The use of genetically encoded Ca2+ indicators allows real-time monitoring of cytosolic Ca2+ levels to study such responses. Here we explored the possibility of using the ratiometric Ca2+ indicator GEM-GECO for monitoring cytosolic Ca2+ concentration ([Ca2+]cyt) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca2+ ATPase Pmc1, which is involved in [Ca2+]cyt control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca2+ depletion, but to excessive Ca2+ signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca2+-free to Ca2+-bound state minutes after external addition of CaCl2. This was followed by gradual return of GEM-GECO to a Ca2+-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca2+]cyt monitoring in budding yeast.Ca In particular, it is involved in neuron activity, muscular contraction, cell cycle regulation, etc. cyt) in different types of animal cells. One of these indicators was used to monitor [Ca2+]cyt in dividing cells of the Schizosaccharomyces pombe yeast cyt in individual cells independently of variation in the GECI production level. Notably, a GECI is effective in this kind of experiments if its presence in predominantly Ca2+-free form switches to predominantly Ca2+ -bound form within the range of the physiological [Ca2+]cyt variations. This means that the Kd of its Ca2+-bound form must be between the resting [Ca2+]cyt level and the level reached after stimulation.Intracellular Caaequorin ,6 or artaequorin ). Among iewed in ,8). Effoiewed in ,10,11) weveloped . This pr2+ [Sch. pombe [2+]cyt monitoring in various species of budding yeasts.Notably, high levels of GECI production were shown to affect neuronal cell physiology due to depletion of cytosolic Ca2+ , which mh. pombe , to our 2+ as a second messenger allowing cells to respond to different external stimuli. This requires maintaining [Ca2+]cyt at a low level (50–200 nM) by pumping it from the cytosol into intracellular depots. Studies of Saccharomyces cerevisiae have demonstrated that the main Ca2+ depot in the yeast cell is the vacuole, which accumulates Ca2+ due to action of the vacuolar calcium ATPase Pmc1 and the Ca2+/H+ exchanger Vcx1. The proton gradient, which empowers Vcx1 action, is generated by Vma1 .2+]cyt, activating the Ca2+ binding protein CaM, which interacts with CaM-dependent proteins, thus modulating their activity. One of these proteins is the protein phosphatase calcineurin which is activated both by binding Ca2+ directly and by interaction with Ca2+-bound CaM. Calcineurin dephosphorylates transcription factor Crz1 to trigger its translocation to the nucleus. This alters the expression of a number of genes including those involved in maintaining Ca2+ homeostasis [2+]cyt to the resting level.Certain extracellular stimuli cause an increase in cyt monitoring in this yeast and characterize the effects of its production on cell physiology in wild-type and pmc1-Δ strains.Previously we have observed that inactivation of the vacuolar Can arrest . Here we2+ using a genetically encoded fluorescent indicator such as GEM-GECO demands that its fluorescence be observable and quantifiable over the background fluorescence of the host cells. To achieve this in O. parapolymorpha, we used a codon-optimized gene coding for GEM-GECO, which was placed under control of a strong regulatable promoter of the O. parapolymorpha MAL1 gene. The latter codes for maltase, which is required for utilization of some disaccharides including sucrose and is strongly induced upon growth on these carbon sources cyt in O. parapolymorpha, apart from the wild-type strain we also used a derivative of DL5 lacking the vacuolar Ca2+ ATPase Pmc1, which is responsible for maintaining [Ca2+]cyt at a low level.To test applicability of GEM-GECO for monitoring cyt? We proposed that the CaM domain of this protein somehow participates in Ca2+ dependent signaling. While it was previously shown that GECIs possessing this domain can affect cell physiology by depleting Ca2+ from cytosol cyt.To analyze GEM-GECO fluorescence in individual cells we used flowcytometry with a 405 nm excitation laser and fluorescence detection with 450 and 525 nm band-pass filters. Although the 450 nm fluorescence of majority of the GEM-GECO-expressing cells in regular conditions surpassed fluorescence of control cells lacking this expression cassette B, the au2+ concentration would lead to some increase in [Ca2+]cyt. This would allow us to test applicability of GEM-GECO for monitoring [Ca2+]cyt changes in yeast cells. To demonstrate this effect with an alternative method we used the fluorescent dye Fluo4-AM, which is often used to detect increases in intracellular [Ca2+] ) possessing an autonomously replicating plasmid with the LEU2 selectable marker. The 1B27-740G1M strain was obtained by transformation of the 1B27 strain cyt in yeast O. parapolymorpha. Production of this protein in cells with wild type Ca2+ homeostasis had a negligible effect on cell physiology. However, inactivation of the vacuolar Ca2+ ion pump Pmc1 manifested a pronounced interaction with GEM-GECO expression, which was revealed as aggravation of pmc1-Δ mutant phenotypes. Although previous studies indicated that physiological effects of such indicators in neuronal cells are related to Ca2+ depletion in cytosol, the effects we observed here were more likely to be related to an enhanced response to [Ca2+]cyt rise. We hypothesize that this is due to the CaM domain of GEM-GECO, which might interact with the host Ca2+-dependent machinery. Nevertheless, this indicator is applicable for monitoring [Ca2+]cyt in yeast cells. In particular, it is predominantly present in the Ca2+-free form in cells during normal conditions and converts to the Ca2+-bound form when [Ca2+]cyt rises in response to external Ca2+ concentration rise. This means that the [Ca2+]cyt in yeast cells is essentially below the Kd of the GEM-GECO-Ca2+ complex and can rise above this value in response to various treatments. We also observed that GEM-GECO expression under control of MAL1 promoter somehow depended on Ca2+ homeostasis, since it became elevated after inactivation of the PMC1 gene encoding the vacuolar Ca2+ ATPase. However, this did not affect [Ca2+]cyt monitoring since this indicator is ratiometric.In this work we have studied the applicability of the ratiometric GEM-GECO Ca


Currently, no information is available regarding the expression and function of SOCE in tumour-infiltrating lymphocytes (TILs) that have been expanded in vitro for adoptive cell therapy (ACT). This study provides the first evidence that SOCE is up-regulated in ex vivo-expanded TILs from metastatic colorectal cancer (mCRC) patients. The up-regulation of SOCE mainly depends on diacylglycerol kinase (DGK), which prevents the protein kinase C-dependent inhibition of Ca2+ entry in normal T cells. Of note, the pharmacological blockade of SOCE with the selective inhibitor, BTP-2, during target cell killing significantly increases cytotoxic activity at low TIL density, i.e., when TILs-mediated cancer cell death is rarer. This study, albeit preliminary, could lay the foundation to propose an alternative strategy to effect ACT. It has been shown that ex vivo-expanded TILs did not improve the disease-free survival rate in mCRC patients. Our results strongly suggest that pre-treating autologous TILs with a SOCE or DGK inhibitor before being infused into the patient could improve their cytotoxic activity against cancer cells.Store-operated Ca2+ entry (SOCE) drives the cytotoxic activity of cytotoxic T lymphocytes (CTLs) against cancer cells. However, SOCE can be enhanced in cancer cells due to an increase in the expression and/or function of its underlying molecular components, i.e., STIM1 and Orai1. Herein, we evaluated the SOCE expression and function in tumour-infiltrating lymphocytes (TILs) from metastatic colorectal cancer (mCRC) patients. (2) Methods: Functional studies were conducted in TILs expanded ex vivo from CRC liver metastases. Peripheral blood T cells from healthy donors (hPBTs) and mCRC patients (cPBTs) were used as controls. (3) Results: SOCE amplitude is enhanced in TILs compared to hPBTs and cPBTs, but the STIM1 protein is only up-regulated in TILs. Pharmacological manipulation showed that the increase in SOCE mainly depends on tonic modulation by diacylglycerol kinase, which prevents the protein kinase C-dependent inhibition of SOCE activity. The larger SOCE caused a stronger Ca2+ response to T-cell receptor stimulation by autologous mCRC cells. Reducing Ca2+ influx with BTP-2 during target cell killing significantly increases cytotoxic activity at low target:effector ratios. (4) Conclusions: SOCE is enhanced in ex vivo-expanded TILs deriving from mCRC patients but decreasing Ca2+ influx with BTP-2 increases cytotoxic activity at a low TIL density.(1) Background: Store-operated Ca Colorectal carcinoma (CRC) represents the third most common cause of malignancy-associated mortality, which is mainly due to the development of liver metastases . SurgicaAdoptive cell therapy (ACT) represents a promising approach to achieve complete and durable cancer eradication by the transfer of ex vivo-expanded cells reactive against tumour ,4. Studi2+ concentration ([Ca2+]i) is of paramount importance to promote the differentiation and effector functions of T cells by driving processes that are key to the immune response, including proliferation, differentiation, cytokine secretion and cytotoxicity ER) is detected by Stromal Interaction Molecule 1 (STIM1), which serves as sensor of [Ca2+]ER and is thereafter prompted to translocate from perinuclear to peripheral ER cisternae, where STIM1 traps and activates the Ca2+-selective Orai1 channels at the plasma membrane i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternately at 340 and 380 nm (shortly termed “ratio”). An increase in [Ca2+]i causes an increase in the ratio [2+]i in the three cell types was monitored by using the Grynkiewicz method, as described in i was significantly (p < 0.001) higher in the TILs than in the cPBTs and hPBTs  activity, in the absence of extracellular Ca2+ (0Ca2+) to induce ER Ca2+ release and thereby promote ER Ca2+ depletion. Extracellular Ca2+ was restored upon recovery to the baseline to selectively measure the SOCE. Careful analysis of the Ca2+ tracings (2+ release was as follows: cPBTs > TILs > hPBTs (p < 0.001) larger in the TILs than in cPBTs, which, in turn, exhibited a larger SOCE than hPBTs (2+ (1.5 mM), the amplitude of the Ca2+ peak was as follows: TILs >> cPBTs > hPBTs (p < 0.001) reduced CPA-evoked SOCE in the TILs , which can both result in SOCE activation in hPBTs i [2+ response in TILs than in cPBTs and hPBTs  hindered the AUC of the Ca2+ signal  46.08% (106/230) of responding cells presented intracellular Ca2+ oscillations that persist for about 40 min from the beginning of the stimulation (termed “oscillatory pattern”) (2+ waves that again lasted for about 40 min (termed “slow pattern”) (2+ transient followed by a slow increase in [Ca2+]i that ultimately achieved a plateau phase (therefore termed “plateau pattern”)  induced all of the TILs to display the “oscillatory pattern”   and presence of BTP-2 (20 µM) using the same batches of cells that were employed for the Ca2+ imaging recordings. Surprisingly, the average cytotoxic activity against autologous mCRC cells at each E:T ratio was increased by pre-treating the ex vivo-expanded TILs with BTP-2 (20 µM) (p < 0.05) at low E:T ratios  A; (2) 34attern”) B; and (3attern”) C, which s; 100%) D, therebresponse E, wherearesponse F, which  (20 µM) G, with tT ratios H. Overal2+-dependent cytotoxic response against cancer cells is of paramount importance to help improve the therapeutic outcome of ACT via autologous TILs. Herein, we demonstrated, for the first time, that SOCE is up-regulated in TILs and cPBTs deriving from mCRC patients compared to hPBTs, mainly due to tonic DGK-dependent modulation. Surprisingly, the increase in SOCE amplitude is associated with a reduction in TILs’ cytotoxic activity against autologous mCRC cells compared to that of CTLs. This unexpected finding could explain their limited therapeutic efficacy in the patients i i . Enhance or RyRs ,17. Howeemptying  and thatin hPBTs . Overall2+-handling machinery could be finely regulated by controlling mRNA stability i that arises in both CTLs and TILs following TCR stimulation at the immune synapse to support cancer cell killing i. The pharmacological blockade of SOCE with BTP-2 significantly increased the percentage of TILs displaying intracellular Ca2+ oscillations (from 46.08% to 100%) and significantly reduced the amount of total Ca2+ introduced into the cytosol during the immune response. Notably, pre-treating the TILs with BTP-2 improved their cytotoxic activity against the mCRC cells at each E:T ratio, and this increase became statistically relevant at a lower E:T ratio, suggesting that this effect was more evident when the TILs-mediated lysis was low. This feature is quite relevant, since, in non-haematological solid cancers, TILs often fail to kill target cells during 1:1 conjugation [2+ entry in CTLs or NK can interfere with multiple steps of the immune response, e.g., migration and release of lytic granules, while it can enhance proliferation and apoptosis [2+ entry in TILs during mCRC cell killing reduces cytotoxic activity, e.g., increased apoptotic rate, is (are) under current investigation.SOCE supports the increase in [Ca killing ,31,50,51gagement ,31,49. Hjugation . Our resjugation . In accopoptosis ,52. The 2+ entry is mainly enhanced by DGK activity, which prevents SOCE inhibition by PKC. The pharmacological blockade of SOCE with BTP-2 enhances TILs’ ability to kill autologous mCRC cells at low-density ratios. This preliminary evidence might suggest an alternative pharmacological strategy to improve the therapeutic outcome of ACT in mCRC patients.We provide the first evidence that SOCE is up-regulated in ex vivo-expanded TILs from mCRC patients. Extracellular Ca


Acute myeloid leukemia (AML) is a molecularly and cytogenetically complex disease that has proven difficult to treat with a plateau in long-term disease-free survival over the last several decades of less than 30% at 5 years. In the past five years, a number of novel agents and combinations have been FDA approved for the treatment of AML. The understanding of novel mechanisms which can be therapeutically targeted for the treatment of AML continues to expand rapidly. In addition attempts to exploit the immune system as a mechanism treat leukemia is expanding beyond allogeneic hematopoietic stem cell transplantation (HSCT) to check point inhibition and T-cell therapies such as BiTE and CAR-T technology. This has led to an increasing number of clinical trials in the treatment of AML as well as in the area of maintenance therapy after intensive induction or consolidative transplantation.The goal of this Research Topic was to explore novel pathways in AML which could be exploited for therapy of either upfront or relapse/refractory AML as well as in the maintenance setting including maintenance therapy post allogeneic transplantation.Alotaibi et al. describe a series of cases of AML treated with FLT3 inhibitors which developed BCR-ABL1 mutations. This mutation provides an alternative therapeutic target and identifying this mutation at relapse after FLT3 inhibitor failure should be a priority. In the second paper focusing on relapsed AML therapy, Watts et al. introduce a CD3xCD123 bispecific antibody (APVO436). This paper explored the therapeutic and pharmacodynamic effects of APVO436 in 14 relapsed/refractory AML/MDS patients who failed treatement with hypomehylating agents +/- venetoclax. With single agent use at a range of dose levels, in vivo immunomodulatory and anti-leukemic activity was demonstrated via T-cell activation as well as a median overall survival of 282 days. This paper supports further investigation of the potential benefits of APVO436.Seven manuscripts in this topic explore a variety of aspects AML therapy, five of which focus on allogeneic transplantation including maintenance. In the first of the papers that focus on non-transplant therapy, Wang et al. presented a case study of stem cell transplantation using a decitabine-containing preconditioning regimen in TP53 mutated myelodysplastic syndrome. Nine TP53 mutated patients received decitabine preconditioning followed by myeloablative allogeneic HSCT. 89% of patients were alive and progression free at a median follow up of 42 months. Given the extremely poor prognosis of TP53 mutated myeloid malignancy patients, this preliminary evidence of efficacy by decitabine containing conditioning regimens should be explored in larger populations.Five manuscripts focus on allogeneic stem cell transplantation ranging from conditioning regimens through DLI to reviews of maintenance options post-transplant. Salhotra and Stein. presented a review article that discussed the role of newer radiation based conditioning regimens such as total marrow irradiation (TMI) in patient with high-risk AML undergoing allogenic HSCT in remission or with active disease. They also discuss mechanisms associated with leukemia relapse and treatment options available after relapse following allo-HSCT using intensified conditioning regimens. This comprehensive review provides background on currently used radiation and chemotherapy preparative regiments as well as describing new techniques to deliver radiation using total marrow and lymphoid irradiation which allow for better disease control and improved clinical outcomes. However, relapse remains the primary cause of transplant failure so they further discuss adoptive immunotherapy and maintenance therapies to improve relapse-free outcomes.Stadler et al., the use of preemptive and therapeutic donor lymphocyte infusion (preDLI and tDLI) in relapsed myeloid malignancies is explored when using a novel, quantitative PCR-based high-sensitivity chimerism (hs-chimerism). This technique allows for redicition of relapse more than a month before clinical diagnosis. They compared a historic cohort of patients who received preDLI or tDLI prior to hs-chimerism to 32 patients who received these therapies after. At 2 years from first DLI the hs-chimerism group had a very impressive overall and disease-free survival, early 3 times that of controls. This was a result of both reduced relapse incidence with a larger proportion of patients receiving preemptive DLI as well as decreased non-relapsed mortality. This paper supports the value of hs-chimerism for directing DLI after alloSCT and warrants exploration in larger populations across multiple centers.In a brief research report by Manobianco et al. discuss the mechanisms of treatment being targeted for post-transplant maintenance therapy for AML. This includes a discussion of FLT3 inhibitors, histone deacetylase inhibitors, IDH1 & 2 inhibitors, hypomethylating agents, bcl2 inhibitors, and checkpoint inhibitors. The review includes data for their use as well as a discussion of recruiting clinical trials. Nayak and Chen review these agents as well as hedgehog inhibitors and other immunomodulatory agents such as lenalidomide.The final two manuscripts in the Research topic provide comprehensive reviews of post-transplant maintenance therapy for AML after allogeneic HSCT. The manuscripts presented in this Research Topic provide hope for novel treatments for acute myeloid leukemia before and after transplant, but also highlight the desperate need for expanding our repertoire to treat this complicated disease with a mortality rate of nearly 70% which has not improved over the last decades despite an array of novel therapies.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although the basic function of the UPS has been extensively elucidated and has been continued to define its mechanism, the precise relationship between the UPS and Ca2+ signaling remains unclear. In the present review, we describe the relationship between the UPS and Ca2+ signaling, including Ca2+-associated proteins, to understand the end point of oxidative stress. The UPS modulates Ca2+ signaling via the degradation of Ca2+-related proteins, including Ca2+ channels and transporters. Conversely, the modulation of UPS is driven by increases in the intracellular Ca2+ concentration. The multifaceted relationship between the UPS and Ca2+ plays critical roles in different tissue systems. Thus, we highlight the potential crosstalk between the UPS and Ca2+ signaling by providing an overview of the UPS in different organ systems and illuminating the relationship between the UPS and autophagy.The ubiquitin–proteasome system (UPS) is the main proteolytic pathway by which damaged target proteins are degraded after ubiquitination and the recruit of ubiquitinated proteins, thus regulating diverse physiological functions and the maintenance in various tissues and cells. Ca The homeostatic maintenance of protein levels or elimination of misfolded or oxidized proteins requires essential quality control processes such as the ubiquitin–proteasome system (UPS) and autophagy ,2,3,4. T2O2-induced protein carbonyl group, which is the indicator of protein oxidation, is increased by the treatment of proteasome inhibitor MG-132 i) is a messenger signal of oxidative pathways. Oxidative stress triggers an increase of [Ca2+]i through the ER membrane Ca2+ channels i is initiated through two pathways via the release of Ca2+ from intracellular stores by various extracellular stimuli and the influx of Ca2+ through plasma membrane-associated Ca2+ channels, including voltage-gated Ca2+ channels (VGCCs), ligand-gated Ca2+ channels (LGCCs), and Ca2+ ATPases [2+]i induces versatile and universal Ca2+ signaling to regulate various cellular physiological functions i level. The crosstalk between the UPS and autophagy with Ca2+ signaling will be discussed in Regulatory channels of Ca+ influx . The ovepression , whereaspression . To mainpression . If one pression . Thus, t2+ channel voltage-gated calcium channel (CaV)2.2, which induces peripheral neuron neurotransmission i increases by the constitutive activation of the epithelial sodium channel, which induces the aggregation and activation of caspase-8 to inhibit the proteasome, and activated caspase-8 induces cellular apoptosis i that regulates the UPS. Acute ER stress increases the degradation of the amyloid precursor protein, a diagnostic marker of AD i, and the treatment with BAPTA attenuates the ubiquitination of MGRN1 and GP78 i homeostasis, regulates the proteasome activity i through the A233187 Ca2+ ionophore increases the proteasome activity via CaMKII- and calpain-dependent mechanisms i in β cells induce the apoptotic pathway through the UPS [2+]i increases through Ca2+ ionophore A23187, or by the supplementation of Ca2+ in the media in pancreatic β cells, the UPS-mediated degradation of islet-brain 1 and c-Jun N-terminal kinase (JNK) interacting protein 1 (IB1/JIP1) is increased [2+]i that occurs through ER stress reduces the proteasome activity in β cells i in pancreatic acinar cells i affects proteasome activity i and induces Ca2+ signaling i and CaMKII subunit β in MDA-MB-231 and MDA-MB-498 cells i via ER stress in HCT116 colon cancer cells [2+ chelation through BAPTA-AM [2+-related protein activity, including calcineurin, and activates autophagy through the calcineurin-transcription factor EB-p62 pathway in cardiomyocytes , Parkinson’s disease (α-synuclein), and amyotrophic lateral sclerosis (superoxide dismutase), are substrates of the UPS [2+ signaling as the checklist of clinical approaches in proteasome modulation might be beneficial to avoid unwanted effects.The studies of the relationship between the UPS and Carial ROS ,175. Fro the UPS . Additio the UPS ), cardio the UPS ), the de the UPS , and isc the UPS . Althoug the UPS . A recen the UPS . However the UPS . Especia the UPS ,184, and the UPS . Thus, w


It has been reported that relapsed/refractory AML patients with t translocation achieved better clinical outcomes with a decitabine-based combination regimen than other AML subtypes; however, the mechanisms underlying this phenomenon remain unknown. Herein, the DNA methylation landscape of de novo patients with the t translocation was compared with that of patients without the translocation. Moreover, the methylation changes induced by decitabine-based combination regimens in de novoThirty-three bone marrow samples from 28 non-M3 AML patients were subjected to DNA methylation sequencing to identify the differentially methylated regions and genes of interest. TCGA-AML Genome Atlas-AML transcriptome dataset was used to identify decitabine-sensitive genes that were downregulated following exposure to a decitabine-based regimen. In addition, the effect of decitabine-sensitive gene on cell apoptosis was examined in vitro using Kasumi-1 and SKNO-1 cells.LIN7A, CEBPA, BASP1, and EMB were identified as critical decitabine-sensitive genes in t AML. Moreover, AML patients with hypermethylated LIN7A and reduced LIN7A expression had poor clinical outcomes. Meanwhile, the downregulation of LIN7A inhibited decitabine/cytarabine combination treatment-induced apoptosis in t AML cells in vitro.A total of 1377 differentially methylated regions that specifically responsive to decitabine in t AML were identified, of which 210 showed hypomethylation patterns following decitabine treatment aligned with the promoter regions of 72 genes. And the methylation-silencing genes, LIN7A is a decitabine-sensitive gene in t AML patients that may serve as a prognostic biomarker for decitabine-based therapy.The findings of this study suggest that The online version contains supplementary material available at 10.1186/s13148-023-01458-0. Administration of intensive chemotherapy with cytarabine (Ara-C) and anthracycline has profoundly improved the clinical outcomes of patients with acute myeloid leukemia (AML) . However2 on days 1–5), Ara-C (10 mg/m2 every 12 h on days 1–5), aclarubicin , and granulocyte colony-stimulating factor (300 μg/d from d0 to neutrophil recovery). All patients were diagnosed and assessed according to the AML guidelines of the National Comprehensive Cancer Network . As described previously [Twenty-eight non-M3 AML patients who were monitored at the Hematology Department of the Chinese People’s Liberation Army General Hospital between August 2014 and June 2016 were enrolled in this study and underwent DNA methylation sequencing, as described previously . Three oeviously , specimeFor the enrichment of mononuclear cells, bone marrow samples were separated using a Ficoll density gradient , and DNA from the lymphoblasts was purified using a Wizard Genomic DNA Purification Kit . MethylC-capture sequencing was performed as described previously . Brieflyhttps://www.bioinformatics.babraham.ac.uk/index.html), Bismark [https://bedtools.readthedocs.io/en/latest/; parameters: intersect -a DMRa.bed, -b DMRb.bed, -wa). The differences in absolute methylation levels in the DMRs were determined by subtracting the methylation level of the non-t samples from that of t or CR samples from de novo samples. DMRs with absolute methylation differences > 0 were defined as being hypermethylated, whereas those with absolute methylation differences < 0 were defined as being hypomethylated. “Promoter” regions were identified as DNA regions between 2200 bp upstream and 500 bp downstream of the transcription start sites (TSS). DNA regions only overlapping the gene body were identified as “gene body,” while those partially overlapping the promoter and partially overlapping gene body were identified as “Promoter/Gene body (P/Gb).” The remaining DNA regions were identified as “Other.”Following the removal of the adapter sequences and poor-quality reads based on FastQC . R (V3.6.1) and the limma package were used to identify differentially expressed genes related to t AML.Clinical and RNA sequencing data from 156 patients with AML (TCGA-AML), including 7 t AML and 149 non-t AML patients, were obtained from the UCSC Xena dataset platform (https://www.ncbi.nlm.nih.gov/gds). The methylation data, defined based on GRCh36Refseq, were remapped to GRCh37Refseq using ANNOVAR [GSE18700, a methylation profile containing data from 24 t and 320 non-t AML patients and constructed using a methylation array targeting the promoter regions, was obtained from the Gene Expression Omnibus database  and Ara-C (Topscience) were dissolved in dimethyl sulfoxide (Thermo Fisher Scientific) and stored at − 80 °C.Kasumi-1, SKNO-1, U937, and K562 cell lines were purchased from the American Type Culture Collection  and maintained in Gibco RPMI-1640  supplemented with streptomycin , penicillin , and 10% fetal bovine serum . As previously described , SKNO-1-LIN7A (shLIN7A).Human embryonic kidney 293 T cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium containing streptomycin (100 µg/mL), penicillin (50 U/mL), and 10% FBS. Short hairpin RNAs  were cotransfected into 293 T cells with pHR and VSVG plasmids to produce shRNA lentiviruses. Kasumi-1 and SKNO-1 cells were transfected with control shRNA lentiviruses (shNC) or lentiviruses targeting treated blank/Abscontrol blank) × 100%.Cell viability was assessed using the MTS assay  following treatment with different concentrations of DAC for 7 days; the optical density was measured at 490 nm. Cell viability was calculated using the following formula: cell viability (%) =  for 15 min at room temperature in the dark. Propidium iodide was added to the samples before analysis to distinguish live cells from dead ones, and the apoptosis rate was determined using a CytExpert flow cytometer . Apoptosis experiments were performed in triplicate using the following treatment groups: DAC alone, Ara-C alone, and DAC followed by Ara-C.After successfully transfecting cells with shNC or shLIN7A lentivirus, the cells were treated with 10 nM DAC for 3 days; the medium was then replaced with fresh medium. On day 4, 2 × 10http://www.urogene.org/methprimer/) and are listed in Additional file Genomic DNA from Kasumi-1 and SKNO-1 cells was isolated using a Wizard Genomic DNA Purification Kit (Promega). Furthermore, genomic DNA was treated with sodium bisulfate  according to the manufacturer’s instructions. Methylation-specific PCR primers were designed using MethPrimer  was used to extract total RNA. cDNA was synthesized using a cDNA kit  according to the manufacturer’s instructions. An ABI PRISM 7500 Sequence Detection System (Thermo Fisher Scientific) was used to perform quantitative reverse transcription-PCR (qRT-PCR) under the following cycle conditions: 95 °C for 30 s to denature the cDNA template, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. Gene expression levels were calculated using the 2Radioimmunoprecipitation assay buffer, supplemented with phenylmethylsulfonyl fluoride and phosphatase inhibitors , was used for cell lysis. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Subsequently, the proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-LIN7A , anti-DNMT3A , and anti-GAPDH . Thereafter, the membranes were washed with Tris-buffered saline with Tween 20 and then incubated with the relevant secondary antibodies  for 2 h at room temperature.t-test and one-way analysis of variance were used to determine the statistical differences among the experimental groups. All statistical tests were two-sided, statistical significance was defined as p < 0.05.All data were expressed as the mean ± standard error of the mean from at least three independent experiments. SPSS software  was used to perform all statistical analyses. Student’s Considering that patients with relapse/refractory t8;21) AML have been reported to more likely benefit from a DAC-based regimen [;21 AML hn = 5393; 28.21%) was much lower than that of hypomethylated DMRs  than DMR1. However, similar to DMR1, hypomethylated DMRs within DMR2  were more frequent than hypermethylated DMRs  cell line SKNO-1 with an AML1-ETO knockdown cell line (SKNO-1-siAE), and the DMRs were collectively designated as the “DMR2” profile. DMR2 contained more DMRs  translocation, we aligned the DMR2 profile with that of DMR1  translocation, we sequentially compared the differential methylation patterns of the two t de novo/CR-paired samples with those of the three non-t de novo/CR-paired samples. These five patients achieved CR after administration of one cycle of the DCA-based regimen  de novo/CR samples were preferentially interrogated  paired samples were collectively designated “DMR5”  AML, we compared the specific methylation profiles of t AML  translocation than that in patients with other karyotypes AML. A total of 128 non-M3 AML samples, including 7 t AML samples, were analyzed. Upon comparing the expression profiles of t and non-t AML samples, 986 downregulated genes in t AML were identified  AML  AML.In addition, to validate the methylation status of the four genes in t AML, a publicly available AML methylation dataset (GSE18700) comprising 24 t AML and 152 non-t AML samples was employed in this study. Through alignment with the 210 hypermethylated DMR7 sequences, a cluster of 10 probes that targeted 7 genes were obtained, among which 3 probes annotated with LIN7A can serve as a predictor of the t AML response to DAC, we conducted an in vitro study exploring the relationship between DAC and LIN7A expression. First, we examined whether DAC affects LIN7A expression in Kasumi-1 and SKNO-1 cells. Notably, LIN7A expression was significantly elevated upon treatment with 10 nM DAC at the mRNA  for methylation-specific PCR analysis. The mapped regions within the LIN7A DMR were methylated in the Kasumi-1 cell line  AML, we knocked down DAC Fig. m. We connes Fig. n, o.LIN7A-knockdown cells after treatment with DAC and Ara-C  AML cells.Given that the sequential combination of DAC and Ara-C has a synergistic pro-apoptotic effect on AML cells , we evala-C Fig. a–d. Consp15, p16, and CDH1) failed to predict DAC efficacy [DAC-based regimens are used as an alternative therapy for patients who are ineligible for intensive chemotherapy. However, the clinical efficacy of DAC-based treatments varies greatly among patients, resulting in the currently unmet clinical need for more predictable biomarkers. Genes directly involved in DNA methylation regulation, such as DNA methyltransferases (DNMTs), have been of interest for decades. However, although DNMT1 is directly inhibited by DAC, its expression is unrelated to the DAC response . Moreoveefficacy . Notablyefficacy , which mHerein, we initially retrieved the DMRs between t and non-t  AML samples. The significant variation observed between AML1-ETO-positive and -negative samples directly indicated the influence of AML1-ETO on the DNA methylation profile. This event may be mediated by DNMT1  and DNMTAlthough DAC is recognized as a demethylation agent, our study showed that a large number of DNA regions acquired methylation modification following DAC-based combination therapy. This might be a consequence of the combination regimen since eliminating leukemia blast cells drives bone marrow cells to exhibit normal phenotype. However, the methylation restoration effect of DAC cannot be ruled out, considering that numerous DNA regions were methylated in cells treated with DAC only.LIN7A may be a key gene that influences DAC treatment efficacy. Furthermore, we demonstrated that LIN7A mediates the synergistic anti-apoptotic effect of DAC and Ara-C treatment in t AML.Moreover, upon mapping DMRs with reference genes, we observed that more DMRs annotated within the gene body compared with those in promoter regions, thus emphasizing the importance of including non-promoter regions in DNA methylation studies. In contrast to the suppressive effect elicited by methylation in promoter regions, DNA gene body methylation has been reported to promote gene expression . MeanwhiCollectively, our study suggests that LIN7A may serve as a specific biomarker for predicting treatment responses to DAC-based therapy in t AML patients. However, these findings warrant further confirmation through in vivo studies and clinical assessment.Additional file 1: Table S1. Primers used in MSP analysis. Table S2. Primers used in qRT-PCR. Table S3. The 72 gene list derived from DMR7. Table S4. 986 downregulated genes in t AML.


Upon perception of cold stress, Ca2+ channels and/or Ca2+ pumps are activated, which induces the Ca2+ signatures in plant cells. The Ca2+ signatures spatially and temporally act inside a plant cell and are eventually decoded by specific Ca2+ sensors. This series of events results in the molecular regulation of several transcription factors (TFs), leading to downstream gene expression and withdrawal of an appropriate response by the plant. In this context, calmodulin binding transcription activators (CAMTAs) constitute a group of TFs that regulate plant cold stress responses in a Ca2+ dependent manner. The present review provides a catalog of the recent progress made in comprehending the Ca2+ mediated cold acclimation in plants.Exposure of plants to low temperatures adversely affects plant growth, development, and productivity. Plant response to cold stress is an intricate process that involves the orchestration of various physiological, signaling, biochemical, and molecular pathways. Calcium (Ca The signal transduction pathways comprise various protein and non-protein elements. The protein elements encompass various enzymes, receptors, and TFs, while the non-protein elements include second messengers such as Ca2+, cyclic AMP, cyclic GMP, inositol triphosphate, diacylglycerol, lipids, and hydrogen ions. Amongst all the reported second messenger molecules, Ca2+ is considered central to several signal transduction pathways , but upon receiving signals to respond, the Ca2+ channels are transiently opened, resulting in the rapid influx of Ca2+ ions inside the cell. This eventually leads to an increase in cytosolic Ca2+ ([Ca2+]cyt) levels. The levels of Ca2+ ion inside the cell fluctuates either due to Ca2+ influx via dedicated channels or Ca2+ efflux via specific pumps , glutamate receptor-like channels (GLRs), stretch-activated Ca2+ channels (OSCAs), and the MID1-complementing activity (MCA) , ER-type Ca2+-ATPases (ECAs), mitochondrial Ca2+ uniporter (MCU), P1-ATPases , Ca2+ exchangers (CAX), two-pore channel (TPC), 1,4,5-trisphosphate receptor-like channel (InsP3R), 1,4,5-trisphosphate (IP3), cyclic ADP-ribose (cADPR)-activator ryanodine receptor-like channel (RyR), slow-activating vacuolar channel (SV), and sodium–calcium exchanger (NCX).Plants sense and respond to distinct environmental and developmental cues pathways . Ca2+ isot hairs . Ca2+ siignaling . Brieflyic pumps . In Arabty (MCA) . Several vacuole . These i2+ signatures that are sensed and recognized by specific Ca2+ sensors. This cascade of events eventually results in transcriptional and metabolic responses  motif. Multiple EF-hand containing proteins are present in plants, and Ca2+ sensors represent just one of the many that translate chemical signals into an appropriate biochemical response. The EF-hand motif is represented by a conserved helix–loop–helix structure that binds to one Ca2+ ion. They occur in pairs as distinct domain, hence, the majority of Ca2+ sensors harbor two, four, or six EF-hands . Three major classes of Ca2+ sensor families have been recognized in plants, namely, (i) Calmodulins (CaMs) and calmodulin-like proteins (CMLs), (ii) calcineurin B-like proteins (CBLs), and (iii) Ca2+-dependent protein kinases (CDPKs)  along with the Ca2+ sensing domain. Accordingly, CDPKs act as sensor responders to directly activate and regulate their TPs upon sensing Ca2+ signals , which is sensed by Ca2+ sensor—CaM. Eventually, CaM in a Ca2+ dependent manner regulates the transcriptional activity of the CAMTA gene, withdrawing an appropriate response by the plant against cold stress. The present review summarizes the progress made in the recent years to comprehend the involvement of Ca2+signaling in cold stress tolerance  . CaMs ar (CDPKs) . CaMs, Ct manner . Differe signals . Thus, tt manner . The CAMolerance .via key components of Ca2+ signaling  is implicated in an accurate development of the cold-acclimation response by regulating the induction of CBF/DREB1 and downstream genes (2+/cation antiporter (CaCA) superfamily proteins have been identified in Saccharum to play pivotal roles in environmental stresses, including cold , and ZjCNGC2 was reported to regulate signaling cascades in response to cold stress  are also implicated in tolerance toward low temperature  genes. It remains a subject of further evaluation whether COLD1 plays a role as a Ca2+-permeable channel or as a mediator promoting Ca2+-permeable channel activity. Taking into account another plasma membrane-bound Ca2+ channel—GLR—mediate Ca2+ fluxes across membranes and is responsive to an array of exogenous and endogenous signals in plants. AtGLR3.4 localizes to the plasma membrane and is stimulated by cold stress in a Ca2+-dependent manner -and cold-responsive genes. The study concluded that GhCAX3 plays an imperative part in the cross-talk of cold and ABA signal transduction  coupled with RICE G-PROTEIN α SUBUNIT1 (RGA1) was reported in cold stress signaling . sativa . The COLt manner . AtGLR1.thaliana . The colon lines . Similareostasis . Next, astresses . Recentlthaliana . The mutd stress . Furthersduction . Likewissduction .2+ sensors that sense and decode rapid and transient fluctuations in the intracellular Ca2+ levels in response to environmental cues. In plants, CaMs and CMLs have been reported to play pivotal roles in developmental and stress biology . Incubation at 4°C or 10°C induced the expression of six CML genes  . All the four splice variants were highly induced under cold stress. Heterologous expression of CML21v2 and VaCML21v4 in A. thaliana increased the survival percentage of the transgenics upon freezing. Cold stress-responsive marker genes: dehydration-responsive element-binding, AtDREB1A and AtDREB2A were induced in VaCML21v2 overexpression lines, while AtCOR47, AtRD29A, AtRD29B, and AtKIN1 genes were induced in VaCML21v4 overexpression lines after freezing stress in the transgenic Arabidopsis plants. Thus, it was established that CML21 acts as a positive regulator of cold stress  were functionally characterized under various environmental stresses. The transcript levels of CsCML16, 18-2, and 42 were significantly induced by low temperature and salt stress (Solanum habrochaites (cold-tolerant wild tomato) ShCML44 gene was functionally characterized under a variety of environmental stresses, including cold stress. The ShCML44 overexpressed plants had higher antioxidant enzymes activity, better gas exchange and water retention capacity, lower malondialdehyde (MDA) accumulation and membrane damage, reduced reactive oxygen species (ROS), and higher relative water contents —CaM antagonist were involved in ABA-induced transcription of OsCML16 and its target genes. OsCML16 and its target genes were triggered by salt, drought, and low-temperature stress  . The samd stress . Likewisd at 3 h . In a sit stress . Previoucontents . Very reolerance . Additioing time . Furthere stress .crlk1 mutants compared to control plants are sensitive to freezing temperatures. The expression of cold-responsive genes, such as, CBF1, RD29A, and COR15a was suppressed in crlk1 mutants, making them more susceptible to cold stress than control plants. CRLK1 protein expression is induced upon low temperature (4°C) exposures and oxidative stress (H2O2). Thus, CRLK1 is considered a positive regulator of cold stress responses in A. thaliana. Additionally, the Ca2+/CaM complex is a requisite for triggering CRLK1 kinase. It has been reported previously that an increase in CaM levels in the presence of Ca2+ elevates the activity of CRLK1 kinase. On the contrary, chlorpromazine (CPZ)—CaM antagonist blocked the CaM mediated CRLK1 kinase activity (C-repeat binding factor (CBF) and COR genes  encoding a plasma membrane-associated serine/threonine kinase has been reported to play a crucial role in cold stress responses , 2014. Tactivity . Expliciactivity . BesidesOR genes . CRLK1 aOR genes . Hence, by CRLK1 ,b.2+ binding proteins and are considered imperative relays in plant Ca2+ signaling pathways. CBL and CBL-interacting protein kinase (CIPK) complex are central to Ca2+ signaling. This complex had been reported to be implicated in a plethora of external stress signals -N,N,N′,N′-tetraacetic acid (EGTA)—Ca2+ chelator and lanthanum chloride—Ca2+ channel blocker significantly altered [Ca2+]cyt in cbl9 mutants (Camellia sinensis (tea plant), it was shown that CsCBL9 and CsCIPK4/6a/6b/7/11/14b/19/20 were upregulated in both mature leaves and young shoots upon cold stress. Results of yeast two-hybrid assay demonstrated that CsCBL1 potentially interacted with CsCIPK1/10b/12 but not with CsCIPK6a/7/11/14b/20. Similarly, CsCBL9 interacted with CsCIPK1/10b/12/14b but not with CsCIPK6a/7/11/20. Thus, the study proposed distinct responses to cold stress mediated by CBL–CIPK complexes  CBL–CIPK signal networks function in responses to abiotic stresses. MdCIPK6L ectopic expression significantly enhanced chilling tolerance in transgenic tomatoes  after 4 h of cold treatment in one of the in-bred lines of Brassica rapa (Chiifu)  . Furtherthaliana . On simid stress .2+ signals , thereby, sustaining higher glutathione levels and phosphorylation. OsCPK24 has been shown to positively regulate cold stress tolerance   MdCPK1a gene was reported to enhance tobacco cold resistance via scavenging ROS accumulation , peroxidase (POD), and catalase (CAT) activities were higher, while electrolyte leakages (EL), MDA content, and ROS were lower. This was suggestive of the fact that the transgenics underwent less chilling injury than control plants  . Overexpcultures . Earlierpathways . On simiin maize . ZmCPK1 d stress . Recentlmulation . The undl plants . Thus, Cn plants .2+ signals are elevated, which might direct Ca2+ to either repress or activate the activity of Ca2+ responding protein. Similarly, the interaction of Ca2+ with Ca2+ sensors either suppresses or enhances the binding to a TF. Depending upon whether the TF itself is a repressor or activator, the transcription of the target gene is repressed or activated. Ca2+/CaM dependent TFs relay cold-induced Ca2+ transients to transcriptional reprograming. CAMTAs are one such group of TFs that regulate plant cold stress responses in a Ca2+-dependent manner. CAMTA proteins have been stipulated to play a direct link between Ca2+ signals and cold acclimation . CM4 and CM6 have negative regulatory activity, while CM2 has both negative and positive activity. The study also revealed that CAMTA3 binds to the CM2 motif and is a positive regulator of CBF2 expression. Moreover, camta1 camta3 double mutant plants were impaired in freezing tolerance. This study exhibited a novel role of CAMTA in cold acclimation and provided a plausible link of low-temperature Ca2+ and CaM signaling with cold-regulated gene expression  . However, the SA levels increased in plants exposed to low-temperatures for more than one week. The study revealed that the isochorismate synthase (ICS) pathway is involved in chilling-induced SA biosynthesis. The accumulation of ICS1, CBP60g, and SARD1 transcripts were suppressed at warm temperatures by these three CAMTAs, but not at low temperatures  are necessary for its activity  were partially restored to control plants. Moreso, the suppression of SA biosynthesis in the mutants was compromised, suggestive of the fact that phosphorylation is necessary for the full functionality of AtCAMTA3  has a central role in transcriptional machinery at low temperatures . However CAMTA3) . CAMTA1 eratures . The anal plants . The sturomoters . Anotherromoters . At low-HP genes . This suactivity . Post-tr protein . The camAtCAMTA3 . It is wAtCAMTA3 . IntriguAtCAMTA3 . NonetheAtCAMTA3 . These oolerance . Thus, C2+ transients (2+ transients are generated via a number of Ca2+ channels and/or Ca2+ pumps. These Ca2+ transients are relayed and decoded by a variety of Ca2+ sensors to regulate gene expression and subsequently confer cold tolerance to plants (2+ signaling network, such as, Ca2+–CBL–CIPK, CDPK, and Ca2+–CaM–CAMTA (2+ signaling pathways to confer cold tolerance in plants. It is still a challenge to deeply decipher the role of Ca2+ signals in the cold stress tolerance mechanism and to ascertain whether cold stress-triggered Ca2+ transients exist in the cell nucleus. Cutting edge techniques such as multi-omics (2+ imaging (2+ signaling pathways. Thus, future research should focus on deciphering the key converging and diverging pathways pivotal to Ca2+ mediated cold signaling. Further, gaining in-depth insights as to how Ca2+ signatures are induced and decoded in response to cold stress can help better comprehend the involvement of Ca2+ ion in cold stress signaling. Nonetheless, efforts should be made to identify low-temperature sensors using biological methods in combination with biochemical and biophysical approaches.The underpinning mechanisms of cold signaling pathways and genes implicated in cold stress have been extensively studied in the past few years. Different signaling pathways converge to allow plants cope with cold stress. Perception of cold stress by the plant is contemplated to be the first event for the induction of Caansients . The colo plants . ConsideaM–CAMTA . Moreoveti-omics , CRISPR/ti-omics , and sen imaging  can provZI drafted and wrote the manuscript. AGM and AA critically revised the manuscript for consistency and content. MSI conceptualized the idea and reviewed the manuscript. All authors reviewed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as “acrosome exocytosis” (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed “acrosome reaction” model. The “acrosome reaction” model involves receptors on the sperm membrane being triggered by physical contact with ligands on the zona pellucida (ZP), resulting in exocytosis of the acrosome, the sperm’s single exocytotic vesicle. This long-dominant model defines two states or conditions for sperm—acrosome-intact or acrosome-reacted. In this model, it is thought that creating pores between the plasma membrane and outer acrosomal membrane (OAM) would result in loss of acrosomal contents in a manner analogous to popping a water balloon. Almost 2 decades of data challenge this thinking, suggesting a more complex, multistep model of “acrosome exocytosis” (AE). However, the acrosome reaction model has proven remarkably persistent, due both to its simplicity and lack of precise understanding of the steps involved in AE. The multistep AE model is based on numerous lines of evidence from different laboratories, including that putative receptors for the ZP are found in the acrosomal matrix  and then2+ dynamics. There are multiple factors underlying this, including (A) sperm are transcriptionally and translationally inactive with no culture systems available to produce/manipulate mature sperm; (B) mature sperm are motile and difficult to image; (C) across mammalian species, only a subset of sperm are actually capable of fertilizing  spedynamics , 17, 18;dynamics , 20; andM1 play a regulatory role in enabling AE through at least 2 complementary pathways in the plasma membrane overlying the acrosome . First, sterol efflux enables proteolytic activation of phospholipase B, which cleaves both tails of a phospholipid molecule  is also a critical regulator of sperm function. Released by cumulus cells and encountered by sperm in the vicinity of the egg, P4 has been reported to facilitate multiple events in sperm including stimulation of capacitation , hyperacto sperm ; these C channel , 38. Pat2+ dynamics in sperm, we generated a novel mouse model with a genetically encoded Ca2+-sensor (GCaMP3) in fusion with mCherry that we targeted to the acrosome. This positions it both to reflect Ca2+ concentrations in that compartment and reflect the status of the acrosomal contents, in live sperm, in real time, under physiological conditions. As a component of the matrix, loss of the photostable and pH-insensitive mCherry fluorescence from the sperm would reflect membrane fusion (MF) and AE akin to the acrosome-targeted GFP mouse line . We targeted the GCaMP–mCherry fusion protein to the acrosome using the acrosin promotor and signaling peptide  or that would mimic sterol acceptors found in the female tract in a reductionist, controlled way , or stimuli that exist on sperm but are released from inhibition provided by the binding of seminal plasma proteins (GM1). Data generated from these AcroSensE mice support a complex, multistep AE model and are inconsistent with the traditional acrosome reaction model, helping resolve the existing controversy and providing new insights into functionally redundant mechanisms regulating sperm AE.Here, we characterize this novel model (Acrosome-targeted Sensor for Exocytosis [AcroSensE]) and then use it experimentally to inform our understanding of AE and sperm CaA). The mCherry and GCaMP3 were coupled by a 23-aa linker  demonstrated that the AcroSensE protein was expressed predominantly in testes . Moreover, we were not able to detect expression of the AcroSensE construct at the protein level in any tissue but sperm using fluorescence microscopy .To generate the AcroSensE plasmid, we replaced the GFP sequence in the pAcr3-EGFP backbone plasmid  with a cFig. 1t linker . TransgeM1, CD, or P4. Preincubation of populations of sperm under capacitating conditions was performed with some stimuli (CD with/without bicarbonate) to mimic the physiological timeline when sterol efflux would occur relative to the effects of those other molecules . To stimulate exocytosis in single cells, a short (10 s) puff of stimulant was applied near an individual sperm via micropipette while recording the changes in GCaMP3 and mCherry fluorescence intensities at image rates of up to 5 frames per second for each wavelength. Concentrations are indicated for the solution in the micropipette, as specified in the Sperm from AcroSensE mice were collected as previously described . FollowiM1 . Under noncapacitating control conditions (no CD or GM1), 10.7% of sperm underwent AE. After 90 min, 36.3% of sperm underwent AE in response to GM1, 45.2% in response to CD, and 47.1% in response to both CD and GM1. At this time point, the value for noncapacitating conditions also increased to 25%. These percentages are similar to our prior observations (2+ rise (ACR) and MF upon stimulation with various reagents mentioned in the following part of the article.To understand baseline effects and aid interpretation, we quantified the percentage of w.t. B6 sperm undergoing AE as a result of initial incubation under noncapacitating and capacitating conditions . Using Crvations . BecauseAfter preincubation for 30 min, the AcroSensE sperm were then imaged. In each trial, sperm from multiple mice were pooled, and typically only 1 to 2 of the experimental conditions described here were tested per trial to ensure that length of preincubation would not be a confounding factor in interpreting results from different conditions. Under these experimental conditions, we observed several steps in the process leading to AE, consistent with ACR, and full MF leading to loss of the mCherry fluorescence and therefore full AE. In some cells, we saw traces similar to prespike foot (PSF) events as frequently observed when using amperometry to study exocytosis; however, these were on a much slower time course than when observed in other cells using amperometry. Although temporally these PSF-like events were the earliest events we observed using AcroSensE fluorescence, they were observed infrequently and only under specific circumstances. Therefore, we will first describe the more common ACR and MF responses to the different treatment conditions.A and D; note that population data are summarized in B and C). Following the addition of ionophore A23187, one subpopulation of sperm demonstrated rapid ACR which was typically quickly followed by MF as evidenced by loss of mCherry signal . In contrast, the remaining 60% of sperm responding to A23187 showed a slow, gradual ACR that did not result in AE within the time frame of image acquisition .i.e., sterol efflux mediated by addition of CD and sterol efflux coupled with exogenous GM1,B and C) typically resulted in a lower proportion of cells responding, but more uniformity in having a rapid ACR, followed quickly by MF in most of the responding sperm. The notable exception was the case of CD stimulation in the absence of extracellular Ca2+ (CD + EGTA), in which no ACR was observed. Interestingly, although the EGTA sufficiently reduced Ca2+ in this condition such that there was no detectable ACR (D), 17.1% of sperm still underwent exocytosis .Stimuli resulting in alterations in membrane lipids . Excepting the CD+EGTA treatment, under all other conditions in which MF and AE were observed, a rise in GCaMP3 signal preceded the loss of mCherry fluorescence. These results combine to suggest that ACR resulted from extracellular Ca2+ entry into the acrosome and occurred prior to exocytosis of the acrosomal contents. This temporal pattern is illustrated in Figure 3A and further discussed.Both low and high P4 conditions (with and without bicarbonate) induced ACR in varying percentages of sperm, with several differences seen in patterns of CaA provides a schematic representation of the typical observations for the AcroSensE sperm, in which time (X axis) is expanded relative to B and C summarize population data of the ACR and MF responses of cells to the various stimulations. In response to the addition of ionophore (A23187) following preincubation with 3 mM CD, 75.3% of the cells exhibited ACR and 30.1% exhibited MF (B). As described earlier in the study, 60% of cells responding to A23187 with ACR did not undergo MF, out of which 10.6% had fast ACR and 82% of cells had slow ACR (89.4% of sperm with fast ACR and 18% with slow ACR did progress into full MF).Figure 3bited MF B. As desM1 (following preincubation with CD) to the sperm’s plasma membrane, or induction of sterol efflux by addition of CD alone (with none added during preincubation), resulted in a lower percentage of cells responding with ACR than that responding to A23187 . However, the vast majority of these responding cells also exhibited MF/AE . Note that the value of 25.3% of sperm undergoing MF in response to single-cell application of CD without prior exposure during preincubation is very close to what we saw when preincubating populations of sperm with CD as noted earlier, giving us confidence that exposure to this mediator of sterol efflux under these conditions of single-cell application had similar effectiveness regardless of the method of exposure. Recall that because preincubation with CD occurred first, and then the GM1 was added, those cells responding to GM1 represent a subpopulation that had not already responded to CD-mediated sterol efflux alone within the preincubation time frame. The combined percentage of sperm exhibiting MF in response to modulation of these two membrane lipids was therefore approximately 45%  but roughly comparable transition rates to that seen with A23187 . However, keep in mind that of sperm that responded to A23187 quickly, 89.4% underwent MF/AE, more consistent with CD alone and GM1 + CD.Comparison of responses under the other conditions revealed very different percentages of sperm experiencing ACR B but rouincrease in the fluorescent signal coming from the GCaMP3 was used to localize the initiation site of the intensity change associated with ACR, whereas the initiation site for AE was noted by the initial site of mCherry signal loss. Changes in fluorescence in both wavelengths were used to track the directionality and progression of Ca2+ rise and AE.Live cell microscopy of AcroSensE sperm enabled us to determine the spatial characteristics of ACR and MF events. As seen in A. We observed 4 main patterns of progression: (1) diffuse—in which the signal initiated evenly over the entire apical acrosome area (D), (2) top to bottom—initiating from a rostral position  and progressing caudally  toward the postacrosomal region (T > B), (3) center outward—initiating at the center and propagating in opposite directions to the top and bottom of the apical acrosome (C > TB), and (4) bottom to top—progression rostrally from near the subacrosomal ring to the top (B > T). Following A23187 and various P4 stimulations, the plurality of responding cells showed diffuse patterns of Ca2+ rise throughout the acrosome. In sperm following the addition of exogenous GM1 or induction of sterol efflux by CD, this pattern was observed less frequently. Particularly, addition of GM1 or CD to cells resulted in increased incidence of signals initiating from the top or top/center (A). Initiation of ACR from the bottom of the acrosome near the subacrosomal ring was consistently lower under all conditions .A summary of the findings for the spatial characteristics of ACR formation is provided in Figure 5p/center A. InitiaB), as determined by the loss of the mCherry signal, spatial patterns were similar with the distinction that instead of a signal propagating from the center to the ends as seen for ACR (C > TB pattern), we observed MF initiating from both ends and moving toward the center . In cells incubated with bicarbonate during preincubation, we observed higher rates of B > T patterns of loss of the mCherry signal . In contrast, higher incidence of T > B were observed when P4 concentration (+CD) was low . All patterns were observed relatively equally upon GM1 stimulation, while addition of CD alone resulted in slightly less than half of the sperm demonstrating a diffuse pattern of release of the AcroSensE during AE .For MF B, as detC). Interestingly, upon A23187 stimulation, ACR transformed into AE almost exclusively if the ACR events initiated at the center of the sperm head (C > TB). Under all conditions, cells were less likely to demonstrate progression from ACR to MF if the ACR events were diffusely localized or had a bottom-to-top progression. Addition of exogenous GM1 to the cells had a higher chance to generate MF (80% of cells) if the ACR events initiated at the center or the top of the sperm head, but not at the bottom (20% of cells). In the case of CD and low P4 with CD and bicarbonate, at least 50% of the sperm undergoing AE demonstrated ACR initiating rostrally (at the top of the sperm head) and moving downward toward the subacrosomal ring .Next we examined the percentage of cells transitioning from ACR to full MF and AE as a function of the initiation site of the rise in the AcroSensE fluorescence intensity C. IntereD provides the percentage of cells in each condition where the ACR and MF initiated at the same location. Interestingly, in AcroSensE sperm stimulated with GM1 or CD, we see the highest rates of both ACR and MF demonstrating similar initiation sites . P4 stimulation demonstrated somewhat lower rates of spatial correlation for ACR/MF initiation, up to 36% (for high P4). In contrast, A23187 demonstrated the lowest spatial correlation, with only 14% of the cells showing ACR and MF initiating at the same location. These data show that AE can initiate independently from the original site of ACR, but a stronger spatial link between that site and MF was associated in membrane lipid–mediated signaling.Finally, we looked at the spatial correlation between the initiation sites of ACR and MF. Figure 5A). Conversely, ACRs were slowest to initiate in sperm stimulated with exogenous GM1 (231 ± 31 s), more than 12 times slower. The trend of onset times between the different conditions was consistent with the values observed for time to peak, in which GM1-mediated ACR was the slowest and P4 was the fastest .Next we examined key temporal characteristics of ACR as they occurred under various conditions . StrikinFig. 6M1 (A and B) corresponded with shorter rise durations (C). The rise duration (C) indicates the timeframe in which the ACR either reached its peak value or diminished by the occurrence of AE. Comparable results were found for GM1 (18.7 ± 5.4 s) and CD stimulation , whereas P4 demonstrated approximately 2-fold longer time-to-peak duration . Compared to all other conditions, the average time-to-peak duration was significantly longer following A23187 stimulation.Surprisingly, these trends were followed in reverse regarding the total rise time (rise duration), in which longer start times and times to peak for CD and GM1 , A and Burations C. The riduration C indicatD summarizes the increase in the GCaMP3 fluorescence signal, which corresponds to the net change in the acrosomal Ca2+ concentration during ACR. Our data show that for A23187 (576 ± 20 ΔF) and High P4 with bicarbonate (577 ± 32 ΔF), the total Ca2+ rise in the acrosome was almost twofold higher than for CD , or High P4 without bicarbonate during capacitation , or Low P4 with bicarbonate . GCaMP3 signal increase in response to GM1 was only marginally lower than the ionophore A23187 (p < 0.0081) or high P4 with bicarbonate (454 ± 43 ΔF).Significant differences of almost 2-fold were also observed between the various conditions for the GCaMP3 signal when looking at the rise amplitude (ΔF). Figure 62+ signal rise  were the highest following GM1 and CD stimulations , with other conditions being approximately two-fold slower (E). However, there was more cell-to-cell variation in this parameter reducing the statistical significance of many differences.Conversely, the kinetic rates of GCaMP3 Cad slower E. HoweveF breaks down the slope (ΔF/sec) for the sperm in each condition that either underwent MF (AE) or did not. The data clearly show that although there was particularly high variance when stimulating with P4, in all conditions, cells that successfully transitioned from ACR to MF had significantly higher rates of GCaMP3 signal increase .Remarkably, the kinetic rate of GCaMP3 fluorescent signal rise was directly associated with the ability of the stimulated sperm to undergo MF and AE. Figure 62+ rise (ACR), and while demonstrating much slower timescale, their occurrence and profile are reminiscent of PSF-like events observed in a variety of secretory cells when measured with amperometric methods using carbon fiber electrodes . The PSF-like events were defined as a Ca2+ signal rise that preceded the major ACR event and which demonstrated reduced amplitude and kinetics  in comparison to that ACR event. Figure 7A shows the application of these criteria for determining a PSF event, where the GCaMP3 signal rises above the baseline but demonstrates a distinctive dissimilar rise rate compared to the ACR. In addition, the PSF-like events were significantly smaller (4–7 fold) in amplitude than the ACR, depending on the condition, and resulted in a biphasic increase. Together, these two criteria were used to define these events. Except for sperm incubated in medium depleted of extracellular Ca2+ (EGTA in medium), PSF-like events were detected under all conditions, suggesting that these miniature elevations in the GCaMP3 signal were dependent on influx of extracellular Ca2+ into the acrosome. Nevertheless, considerably higher incidence of PSF-like events was observed in cells stimulated by A23187  and in cells stimulated with high P4 following capacitation in the presence of CD and bicarbonate .Unexpectedly, the AcroSensE sperm revealed what is typically a subtle intermediate step in the process of MF and cellular exocytosis . These eectrodes . Figure C). The amplitude values of the PSF-like events  were independent of the duration parameters, demonstrating the highest values in cells stimulated with high P4 with bicarbonate and in cells stimulated with ionophore A23187 . Nonetheless, by definition, the average amplitude was markedly lower than the amplitude of ACR (4–7 fold), indicating the limited amount of Ca2+ ions able to enter the acrosome via the PSF-like events.Although their manifestation in response to high P4 stimulation was lower in the absence of bicarbonate, PSF-like event duration in this condition was roughly 3 times longer (42.5 ± 4.6 s) than that of low P4 or high P4 with bicarbonate . In contrast, following the addition of GM1 or A23187, cells demonstrated the longest delay to the onset of the MF responses . Note that the trends of start times between the different conditions were comparable for ACR and MF.In mice expressing GFP in the acrosome, loss of fluorescence results from loss of acrosomal matrix contents due to MF events . As showFigure 82+ conditions demonstrated the shortest duration times of the mCherry signal dispersal from the acrosome . On the other hand, stimulation of cells with P4 resulted in the longest duration periods regardless of the presence or absence of bicarbonate during capacitation . All other conditions demonstrated similar times for the diffusion of the AcroSensE out of the acrosome ranging from 11 to 15 s on average.Next, we determined the duration of MF events, as calculated from the time frame of the start of mCherry signal loss to complete loss of signal, consistent with the completion of AE. Interestingly, cells stimulated with CD under zero CaD summarizes the ACR–MF time interval, as calculated from the time between the onset of the ACR  and the initiation of the MF . The ACR–MF time interval showed little variability among the various conditions, except for cells stimulated with CD where the delay between Ca2+ rise and MF was considerably shorter than all other conditions (6.1 ± 3 s). Importantly, no significant differences were observed for the total amount of mCherry fluorescent signal lost as demonstrated by the ΔF/F0 analysis provided in Figure 8E, suggesting that under all conditions tested here, once AE occurred, the loss of protein content (matrix) from the acrosome vesicle was to a similar extent. Conversely, the rates of protein diffusion out of the acrosome during AE were dependent on the stimulation, where P4 signaling resulted in slower protein release rates and lipid modulation of the sperm or addition of A23187 induced generally faster rates of protein diffusion out of the matrix within the acrosome vesicle (F).As a single fusion protein comprising both the GCaMP3 and mCherry elements, the AcroSensE construct also allowed us to look at the delay time between ACR and full MF. Figure 8 vesicle F.2+ dynamics in the compartment of the acrosome and MF between the plasma membrane and OAM. Differences in response to varied stimuli in turn revealed important insights into the mechanisms and pathways leading to AE, enabling us to generate a working model (shown below) that summarizes current understanding.Sperm from the AcroSensE mice demonstrated spatiotemporal relationships between Ca2+ for ACR. Interestingly, some exocytosis was observed under (nonphysiologic) Ca2+-depleted conditions. These were the only conditions under which MF occurred without ACR. This observation could be explained by some combination of two known mechanisms that are not physiologically relevant: EGTA causes membrane ruffling/perturbation (2+-independent PLA2β has been shown to be responsible for spontaneous (unstimulated) AE  AE .M1). The methodological soundness of delivering these stimuli via puffing by micropipette was supported by the similarity of results with puffed CD to those seen when we preincubated sperm with CD in the medium (M1 closely matched those when incubating sperm in media in prior studies (Based on the current understanding that most sperm undergo AE before they reach the zona pellucida (which had been the main stimulus in the acrosome reaction model) , 7, 48, e medium . Also, c studies . One intvia the increase in GCaMP3 signal mediated by Ca2+ flux into the acrosome. A limited number of FPs, FPs of a transient nature, and/or FPs of very small diameter could all account for the PSF-like events. As the number/size of FPs would increase, this would lead to more rapid ACR, and simultaneously the increase in MF events would be predicted to result in quicker AE. This relationship would account for our finding that the rate of ACR in sperm undergoing MF/AE was higher than that in cells that did not demonstrate MF (F). However, mechanisms for Ca2+ entry into the acrosome other than FPs must also be considered:1.GCaMP3 signal increase was a result of Ca2+elevation in the acrosome generated by mobilization from other internal stores. Mobilization of Ca2+ ions from one of the sperm’s internal stores , demonstrating the dependence of the acrosomal GCaMP3 signal on extracellular Ca2+. Moreover, this alternative is also spatially incompatible with our observations, as both the putative Ca2+ stores in the RNE and mitochondria are located in the connecting piece/midpiece of the sperm, whereas we frequently observed high occurrence of ACR initiating at the top  or center of the sperm (A).pe [RNE] ) into thhe sperm A.2.The ACR was mediated via transporters and/or channels localized on the OAM. In this alternative model, Ca2+ entry to the acrosome occurs in a two-step process, where cytosolic Ca2+ increase (via channels/transporters on the APM or release from RNE/mitochondria stores) is followed by Ca2+ entry into the acrosome through either a channel or a transporter on the OAM. Indeed, several proteins have been reported to be involved in acrosome Ca2+ mobilization including Ip3R  has been shown to be expressed in mammalian sperm (2+ uptake into the acrosome (2+ ATPases (SPCAs) have been reported to function in mammalian sperm –Orai channel complex involved in store-operated Ca2+ entry . Bean sperm  and reguacrosome . There aacrosome , suggestan sperm . Althougand Ford ]. In addchanisms ), it is 2+ entry , 58, 59.2+ entry  and mous2+ entry  by Westey , and Cl−/HCO3− exchanger . Duringxchanger . Thus, tacrosome , and the4.Release of sequestered Ca2+from Ca2+-binding proteins. Several types of Ca2+-binding proteins have been detected in spermatozoa, including calmodulin, calreticulin, CABS1, and glucose-regulated protein 78 (BiP), although the latter demonstrates only low Ca2+-binding capacity , which were not coupled to mechanisms promoting MF/AE. Second, this subpopulation of cells might have had other differences that decoupled acrosomal Ca2+ concentrations and MF/AE. For example, differences in membrane lipid composition such as in local sterol abundance could render them less fusogenic. Regardless, the retention of mCherry signal showed that AE was not triggered in these cells. Under all conditions, the rate of ACR was much higher in sperm that underwent MF/AE than in sperm that did not (F). Whether the rate of ACR itself regulates MF such as through stimulation of MF machinery  or whether a faster rate of ACR results from pre-existing membrane differences that would have made them more fusogenic cannot be determined by these correlations and will be a topic of future study.Our results showed clearly that it was the rate of ACR, and not how long it takes to induce it or the absolute amounts of Cago MF/AE . This wa did not F. WhetheM1, or the removal of sterols. The architecture of the sperm head and localization of microdomains highly enriched in sterols can also lead to mechanical resistance to fusogenicity . This would make sense if this sphingolipid acted as previously proposed, by inserting into the plasma membrane and then causing a focal enrichment, leading to a transient that would then locally prime MF machinery, facilitating AE in response to a subsequent Ca2+ wave (D). This would be consistent with a mechanism of action involving activation of PLB, which would cause immediate local changes in membrane curvature through its hydrolysis of both fatty acid tails at the sn-1 and sn-2 sites, leading to MF events (C). If broken out by suppopulation, those sperm responding to A23187 with a fast ACR had a similar transition percentage to GM1 and CD as noted earlier in the study.Mechanistically, some differences in the patterns and kinetics of ACR can be explained by the hypothesized mechanisms of action for each stimulus. For example, G to peak , A and Ba2+ wave . In conta2+ wave D. This wF events . Both ofF events C. If brosurprisingly showed a lack of correlation between the sites of initiation of ACR and MF. GM1 and CD showed the highest levels of correlation, but even these were below 50%. This very simple finding provides compelling evidence against the acrosome reaction model in which fusion between the plasma and OAMs results in explosive exocytosis akin to popping a water balloon.Across all stimulation conditions excepting the small subset of cells responding nonphysiologically in the presence of EGTA, ACR was required for, and preceded, MF and AE. Of critical importance in trying to resolve existing controversy over the process of AE, the AcroSensE model enabled moderately fast imaging with high resolution. Spatiotemporal analysis of rise in GCaMP3 signal and loss of mCherry fluorescence 2+ propagate in different directions  can leak out. Over time, the number of FPs increases in response to the one or more stimuli, and eventually, they coalesce to make hybrid vesicles resulting in full AE. Note that although our data (E) show loss of mCherry signal occurring to the same degree under all conditions, both the length of time over which it is lost (C) and the rate at which it is lost (F) differ between the membrane lipids and A23187 and the progesterone treatments. Given that the fusion construct is a soluble protein, this difference in the rate of loss likely reflects differences in the size and/or number of FPs through which the protein would diffuse out.The most parsimonious explanation for all our findings combined is shown as a model in our data E show lo is lost C and the is lost F differ Overall, the combined data provided by the AcroSensE mice clearly support a regulated, multistep model of AE instead of the prior dogma of the acrosome reaction. Although AE is a common endpoint, different stimuli were found to induce it with different spatial and temporal dynamics underscoring functional redundancy in mechanisms for this critical step, which is essential for the transmission of life.All reagents were purchased from Sigma, unless otherwise noted. Male and female B6129SF/J mice were purchased from Jackson Laboratories. All animal procedures were performed under the guidelines and approved by the Institutional Animal Care and Use Committee at Cornell University.Plasmid construction was performed by BlueSky BioServices. The pAcr3-EGFP backbone plasmid was a gift from Dr Masahito Ikawa (Osaka University). The GCaMP3-expressing vector (#63885) and mCherry were obtained from Addgene. The mCherry was first inserted downstream of the GCaMP3, and then the GCaMP3–mCherry cassette was inserted into the Acr-EGFP plasmid (pBluescript II SK+), which had had the GFP portion excised using EcoRI. The full construct contained a 2.4-kb mouse genomic region carrying the acrosin gene with the sequences of a signal peptide and an N-terminal peptide (KDNTT). The plasmid was verified by sequencing using the following primers: GTGGAGCATTGTGAGGTCACAG and GAGCTTGCCGGTGGTGCAGATG.Acr-GCaMP3-mCherry transgenic mice were generated by microinjection of gel-purified linearized DNA into a pronucleus of 100+ fertilized embryos collected from superovulated mice using standard techniques. Embryos for injection were obtained by mating (C57BL6/J and CBA) F1 hybrids. Transgenic founders were backcrossed to C57BL/6 mice. Primers for genotyping and RT-PCR were GTGGAGCATTGTGAGGTCACAG and GAGCTTGCCGGTGGTGCAGATG. Immunoblotting was performed on tissues collected from the AcroSensE mice including the brain, heart, muscle, testis, and liver. Protein was quantified, and equal amounts of protein were loaded for SDS-PAGE. Blots were blocked with 10% milk overnight at 4 °C. The primary rabbit GFP antibody (Abcam) was diluted 1:2500 in Tween Tris buffered saline to 2.5 μg/μl and incubated for 2 h at room temperature. Anti-rabbit IgG linked to horseradish peroxidase was used as a secondary antibody (Species-Specific F(ab’)2 fragment from donkey; GE Healthcare) and was diluted 1:3000 in Tween Tris buffered saline and incubated at 4 °C overnight.2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid hemi-Ca2+ salt, pH 7.35; (3 (10 mM), and CD (3 mM) were supplemented as needed. Cauda epididymal murine sperm collection was described previously  was useeviously . All ste2 was added. The dish with the sperm was then mounted on a Zeiss 510 microscope with a heated 100X oil immersion objective (37 °C). GCaMP3 was excited using the 488-nm line of a krypton/argon laser and viewed with a 505− to 550-nm BP filter. The mCherry was excited using the 555-nm line of a HeNe laser and viewed with a 575− to 615-nm BP filter. Selected cells were approached with a ∼5-μm-diameter pulled-glass capillary, prefilled with stimulating solution, and positioned within 100 μm from the sperm head. The stimulating solutions were made at 5X the normal concentration  to compensate for diffusion in the volume/space between the capillary and the cell. Sperm cells were imaged at 2 to 4 Hz while applying a puff of 10 s/5 psi from the pipette, controlled by a Picospritzer III . Unless otherwise stated, all data are presented as mean ± SEM. For each of the experiments, the number of cells analyzed (n) is presented in the text or legend. Data were processed and plotted using Origin 8 (OriginLab) and Excel (Microsoft). Statistical comparisons (Student’s t test or χ2) were performed using Excel or JMP Statistical Software and are provided in For microscopy, sperm were plated on poly-D-lysine–coated 35-mm coverslip dishes (MatTek Corp), and 3 ml of warm MW base media supplemented with 10 mM CaClOffline image analysis was conducted using Zeiss LSM image analysis software and ImageJ . Changes in fluorescence (F) were normalized by the initial fluorescence (F0) and are expressed as ΔF.The data that support the findings of this study are available from the corresponding author upon reasonable request.This article contains A. J. T. discloses that he is a founder and an officer of Androvia LifeSciences, LLC, a biotechnology company investigating solutions for male infertility, in which he holds a minor equity stake. There are no conflicts with the current work. All other coauthors declare that they have no conflicts of interest with the contents of this article.