thermophilus HB8 GrpE genetic sequence was synthesized commercially (Genewiz), based on sequence information from PDB . The synthesized GrpE, nucleotides 2222-2734, was PCR amplified and cloned between SacI and NotI sites of pET28a vector (Qiagen) to generate the His6-tagged GrpE expression construct. The cDNA encoding mouse EFSAM sequence (STIM1 residues 58-209) was PCR amplified and cloned at the NdeI and SacI sites of pET28a vector at the 5’ end of the GrpE sequence for the His6-tagged EFSAM-GrpE expression clone, resulting in a construct encoding EFSAM(58-209)–LELSRANSSTLAAVTSGSEL–GrpE(1-171). The D76A mutation was introduced into the pET28-EFSAM-GrpE plasmid using the QuikChange site-directed mutagenesis kit (Agilent). The single-cysteine EFSAM constructs used for energy transfer experiments were generated by PCR amplification of EFSAM-GrpE, introducing nine nucleotides encoding the protein sequence –CGG– immediately N-terminal to EFSAM residue 58, and cloned into pET28a vector. The 100-residue Single Alpha Helix (SAH) construct (pBIEX1-SAH) from Sus scrofa myosin VI is a kind gift from Dr. Sivaraj Sivaramakrishnan, University of Minnesota. The SAH domain was introduced between EFSAM and GrpE sequences using BstBI and AvrII to generate pET28-EFSAM-SAH-GrpE. Expression constructs encoding EFSAM mutated in the surface sites of regions 1, 2, and 3 were generated by mutagenesis of pET28-EFSAM-GrpE plasmid using a commercial service (Genewiz). The 1NQ variant of EFSAM-GrpE contains the mutations E59Q, D60N, E61Q, and E66Q; the 11 mutations of the 2NQ variant are D77N, D82N, E86Q, D89N, E90Q, E94Q, D100N, E111Q, D112N, E118Q, and D119N; and the six mutations of the 3NQ variant are E128Q, D135N, E136Q, E151Q, E152Q, and D183N. The single-cysteine EFSAM-GrpE mutants used for biotinylation experiments were generated by mutagenesis of the pET28-EFSAM-GrpE plasmid using the QuikChange site-directed mutagenesis kit (Agilent).28. Two restriction sites, BsiWI at the 5’ end and MluI at the 3’ end of the mutated regions, were introduced into the pET28-EFSAM-GrpE to facilitate subcloning of the surface variants. Segments of DNA encoding the surface mutations were spliced into the mammalian vectors using PCR amplification and standard subcloning steps. The single-cysteine eGFP-STIM1 mutants used for biotinylation experiments in cellular membranes were generated by mutagenesis of the eGFP-STIM1 plasmid using the QuikChange site-directed mutagenesis kit (Agilent).The HA-tagged STIM1 construct and eGFP-STIM1 are described in detail in ref The cDNA encoding STIM2(1-434) was PCR amplified and cloned into the pEGFP-N1 vector using XhoI and BamHI sites to generate STIM2(1-434)-WT-GFP. Subsequently, the D167A mutation was introduced into the STIM2(1-434)-WT-GFP plasmid using the QuikChange site-directed mutagenesis kit (Agilent). The STIM2(1-434)-2NQ-GFP insert was synthesized using commercial services (Genewiz) and encodes the following ten mutations: D168N, E177Q, D180N, E181Q, E185Q, D191N, E202Q, D203N, E209Q and D210N. pET28-STIM2-EFSAM-GrpE was generated by cloning the cDNA corresponding to STIM2(149-299) between NdeI and SacI sites of the pET28-EFSAM-GrpE plasmid.The pEX-SP-CFP/YFP-STIM1(23-685) constructs used for live-cell FRET measurements were a kind gift from Dr. Tobias Meyer, Stanford University. pEX-SP-CFP/YFP-EFSAM-TC-GrpE plasmids were made by subcloning PCR-amplified fragments of EFSAM-TM-CC1 (encoding human STIM1 residues 58-343) and GrpE into KpnI-AgeI and AgeI-NotI sites, respectively, at the 5’ ends of the CFP/YFP coding sequences in place of the full-length STIM1 inserts.The oligonucleotide primers utilized for PCR amplifications and mutagenesis are listed in Supplementary Table E. coli strain BL21 (NEB) for efficient protein expression. The transformed cells were grown at 37 °C. Protein expression was induced by addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when OD600 of the culture reached 0.6, followed by incubation for 12 h at 16 °C. Harvested cells were resuspended in lysis buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 20 mM CaCl2, 5% (vol/vol) glycerol, 1 mM β-mecaptoethanol, and Roche protease inhibitors (1 tablet per 50 ml buffer), and sonicated. Cell debris was removed by centrifugation, and the lysate was applied to Ni2+-nitrilotriacetic acid (Ni-NTA)-agarose resin (Qiagen). Bound recombinant proteins were eluted in lysis buffer containing 250 mM imidazole. Proteins were further purified by gel filtration on a Superdex 200 column  in the presence of 20 mM CaCl2, 5 mM EGTA, or Chelex-treated Ca2+-free buffer , with 2 mM TCEP (tris(2-carboxyethyl)phosphine). Eluted proteins were concentrated using Amicon Ultra 30 kDa concentration devices (EMD Millipore).Plasmids encoding EFSAM-GrpE variants were transformed into 5 maleimide (Thermo), or with both fluorophores, for in vitro energy transfer measurements. Each protein sample, ~100 μM in 50 mM Tris pH 7.5, 150 mM NaCl, was first treated with 10 mM TCEP for at least 1 h to reduce any disulfide bonds. Excess TCEP was removed by passing the fully reduced protein sample through an Amicon 30 kDa device with a quick buffer exchange using 50 mM Tris pH 7.5, 150 mM NaCl buffer. Dye working stocks (10–20 mM) were prepared in anhydrous DMF immediately prior to use. A 10-fold molar excess of the fluorescent dyes was added slowly to the protein and incubated at 4 °C for 1 h. The labelled protein was passed through a PD-10 desalting column  and further concentrated. Labelled proteins yielded a ~ 0.8-0.9:1 molar ratio of fluorophore to protein, based on measuring the absorbance at 495 nm , 588 nm , and 280 nm . Dye conjugation to each protein was further confirmed by resolving the labelled proteins on a 4–12% NuPAGE gel (Life Technologies) and imaging under a broad-range UV light using Gel Doc EZ (Bio-Rad).Single-cysteine-containing EFSAM-GrpE variants were labelled either singly with fluorescein-5-maleimide (Thermo) or Alexa Fluor 594 CFor in vitro FRET measurements, fluorescence spectra were acquired at 22 °C using a QuantaMaster 40 spectrofluorometer (PTI). Protein concentration was 250 nM. The spectra were collected from 450 to 650 nm with the excitation set at fluorescein dye excitation wavelength (420 nm) and the slit widths set at 12 nm.λex = 370 nm) with the specified amounts of CaCl2 at 22 °C in dark. The slit width was set to 8 nm and the fluorescence emission spectra were acquired from 440 to 600 nm.In the ANS dye-binding assay, 1.4 μM of the specified EFSAM-GrpE variant was mixed with 40 μM ANS  dye (Thermo) in a Chelex-treated buffer containing 50 mM Tris-Cl pH 7.5, 150 mM NaCl, and 5% glycerol. The dye-mixed protein was incubated for 5 min before each spectral scan (λex = 295 nm) were recorded from 310 to 420 nm for 1 μM wild-type EFSAM-GrpE and its variants, at 4 °C. The slit widths were set at 2 nm. Proteins incubated with 6 M GdCl were used to collect the denatured protein emission spectra.Tryptophan emission spectra (36). 150 nM D4 sensor, resuspended in Chelex-treated buffer containing 50 mM Tris-Cl pH 7.5, 150 mM NaCl and 5% glycerol, was titrated with specified amounts of Ca2+, in the presence or absence of 35 μM EFSAM-GrpE variants. Emission spectra were recorded from 450 to 600 nm at λex = 410 nm with 2 nm slit width, at 22 °C.D4 cameleon sensor expression vector, pBAD-D4, was a gift from Amy Palmer and Roger Tsien  at 20 °C. Chelex-treated buffer containing 50 mM Tris-Cl pH 7.5, 150 mM NaCl, and 5% glycerol was used to dilute the proteins and Ca2+ stock. The buffer contained 2 mM Mg2+ where indicated. EFSAM-GrpE concentrations were calculated from A280 (measured with a NanoDrop spectrophotometer), after subtracting the buffer blank reading, using the tryptophan and tyrosine molar extinction coefficients determined by Edelhoch38 for tryptophan and tyrosine model compounds. (EFSAM-GrpE contains no cysteine/cystine.) Ca2+ was injected 39 times, 1 µl each, with 150 s intervals between injections. The background data obtained from the buffer sample were subtracted before the data analysis. The data were analyzed with the Origin7 software package .Can of binding sites from a ‘Wiseman plot’ of the ITC data, such as the one in the right-hand panel of Fig. 39 showed that under the experimental conditions used in ITC measurements, and for a protein having a single class of binding sites , n corresponds approximately to the molar ratio of ligand to protein at the midpoint of the titration curve. This feature carries over when there is more than one class of sites, taking for n the molar ratio at the midpoint of the final phase of the titration curve, as illustrated by a collection of Wiseman plots in the literature41.It is possible to read the approximate number 2+-binding sites, the interdependence of binding at the EF-hand and the other Ca2+-binding sites, and the coupling of Ca2+ binding to a change in intradimer EFSAM–EFSAM interactions. A quantitative description of binding would entail fitting a large number of Kd values, ΔH values, parameters for coupling between the sites, and parameters for coupling between binding at individual sites and the dimer > monomer conformational change. Mathematically, the data do not provide adequate constraints to fit so many parameters.Fitting ITC data quantitatively requires adoption of a specific model, such as binding to a single class of sites, binding to two classes of independent sites, or in some cases a more complicated model. We did not attempt to fit the EFSAM ITC data to a detailed binding model because of the large number of Cad and ΔH are not meaningful. However, the estimate for n may be credible, because any fit has to regress to the baseline in the region around the true value of n, independent of the model chosen. Using the MicroCal software to fit the data to a model with two classes of independent sites consistently yielded an estimate of 5–6 binding sites per EFSAM monomer.The standard software supplied with the MicroCal ITC-200 microcalorimeter has options to fit data to models with one or two classes of independent sites. We stress that these models are not appropriate for EFSAM, and that any estimated values for K2+ where indicated. All spectra were obtained as the average of at least six scans with a scan rate of 50 nm/min. The ellipticity was measured from 190 nm to 260 nm and converted to mean residue ellipticity (deg cm2 dmol-1 res-1). For the experiment of Supplementary Fig. CD spectra of EFSAM-GrpE variants were recorded in an Aviv 62 DS spectropolarimeter at ambient temperature (~22 °C), using a 1-mm path length quartz cell with the protein concentration at 5 μM in Chelex-treated buffer containing 50 mM Tris pH 7.5, 100 mM NaCl, 5% glycerol, and 1 mM or 10 mM Ca2O (Cambridge Isotopes). In pilot experiments, the number of EFSAM peptides reliably detected by MS after pepsin digestion decreased at Ca2+ concentrations higher than 30 μM. This concentration was acceptable for the HDX–MS experiments, since the FRET decrease of donor/acceptor-labelled EFSAM-GrpE was nearly complete at 30 μM Ca2+, and hence the defining conformational change induced by Ca2+ could be captured at 30 μM Ca2+. EFSAM proteins, in the presence or absence of 30 μM Ca2+, were diluted to 10 μM in the deuterated Chelexed buffer, quenched after 0.5, 1, 2, and 5 min with 125 mM sodium phosphate monobasic, pH 2.6 for 1 min, and injected into a Waters G2-Si HDX-MS system (Waters Corp.) by a LEAP H/DX PAL autosampler. Non-deuterated protein controls were processed in parallel. Protein samples were digested on-column with immobilized pepsin (Pierce) at 0 °C, then separated by liquid chromatography on a Waters NanoACQUITY UPLC BEH C18 column with a 7–85% acetonitrile gradient in 0.1% formic acid. Peptides were analyzed by electrospray ionization mass spectrometry with ion mobility separation in a Synapt G2-Si quadrupole time-of-flight mass spectrometer. Peptides were identified with ProteinLynx Global Server (Waters Corp.). Mass spectra were assigned and H/D exchange was determined with DynamX 3.0 (Waters Corp.). The data were corrected for a back exchange loss of < 26%.The pepsin digestion profile of each EFSAM-GrpE variant was determined using 10 μM protein. For the deuterium exchange, deuterated Chelexed buffer, pD 7.5, was prepared by lyophilization of Chelex-treated buffer containing 50 mM Tris pH 7.5, 100 mM NaCl, 5% glycerol, and resuspension in D6 cells per construct) were transfected with the specified single-cysteine constructs using Lipofectamine 2000 and grown under 10% CO2, in DMEM  containing 10% heat-inactivated FBS for 24 h. The cells were scraped from the substrate and resuspended in lysis buffer containing 25 mM Tris-Cl (pH 6.8 with NaOH), 25 mM NaCl, 12.5 U/ml DNase I , protease inhibitors , 1 mM DTT, with either 0.5 mM EGTA (low Ca2+) or 2 mM Ca2+ (high Ca2+). Cellular membranes were prepared and resuspended in 200 μl resuspension buffer containing 25 mM Tris-Cl (pH 6.8) and 150 mM NaCl, with either 0.5 mM EGTA or 2 mM Ca2+. 300 μM biotin-maleimide reagent  was added to the resuspended membranes and the reaction was carried out for 1 h at room temperature. The reaction was quenched using 5 mM DTT and 200 mM Tris-Cl (pH 8.5). The membranes were collected by centrifugation at 167,000 g for 20 min in an airfuge (Beckman Coulter) at 4 °C. The pellets were resuspended in 200 μl pulldown buffer containing 50 mM Tris-Cl, pH 8.5, and 150 mM NaCl. Once thoroughly resuspended, the membranes were solubilized with 0.5% Triton-X 100, 0.5% NP-40 and 0.5% SDS. The solubilized membranes were diluted further with 1200 μl of pulldown buffer and incubated with MyOne streptavidin T1 Dynabeads (Thermo Fisher). The beads were thoroughly washed thrice with pulldown buffer, boiled in 5x reducing loading/dye buffer to elute the biotinylated proteins and analyzed by SDS-PAGE using 4–12% NuPAGE Bis-Tris gels (Invitrogen). Immunoblotting was performed using rabbit anti-STIM1  and HRP-labelled goat anti-rabbit secondary antibody  and the blots were developed using ECL substrate (Perkin Elmer).HeLa cells ( ~ 6 × 10E. coli strain BL21 (NEB) and grown at 37 °C. Proteins were purified using Ni-NTA affinity purification as described above, without any added Ca2+. The purified proteins were passed through PD Minitrap G-25 desalting columns  to remove β-mecaptoethanol and imidazole. The proteins were eluted in biotinylation buffer containing 50 mM Tris-Cl (pH 6.8) and 150 mM NaCl. 24 μM of the specified protein, containing either 0.5 mM EGTA or 2 mM Ca2+, and under native conditions (diluted using biotinylation buffer) or under denaturing conditions , was treated with an equimolar amount of biotin-maleimide reagent for 5 min at room temperature. The biotinylation reactions were quenched directly with 5x reducing loading/dye buffer and the samples analyzed by SDS-PAGE using 4-12% NuPAGE Bis-Tris gels (Invitrogen). Immunoblotting was performed using HRP-labelled goat anti-biotin antibody  and the blots were developed using ECL substrate (Perkin Elmer).For assessing the biotinylation of EFSAM-GrpE single-cysteine mutants, the specified proteins were overexpressed in 2, in DMEM  containing 10% heat-inactivated FBS for 24 h before imaging or crosslinking experiments. HeLa cells, transiently expressing wild-type eGFP-STIM1 or specified variants, were grown on 35-mm glass-bottom dishes (MatTek). Imaging was performed using an Olympus Fv10.1 confocal laser-scanning microscope with a 60×, 1.35 NA, oil-immersion objective. Cells were imaged at room temperature, first in Ringer’s buffer containing 20 mM HEPES (pH 7.4 with NaOH), 125 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 1.5 mM CaCl2, and 10 mM D-glucose, for the resting-state images. This solution was replaced with a modified Ringer’s buffer lacking CaCl2 in the presence or absence of 1 μM thapsigargin (Life Tech), as specified, and incubated at least 5 min before obtaining the activated state images.HeLa cells (ATCC) were transfected with the specified constructs using Lipofectamine 2000 and grown under 10% CO42.HeLa cells co-expressing CFP- and YFP-tagged EFSAM-TC-GrpE protein pairs, corresponding full-length STIM1 protein pairs, or a tandem CFP-YFP construct were grown on 35-mm glass-bottom dishes (MatTek) overnight. The pCMV-CFP-YFP control plasmid was a gift from Liusheng He and Jehonathan Pinthus (Addgene plasmid # 24520). A Zeiss LSM 780 confocal microscope equipped with 405-nm and 514-nm laser lines was used to collect CFP, YFP, and FRET images. The apparent FRET efficiency (expNFRET) between CFP- and YFP-labelled proteins was calculated with the help of PixFRET, an ImageJ-based plugin2 and 10 μM LaCl3 on 35-mm glass-bottom dishes (MatTek). Imaging was performed using an Olympus Fv10.1 confocal laser-scanning microscope with a 60 ×, 1.35 NA, oil-immersion objective. Care was taken to obtain confocal sections at the mid-nuclear level of the cells, showing plasma membrane as well as cytoplasmic compartments. Cells were imaged at room temperature, first in Ringer’s buffer with no additional Ca2+, followed by Ringer’s buffer with 12 mM Ca2+. Fluorophore intensities were quantitated in regions of interest at the plasma membrane and cytoplasmic compartments using ImageJ. Statistical significance of the differences was determined using unpaired, two-tailed Student’s t-test.HeLa cells, co-transfected with mCherry-CAD and the specified variants of STIM2(1-434)-eGFP, were grown overnight in a modified DMEM medium containing 0.2 mM CaCl2+ titration in Supplementary Fig. 2, 11.5 mM glucose, 20 mM HEPES adjusted to pH 7.2 with NaOH. After incubation in the Ca2+-free imaging solution for 10 min, live-cell imaging was carried out on a Nikon Eclipse Ti-E microscope (Nikon Instruments) equipped with an A1R-A1 confocal module with LU-N4 laser sources , CFI (chrome-free infinity) plan Apochromat VC series objective lenses (40 × oil), and a live-cell culture cage to maintain the cells at 37 °C and 5% CO2. Time-lapse imaging was performed to monitor the subcellular localization of mCherry-CAD upon addition of increasing amounts of external Ca2+ (0–25 mM). The cytosolic intensity of mCherry-CAD was analyzed by using the NIS-element AR software (version 4.0).For the extracellular Ca28. Briefly, HeLa cells expressing specified variants of STIM1(A230C) (~12 × 106 cells) were scraped from the substrate and resuspended in Chelex-treated  dilution buffer with no added EGTA or Ca2+. Cellular membranes were prepared and resuspended in 130 μl Chelex-treated resuspension buffer containing 25 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 0.3 mM DTT. Equal volumes of membranes were apportioned to twelve wells containing resuspension buffer supplemented to give either a final EGTA concentration of 0.5 mM or final Ca2+ concentrations ranging from 0.3 μM to 2 mM. Iodine oxidation, SDS-PAGE analysis, immunoblotting with anti-STIM1 antibody , and quantitation were as described in ref 28.STIM1 crosslinking assay was performed as described in ref. 2+ imaging was performed using HeLa cells that had been co-transfected with eGFP-STIM1 variants and mCherry-ORAI1 (ref. 43), FACS-sorted for medium-level GFP-expressing cells, and plated on 18-mm coverslips. The cells were loaded with 5 μM Fura-2-acetoxymethyl ester for 45–60 min at 37 °C in DMEM containing 0.02% Pluronic F-127 and 10 mM HEPES pH 7.4, washed twice with fresh media, and analysed immediately. Modified Ringer’s solution used in this assay consisted of 125 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 10 mM D-glucose, and 20 mM HEPES (pH 7.4 with NaOH), with the addition of either 1 mM or 2 mM CaCl2, or 1 μM TG, where indicated. Coverslips were assembled into a chamber on the stage of an Olympus IX 71 microscope equipped with an Olympus UPLSAPO 20 × , NA 0.75, objective. Cells were alternately illuminated at 340 nm and 380 nm with the Polychrome V monochromator (TILL Photonics) using ET - Fura2 filter . The fluorescence emission at λ > 400 nm  were captured with a CCD camera . Ratio images were recorded at intervals of 4 s. Ca2+ concentration was estimated from the relation44Kd = 220 nM, and the values of Rmin, Rmax, and (Sf2/Sb2) were determined from an in situ calibration of Fura-2 in HeLa cells. Data were analyzed using TILL Vision (TILL Photonics).Single-cell Cat-test.Data in Figs. Supplementary InformationPeer Review File


This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101’s action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca The anti-CD20 monoclonal antibody (mAb), rituximab (RTX), combined with chemotherapy, became the standard regimen for treating non-Hodgkin lymphomas (NHL), such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL) ,2,3. HowCurrently, anti-CD20 mAbs are generally divided into two distinct classes. Type I mAbs, such as rituximab, induce antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and low levels of direct programmed cell death (PCD). Conversely, type II mAbs potently trigger ADCC and PCD, but only weakly elicit CDC . GA101/o2+) are of fundamental importance to signal transduction in animal cells, triggering various cellular processes, such as gene transcription, secretion, cell proliferation, migration, and apoptosis i of 10 CLLs responsive to GA101-induced cell death. GA101 triggered an intracellular Ca2+ increase in all CLLs tested. These Ca2+ responses were due to both intracellular Ca2+ mobilization and extracellular Ca2+ entry . In four CLL samples , Ned-19 statistically inhibited cell death  GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in the three studied NHL cell lines; (2) lysosomal Ca2+ release and subsequent LMP were responsible for the cell death induced by GA101 in BL2 and Raji but not in SU-DHL-4 cells; (3) in SU-DHL-4 cells, cell death was repressed by Orai1-dependent Ca2+ influx and, consequently, enhanced by Orai1 inhibitors through the activation of UPR in response to ER stress; (4) in B-CLL, GA101 triggered an intracellular Ca2+ increase involving mobilization of either lysosomal or ER Ca2+ pools and a Ca2+ influx, which are differentially implicated in GA101-induced cell death. Taken together, these results revealed the central role of GA101-induced Ca2+ signaling in its action mechanism.The role of Castigated ; however2+ responses: one similar to that described with RTX i measurement cells were loaded with 7.5 µM Fluo2- LR-AM  calcium dye, which exhibits limited compartmentalization in intracellular stores and is leakage resistant, in the presence of 0.02% pluronic F127 in HBSS at room temperature for 25 min. The cells were rinsed with HBSS and incubated without Ca2+ probe for 15 min to complete de-esterification of the dye. For B-CLL, cells were previously labeled with anti-human CD19 APC (Allophycocyanin) at room temperature for 20 min, to ensure that Ca2+ recordings originated from B cells. In some experiments, cells were placed in a Ca2+-free HBSS medium, to which 100 µM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) was added to chelate any residual Ca2+ ions. This medium was added to the cells just before recording to avoid leaks from the intracellular calcium stores.For single-cell [Ca2+ probe concentration between cells, we normalized the fluorescence signal (F) of each cell by its starting basal fluorescence (F0). Data were processed using OriginPro 7.5  and Prism 6.0  software. Fluorescence was recorded using an inverted fluorescence confocal microscope  with a 25× oil immersion objective. Images were recorded at constant 10-s intervals. Regions of interest corresponding to cells recorded were drawn to analyze the fluorescence signal. To overcome putative differences of CaLysosomal membrane permeabilization (LMP) and lysosomal cathepsin B release were assessed by lysotracker staining and immunofluorescence, respectively, and analyzed by fluorescence microscopy. For LMP detection, cells were stained with lysotracker red (100 nM) in HBSS at 37 °C for 45 min. Cells were then rinsed twice in HBSS. Fluorescence micrograph images were obtained using an inverted fluorescence microscope with an ApoPLAN 63× objective. Images were recorded at constant 1-min intervals. Regions of interest corresponding to cells recorded were drawn to analyze the fluorescence signal, and its slope was calculated using GraphPad Prism software. w/v paraformaldehyde at 4 °C for 10 min and then permeabilized in PBS supplemented with 5% Bovine Serum Albumin (BSA)/0.1% saponin for 5 min. Cells were incubated with anti-cathepsin B in PBS/1% w/v BSA at 4 °C overnight. Cathepsin B was revealed using secondary Alexa594-coupled donkey anti-goat Ab. Nuclei were stained using Hoechst 33258. Images were acquired using a Zeiss LSM 510 meta confocal microscope with an ApoPLAN 63× objective.To visualize cathepsin B release in the cytosol, cells were untreated or treated with GA101 or siramesine (a lysosomotropic agent), and immunofluorescence was performed. Cells were fixed in Phosphate Buffer Saline (PBS) containing 4% Cell lysates were prepared using a lysis buffer containing protease and phosphatase inhibitors . The lysates were separated by SDS-PAGE and transferred to Polyvinylidene fluoride (PVDF) membrane using an iBlot Gel Transfer System (Thermofisher Scientific). The membranes were incubated with 5% nonfat milk w/v in PBS buffer for 1 h and then reacted with the primary Ab in PBS/Tween buffer with 5% nonfat milk w/v by shaking at 4 °C overnight. The appropriate second Abs conjugated to HRP was used to detect the protein of interest via Enhanced Chemiluminescence (ECL) (Millipore). Total RNA was isolated using the RNeasy kit  and its concentration was determined by Nanodrop. Quality and integrity of total RNA was assessed using the Bioanalyzer . For real-time PCR, the reverse transcription (RT) reaction was carried out with 1 µg of total RNA in a 20-µL mixture to generate first-strand complementary DNA (cDNA) using PrimeScript RT reagent kit with genomic DNA (gDNA) Eraser . All real-time PCR reactions were performed in a 20-µL mixture containing 1/2 volume of cDNA preparation using PowerUp Sybr Green Master mix (Thermofischer Scientific) and Taq Ozyme HS (Ozyme) according to manufacturer’s protocol. Quantitative PCR was performed using appropriate primers . Real-time quantifications were performed using the StepOnePlus Real-Time PCR System . The fluorescence threshold value was calculated using StepOne software (Applied Biosystems).t-test or one-way ANOVA test or the non-parametric Mann–Whitney U or Wilcoxon tests, as appropriate (* p < 0.05).All data were expressed as means ± SE. The significance of differences was calculated using the parametric 2+ signaling. The source of Ca2+ increase, as well as its role in GA101-induced cell death, differed according to the cell type studied. Further investigations are required to identify the biomarkers of Ca2+ involvement in GA101-induced cell death, in order to design new, rational drug combinations to improve the clinical efficacy of GA101.In conclusion, this study provides new mechanistic insights into GA101-induced cell death, including the important, complex role of Ca