Cytosolic concentrations of Ca2+ can signal dysregulation in organelles including the mitochondria, nucleus, and sarcoplasmic reticulum in skeletal muscle. In this work, a treatment is applied to mimic the Ca2+ increase associated with these atrophy-related disease states, and broadband impedance measurements are taken for single cells with and without this treatment using a microfluidic device. The resulting impedance measurements are fitted using a single-shell circuit simulation to show calculated electrical dielectric property contributions based on these Ca2+ changes. From this, similar distributions were seen in the Ca2+ from fluorescence measurements and the distribution of the S-parameter at a single frequency, identifying Ca2+ as the main contributor to the electrical differences being identified. Extracted dielectric parameters also showed different distribution patterns between the untreated and ionomycin-treated groups; however, the overall electrical parameters suggest the impact of Ca2+-induced changes at a wider range of frequencies.Many skeletal muscle diseases such as muscular dystrophy, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and sarcopenia share the dysregulation of calcium (Ca The Ca2+ within the cytoplasm skeletal muscle cells has a variety of deeply important functions, including regulating the mitochondrial function and controlling signaling pathways between cells. In skeletal muscle systems, Ca2+ has roles in the excitation–contraction (EC) coupling, mitochondrial function, and as a signal regulator for protein and degradation i), the cells were seeded in a 96-well plate in the culture media described previously at a density of 1000 cells per well for three days at 37 °C with 5% CO2. The cells were incubated with physiological solution containing (mM) NaCl 140, KCl 2.8, CaCl2 2, MgCl2 2, glucose 10, Hepes–NaOH 10, pH 7.4, named normal external solution (NES) containing 10 mg mL−1 bovine serum albumin and 5 µM Fura-2 AM  for 45 min. Then, the cells were rinsed by incubation in NES with Ca2+ for 40 min at 37 °C to allow de-esterification of the dye inside the cells. Ca2+ imaging was performed using epifluorescence microscopy that assists in the acquisition of the Fura-2 emission fluorescence at both an excitation of 340 nm and then at an excitation of 380 nm. These excitations and measurements were accomplished using a Xenon lamp and monochromator wavelength switcher Polychrome II  to isolate the wavelength of interest. The emission fluorescent images were collected using a 40× oil objective lens, acquired by an intensified CCD camera or a C6790 cooled CCD camera , digitized and stored in an interfaced computer. The software Aquacosmos (version 2.0) was used to analyze the resulting emission fluorescence ratio (f[340/380]) on selected representative cellular areas after the subtraction of background fluorescence. After an initial baseline was established, 1 µM Ionomycin in 1.5 mM Ca2+ was added for 10 min while measuring fluorescence. These measurements were accomplished in a temporal sequence generating spectral measurements showing the cell fluorescence ratio prior to ionomycin treatment, during treatment of 10 min, following a wash Ca2+-free PBS, and after incubation in an 8.5% sucrose and 0.3% dextrose solution. The mean initial values and post-treatment values were calculated using 25 time points in the initial plateau and the plateau following the wash, which were used to calculate the ratio. The acquisition time for each fluorescence emission was 0.5 s and the overall imaging lasted 1.5 h. Additional experiments were performed using sucrose solution with 10 mM EGTA, a calcium chelator, in order to prevent any possible external Ca2+ interference post-ionomycin treatment, showing similar results.For measurements of the intracellular calcium concentration  electrode device was assembled by attaching a polydimethylsiloxane (PDMS) channel to the CPW surface aligned with a series gap. Further details on the CPW and channel design can be found in previous publications ,22,23 anOnce all components were in place, cells were introduced to the channel at a sample flow rate of 0.1 µL/min along with a stream of sheath sucrose at a rate of 0.2 µL/min. An electrical dielectrophoretic (DEP) signal of −9 dBm at a frequency of 500 MHz was introduced between the series gap in the electrodes to trap the cell. Then, a broadband sweeping signal of −15 dBm from 9 kHz to 9 GHz was applied to measure the S-parameters with and without the cell present using a virtual network analyzer . The S-parameters include the reflection coefficient (S11) and the transmission coefficient (S21). S11 is representative of the power reflected back to the power input port 1 by the system, while S21 is representative of the power that passes through the system to port 2 . These mThe raw signals of ΔS11 and ΔS21 taken from the Keysight network analyzer for each cell at all 201 frequencies appear as spectra. Using the impedance measurements for these spectra, curve fitting using simulated circuit modeling was used to obtain dielectric parameters.Simulation of a single-shell circuit model was performed to find the optimal fit of the cytoplasm resistance and capacitance for each electrically measured cell using Advanced Design System  software by Keysight. The circuit designs for the simulations can be seen in c and Cc. From the extracted variables, the dielectric permittivity (σ) and conductivity (ε) of the cytoplasm were calculated using Equations (1) and (2), as seen in previous single-shell fittings  in the periods prior to ionomycin addition and after the maximum determined experimental length of 1.5 h, based on viability in sucrose, as shown in 2+ concentration, which is shown in p = 0.012). However, looking at the distribution in the middle of the time period during which electrical measurement occurs, there is a significant increase in mean to 0.102 ± 0.041 (p < 0.0009) and a significant shift in the distribution (p < 0.0008). This indicates that although the cells return eventually to the original Ca2+ level, there is an increase for the period wherein measurement occurs. By treating L6 skeletal muscle cells with ionomycin for a short period of time, Ca2+ mobility led to an extended increase in cytoplasmic Ca2+ by shifting the necessary electrical potential for gated channels [2+-activated K+ current (IKCa), as demonstrated in our previous paper [To determine the Cachannels . Based ous paper . However2+ in the L6 muscular cell line, broadband impedance spectroscopy was applied as a method for rapid cell capture and characterization of intracellular properties. The collection of spectra for each cell was examined, then compiled for statistical comparison of the mean curves and deviations in signals among treatment types, as shown in Following the establishment of the ionomycin treatment as a method of increasing the intracellular Ca2+ distribution, shown in −4 compared to the TRT population, which had a significantly higher signal of 8.1 ± 4.5 × 10−4 when measured at 190 MHz (p = 0.021). At this frequency, there is a significant shift in distribution mirroring the Ca2+ shift, which indicates a sensitivity to ionic changes (p = 0.017). In the MHz range, the electrical signal can penetrate the cell membrane for measurements of the cytoplasm, and in the lower MHz range, there is minimal measurement interference from organelle structure or large molecules within the cell, making 190 MHz an ideal frequency to look at the dielectric properties of the cytoplasm itself [The overlap due to the subtle differences between the UNT and TRT populations can also be visualized from the impedance measurements similarly to the fluorescent Cam itself ,16.c) and capacitance (Cc). The circuit model relies on the assumption that the induced increase in Ca2+ has no effect on the membrane characteristics or the membrane returns to its typical homeostasis quickly. The assumption is supported by the gradual return of Ca2+ over time to the initial concentration as the homeostasis processes regulate the individual cells. By applying the model to each curve, the way properties impact the spectra can be examined. In the spectra found in c for both treatment types and 3.89 fF and 3.76 fF for the Cc for the UNT and TRT cells, respectively. While the differences were minimal in the circuit values themselves, the dielectric parameters equate to have significantly different fitting values.To better understand the implications of the collected ΔS11 and ΔS21 spectra, a single-shell circuit model fits the electrical properties of the cytoplasm resistance (Rc) and capacitance (Cc) of the cytoplasm can also be extended to calculations of the cytoplasmic permittivity (cε) and conductivity (cσ). Based on the extracted parameters, the cσ for the UNT and TRT populations were 0.1929 ± 0.0002 S/m and 0.1933 ± 0.0008 S/m, respectively. Though small in magnitude, the TRT cells had a significantly higher conductivity (p = 8.3 × 10−5). The extracted εc of UNT cells was 11.3 ± 0.34 ε0 and had a significantly lower (p = 0.0063) average of 10.9 ± 0.13 ε0 for TRT. Looking at the distribution of extracted cσ and cε in Using the single-shell model and Equations (1) and (2), the fit resistance , which deliver a small electrical stimulant to generate pores in the cell plasma membrane, depending on the signal magnitude and polarity [2+ increase within cells has shown that the presence of sucrose molecules can delay the swelling response typically associated with an increase in intracellular Ca2+; however, this can depend on the external concentration introduced and the voltage-gated channels present on the cell type of study [2+, there are less understood effects on deeper membranes and compartments within the cell after treatment [2+ function in cells, regulation of levels in the cytosol is carefully controlled by several proteins through storage and release in the sarcoplasmic reticulum. Based on this crucial system, it is anticipated that while the overall distribution increased, some cells returned to fluorescence consistent with their initial values. The rapid peak of fluorescence and accompanying plateau is consistent with previous work observing the rapid flux of cytosolic calcium before slower recovery after treatment with ionomycin [In polarity . Recent of study ,31. As wreatment . While treatment ,34 or CAreatment ; howeveronomycin .2+ increase and the extracted values are consistent with previous measurements of similar cellular changes. The values from the UNT cells are comparable in scale to the previously published fitting value of 0.22 S/m and 9.49 ε0 [n = 51) and TRT (n = 20) cells shows that a significant increase exists for conductivity and a significant decrease exists for permittivity. The small magnitude of change is consistent with the expected pattern of recovery after the removal of ionomycin transport complexes. For the maintenance of cell viability, the change in calcium while established would be minimal in a healthy population. The observed change in conductivity and permittivity is consistent with an increase in ions, making a more even distribution of charge throughout the cell cytoplasm. It has been noted earlier that based on the initial Ca2+ imaging data, not every ionomycin-treated cell sustains the increased intracellular Ca2+ concentration, leading to the significant overlap between the UNT and TRT groups. It is also important to note that while this work focuses on the cytosolic changes, the homeostasis of calcium management also occurs in the sarcoplasmic reticulum. However, due to the broadband nature of the reported measurements, changes in other compartments are captured in a wide range of frequencies.Similarly, looking at the electrical measurements, the increase is consistent with cytosolic Ca 9.49 ε0 . The sta2+ prior to the start of apoptosis when exposed to chronic oxidative stress [2+ levels in skeletal muscle and the ability to maintain the balance of ROS and mitigate the effects of oxidative stress. Not surprisingly, the spectra alteration observed here from intracellular Ca2+ elevation, an increase in ΔS11 in the MHz range followed by a dip in the GHz range and a less negative value of ΔS21 in the kHz range, is comparable to those from L6 cells exposed to long-term oxidative stress [2+ as a key factor differentiating cells that experienced exposure to oxidative stress, while in this work, the ability to differentiate calcium through modelling shows the milder but noticeable contributions Ca2+ makes to the internal dielectric properties. Alternatively, work using the ratio of impedance at 1 MHz to 300 kHz to characterize individual cell opacity also showed the ability to measure the changes in neutrophils due to calcium ionophore exposure [2+ levels induced by ionomycin.The previous work posits oxidative damage to the mitochondria and flooding of intracellular reactive oxygen species (ROS) and Cae stress . Physiolexposure . This wo2+ changes has the potential to aid in improving our understanding of many associated skeletal muscle diseases such as DMD, cachexia, and the process of sarcopenia development [2+ concentration, the effects of other ions or molecules may also contribute to the observed changes in the electrical signal. This limitation of ion sensing selectivity has been reported in an aqueous solution at high frequencies [2+ concentration have implications for many more diseases [2+ in vivo relies on the inclusion of fluorescent agents such as the Fura-2 in this work to look at cytosolic Ca2+ or Mag-Fluo-4 to look at Ca2+ in the endoplasmic reticulum [2+ for disease diagnosis or monitoring applications, which shapes our approach in the future. Going forward, the spectral changes seen in this work will be used in a further study of ME/CFS clinical samples looking at how skeletal muscle electrical properties at a variety of frequencies can be correlated with biological changes to further our understanding of this rare disease.The ability to identify cytoplasmic Caquencies ,40. Becadiseases ,42. Typieticulum ,43. Howe2+ levels. The electrically measured differences can be further described by changes to the dielectric parameters of cytoplasmic permittivity (cε) and conductivity (cσ). In this work, it was found that increased cytoplasmic Ca2+ concentration can be associated with a significant increase in cytoplasmic conductivity and a decrease in cytoplasmic permittivity. This monitoring system improves the depth of information available about intracellular conditions and ion study in the cytoplasm. The work presented here is limited by the lack of comparison with true concentration correlation and selective sensing of the measurement system to particular ions; therefore, further exploration is necessary to develop a true system for disease monitoring. That being said, understanding these Ca2+ levels can help generate understanding and evaluate skeletal muscle disease progression and treatment effectiveness. In addition, by modeling these changes in the context of pre-evaluated oxidative changes, important deductions can be made about how different properties associated with ME/CFS contribute to an overall electrical profile to move toward a unique and rapid diagnostic tool.Based on the electrical measurements and corresponding extracted parameters, there is a subtle change in MHz and GHz spectral patterns that can be correlated with fluorescent-image-based cytoplasmic Ca


Microenvironment and Therapy-Resistance in Leukemias”.The tumor microenvironment (TME) can mediate resistance of acute leukemias to chemotherapy, however few therapies targeting this niche interaction are available. This Research Topic highlights recent findings regarding “Hellmich et al. the authors focus on the role of senescence in the aging bone marrow (BM) microenvironment in acute myeloid leukemia (AML) and multiple myeloma. Senescence is the irreversible arrest of stem cell proliferation. Through senescence, the senescence associated secretory phenotype (SASP) is activated, causing manipulation in growth and proliferation by pro-inflammatory cytokines, chemokines, proteases and growth factors. Though evolutionarily protective as an anti-cancer mechanism promoting repair, the system of senescence can become maladaptive, and instead create a pro-inflammatory, pro-tumor growth, and chemotherapy resistant environment. The prior publication by this group (Blood 2019) , deTabe et al., the authors present the role of fatty acid metabolism as a targetable mechanism of AML chemoresistance in adults whose BM content is progressively replaced by adipocytes. Oxidative phosphorylation (OXPHOS) is utilized by Leukemic stem cells (LSCs) as an energy source to promote growth and survival of the tumor cells. The authors describe the potential for combinatorial regiments utilizing fatty acid oxidation (FAO) inhibitors to target the critical energy source for the chemo-resistant leukemia cells and LSCs. FAO produces twice as much ATP per mole compared to the oxidation of glucose, making it a more effective energy source. LSCs obtained from relapsed AML patients acquire a compensatory ability to overcome the loss of amino acid metabolism (targeted by chemotherapies) by increasing FAO. While FAO inhibition sounds promising, tumor cells have several mechanisms for adapting to nutrient deprivation. Therefore, FAO alone is unlikely to be useful; rather combination with chemotherapy or targeted therapies have potential for synergistic effect.In the review by Nehrbas et al. focuses on the role of Extracellular Vesicles (EVs) used to mediate crosstalk between AML cells and the BM microenvironment, which facilitates AML chemoresistance through the transport of protein, RNA, and DNA. EVs are membrane-bound secreted particles containing a variety of nucleic acid/protein material. Exosomes, an EV subtype, can traffic proteins, lipids, mRNAs, multiple types of non-coding RNAs, DNAs and chemokines/cytokines. Co-culture experiments showed that chemo-resistant AML cells could induce chemoresistance in chemo-sensitive AML cells by upregulating the anti-apoptotic protein BCL-2. EV-mediated transfer of miRNAs is known to increase chemoresistance in several cancer types. Specific microRNAs are described as inducing resistance through tyrosine kinase inhibitor resistance and overexpression of drug efflux pump multidrug resistance protein 1(MRP-1). EVs also mediate resistance to immunotherapy reducing the efficacy of adoptive Natural Killer (NK) cell therapy cells through transport of inhibitory ligands. AML cells release exosomes containing programmed death-receptor ligand 1 (PD-L1) causing T-cell suppression. It also sequesters the anti-PDL-1 antibodies leading to checkpoint inhibitor resistance. Further characterization of EV cargo, mechanisms of downstream chemoresistance and ultimately development of novel approaches to control EV biogenesis or uptake will be a potential target to improve treatment outcomes in leukemia.The review by Bhatnagar and Garzon review the role of MicroRNAs (miRs), short non-coding RNAs, in AML as a prognostic factor as well as therapeutic target. Dysregulated miRs can function as oncogenes or tumor suppressors, and the same miR can function as a tumor suppressor or oncogene depending on its context. The review focuses on potential clinical applications of miRs in adult AML and discusses unique miR signatures in specific AML subtypes. Though several miRs have been identified as promising targets in AML, clinical trials have not been conducted. The limiting factors include off-target effects, toxicity, and specific delivery to blasts.Cancilla et al. review the chemokine receptor CXCR4 and its interaction with CXCL12 as a key player in the survival and migration of malignant stem cells to the BM. High expression of CXCR4 on AML and ALL blasts has been identified as a predictor of poor prognosis. Given its role in retaining AML blasts in the BM, inhibiting CXCR4 can disrupt its pro-survival signaling, induce cytotoxicity and mobilize the leukemia cells from the marrow into the vasculature where they would be more susceptible to chemotherapy. The data review the four categories of CXCR4 targeting drugs, including small molecule CXCR4 agonists, peptide-like CXCR4 antagonists, antibodies to CXCR4, and CXCL12 antagonists. The use of CXCR4 inhibitors in hematopoietic transplant myeloablation, enhancement of donor engraftment post-transplant, and the combination with checkpoint inhibitors is described.Kim et al. reviews the role of cadherins, selectins, and integrins in cell adhesion mediated drug resistance (CAM-DR), and the results of clinical trials targeting these molecules. CAM-DR has been demonstrated in many subsets of leukemia including B- and T- acute lymphoblastic leukemia. Cell adhesion molecules (CAMs) not only recognize ligands for binding but also initiate the intracellular signaling pathways that are associated with cell proliferation, survival, and drug resistance upon binding to their ligands.Epperly et al. review the immunosuppressive AML microenvironment as an underlying mechanism for the failure of chimeric antigen receptor T (CAR-T) cell therapy in AML. The review addresses the various cell types in the AML microenvironment which can lead to T-cell exhaustion, including AML blasts, myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), macrophages, and dendritic cells. Other mechanisms for immune evasion are explored such as the downregulated expression of major histocompatibility complex class I and II, the increased expression of inhibitory ligands as well as structural components of the BM and metabolic alterations including amino acid concentrations. This review sheds light on ways to enhance the AML response to CAR-T cells through targeting the immunosuppressive and anti-inflammatory aspects of the microenvironment.In summary, this Research Topic of outstanding articles illustrates the biological and therapeutic significance of the microenvironment of therapy-resistant leukemias.MK and RF wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


Despite the central importance of MAM Ca2+ dynamics in diverse biological processes, directly and specifically measuring Ca2+ concentrations inside MAM is technically challenging. Here, we develop MAM-Calflux, a MAM-specific BRET-based Ca2+ indicator. The successful application of the bimolecular fluorescence complementation (BiFC) concept highlights Ca2+-responsive BRET signals in MAM. The BiFC strategy imparts dual functionality as a Ca2+ indicator and quantitative structural marker specific for MAM. As a ratiometric Ca2+ indicator, MAM-Calflux estimates steady-state MAM Ca2+ levels. Finally, it enables the visualization of uneven intracellular distribution of MAM Ca2+ and the elucidation of abnormally accumulated MAM Ca2+ from the neurons of Parkinson’s disease mouse model in both steady-state and stimulated conditions. Therefore, we propose that MAM-Calflux can be a versatile tool for ratiometrically measuring dynamic inter-organellar Ca2+ communication.Mitochondria-associated ER membrane (MAM) is a structure where these calcium-regulating organelles form close physical contact sites for efficient Ca 2+ dynamics play an important role in diverse biological processes, but directly and specifically measuring Ca2+ concentrations in this region is technically challenging. Here the authors report a MAM-specific BRET-based Ca2+ indicator called MAM-Calflux, which works as both a Ca2+ indicator and a structural marker due to its ratiometric nature.MAM Ca MAM is a signaling hub that governs ER-mitochondria Ca2+ transfer6, lipid synthesis and exchange7, and mitochondrial structure dynamics2 to regulate autophagy8, ER stress response9, inflammasome activation10, and apoptosis11. Accumulating evidence implies that MAM is rearranged in response to diseases such as Parkinson’s disease (PD)13, Alzheimer’s disease (AD)15, amyotrophic lateral sclerosis (ALS)17, and type 2 diabetes mellitus (T2DM)18; yet the underlying mechanisms are largely unexplored.Mitochondria and endoplasmic reticulum (ER) form close contact sites spanning ~10–30 nm and are called mitochondria-associated ER membranes (MAM) or mitochondria-ER contacts (MERC)2+ transfer from the ER to mitochondria, thus playing an important role in diverse biological processes. Ca2+ indicators, which are located at the mitochondrial matrix or cytosolic part of the outer mitochondrial membrane (OMM), determined that, upon Ca2+ release from the ER, the mitochondria are then exposed to a higher concentration of Ca2+ compared with that in the cytosol; this microdomain with high-Ca2+ concentration is the MAM14. The MAM is concentrated with several proteins responsible for MAM Ca2+ transfer, including inositol triphosphate receptors (IP3Rs) and polycystin-2 (PKD2), which are involved in Ca2+ release from the ER, and voltage-dependent anion channels (VDACs), which are involved in Ca2+ influx into the mitochondria. In addition, the MAM structure is maintained by tethering proteins such as glucose-regulated protein 75 (GRP75) and DJ-113, which also participate in regulating MAM Ca2+ transfer. Accumulated Ca2+ on the mitochondrial surface activates enzymes responsible for cellular bioenergetics and mitochondrial transport20. MAM Ca2+ transfer is also important for inhibiting autophagy8, stimulating Drp1-dependent mitochondrial division and pro-apoptotic signaling21, and buffering local cytosolic Ca2+ concentration5. Despite its importance, there is a limitation in the methodology used to directly and specifically measure MAM Ca2+, resulting in a poor understanding of MAM Ca2+ physiology.MAM serves as a ground for dynamic Ca2+-mediated cellular functions, particularly in cell biology and neuroscience. GCaMP, one of the most abundantly utilized GECI, is composed of a calmodulin-binding sequence of myosin light chain kinase (M13), circularly permutated green fluorescent protein (GFP), and calmodulin. The binding of Ca2+ to calmodulin and M13 induces conformational changes, which increase the GFP fluorescence intensity22. Over the past decades, significant progress has been made in the development of GECIs with longer wavelengths, higher signal-to-noise ratios, and faster kinetics24. In addition, ratiometric versions of GECIs were invented to overcome the limitations of GCaMPs whose basal signal intensities are multiplied in an expression-level-dependent manner. Ratiometric GECIs enable the quantitation of steady-state Ca2+ concentrations, such as in GCaMP-R based on the GCaMP/RFP ratio25, Twitch based on fluorescence resonance energy transfer (FRET)26, and Nanolantern or CalfluxVTN based on bioluminescence resonance energy transfer (BRET)27. Efforts have been made to understand intracellular Ca2+ distribution by developing organelle- or compartment-specific GECIs, such as for mitochondria28, ER29, and synapses30. However, limitations exist in directly and specifically investigating MAM Ca2+, and hence, indirect estimations are made by comparing Ca2+ dynamics separately measured from the ER and mitochondria31. Thus, the development of a MAM-specific Ca2+ sensor represents the effort to better understand the physiology of MAM Ca2+ dynamics.Genetically encoded calcium indicators (GECIs) are powerful tools for understanding Ca2+ concentration by applying bimolecular fluorescence complementation (BiFC). Venus fluorescence is only operative where the ER and mitochondria are closely tethered, allowing the MAM-specific observation of Ca2+-responsive BRET signals. The specificity of the Venus to the MAM also enables the use of this sensor as a quantitative structural marker, facilitating the dual functionality. MAM-Calflux demonstrates an uneven distribution of MAM Ca2+ dynamics. Furthermore, we apply this sensor to estimate MAM Ca2+ dynamics in the neurons of multiple neurodegenerative disease mouse models.In this study, we devise a modified version of CalfluxVTN which is specific for MAM, named MAM-Calflux. MAM-Calflux can directly probe MAM-specific Ca2+ indicator, we applied the concept of bimolecular fluorescence complementation (BiFC)32 to a BRET-based Ca2+ indicator, CalfluxVTN27, with organelle targeting sequences. CalfluxVTN was separated into two fragments: 1–173 aa Venus fluorescence domain (VN173) and 156–228 aa Venus domain (VC155) conjugated with troponin C and Nanoluciferase (NanoLuc) domains. Linker sequences of optimized lengths were used to insert the ER-targeting sequence (Sac1 521–587 aa) and mitochondria-targeting sequence (AKAP 1–30 aa) into VN173 and VC155 fragments, respectively  with the soluble form of VC155 resulted in a Venus fluorescent pattern that significantly overlapped with that of Sec61β-mCherry, an ER marker  to acquire images of the bioluminescence Em460) and BRET-based Venus (Em525) signals simultaneously in a single frame . This indicates that the restored BRET ratio after histamine treatment presumably demonstrated active Ca2+ restoration in MAM.Next, we attempted to validate the MAM-Calflux response to changes in MAM Catio Fig. . Meanwhilux Fig. . The BRE2+ concentration is calculated as ratiometric signals26. This advantage allowed us to estimate steady-state MAM Ca2+ levels via MAM-Calflux imaging without any external Ca2+ stimulation. Thus, we introduced the conditions that were reported to regulate MAM contact size and examined the basal levels of the BRET ratio and Venus excitation of the MAM Ca2+ sensor and MAM marker, respectively. VAPB and PTPIP51 form a tethering complex that mediates the formation of the MAM structures16. The over-expression of VAPB and PTPIP51 increased both Venus excitation and BRET ratio in HeLa cells and primary cultured mouse neurons  2 h treatment , the primary cultured 5xFAD hippocampal neurons had a decreased BRET ratio and Venus excitation intensities, indicating that both MAM Ca2+ levels and MAM extent were affected  is the second most frequent gene associated with familial ALS, and the accumulation of mutant SOD1 proteins in the MAM has been reported17. SOD1*G93A transgenic mouse neurons, which ectopically express human SOD1 with ALS-linked mutation, had a minimal effect on the Ca2+ dynamics and MAM structure  protein, which is located at the presynapse and regulates synaptic vesicles, is one of the most prominent causes of PD, as evidenced by its presence in Lewy bodies and missense mutations discovered in familial PDced Fig. . In contced Fig. , which iure Fig. . These r2+ transfer was assumed to be one of the major functions of MAM, specific and direct measurement of Ca2+ concentration in the MAM was not possible due to technical limitations. Instead, MAM Ca2+ has been evaluated by measuring cytosolic Ca2+ levels near the OMM36 or by comparing Ca2+ dynamics in the ER and mitochondria after stimulation of Ca2+ crosstalk31. Csordas et al. presented an advanced methodology utilizing rapamycin-inducible bridge-forming modules (RiBFM) to guide OMM-targeted Pericam toward the ER; thus, the signal was more specific to MAM, although this still could not exclude non-MAM Ca2+ signals and had a chance of artificial increase of MAM structures53. In this study, we modified the fluorescent domain of the ratiometric calcium indicator to be specifically located inside the MAM, which could directly and specifically measure steady-state MAM Ca2+ levels without external stimuli. Its ratiometric character enables the estimation of MAM Ca2+ physiology with cell-to-cell or region-to-region comparisons. For instance, when we compared steady-state BRET ratios of different cell types which were almost simultaneously evaluated, such as HeLa control groups and primary cultured hippocampal neuron control groups in Fig. 2+ signals than that of HeLa cells, although the MAM structure intensities (Venus excitation) were similar. This implies that MAM in neuronal cells contains more activated Ca2+ transfer machinery or a higher frequency of MAM Ca2+ crosstalk. Therefore, the availability of data on the MAM-Calflux BRET ratio values corresponding to Ca2+ concentrations is critical for the precise measurement of MAM Ca2+ concentrations. Further applications of MAM-Calflux, such as comparisons of MAM Ca2+ concentrations in various cellular compartments and in response to various extracellular conditions, would help comprehend MAM Ca2+ physiology.Although Ca2+ indicator and MAM structural marker , an activator of voltage-dependent anion-selective channel protein 1 (VDAC1), decreased IP3R-mediated ER-mitochondria Ca2+ transfer. In contrast, the MAM structure was not affected57. We introduced MAM-Calflux into SNCA*A53T mouse neurons, indicating that the steady-state MAM Ca2+ level was significantly higher than that of the wild-type, whereas the MAM structural intensity decreased  and increases cytosolic Ca2+ level at a later stage59. Therefore, based on results from MAM-Calflux, we hypothesize that the pathogenic α-syn decreases Ca2+ uptake by mitochondria, resulting in the abnormal accumulation of Ca2+ in the MAM regardless of its loosened structure. Likewise, defining both MAM structure and MAM Ca2+ by the dual functionality of MAM-Calflux can provide more insights into understanding MAM Ca2+ physiology in various cellular or disease-relevant conditions.In addition, the dual functionality of MAM-Calflux could help distinguish whether the change in MAM Cased Fig. . These u2+ response of MAM-Calflux proves that this strategy can be employed in developing calcium indicators specific for other inter-organelle contacts by making appropriate substitutions for organelle-specific localization signals of MAM-Calflux and optimizing the linker lengths60. For instance, the plasma membrane (PM)-ER contact serves for the efficient transfer of extracellular Ca2+ to the ER lumen and activates ER Ca2+ release under specific conditions such as muscular contraction61. Thus, interrogating Ca2+ crosstalk between the PM and ER would be informative. Dolman et al. determined that the mitochondria may be responsible for the Ca2+ gradient in trans- and cis-Golgi by close contact with Golgi apparatus62. Since Ca2+ dynamics in the Golgi affect its structures and functions, especially secretory pathways and local Ca2+ buffering63, the interaction between mitochondria and the Golgi apparatus is another possible candidate to examine Ca2+ crosstalk. Calcium indicators specific to these inter-organelle structures may advance our understanding of organellar Ca2+ dynamics and organelle-to-organelle Ca2+ crosstalk within the cell.The MAM-specific localization and sustained Ca2+ physiology, a number of further improvements can be made. First, the split fragments of Venus by BiFC have been reported to undergo spontaneous self-assembly, although this was reduced when each fragment was fused to other proteins65. Self-assembly of Venus in MAM-Calflux would ectopically enhance MAM structure formation, resulting in the over-estimation of MAM Ca2+ basal levels. Indeed, we observed that expression of MAM-Calflux for long periods can affect MAM structure formation, requiring careful design of experimental conditions, such as expression period and levels. This may be partially overcome by modifying the strategy for Venus fragmentation in MAM-Calflux, for instance, introducing mutations in Venus67, changing Venus split residues68, and utilizing dimerization-dependent fluorescent protein69. Employing low-expression promoters or inducible expression systems will also be beneficial. Also, a relatively low bioluminescence signal and BRET-based Venus signal can restrict the frame-to-frame interval of time-lapse imaging. In our imaging condition, we achieved the shortest interval of 5 sec to observe MAM Ca2+ dynamics against external stimuli. Still, this interval may be insufficient to precisely analyze Ca2+ dynamics in some conditions, indicating room for further improvement in conjunction with the conventional BRET system27.Although the MAM-Calflux is a versatile tool for studying MAM-specific CaAll animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Pohang University of Science and Technology (POSTECH-2022-0085). All experiments were performed in accordance with the approved guidelines.52. PINK1-deficient mice were a generous gift from Dr. Xiaoxi Zhuang (Chicago University) and Dr. Sang Myun Park (Ajou University School of Medicine)70. 5xFAD mice were a generous gift from Dr. Kyong-Tai Kim (Pohang University of Science and Technology)45. SOD1*G93A mice71 were purchased from The Jackson Laboratory (Stock No. 002726) and maintained on a C57BL/6 background. The animals were group-housed under diurnal light conditions  and had free access to food and water. .Pregnant B6 mice used for primary hippocampal neuron culture were purchased from Hyochang Science . SNCA*A53T mice were a generous gift from Dr. Won-Jong Oh (Korea Brain Research Institute)Anti-TOM20 mouse monoclonal antibodies , anti-Calnexin , and anti-Calreticulin  were used for immunocytochemistry experiments for 1:50–1:200. For immunostaining, Alexa Fluor 568 or 647 conjugated goat anti-mouse IgG  and Alexa Fluor 647 conjugated goat anti-rabbit IgG  were used as secondary antibodies for 1:200.27, was served as the template to design MAM-Calflux. A 173 aa N-terminal fragment of Venus domain of CalfluxVTN, VN173, was conjugated with a 103 aa linker and the ER-targeting sequence (mSac1 521–587 aa) at its C-terminus. A fragment containing the C-terminal part of Venus, VC155, and following Troponin C and NanoLuc domains was conjugated with the mitochondria-targeting sequence (mAkap1 1–30 aa) and a 57 aa linker at its N-terminus. Sec61b-mCherry was a gift from Gia Voeltz (Addgene plasmid # 49155)72. pCMV R-CEPIA1er was a gift from Masamitsu Iino (Addgene plasmid # 58216)29. The core sequence of human MFN2 shRNA was GGAAGAGCACCGTGATCAATG73.pT7-CalfluxVTN, which was a gift from Carl Johnson (Addgene plasmid # 83926)5 cells/mL of cells were seeded on the 18-mm or 25-mm cover glass in 12-well or six-well plates, respectively. The cells were transfected using transfection reagents, either polyethylenimine (PEI) or Lipofectamine 2000 (Thermo Fisher Scientific). For transfection in a 12-well plate, 300–400 ng of MAM-Calflux-P2A construct and each 400-600 ng of other plasmids, such as EBFP2-N1, EBFP2-hVAPB, FLAG-hPTPIP51, MYC-hGRP75, and pLL3.7-mRFP-MFN2 shRNA, were mixed in 100 μL Opti-MEM with 4 μL PEI or 1 μL Lipofectamine 2000.HeLa (ATCC # CCL-2) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco). The cell-line was authenticated using STR profiling method and tested negative for mycoplasma contamination. For imaging samples, ~1.5 × 105 cells/mL. The cells were plated on glass coverslips pre-coated with poly-D-lysine and laminin. After 2 h of plating, the cell medium was replaced with Neurobasal medium containing 2 mM glutamine, 2% (v/v) B27 supplement (Gibco), and 1% (v/v) penicillin/streptomycin. The neurons were transfected with Lipofectamine 2000, and the medium was replaced with the culture medium 2 h after transfection. For transfection in a 12-well plate, 500–600 ng of MAM-Calflux-P2A construct and each 800–1000 ng of other plasmids were mixed in 100 μL Opti-MEM with 2 μL Lipofectamine 2000.Primary cultures of hippocampal neurons were established by isolating E16 B6 mouse embryo hippocampal tissues in Hanks’ balanced salt solution (HBSS) (Gibco) and dissociating the tissues in 0.25% trypsin (Sigma-Aldrich) and 0.1% DNase I (Sigma-Aldrich) for 10 min at 37°C. Cells were resuspended in Neurobasal medium (Gibco) supplemented with 10 mM HEPES (pH 7.4) and 10% (v/v) horse serum to a final cell concentration of 4.0 × 1074. Images were deconvolved using the advanced constrained iterative (CI) algorithm-based deconvolution program of cellSens software. Colocalization between ER and mitochondrial markers was quantitated using cellSens software, Imaris 9.2 software (Bitplane), or Fiji software.For immunocytochemistry, the cells were fixed with 4% paraformaldehyde in PBS or 4% paraformaldehyde and 4% sucrose in PBS for 20 min and washed with PBS for three times. Cells were permeabilized with 0.2% Triton-X-100 in PBS for 5 min and blocked with 5% goat serum or 5% BSA in PBS for >30 min. For protein staining, the cells were incubated with primary antibodies diluted in the blocking solution for 1 h at room temperature or overnight at 4°C, washed with PBS for three times, and treated with secondary antibodies diluted in the blocking solution for 1 h at room temperature. Cell images were acquired using FV3000 confocal laser scanning microscope (Olympus) and processed using cellSens 2.3 software (Olympus) and ImageJ 1.52p (Fiji) software 600nm of 0.6 was achieved. Subsequently, a 16-hour induction was performed using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C, followed by cell harvesting via centrifugation and snap-freezing in liquid nitrogen. The resulting cell pellets were resuspended in lysis buffer , incubated for 30 min at 37 °C, and sonicated . Cellular debris was removed through centrifugation at 7000 × g for 15 min at 4 °C, and the resulting clear supernatant was combined with Ni-NTA agarose beads in phosphate-buffered saline (PBS), followed by a 4-hour incubation at 4 °C. The beads were subsequently washed with a washing buffer , and the His-tagged proteins were eluted using an elution buffer . Finally, the proteins were dialyzed in a dialysis buffer . In vitro purified Calflux-N term and Calflux-C term were mixed to calcium calibration buffer being 1 μM and 0.05 μM for final concentrations, respectively. Calcium calibration buffer was made by mix of Ca-EGTA  and zero calcium buffer .To obtain in vitro purified Calflux proteins, the His6-tagged split-version of CalfluxVTN  was introduced to the BL21 strain and cultured at 37 °C in LB medium containing 100 mg/mL ampicillin, until an OD2+-free HBSS  with 2 μM ionomycin and either 5 mM EGTA or variable concentrations of CaCl2 from 0.001 to 100 mM for 10–15 min.For live cell samples, HeLa cells in six-well plate were transfected with MAM-Calflux-C or MAM-Calflux. After 6 h, the cells were detached using trypsin-EDTA, resuspended in complete DMEM, and transferred to a white-bottom 96-well plate. For emission wavelength scan, after 12-16 h, the medium was replaced with CaLuciferase was activated by treating the cells with 50 μM of furimazine  substrate. After stabilization for 2 minutes, the emission intensities of wavelengths from 400 to 600 nm with 2 nm intervals were measured at 37 °C using a multi-functional microplate reader (Tecan Infinite M200 pro).2+-free HBSS with 10 mM HEPES and 50 μM furimazine. To maintain a sufficient concentration of furimazine, at least 250 μL of buffer was used for the 18-mm coverslip. After 5 min of stabilization with furimazine, BRET imaging was performed with an inverted fluorescence microscope  using ×20 (UApoN340 ×20/0.70 W infinite/0.17/FN22), ×40 (UPlanSApo ×40/0.95 infinite/0.11-0.23/FN26.5), and ×60 (UPlanSApo ×60/1.35 Oil infinite/0.17/FN26.5) objective lenses at 37 °C. To get a 525/460 nm ratio image, a dual-view beam splitter (Photometrics) was installed in front of the EMCCD camera . To obtain the BFP/RFP and Venus images separately, a triple dichroic mirror  with dual-view emission filters for 525 nm  and for 460 nm and 600 nm  were used. Images were acquired using MetaMorph 7.7 software (Molecular Devices) with multi-dimensional acquisition for the Em525 and Em460 dual image, Venus excitation image, and BFP excitation image, sequentially. Each image was obtained at 690 kHz, 11×, gain 100, binning 1, 10 s for Em460/Em525 imaging and 690 kHz, 1×, 500 ms for Venus excitation imaging. These conditions prevented the crosstalk of signals from Em525 into Venus excitation images. The BFP excitation imaging was obtained at 2.75 MHz, 1×, 300 ms. The images were analyzed using Fiji with the use of “Template Matching” plug-in to align Em460 and Em525 images, “Subtract Background” function or “BRET Analyzer 1.0.7” plug-in to subtract background signals, and “BRET Analyzer 1.0.7” plug-in to produce BRET ratio images. To calculate mean BRET ratio after masking MAM regions, “Threshold” and “Analyze Particles” functions were used over Venus excitation images to determine MAM-specific region-of-interests (ROIs).HeLa or primary cultured neurons on the cover glass were transfected with MAM-Calflux and EBFP2-N1, EBFP2-hVAPB and FLAG-hPTPIP51, and Myc-hGRP75. For MFN2 knockdown, HeLa cells were transfected with pLL3.7-mRFP-MFN2 shRNA, moved to a cover glass after 24–30 h of transfection, and again transfected with MAM-Calflux after 24–30 h of seeding. After 18–24 h, the cover glass was transferred to a live imaging chamber, and the cell medium was replaced with Ca75. To measure the Ca2+ responses of subcellular organelles, HeLa cells or primary cultured hippocampal neurons were transfected with MAM-Calflux and EBFP2-N1, cyto-GCaMP6s and R-CEPIA1er, or mito-GCaMP6s and R-CEPIA1er constructs on DIV6. The cells were loaded with Ca2+-free HBSS and sequentially exposed to 10 μM histamine and 10 μM Ca2+-free ionomycin  with 1 mM CaCl2. For a single treatment of histamine, cells were loaded with HBSS containing 1 mM MgCl2 and 2 mM CaCl2. To maintain a sufficient concentration of furimazine during time-lapse imaging, at least 250 μL of buffer was used for the 18-mm coverslip, and images were acquired within 20–25 min.Live calcium imaging was performed as previously described, with some modificationsB/B0) of each neuron was calculated as (B−B0)/B0, where B0 is the baseline BRET ratio signal averaged over five frames before stimulation and B is the peak intensity of the BRET ratio in the response.BRET images were recorded in MetaMorph software with multi-dimensional acquisition and time-lapse functions for the Em525 and Em460 dual image, the Venus excitation image, and the BFP excitation image, sequentially. In total 25–35 frames were acquired, and imaging conditions and interval times were adjusted to finally obtain ~15 seconds between frames. After image processing, the amplitude  with UPLSAPO ×20/0.75 NA objective. Background fluorescence was subtracted and the amplitude (ΔF/F0) of each neuron was calculated as (F−F0)/F0, where F0 is the baseline mito-GCaMP6s, cyto-GCaMP6s, or R-CEPIA1er fluorescence signal averaged over 20 seconds before the stimulation and F is the peak intensity of fluorescence in the response.The fluorescence intensities were recorded at 37 °C with supplying 5% COt test for comparisons between two groups and one-way ANOVA followed by Bonferroni’s post-hoc test for comparisons among multiple groups. All statistical analysis, correlation analysis, and fitting with a sigmoidal curve model or a simple linear regression model were calculated using Prism 9.4 (GraphPad).All graphs are presented as the mean ± SEM or box plots representing the median and interquartile range with whiskers min/max value and the cross representing the mean value. The statistical significance of the data was analyzed using two-tailed Student’s 60. No data were excluded from the analyses. All sample images were acquired in random order within the sets and all cells were chosen at the random site. Experimenters were blinded to each group allocation during the image analysis by removing any sample information in file names.Sample sizes for all statistical evaluations are indicated in Figure legends. No statistical method was used to predetermine sample size. Number of cells per group was chosen based on sample size in the calcium measurement experiment with microscopic images which meets or exceed previous studiesFurther information on research design is available in the Supplementary InformationPeer Review FileReporting summary


One of the distinguishing properties of hematopoietic stem cells is their ability to self-renew. Since self-renewal is important for the continuous replenishment of the hematopoietic stem cell pool, this property is often hijacked in blood cancers. Acute myeloid leukemia (AML) is believed to be arranged in a hierarchy, with self-renewing leukemia stem cells (LSCs) giving rise to the bulk tumor. Some of the earliest characterizations of LSCs were made in seminal studies that assessed the ability of prospectively isolated candidate AML stem cells to repopulate the entire heterogeneity of the tumor in mice. Further studies indicated that LSCs may be responsible for chemotherapy resistance and therefore act as a reservoir for secondary disease and leukemia relapse. In recent years, a number of studies have helped illuminate the complexity of clonality in bone marrow pathologies, including leukemias. Many features distinguishing LSCs from normal hematopoietic stem cells have been identified, and these studies have opened up diverse avenues for targeting LSCs, with an impact on the clinical management of AML patients. This review will discuss the role of self-renewal in AML and its implications, distinguishing characteristics between normal and leukemia stem cells, and opportunities for therapeutic targeting of AML LSCs. Hematopoiesis is organized as a hierarchy, with hematopoietic stem cells (HSCs) at the apex. Blood homeostasis relies on the regulated differentiation of HSCs into the diverse cell types that constitute blood. In normal hematopoiesis, blood production is sustained by HSCs, which create committed progenitor cells that multiply and differentiate into functional blood cells of distinct lineages . Centralin vivo xenotransplantation assays alternative techniques to study human AML-LSCs were also developed. For example, an ex-vivo bone marrow stromal co-culture system using CD34+ cells from peripheral blood samples from AML patients allowed long-term cultures to be maintained over 20 weeks , hematopoietic stem or progenitor cells acquire genetic alterations that confer pre-leukemic features of heightened competitive fitness, including proliferative expansion and enhanced survival . The cel20 weeks . This asherapies . HoweverSeveral groups have sought to investigate the overlapping and distinct properties of normal HSCs and AML-LSCs in order to understand the means by which normal stemness attributes are co-opted in AML. In human AML, several studies showed that LSCs can share certain cell-surface markers that are used to define normal HSCs, including non-expression of lineage (Lin) markers, expression of the CD34 surface marker, and a CD38 negative (CD38-) immunophenotype. Later studies using immunodeficient mice – which are more permissive for engraftment of human cells – showed that human AML-LSCs from most patients reside in the CD34+/CD38- or CD34+/CD38+ population, and in a few AMLs such as NPM1 mutant AML, may also be present in the CD34- population , 8. TherSeveral different surface markers have been shown to be enriched in the LSC compartment including CD96 , CD123 , CD44 22, CD47  and GPR5Some of the most common surface-associated antigens that have been pursued in clinical trials are CD33, FLT3 and CD123 (IL3-Ra). In each of these cases, trials have been discontinued due to unforeseen toxicities, raising questions about the selectivity of these surface markers on AML cells and particularly on AML-LSCs. Despite substantial toxicities, the ADC gentuzumab ozogamicin (GO) - a CD33 antibody conjugated to the potent enediyne DNA-binding cytotoxic antibiotic calicheamicin - is approved by the Food and Drug Administration for treating certain subtypes of AML. In core-binding factor AML, GO treatment in induction therapy increased long-term survival rates . AnotherIn addition to the use of antibody-based targeting approaches, there are also several ongoing efforts to engage T-cells using bispecific T-cell engaging antibodies (BiTEs), which recruit CD3 effector T cells to target tumor cells. Some of the early BiTEs tested in AML included AMG-330 AMG673, and AMV564, all of which are dual-targeting agents for CD33 and CD3. Each of these BiTEs showed early evidence of anti-leukemia activity in patients with relapsed/refractory AML , 36. Simde novo AML patients showed durable efficacy in AML, including in p53-mutant AML, which is one of the most treatment-resistant subtypes of AML  genes, the three amino acid loop extension (TALE) domain proteins MEIS1 and PBX1 , 55, theHOX genes . Recent studies have demonstrated that blocking DHODH activity, resulting in pyrimidine deprivation, induces the differentiation or death of AML blasts , a flavoprotein located in the inner mitochondrial membrane. DHODH is interesting because it connects two important nodes recognized to be important in AML. It plays a crucial role in L blasts –106. It  or TET2 . IDH1/2 HSCs reside in the bone marrow niche, a specialized environment that protects their quiescent state. Cell cycle quiescence allows for the limitation of oxidative stress from mitochondrial respiration and avoiding exhaustion from excessive cell cycling and proliferation –117. TheThe hypoxic state of the bone marrow niche also contributes to the therapy resistance features of LSCs , 121. A As mentioned previously, early studies on normal and malignant hematopoiesis relied on the characterization of cell-surface markers by flow cytometry and limiting dilution experiments to determine long-term reconstitution of the entire heterogeneity of normal or leukemic hematopoiesis in mouse models. More recently, emerging techniques including next-generation sequencing (NGS) and the computational deconvolution of complex clonal hierarchies have allowed new insights into the hematopoietic system. Among these is the understanding of age-related clonal hematopoiesis (CH), a phenomenon in which the progeny of certain HSC clones expands progressively, due to selective advantage conferred by accumulated mutations in the HSC , includemission , 159. MRemission . The tecemission ). The moemission  and highSince relapse may arise from genetically diverse dormant populations that can take on LSC properties, therapeutic approaches that target only the features of the dominant clone would prove ineffective. Therefore, any anti-LSC therapies would have to comprehensively address these recently appreciated features of LSC biology in order to achieve long-lasting cures in AML.HOX/MEIS activation in AML was found to have modest efficacy in a clinical trial for patients with MLL-rearranged AML. The efficacy of other epigenetic regulators that can control the transcription of self-renewal associated genes in AML, such as inhibitors of the MLL-Menin interaction is promising (Research over the last two decades has enabled a better understanding of the concept of LSCs, their importance on AML progression, pathogenesis, and disease refractoriness. The main challenge in the near future is to determine how this rapidly growing information can be harnessed to develop effective LSC-targeting therapies. To this end, it is critical to expand on the search for clinically actionable LSC-specific targets. Rapid strides have been made in this direction as mentioned previously in this review. Immunophenotypic markers, including CD96 , 163, CDromising , 167–172romising –175. TheKB and AD conceived, wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.


In particular, the original articles harboured by this Topic describe how mechanisms regulating Ca2+ responses underlie the remarkable capacity of neurons and astrocytes to undergo changes in response to physiological activation and plasticity, promote learning and memory, or mediate memory loss, cell death, or oxidation-associated neurodegeneration.This Research Topic presents cellular and molecular mechanisms that shape cytoplasmic Ca2+ concentration is a powerful signal for the initiation of a variety of cellular processes in neurons and glial cells. As an example, astrocytes can sense their environment and respond quickly to the changes caused from nearby dying cells, as described in this Topic by Gomez-Godinez et al.. These authors used laser nano-surgery to trigger cell photolysis while evaluating the response of neighboring astrocytes to this treatment. Interestingly, responsive astrocytes display cytoplasmic Ca2+ transients largely dependent on the endoplasmic reticulum and mediated by inositol 1,4,5-trisphosphate (IP3) and Ryanodine receptors (RyR) Ca2+ channels, which are distinguishable from the spontaneous Ca2+ oscillations occurring in resting neuronal cells.The increment in cytoplasmic Ca2+-dependent signaling pathways can be switched on by Ca2+ influx triggered by depolarizing stimuli or neurotransmitter release. The resulting cytoplasmic Ca2+ signal is rapidly coordinated with and amplified by Ca2+ release from intracellular stores through Ca2+-induced Ca2+ release (CICR) by type-2 Ryanodine Receptor (RyR2) Ca2+ release channels. In this Topic, Valdés-Undurraga et al. report that LTP induction promotes RyR2 Ca2+ release channel expression and that suppression of RyR Ca2+ release channel activity abolishes LTP induction and the enhanced expression of these Ca2+ release channels in CA3-CA1 synapses in hippocampal slices. Notably, training in the Morris water maze induces the expression of RyR2 Ca2+ release channels in rats. Based on these findings, the authors propose that the increments in the protein content of RyR2 Ca2+ release channels play a significant role in hippocampal synaptic plasticity and spatial memory consolidation.In neurons, Ca2+-induced enzyme activities, Vergara et al. report that Ca2+-dependent Calmodulin Kinase II (CaMKII) plays a key role in homeostatic synaptic plasticity processes, which are triggered by prolonged changes in neural activity and allow neuronal networks to operate within functional ranges. These authors report that transient preincubation with a specific CaMKII inhibitor either blocks or occludes amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) in synapses of CA1 pyramidal neurons in hippocampal slices, an indication of the involvement of CaMKII in homeostatic synaptic plasticity processes. Vergara et al. propose that their results may contribute to understand fast-developing homeostatic or pathological functional changes after brain injury.Regarding CaZhuang et al. report that inhibition of CaMKII and N-methyl-D-aspartate (NMDA) receptors in the lateral habenula, impact allodynia and anxiety-like behavior in mice. Moreover, activation of NMDA receptors in the lateral habenula increases the expression of the phosphorylated forms of the NR2B subtype of NMDA receptors and CaMKII and induces orofacial allodynia and anxiety-like behaviors in naive mice. Based on these findings, Zhuang et al. suggest that suppressing CaMKII activity in bilateral habenula neurons represent a novel strategy for pain treatment and anxiety associated with trigeminal neuralgia.Along the same lines, 2+ pumps located at the plasma membrane act rapidly and efficiently to remove Ca2+ from the cytoplasm and to switch off Ca2+ signals. The plasma membrane Ca2+-ATPases (PMCAs) are P-type integral membrane pumps with ATPase-consuming activity that expulses cytoplasmic Ca2+ toward the extracellular space, in isoelectric exchange for extracellular H+  , an incurable neurodegenerative disease characterized by loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. These authors argue that while oxidative stress signs in postmortem neuronal tissue, cerebrospinal fluid, plasma, and urine of ALS patients are evident, information is lacking on specific processes in motor neurons. Hence, they investigated the relevance of reactive oxygen species (ROS) detoxification mechanisms in the Wobbler mouse ALS model. They report elevated ROS and DNA damage response proteins (p53bp1 and gH2ax) levels in cultured Wobbler spinal cord motor neurons. They also report altered expression of antioxidant molecules in the spinal cord of Wobbler mice and propose that maintenance of redox homeostasis may play a key role in the therapy of ALS. Albeit Ca2+ signaling was not directly tested in this article, the significant crosstalk between Ca2+ and ROS signaling (Hidalgo and Donoso, 2+ signaling in motor neurons of the ALS rodent model.The article by 2+ signals and Ca2+-promoted signaling pathways control the response of neurons and astrocytes under physiological or pathological conditions. Alterations in these key regulatory mechanisms could result in oxidative damage, abnormal synapse function, and ultimately, memory loss or neurodegeneration.In conclusion, the original articles presented in this Topic are clear examples of how cytoplasmic CaTA, CH, and RH-M were guest editors of the Research Topic, emerging views and players in neuronal calcium signaling, synaptic plasticity, learning/memory, aging and neuroinflammation, and wrote this editorial. All authors contributed to the article and approved the submitted version.


TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification.We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’. These have involved channel proteins including those encoded by RYR2, CALM1, CALM2 and KCNJ2 and SCN5A  or result in a tendency to store overload-induced Ca2+ release. The enhanced SR diastolic Ca2+ release associated with RYR2 gain of function causes an intracellular Ca2+ overload during exercise or emotional stress i is a ubiquitous intracellular second messenger. Pathological unregulated SR Ca2+ release is a potential cause of life-threatening arrhythmias i termed Ca2+ oscillations (COs) i and systolic, and abnormal diastolic [Ca2+]i increases accompanying AP activity reflecting the consequent influx of extracellular Ca2+ or Ca2+ release from intracellular stores i-oscillations i abnormalities, and that this was exacerbated by the presence of the SCN10A-Q1362H variant. The latter was reflected in the increased severity of the resulting phenotype in the proband compared to the proband-F iPS-CMs. By contrast, the SCN10A-Q1362H variant alone in the proband-M iPS-CMs was without effect.Secondly, established reports associate aberrant SR Caractions . The preSCN10A loci with altered cardiac conduction reflected in PR and/or QRS intervals, AF i through the resulting increased net [Ca2+] influx, and decreases efflux owing to the shorter diastolic intervals [Fourthly, cardiac AP waveforms vary with, thereby adapting to, altered heart rates following increased adrenergic drive during exercise ,45. At t courses . Increasntervals . This mantervals . In the . 5INa-L increases the probability of EADs, which may trigger arrhythmias. In addition, enhanced INa-L may result in NCX-mediated Ca2+ release events. These could promote depolarizing transient inward currents (Iti), resulting in similarly pro-arrhythmic DADs [+ current, pro-arrhythmic diastolic Ca2+ release, as well as spontaneous APs. This led to suggestions that Nav1.8-dependent selective INa-L reduction could offer a novel antiarrhythmic therapeutic approach [in vitro remains an important issue when using these as a platform for such studies. Future studies could use the Nav1.8-blocker A-803467 and selective late sodium current inhibitors  in different experiment platforms for PVT.The present study using iPS-CMs has several limitations that are worth mentioning. By prolonging the duration of the AP, mic DADs . In addiapproach . However. 62+ handling caused by the double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants associated with a clinical pro-arrhythmic phenotype. It associated the RYR2-A1855D but not the SCN10A-Q1362H variant when either was expressed alone, with abnormal Ca2+ homeostasis and APD prolongation. However, the compound heterozygous RYR2-A1855D and SCN10A-Q1362H variants showed an accentuated Ca2+ homeostasis and APD phenotype, suggesting a possible basis for the clinical features of the originating patient family.In summary, the study investigates acute abnormalities of myocardial Ca


Chemoresistance often complicates the management of cancer, as noted in the instance of acute myeloid leukemia (AML). Mitochondrial function is considered important for the viability of AML blasts and appears to also modulate chemoresistance. As mitochondrial metabolism is aberrant in AML, any distinct pathways could be directly targeted to impact both cell viability and chemoresistance. Therefore, identifying and targeting those precise rogue elements of mitochondrial metabolism could be a valid therapeutic strategy in leukemia. Here, we review the evidence for abnormalities in mitochondria metabolic processes in AML cells, that likely impact chemoresistance. We further address several therapeutic approaches targeting isocitrate dehydrogenase 2 (IDH2), CD39, nicotinamide phosphoribosyl transferase (NAMPT), electron transport chain (ETC) complex in AML and also consider the roles of mesenchymal stromal cells. We propose the term “mitotherapy” to collectively refer to such regimens that attempt to override mitochondria-mediated metabolic reprogramming, as used by cancer cells. Mounting evidence suggests that mitotherapy could provide a complementary strategy to overcome chemoresistance in liquid cancers, as well as in solid tumors. Since 2017, the U.S. Food and Drug Administration has approved eleven new drugs or combinatory regimens, and AML’s 5-year overall survival (OS) rate has been greatly improved . HoweverRecent years have seen the development of some targeted therapies, thanks to the delineation of abnormalities in the molecular pathways leading to AML. For example, novel therapeutic options are being tested in AML using inhibitors of Fms-Like Tyrosine kinase-3 (FLT3), isocitrate dehydrogenase (IDH), B-cell lymphoma 2 (BCL-2), FOXO3A (a forkhead transcription factor) inhibitor, or with stabilizers of p53 –20. HoweIncreasingly, multiple evolving data have shown that the altered bone marrow (BM) niche in AML patients plays a significant role in disease ontogeny; but also provides opportunities for efficient anti-leukemia immunotherapies . AML immIn the BM microenvironment, AML cells are metabolically reprogrammed and may use mitochondrial oxidative phosphorylation (OXPHOS) as important energy sources . In addiBesides intrinsic cellular metabolic factors, intercellular communications, such as transfer of mitochondria from mesenchymal stromal cells (MSCs) to AML cells, have also been shown to be vital for development of chemoresistance . To elabHere, we coin the word “mitotherapy” to collectively refer regimens that attempt to override the mitochondria mediated aberrant cellular processes used by AML cells.2de novo nucleotide biosynthesis by “metabolons”. These “metabolons” are alternatively termed “purinosomes’ and comprise of dynamic bodies associated with mitochondria, containing enzymes underpinning purine biosynthesis  and ynthesis . These aEstablished links between the purine biosynthesis pathway and mitochondrial metabolism are important for coordinating energy metabolism and nucleotide synthesis within the cells. The purine biosynthesis pathway requires high-energy inputs, which exist in the form of ATP. ATP is synthesized mainly in mitochondria, largely through OXPHOS. Moreover, several enzymes involved in purine biosynthesis are associated with, or localized within mitochondria, such as phosphoribosyl pyrophosphate amidotransferase and adenylosuccinate lyase . Purine When compared with normal hematopoietic progenitors, AML cells exhibit unique mitochondrial signatures, including higher copy numbers of mitochondrial DNA (mtDNA), greater mitochondrial mass, paradoxical dependency on OXPHOS, increased mitochondrial biogenesis and altered translation systems required for survival Figure 1Mitochondria transfer is another important mechanism underpinning the higher mitochondrial mass in AML. In a co-culture system mimicking the BM niche, cultured AML cells increase their mitochondrial mass up to 14% by transfer of functional mitochondria from BM stromal cells . As a re2+ release from endoplasmic reticulum (ER) and enhanced mitochondrial respiration, which is crucial for LSCs survival. LYZ-81, an inhibitor of ORP4L, is designed to selectively eradicate LCSs in vitro and in vivo  -related proteins (ORPs), transient receptor potential melastatin 2 (TRPM2), and neurokinin-1 receptor (NK-1R). The increased expression of OSBP-related protein 4 L (ORP4L) in AML cells favors the synthesis of inositol-1,4,5-trisphosphate (IP3). This mediator activates IP3 receptors, leading to Ca in vivo . TRPM2 ies (ROS) . High TRes (ROS) . NK-1R i in vivo .FLT3-internal tandem duplication (FLT3-ITD) is a genomic marker linked with poor clinical prognosis. FLT3-ITD-caused ROS overproduction drives defective DNA damage repair, leading to high mutation burden and the appearance of new leukemic clones (IDH1 and IDH2 genes result in the production of oncometabolite (R)-2-hydroxyglutarate ((R)-2-HG), exacerbating accumulation of ROS -dependent pathway and phosphorylation of nuclear factor kB (NF-kB)  . However (NF-kB) .In a comparable manner to the role of cancer stem cells in mediating drug-resistance, distant metastasis and tumor recurrence , increas3Given the metabolic reprogramming in AML and the specific reliance on OXPHOS in LSCs, targeting the related pathophysiological processes provides the potential possibilities to overcome chemoresistance in AML. Here, we review those strategies targeting IDH2, CD39, nicotinamide phosphoribosyl transferase (NAMPT) and OXPHOS are putative avenues to regulate the intrinsic mitochondrial-mediated cellular processes hijacked by AML cells Figure 23.1IDH2 mutations serve as possible mitotherapy candidates in AML. IDH1 and IDH2 enzymes are nicotinamide adenine dinucleotide phosphate (NADP+-)-dependent and oxidatively catalyze isocitrate decarboxylation into α-ketoglutarate (α-KG) in TCA cycle. In AML, mutations in IDH1 and IDH2 occur in about 5%-10% and 10%-15% of patients, respectively  can induce the differentiation of AML cells, yet they have limited clinical efficacy as a monotherapy (IDH2 mutant enzymes. The Phase 1/2 study showed that median OS in R/R AML patients was 9.3 months. For the 34 (19.3%) patients with complete remission (CR), OS was 19.7 months .By lowering (R)-2-HG content, otherapy . When cohs  and additional metabolites (i.e. formate and carbon dioxide) when compared to the salvage pathway  is the dominant and rate-limiting ectonucleotidase that hydrolyzes ATP and adenosine diphosphate (ADP) to adenosine monophosphate (AMP) . Under n pathway . In addi pathway –72.via trogocytosis from host BM niche onto otherwise Cd39-non-expressing TIB-49 cells resulted in decreased survival of mice inoculated with cancer cells . The role of these anti-CD39 mAbs in AML has not been tested and elucidated.Thus, targeting CD39 offers a new promising therapeutic strategy to restore metabolic vulnerability in drug-resistant AML cells, at least in part by selectively dampening OXPHOS. Pharmacologic inhibition of CD39 ectonucleotidase activity by POM-1 suppresses mitochondrial reprogramming and enhances the sensitivity of AML blasts to cytarabine . Monocloin vivo, and are in Phase I clinical trial .Instead of blocking CD39 ectonucleotidase activity, our colleagues and we have glyco-engineered anti-mCd39 antibodies (αCd39 mAb) to promote Fc receptor interactions. This approach thereby augments antibody-dependent cellular cytotoxicity and promotes trogocytosis. These mAbs promote anti-cancer responses by depleting Cd39-positive myeloid suppressor cells and inhibiting angiogenesis in murine colon cancer models . The antTo explore the efficacy of such glyco-engineered αCd39 mAbs in AML therapy, we established TIB-49 aggressive AML models in mice. Current data showed that pretreatment with glyco-engineered αCd39 mAb effectively boosted daunorubicin chemotherapy cytotoxicity . The eff3.3+) is a metabolite that plays a role in maintaining the mitochondrial membrane potential. NAD+ is also central to energy metabolism as a coenzyme for redox reactions, carrying electrons from one reaction to another. Moreover, NAD+ is an indispensable cofactor for NAD+-dependent enzymes in non-redox reactions, such as sirtuins, CD38 and poly(ADP-ribose) polymerases , providing metabolic substrates as an alternative to glucose  and rewiring metabolic programs . Here, w4.1MSCs have been documented to transfer their mitochondria to AML cells in the BM microenvironment . One wayvia various mechanisms. One mechanism is mediated by tunneling nanotubes (TNTs), which help cells to “intercommunicate” directly with each other across lengthier spaces  against chemoresistance empowered by donor mitochondria. As normal CD34 effects . Curious effects . Daratum effects . Targeti octanol , they no octanol . Miro1 ito Miro1 . Miro1 oto Miro1 . Disrupttabolism . Targetitransfer .4.2FLT3, IDH1/2, cKIT and RAS also seem to drive ROS production in AML cells  within blast cells. Therapeutic strategies that address mitochondria biology and metabolic reprogramming (mitotherapies) may provide more specific and potent regimens for AML, particularly when used in combination with chemotherapy and potentially also immunotherapy.Conceptualization, LF, JY, and SR; writing – original draft preparation, LF and PZ; writing – review and editing, WG, JY, and SR; visualization, LF; supervision, JY and SR; funding acquisition, SR. All authors contributed to the article and approved the submitted version.


In mammals, the Ca2+ release is periodical, known as Ca2+ oscillations, and mediated by the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). Another divalent cation, zinc (Zn2+), increases exponentially during oocyte maturation and is vital for meiotic transitions, arrests, and polyspermy prevention. It is unknown if these pivotal cations interplay during fertilization. Here, using mouse eggs, we showed that basal concentrations of labile Zn2+ are indispensable for sperm-initiated Ca2+ oscillations because Zn2+-deficient conditions induced by cell-permeable chelators abrogated Ca2+ responses evoked by fertilization and other physiological and pharmacological agonists. We also found that chemically- or genetically generated eggs with lower levels of labile Zn2+ displayed reduced IP3R1 sensitivity and diminished ER Ca2+ leak despite the stable content of the stores and IP3R1 mass. Resupplying Zn2+ restarted Ca2+ oscillations, but excessive Zn2+ prevented and terminated them, hindering IP3R1 responsiveness. The findings suggest that a window of Zn2+ concentrations is required for Ca2+ responses and IP3R1 function in eggs, ensuring optimal response to fertilization and egg activation.Changes in the intracellular concentration of free calcium (Ca Cdc20 (2+)  (2+ release from the egg’s 2+3 main Ca reservoir (2+ via plasma membrane channels and transporters ensures the persistence of the oscillations (Vertebrate eggs are arrested at the metaphase stage of the second meiosis (MII) when ovulated because they have an active Cdk1/cyclin B complex and inactive APC/CCdc20 . ReleaseCdc20 . The unic20 (2+) . Ca2+ retic exit . In mammterphase . The spehase (3)  that bineservoir . The intllations , 2013.2+ release machinery to produce timely and protracted Ca2+ oscillations following sperm entry  also remarkably increase during maturation, amounting to a 50% rise, which is necessary for oocytes to proceed to the telophase I of meiosis and beyond -1,2-ethylenediamine (TPEN) to create Zn2+-deficient conditions buttressed the importance of Zn2+ during meiotic transitions . The nurm entry , includisitivity . The totd beyond . RemarkaMII exit . Followi species . The usensitions . However3Rs are the most abundant intracellular Ca2+ release channel in non-muscle cells , a technique that allows peering into the structure of IP3R1 with a near-atomic resolution, have revealed that a helical linker (LNK) domain near the C-terminus mediates the coupling between the N- and C-terminal ends necessary for channel opening  formation. To this end, ICSI-fertilized eggs were cultured in the presence of 10 μM TPEN for 8h, during which the events of egg activation were examined  of SrCl2 is unclear, although its actions are reportedly directly on the IP3R1  that, after short UV pulses, releases IP3 into the ooplasm  overcame TPEN’s inhibitory effects only if added before , a Sarcoplasmic/ER Ca2+ ATPase pump inhibitor . To test whether the reduced response to Tg means that TPEN prevented the complete response of Tg, leaving a temporarily increased Ca2+ content in the ER, we added the Ca2+ ionophore ionomycin (Io), which empties all stores independently of IP3Rs. Io-induced Ca2+ responses were 3.3-fold greater in TPEN-treated cells, supporting the view that TPEN interferes with the ER Ca2+ leak (P < 0.05). We further evaluated this concept using in vitro aged eggs that often display reduced Ca2+ store content than freshly collected counterparts . We confirmed that this effect was independent of IP3R1 degradation because TPEN did not change IP3R1 reactivity in unfertilized eggs (P <0.05).Our above cIPthe IP3R . Additio1 gating , led us nhibitor  that unmf the ER ; in eggs ER . TPEN had even more pronounced effects following the addition of Ach, leading to a reduced- and prematurely terminated- Ca2+ release from the ER in treated eggs (P <0.05).Next, we used the genetically encoded FRET sensor D1ER  to asses-ER Ca2+ , E. For o groups ; P <0.05ted eggs ; P <0.052+ occurred without pharmacology. To this end, we examined a genetic model where the two non-selective plasma membrane channels that could influx Zn2+ in maturing oocytes have been deleted . These results in dKO eggs show a changed dynamic of the Tg-induced Ca2+ release, suggesting that lower levels of labile Zn2+ modify ER Ca2+ release independently of chelators.Lastly, we sought to use a cellular model where low labile Zn deleted , 2013, n deleted . We founeted  rapidly increases cellular Zn2+ upon extracellular addition /transporter, contrasting with the abrogation of Ach-induced ER emptying from the outset. Remarkably, independently of Zn2+ chelators, emptying of Ca2+ ER was modified in a genetic model of Zn2+-deficient oocytes lacking two TRP channels, confirming the impact of Zn2+ on Ca2+ release. It is worth noting that TPEN did not reduce but maintained or increased the mass of IP3R1, which might result in the inhibition of Zn2+-dependent ubiquitin ligase Ubc7 by the Zn-deficient conditions  and diethylenetriaminepentaacetic acid (DTPA) were reconstituted with double-sterile water at 0.5M and 10 mM, respectively, and the pH was adjusted to 8.0. Tris(2-pyridylmethyl) amine (TPA) was diluted in DMSO at 100 mM and stored at −20°C until use. Acetylcholine chloride and Thimerosal were dissolved in double-sterile water at 550 mM and 100 mM, respectively. Acetylcholine was stored at −20°C until use, whereas Thimerosal was made fresh in each experiment.N,N,N′,N′-tetrakis (2-pyridinylmethyl)-1,2-ethylenediamine (TPEN) and Zinc pyrithione (ZnPT) were dissolved in dimethyl sulfoxide (DMSO) at 10 mM and stored at −20°C until use. SrClTrpm7-floxed (Trpm7fl/fl) Gdf9-Cre and Trpv3−/− mice were bred at our facility. Trpm7fl/fl mice were crossed with Trpv3−/− to generate Trpm7fl/fl; Trpv3−/− mouse line. Female Trpm7fl/fl; Trpv3−/− mice were crossed with Trpm7fl/fl; Trpv3−/−; Gdf9-cre male to generate females null for Trpv3 and with oocyte-specific deletion for Trpm7. Ear clips from offspring were collected prior to weaning, and confirmation of genotype was performed after most experiments.The University of Massachusetts Institutional Animal Care and Use Committee (IACUC) approved all animal experiments and protocols. All gamete handling procedures are as previously reported by us . MII egg2+ monitoring or cultured in KSOM to evaluate activation and development at 36.5°C in a humidified atmosphere containing 5% CO2.ICSI was performed as previously reported by us  using dePlcζ-mEGFP, pcDNA6-CALR-D1ER-KDEL, and pcDNA6-humanERp44-HA were linearized with the restriction enzyme PmeI and in vitro transcribed using the T7 mMESSAGE mMACHINE Kit following procedures previously used in our laboratory (in vitro synthesized RNA (mRNA) using Tailing Kit followed by quantification and dilution to 0.5 μg/μL in nuclease-free water and stored at −80°C until use. Before microinjection, mPlcζ, D1ER, and ERp44 mRNA were diluted to 0.01, 1.0, and 0.5 μg/μL, respectively, in nuclease-free water, heated at 95°C for 3 min followed by centrifugation at 13400×g for 10 min at 4°C. Cytoplasm injection of mRNA was performed under microscopy equipped with micromanipulators . The zona pellucida and the plasma membrane of MII eggs were penetrated by applying small pulses generated by the piezo micromanipulator . The preparation of the injection pipette was as for ICSI . Eggs were monitored simultaneously using an inverted microscope  outfitted for fluorescence measurements. Fura-2 AM, FluoZin3-AM, and Rhod2-AM fluorescence were excited with 340 nm and 380 nm, 480 nm, and 550 nm wavelengths, respectively, every 20 sec, for different intervals according to the experimental design and as previously performed in the laboratory. The illumination was provided by a 75-W Xenon arc lamp and controlled by a filter wheel . The emitted light above 510 nm was collected by a cooled Photometrics SenSys CCD camera . Nikon Element software coordinated the filter wheel and data acquisition. The acquired data were saved and analyzed using Microsoft Excel and GraphPad using Prism software  was performed using a CFP excitation filter, dichroic beamsplitter, CFP and YFP emission filters . To measure Ca2+-ER and cytosolic Ca2+ simultaneously, eggs that had been injected with D1ER were loaded with Rhod-2AM, and CFP, YFP, and Rhod-2 intensities were collected every 20 sec.To estimate relative changes in Ca3/PM (cIP3) was reconstituted in DMSO and stored at −20°C until use. Before injection, cIP3 stock was diluted to 0.25 mM with water and microinjected as above. After incubation in KSOM media at 37°C for 1-hr., the injected eggs were loaded with the fluorophore, 1.25 μM Fluo4-AM, and 0.02% Pluronic acid and handled as above for Fura-2 AM. The release of cIP3 was accomplished by photolysis using 0.5 to 5-sec pulses at 360 nm wavelengths. Ca2+ imaging was as above, but Fluo4 was excited at 488 nm wavelength and emitted light above 510 nm collected as above.Caged-IP3R1 . After blocking with 5% fat-free milk + TBS, membranes were probed with the rabbit polyclonal antibody specific to IPImmunostaining was performed according to our previous study . After iP < 0.05.Comparisons for statistical significance of experimental values between treatments and experiments were performed in three or more experiments performed on different batches of eggs in most studies. Given the number of eggs needed, WB studies were repeated twice. Prism-GraphPad software was used to perform the statistical comparisons that include unpaired Student’s t-tests, Fisher’s exact test, and One-way ANOVA followed by Tukey’s multiple comparisons, as applicable, and the production of graphs to display the data. All data are presented as mean±s.d. Differences were considered significant at 1