We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs evoked by photolysis of caged IP3 or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca2+ puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged IP3 evoked Ca2+ puffs at a similar number of sites (100±35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential stimulation with carbachol or photolysed caged IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites and the sites become more likely to fire as the IP3 concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.The building blocks of intracellular Ca Summary: Ca2+ puffs are the building blocks for IP3-evoked Ca2+ signals. Ca2+ puffs evoked by caged IP3 or via endogenous signalling pathways initiate at the same fixed intracellular sites. IP3 then binds to IP3 receptors (IP3Rs) to stimulate Ca2+ release from the endoplasmic reticulum (ER). Vertebrates express three subtypes of IP3R and numerous splice variants but each contributes to formation of tetrameric Ca2+-permeable channels that open when they bind both IP3 and Ca2+ (2+ concentration ([Ca2+]c) than those required for IP3R activation inhibit IP3Rs. Within ER membranes, IP3Rs form small clusters  that last just a few milliseconds and reflect the opening of single IP3Rs. Higher concentrations of IP3 evoke longer-lasting local Ca2+ signals (‘Ca2+ puffs’) that are typically 5−10 times larger than blips and result from the almost simultaneous openings of a few IP3Rs  or by IP3 produced after activation of endogenous signalling pathways have been observed in numerous cell types. A common theme is the suggestion that Ca2+ puffs recur at the same small number of subcellular locations and that these sites often initiate global cytosolic Ca2+ signals  have also been restricted to a single cell type (SH-SY5Y neuroblastoma cells) responding to photolysis of ci-IP3 , we show that both stimuli evoke Ca2+ puffs at abundant immobile intracellular sites. We demonstrate that IP3 produced in response to stimulation with carbachol, and i-IP3 produced by photolysis of ci-IP3 are delivered to the same intracellular Ca2+ release sites. Increasing the stimulus intensity causes an increase in the frequency of Ca2+ puffs without recruiting additional Ca2+ release sites.Here, we use TIRFM to image human embryonic kidney 293 (HEK293) cells loaded with a fluorescent Ca2+ signals evoked by different stimuli and stimulus intensities. This aim effectively restricts the field of view to ∼82 µm×82 µm, within which there are typically about six HEK293 cells and rarely more than three HeLa cells. It was, therefore, important to establish that the submaximal stimuli that evoke local Ca2+ signals stimulate responses in most cells. In populations of HEK293 cells, carbachol evoked Ca2+ signals with a half-maximal effective concentration (EC50) of ∼40 µM .Our aim was to define the spatial distribution of the local Caf ∼40 µM . In sing2+ puffs were very infrequent in unstimulated HEK239 cells, but carbachol (10 µM) evoked a flurry of Ca2+ puffs beginning 3.2±0.2 s after carbachol addition  . Within a few seconds most Ca2+ signals then propagated across the cell as a Ca2+ wave, as reported for many other cell types , we confirmed the significant increase in the frequency of Ca2+ puffs in HEK293 cells stimulated with carbachol and in HeLa cells stimulated with histamine  detected during a 10-s period of stimulation are similar in HEK293 and HeLa cells  (2+ puffs (3 evoked frequent Ca2+ puffs from relatively abundant sites (2+ puffs originate in HEK293 cells.We suggest that Caspotter)  to ident2+ puffs . Here, tnt sites . Subsequ2+ puffs initiate at the same sites during repeated stimulation with carbachol. The optimised protocol, developed to maximise puff frequency and recording intervals while allowing complete recovery between successive stimuli, is shown in 2+-free HBS, followed by addition of carbachol (10 µM) for 10 s; the carbachol was then removed and replaced with normal HBS. After a 10-min recovery period in normal HBS, the medium was replaced with Ca2+-free HBS (20 s) and the recording and stimulation sequence was repeated . The results demonstrate that three sequential challenges with the same carbachol concentration evoke the same increase in Ca2+ puff activity and with the same frequency distribution of Ca2+ puffs across release sites , the shortest interval between detection of the first Ca2+ puff and initiation of a global Ca2+ signal was 2 s. In subsequent quantitative analyses we, therefore, matched the analysis interval for all recordings by analysing puff activity during only the last 2 s before either initiation of the global Ca2+ signal or termination of the carbachol exposure.We investigated whether Case sites B,C. With2+ waves . In all 2+ puffs initiate at many sites but, even this, may underestimate their number because brief periods of recording may detect too few puffs to reveal the locations of all sites. We would then expect each carbachol challenge to discover new sites, but fewer with each successive stimulus. n=16) of the active Ca2+ release sites had also responded during the first challenge (Table S1). These results suggest that the second challenge, during which 17±1.8 sites/cell were identified (Table S1), reveal only 26% of the sites capable of initiating Ca2+ puffs. From this, we estimate the total number of Ca2+ release sites in a cell (N) to be 65±7. An alternative approach uses the Poisson distribution to estimate the mean number of puffs/site (µ) and then estimates N from µ (Table S2). This Poisson analysis applied to the first carbachol stimulation suggests that there are 70±13 sites/cell. The two analyses are consistent in suggesting that there are ∼70 carbachol-sensitive Ca2+ release sites/cell. Henceforth, we estimate N from comparisons of responses to the first and second stimuli because it is less prone than the Poisson analysis to errors arising from the small numbers of sites that respond twice during our short analysis intervals (Table S2).The results so far suggest that Ca2+ release sites . During the 6 s of stimulation used for analysis of the three sequential carbachol challenges, 41±5 distinct sites/cell (n=16 cells) were directly identified, whereas our extrapolated analyses suggest 65±7 sites/cell. Hence, both the number of directly identified sites and the extrapolated estimate of N suggest that Ca2+ release sites are more abundant than hitherto suggested. We conclude that carbachol evokes Ca2+ release from a large number of distinct sites in HEK293 cells.We obtained matched records from all cells for quantitative analysis by selecting only 2 s from each 10-s recording for analysis, but at the expense of having to extrapolate data to estimate the total number of Ca2+ puffs initiate and the frequency with which new sites are discovered during successive stimulations with carbachol  and between  carbachol challenges suggest that movement of sites is probably not a major factor. Nevertheless, to distinguish between the two possibilities we used the method shown in 2+ release sites within a short (<10 s) stimulation with carbachol to the rate of discovery after a 10-min or 20-min recovery period. For each cell, all Ca2+ puffs during the first carbachol stimulation were counted before the recording was terminated at either 10 s or by initiation of a Ca2+ wave. This first period of stimulation (C1) was then split to provide two intervals (T1 and T2), each with an equal number of Ca2+ puffs (n). For the same cell, we then examined the second (C2) and third (C3) response to carbachol and identified the interval (T2) during which n Ca2+ puffs occurred , 10 min (C1T1 versus C2T2) or 20 min (C1T1 versus C3T2). For the latter, sites have more than 120-times longer to move than during the first comparison (CIT1 versus C1T2). The results demonstrate that the Ca2+ puffs discover new sites at similar rates whether the comparisons span a few seconds or more than 20 min (The large number of sites at which Caarbachol A-D suggearbachol A. The cooccurred E. This an 20 min F.3Rs respond to IP3 (2+ release sites (65±7 sites/cell) than hitherto supposed , we may underestimate the total number of sites in a cell. However, both we for HeLa cells  to the cytosol by photolysis of ci-IP3. Photolysis of ci-IP3 evoked a flurry of Ca2+ puffs after a latency of 0.42±0.09 s (Movie 2). We used a stimulus regime similar to that used for sequential stimulation with carbachol, but with the recording period reduced to 8 s (to match the 8 s available for recording after the ∼2 s taken to add carbachol), and with the recovery period extended to 15 min (since i-IP3 is less rapidly metabolised than IP3) . The incomplete recovery is probably due to i-IP3 being less rapidly degraded than IP3 . We again analysed only the 2 s immediately preceding either initiation of a global Ca2+ signal or termination of the 8-s recording interval and used the rate of discovery of new sites between successive challenges to estimate the total number of Ca2+ release sites/cell  (Table S3). Counting all distinct Ca2+ release sites detected throughout the three 8-s recordings after photolysis of ci-IP3  identified 63±7 sites/cell (2+ release sites accessible to carbachol (65±7 sites/cell) and to i-IP3 delivered uniformly to the cytosol (100±35). We conclude that both carbachol and photolysis of ci-IP3 evoke Ca2+ release from a large number of immobile sites in HEK293 cells (We next examined the distribution of Cahan IP3) A. It prothan IP3 , and thetes/cell E. There 93 cells E.Fig. 52+ puffs and the number of sites detected  and a photolysis flash (120 ms)  A. The twresented B,C. We tstimulus D,E. The 2+ puffs and assigns them to Ca2+ release sites in order to examine the spatial organisation of IP3-evoked Ca2+ signals in human cell lines . In both HEK293 and HeLa cells, stimuli that evoke formation of IP3 through endogenous G-protein-coupled receptors (GPCRs) triggered a flurry of Ca2+ puffs whose properties were similar to those of Ca2+ puffs observed in other cells . However, the sites at which these Ca2+ puffs occurred were about 10-fold more abundant than reported previously for other cells (Table S1), and our less-comprehensive analysis of HeLa cells suggests at least 87 sites/cell  contribute to the Ca2+ release sites.We used a custom-written algorithm that automatically detects Caer cells . In HEK2tes/cell . The latites see . We concsite see  and abou293 cell , it is c3Rs can move within ER membranes  that include only a small fraction of the IP3Rs expressed in a cell. The number of sites appears not to be affected by the intensity of the stimulation, and there is no evidence to suggest that IP3 is selectively delivered to them from endogenous signalling pathways.Our quantitative analyses of the spatial distribution of Ca3-PM  was from SiChem GmbH .Cal520-AM was from AAT Bioquest . Fluo-4-AM, Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F-12 (50:50) supplemented with GlutaMAX, and TrypLE were from Invitrogen . Pluronic acid F-127, dimethyl sulfoxide (DMSO), poly-L-lysine, foetal bovine serum (FBS), histamine and carbamylcholine chloride (carbachol) were from Sigma-Aldrich . Fibronectin and EGTA-AM were from Merck Millipore . Caged IP2+ signals  and HEK293 cells . Regular screening confirmed that all cells were free of mycoplasma infection. Cells were grown on either round glass coverslips for low-resolution measurements  or glass-bottomed dishes for high-resolution measurements . The glass was coated with fibronectin (10 µg/ml) for HeLa cells or poly-l-lysine (10 µg/ml) for HEK293 cells. Coated coverslips in 6-well culture plates were seeded with HEK293 cells (8×105 cells/well) or HeLa cells (9×104 cells/well) and used for imaging after 48 h, when they were still subconfluent.We used two cell lines, HEK293 and HeLa cells (ATCC), neither of which express functional ryanodine receptors (RyRs). There is detectable mRNA for RyRs in HeLa cells, but caffeine does not evoke Ca signals . HEK293  signals . HEK293 2+ signals, cells were loaded with EGTA to restrict Ca2+ diffusion  and, in some cases, with ci-IP3. Cells were washed with HEPES-buffered saline  and then incubated in darkness with pluronic acid F-127 (0.02% w/v) and either ci-IP3-PM (1 µM) or DMSO (0.02% v/v) at 20°C. After 45 min, Cal520-AM (2 µM) was added and after a further 45 min the medium was replaced with fresh HBS supplemented with EGTA-AM (10 µM). Cells were used for imaging after a further 45 min. Similar loading methods, but without EGTA-AM or ci-IP3-PM, and with Fluo-4 as the Ca2+ indicator, were used for low-resolution measurements of [Ca2+]c.To allow recording of local Caiffusion , a Ca2+ 2+ signals were measured using total internal reflection fluorescence microscopy (TIRFM). A glass-bottomed dish containing loaded cells was immobilised on the motorised stage of an Olympus IX83 inverted microscope. Imaging used a 100× TIRF objective  with excitation (488 nm) provided by a diode-pumped solid-state laser  and a band-pass filter . The angle of the excitation beam was adjusted to achieve TIRF with a penetration depth of ∼62 nm. Emitted light was captured through a band-pass filter  with an Andor iXon 897 EMCCD camera . With the 100× objective, each image pixel had dimensions of 160 nm×160 nm. Images were acquired at 25 Hz.Local Ca2+-free HBS immediately before recordings. Extracellular stimuli were applied in Ca2+-free HBS after careful aspiration of half the medium and its replacement with histamine or carbachol at twice its final concentration in Ca2+-free HBS (1 ml). Medium exchanges took ∼1−2 s. Photolysis of ci-IP3 was achieved using a light-emitting diode , with the duration of exposure determined by a shutter controlled from within MetaMorph . A 50:50 mirror in the excitation light path allowed simultaneous excitation with the 488-nm laser  and the 395-nm LED (for photolysis of ci-IP3). Images were captured in Metamorph and saved as tif files for analysis.Cells were kept in HBS  in the intervals between stimulation. The HBS was replaced by Ca2+]c in cells grown on glass coverslips, loaded with Fluo-4, and mounted in a stainless steel chamber (∼1 ml) on the stage of an Olympus IX81 inverted microscope with a 40× objective . Fluo-4 was excited (488 nm) by light from a diode-pumped solid-state laser  and a band-pass filter . Emitted light was captured with an Andor iXon 897 EMCCD camera (each image pixel had dimensions of 400 nm×400 nm) after passing through a band-pass filter . Images (45-ms capture time) were acquired at 2 Hz.Similar methods were used for low-resolution measurements of . As stimulation proceeds, Ca2+ puffs become more frequent, the background [Ca2+]c increases and, eventually, a global Ca2+ signal initiates (Movies 1 and 2). To allow detection of Ca2+ puffs against this creeping (and then explosive) increase in F/F0, PuffFinder calculates the average ΔF value across the entire cell for each frame . The Fn) to create a new image stack . For each pixel, the mean ΔΔF value  and the standard deviation (s.d.) are determined over time. The thresholds  are set at T1=µ+(3.4×s.d.), and T2=µ+(2.45×s.d.), to provide two additional frames (T1 and T2). Every frame in an image stack is now compared with the T1 frame to identify all pixels that exceed T1. Since many of these pixels will be part of the same Ca2+ puff, the next step is to identify the pixel that is likely to be at the centre of a puff by finding the pixel with the largest ΔΔF and surrounded by pixels with the largest ΣΔΔF. This is achieved by ranking pixels in each frame in descending order of ΔΔF and by considering the largest first before progressing to the next ranked pixel . The first pixel is placed at the centre of a 5×5 binary matrix, and each of the 25 pixels is then assigned a binary value (V): V=1 if ΔΔF>T2, and V=0 if ΔΔF≤T2. We chose a 25-pixel area for this analysis because its width (800 nm) would be expected to capture the core of most Ca2+ puffs  to include only columns or rows in which more than 10% of pixels have ΔΔF>T2. The process is then repeated on the next ranked matrix for which the central pixel has not already been placed within an expanded matrix. The output from this processing is a rectangular map enclosing each puff event. The reliability of The ΔΔF values within each rectangular map of a puff are now fitted to a 2-dimensional Gaussian fit using the GAUSS2FIT function in IDL. This provides the radii of the elliptical fit, its rotation angle , and the coordinates of the centre of the puff with sub-pixel resolution. Comparison of these features across sequential ΔΔF frames provides temporal properties and the maximal amplitude of the puff.SiteMapper to identify the different sites at which Ca2+ puffs occur  within a time series to decide whether two puffs occurred at different sites. A similar proximity-based option is provided in the automated analyses described by Parker's group , and then visually confirming the number of enclosed puffs and the location of each site centre with the output from SiteMapper.t-tests, with the Bonferroni correction used for multiple comparisons. Significance is denoted by: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.Results are presented as means±s.e.m. unless indicated otherwise. Statistical comparisons used ANOVA and Student's


Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT1R signaling and whether there is a reciprocal regulation of AT1R signaling by CaV1.2.Blockers of angiotensin II type 1 receptor  AngII concentration resulted in an enhancement of the Ca2+ response. The up-regulation of the Ca2+ response to repeated 1 nM AngII doses and the down-regulation of the Ca2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT1R plasma membrane expression. The Ca2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the CaV1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT1R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT1R is sensitive to calcium influx.Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca2+ channel blockers as mono-therapy in hypertension.Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca Binding of AngII to AT1R results in activation of phospholipase C, release of inositol 1,4,5-triphosphate (IP3) and Ca2+ mobilization from intracellular stores. It is well documented that AngII activation of AT1R can be followed by desensitization i) measurements carried out in HEK cells were performed using the fluorescent Ca2+ indicator Fura Red (Invitrogen). Cells were loaded with Fura Red using a buffer consisting of 5 μM Fura Red and 1 μl of 20% Pluronic F-127 (Invitrogen) diluted in KREBs . Cells grown on #1.5 18 mm diameter coverslips (Warner Instruments) were rinsed with KREBs buffer and loaded for 45 min with the loading buffer at 37 °C with 5% CO2. After 45 min cells were washed again with KREBs prior to being mounted in a perfusion chamber (Chamlide). Through the use of heated optical table and in-line perfusion feed heater the cells were kept at 37 °C throughout experiments. Loading protocol was evaluated by testing different loading times, both at room temperature and in incubator. The minimal dye concentration usable was identified through Ca2+ calibration of serial dilution of dye.Intracellular CaV1.2 inhibitors, nifedipine and verapamil, were both mixed fresh for each experimental day. Solutions were kept in fridge and protected from light. We chose to use a high concentration of nifedipine, 100 μM, to compensate for loss of active drugs during the longer experiments. Degradation was unavoidable within each experiment with cells and solutions kept at 37 °C. UV and 488 nm laser excitation also contributed to some drug degradation throughout experiments.Ca2+] calibration was done by the following protocol, first a 3 min long washout of the KREBs buffer using a 0 [Ca2+] containing buffer  followed by 1 μM ionomycin in 0 [Ca2+] buffer for 4 min. Calibration ended with 7 min of 1.5 mM [Ca2+] KREBs buffer perfusion.i, was calculated with kd provided by Invitrogen as:The strongest AT2+ measurements of RVCM were performed using the fluorescent Ca2+ indicator Oregon Green BAPTA-1 (Invitrogen) with the same protocol as described for Fura Red. Oregon Green BAPTA1 was chosen as its weaker binding affinity compared to Fura Red made it insensitive to background Ca2+ sparklets in RVCM. Calibration was not possible for the RVCM Ca2+ experiments, thus the non-calibrated Ca2+ response for each cell was obtained by the mean intensity within each selected cell.CaNifedipine effects on bradykinin activation of bradykinin B2 receptors were evaluated through comparing signaling strength with or without pretreatment of 100 μM nifedipine on 100 nM bradykinin (Abcam) treatment. Experiments were performed on Oregon Green Bapta 1 loaded HEK cells. Cells were loaded in the same manner as for Fura Red but with 5 μM Oregon Green BAPTA-1. All other parameters were kept identical as for the 1 nM AngII experiments described above. Each experiment was calibrated as per the protocol described above.Peak amplitudes were in all experiments calculated as peak above baseline. Baseline was defined as the mean intensity of a stable part of the trace prior to the peak. This calculation reflects the cytosolic increase in concentration rather than the absolute concentration.1R and Gαq-Gβγ FRET sensor were exposed to 1 nM AngII with or without 3 min pretreatment of 100 μM nifedipine. FRET was measured using a Zeiss LSM 510 LIVE microscope with a 40×/1.2 water immersion objective and excitation of 405 nm. YFP signal was corrected for mTurquoise bleedthrough and used as FRET intensity. Transfecting HEK cells with GαqmTq allowed for determination of donor bleed through into FRET channel. Bleed through coefficient, b, was calculated as ImTq/IFRET. In experiments, FRET channel intensities were then corrected as: FRETcorrected = IFRET – b*ImTq. FRET ratios were finally calculated as ImTurquoise / IFRETcorrected. Increase in FRET ratio corresponds with increased Gq-protein dissociation.HEK cells expressing ATStatistical analysis was performed using Matlab, one way Anova analysis and subsequent multiple comparison tests based on studentized range distribution. Quantified data is shown as mean value +/− SEM.1R signaling have used AngII concentrations that are at least three orders of magnitude higher than the circulatory levels i responses to serial AngII exposure are shown in Fig. 2+]i, response relative the 1st exposure to 1 nM AngII .The majority of studies on ATy levels , 17, 18,y levels . Repeatey levels –21. Heretization , 17. ThengII Fig . The 2ndsure Fig . Applica2+]i response to AngII could be attributed to effects on AT1R plasma-membrane expression. For this purpose, we expressed AT1R with an extracellular hemagglutinin (HA) tag in addition to a Venus tag at the intracellular C-terminus of HEK cells. Cells were fixed and labeled with an Alexa-594 conjugated anti-HA antibody 3 or 30 min after exposure to AngII. The AT1R fraction inserted in the plasma membrane was calculated as the ratio between the fluorescence intensity of Alexa-594 (plasma membrane AT1R) and Venus   Fig . The effV1.2 subtype i signaling from the bradykinin receptor B2 (B2 receptors), which are constitutively expressed in HEK cells. Exposure of cells to 100 nM bradykinin resulted in similar [Ca2+]i response as for 100 nM AngII while in the presence of nifedipine a weaker [Ca2+]i response was observed suggesting that not all GqPCRs are potentiated by nifedipine.To test whether the sensitivity for calcium influx observed for the AT1R signaling is enhanced following inhibition of Ca2+ influx may have implications for AngII regulation of cardiovascular function, since AT1R inhibitors are commonly used to treat cardiovascular disease i response to 1 nM AngII exposures are shown in Fig. V1.2 by nifedipine or verapamil would affect the AngII response in ASMC. Nifedipine and verapamil doses were selected based on blood plasma levels reported in patients [2+]i response in presence of nifedipine and verapamil are shown in Fig. 2+]i response to AngII  and D2 receptors [2+ influx was not shared by the bradykinin receptor, and is thus not a common phenomenon for all GPCRs. The molecular mechanism by which calcium influx modulates the strength of AT1R signaling remains elusive. We found no evidence for a recruitment of receptors to the plasma membrane or altered Gq-protein activity.Voltage-gated calcium channels serve an important role for the control of [Cae CaV1.2 –10, 39. rtension , 33, 34.ntration , 12. A rntration . The seneceptors  to Ca2+ 1R signaling. Notably these effects were observed with physiological and therapeutically relevant concentration of AngII, nifedipine, verapamil. Calcium channel blockers are commonly used to treat hypertension, either in combination with other drugs such as inhibitors of AT1R or ACE, or as mono-therapy. When calcium channels blockers are used as mono-therapy, the possibility for stimulation of AT1R signaling should be taken into account. Notably, a recent meta-analysis, based on more than 40 studies, has shown that combination therapy that targets both the angiotensin system and the voltage gated calcium channels, will provide a better blood pressure control than mono-therapy that acts at one of these targets [In conclusion, this study provides novel and clinically relevant information about how inhibition of CaV1.2 upregulate AT


However, the molecular origin of αSyn aggregation mediated by the metal ions is not fully elucidated. In this study, we revealed that hard divalent metal ions had almost identical influences on αSyn aggregation. Based on these similarities, the molecular role of Ca2+ was investigated as a representative metal ion. Herein, we demonstrated that binding of multiple Ca2+ ions induces structural transition of αSyn monomers to extended conformations, which promotes rapid αSyn fibrillation. Additionally, we observed that Ca2+ induced further interfibrillar aggregation via electrostatic and hydrophobic interactions. Our results from multiple biophysical methods, including ion mobility-mass spectrometry (IM-MS), synchrotron small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), provide detailed information on the structural change of αSyn and the aggregation process mediated by Ca2+. Overall, our study would be valuable for understanding the influence of Ca2+ on the aggregation of αSyn during the pathogenesis of α-synucleinopathies.α-Synuclein (αSyn) is an intrinsically disordered protein, the aggregation of which is highly related to the pathology of diverse α-synucleinopathies. Various hard divalent metal cations have been shown to affect αSyn aggregation. Especially, Ca During the fibrillation of amyloidogenic proteins, monomeric proteins are converted into oligomeric intermediates, and finally, to highly ordered, unbranched β-sheet structures3. Amyloid fibrillation is not thoroughly understood yet; however, it is considered to have a correlation with protein misfolding4. Therefore, understanding the mechanisms of amyloid fibrillation, which is associated with protein misfolding, is necessary for developing therapeutic strategies for amyloidosis.A number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are associated with the formation of amyloid fibrils7. αSyn is considered to modulate the synaptic vesicle cycle, which is involved in neurotransmission8; however, it has received attention because of its pathological significance as the main component of Lewy bodies and Lewy neurites, observed in patients with α-synucleinopathies such as Parkinson’s disease (PD), multiple system atrophy (MSA), and Lewy body dementia (LBD)9. αSyn is an intrinsically disordered protein (IDP), and comprises 140 amino acid residues, which constitute the amphipathic domain at the N-terminal region (residues 1–60), the hydrophobic non-amyloid-β component (NAC) region (residues 61–95), and the acidic domain at the C-terminal region (residues 96–140)  is a small amyloidogenic protein, which is abundant, particularly, at the presynaptic nerve terminals40) Fig. . The str15. Cu2+, a divalent transition metal ion, has been proposed as a potential cause of αSyn fibrillation with its high binding affinity (Kd ~10−6–10−10 M) and unique binding site (The N-terminus and His-50 are involved)18. Unlike Cu2+, hard divalent metal cations have been shown to promote the aggregation of αSyn20 with binding to the acidic C-terminal region (Kd ~ 10−3 M)21. The metal cations also induce the formation of Lewy body-like large assemblies comprising αSyn fibrils22. However, the mechanistic details on the metal-associated fast aggregation and Lewy body-like interfibrillar aggregation of αSyn have not been fully understood. Among the hard divalent metal ions, converging evidence suggests that Ca2+ is a crucial physiological factor related to αSyn aggregation. Firstly, the dysregulation of Ca2+ has been observed in aged animals23 and mice model of α-synucleinopathies24. Abnormally increased intracellular Ca2+ concentration, which is normally regulated to be ~100 nM (while its extracellular concentration is ~1.4 mM)25, can cause aggregation of αSyn28, and finally induce neurodegeneration29. Secondly, significant amount of Ca2+ has been detected in Lewy bodies of patients with PD30. This suggests the possibility of the involvement of Ca2+ in the formation of Lewy bodies. Furthermore, when αSyn is secreted to the extracellular space (secretion of αSyn is commonly observed in PD model systems33), αSyn can be exposed to and influenced by high level of Ca2+ (~1.4 mM). Taken together, the interaction between αSyn and Ca2+ appears to be closely related to αSyn aggregation in α-synucleinopathies.The aggregation of αSyn is believed to be associated with environmental factors, such as an imbalance in metal homeostasis2+, a representative hard divalent cation, was proposed by monitoring the structural transition of αSyn from the monomeric state to the large interfibrillar aggregate state. Our structural and kinetic results, which were obtained using multiple biophysical methods, including ion mobility-mass spectrometry (IM-MS), transmission electron microscopy (TEM), synchrotron small-angle X-ray scattering (SAXS), and inductively coupled plasma optical emission spectroscopy (ICP-OES) demonstrated that Ca2+ mediates the rapid formation of αSyn fibrils via the structural transition of monomeric αSyn into an extended conformation, which is prone to aggregation. We probed that direct interaction between Ca2+ and αSyn fibril induces the subsequent association of the fibrils with secondary structure changes to form large interfibrillar αSyn aggregates through electrostatic and hydrophobic interactions. Moreover, we observed that αSyn aggregates formed through Ca2+ mediation are toxic to SH-SY5Y neuroblastoma cells. We believe that the aggregation mechanism of αSyn mediated by Ca2+ provides an insight into the formation mechanism of the inclusion bodies that is commonly observed in α-synucleinopathies.Herein, we have reported the unique αSyn aggregations mediated by hard divalent cations to form large interfibrillar aggregates. Then, the mechanism of αSyn aggregation mediated by Ca2+ using the thioflavin T (ThT) assay 34 compared with the control group, αSyn incubated without Ca2+ (t1/2 = 28.1 h). Furthermore, the ThT fluorescence intensity of Ca2+-mediated αSyn aggregates was almost twice as high as the control group, which implied that Ca2+ promoted the conversion of more monomers to fibrils. Then, the morphology of aggregates was observed using transmission electron microscopy (TEM). In the absence of Ca2+, normal amyloid fibrils were formed  spectroscopy. The control group without Ca2+ exhibited random coil conformation at 0 h; then, the initial conformation was gradually converted to a β-sheet conformation (negative band at 218 nm) after incubation for 7 d  measurements  spectroscopy. The IR spectra indicated that the αSyn aggregates formed through Ca2+ mediation consisted of a low structural portion of β-sheets, compared with the conventional fibrils ·q2 vs q) from the SAXS profiles38 showed the typical curves of unfolded proteins (i.e. the lack of bell-shaped curves and increase in the q range)39 for both αSyn in the presence and absence of Ca2+  of αSyn, which were obtained from the Guinier analysis, showed that Rg increased when Ca2+ was added. The Rg value of αSyn was measured to be 31.8 ± 0.7 Å, which was similar to the value obtained in the previous study . As Ca2+ concentration increased, the maximum number of Ca2+ bound to αSyn and the relative abundance of Ca2+-bound αSyn peaks in the mass spectrum increased  ~2 kcal/mol41 and weak affinity (Kd ~ 1 mM)41. Because four binding sites were previously reported41, based on Kd ~ 1 mM and the exact Ca2+ concentration, 0.71 and 0.95 Ca2+ per αSyn molecule  could bind on average. However, the electrostatic interactions between a protein and metal cations can be further enhanced in the gas phase, because solvent molecules evaporate during the ESI process42. Thus, despite the low binding affinity, a large number of bound Ca2+ ions, which were weakly associated with the C-terminal region via long range attractive interaction, were ultimately observed in the gas phase. In addition, the increased number of Ca2+ ions bound to αSyn ions in the sample with 330 μM Ca2+ than in the sample with 230 μM Ca2+  detected that multiple Cased Fig. . In parta2+ Fig.  was cons2+-bound αSyn showed multiple ion mobility peaks with collision cross-section (CCS) values ranging between 1400~2700 Å2 for +8 charged αSyn  simulation of Ca2+-bound αSyn was performed to match our SAXS and IM-MS data. Representative structures of αSyn in solution were obtained from the structure pool of αSyn generated using replica exchange MD simulations46, based on the ensemble optimization method (EOM), which identifies the best ensemble by fitting sum of multiple theoretical SAXS profiles to the experimental SAXS profile48. Using the obtained representative structures as initial structures, gas-phase MD simulations were also performed to obtain gas-phase structures having theoretical CCS values (CCStheo) corresponding to the experimental values. The CCStheo value of each structure was estimated using the exact hard sphere scattering (EHSS) method49, and compared with the experimental CCS values  showed differences in Rg distributions. The αSyn ensembles that were obtained in the absence of Ca2+ and in the presence of Ca2+ had distributions ranging from 15 to 60 Å; however, the former had a distribution with an average of 33.6 ± 6.4 Å, while the latter had a distribution with an average of 38.5 ± 7.7 Å , it was observed that the overall structures of the αSyn that were unbound and bound to Ca2+ (8+) were similar if their CCStheo values were similar. To understand why compact conformation was preferred in Ca2+-bound αSyn, we investigated the representative compact structure of Ca2+-bound αSyn (CCStheo = 1717.9 Å2)  that were obtained using the gas-phase MD simulation . In contrast, the αSyn samples that were incubated for 30 h and 60 h without Ca2+ showed the presence of conventional fibrils  increased gradually to approximately 0.05. However, when Ca2+ was added after 60 h of the incubation of αSyn, OD600 increased instantaneously, and reached approximately 0.20 in a few hours of incubation  to measure the amount of Ca2+. For the ICP-OES experiment, incubated αSyn samples were centrifuged at 18,000 × g, and the amounts of Ca2+ were measured in supernatant and insoluble αSyn aggregates. Figure 2+ with αSyn monomer and fibril both include significant amount of Ca2+. Because Ca2+ enhanced the affinity between the C-terminal region of fibrils in both cases, it was considered that Ca2+ reduced the charge–charge repulsion between acidic residues of adjacent fibrils by binding to the acidic residues. In addition, we observed that the amounts of incorporated Ca2+ were similar in both aggregates  the interfibrillar interaction may be further stabilized through the chelating Ca2+ between two acidic residues (i.e. originated from each fibril). In addition, our IR spectra showed the dramatic decrease in β-sheet abundance of Ca2+-mediated fibrils  theils Fig. . Howevera2+ Fig. ; at firs2+, which is similar to that of extracellular fluid, indicates that the observed Ca2+-mediated αSyn aggregation could be induced in the cells undergoing dysregulation of Ca2+ homeostasis or in the Ca2+-rich extracellular space. Because of the relationship between dysregulated Ca2+ homeostasis and α-synucleinopathies, we investigated whether aggregates formed through Ca2+ mediation have cytotoxicity, using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay with SH-SY5Y neuroblastoma cells. Compared to the control groups (buffers and monomer), the αSyn aggregates, which were formed by the initial addition of Ca2+ were as toxic as normal αSyn fibrils (cell viability of 61%), while the aggregates that were formed through Ca2+ mediation from preformed fibrils had slightly reduced cytotoxicity (cell viability of 73%)  and is a critical physiological factor for αSyn fibrillation among hard divalent ions, our results suggested the importance of the interaction between Ca2+ and αSyn in α-synucleinopathies. Furthermore, the detailed examination of the structures and the molecular interactions during αSyn aggregation would be valuable to understand the pathology of α-synucleinopathies.We have observed the formation of large interfibrillar aggregates of αSyn associated with hard divalent metal cations. Then, we characterized the pathway of αSyn aggregation mediated by CaAll data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Our results showed that NF significantly increased myocyte contraction and facilitated relaxation. Moreover, NF increased the amplitudes of diastolic and systolic Ca2+ transients ([Ca2+]i), abbreviated time constant of [Ca2+]i decay (tau), and prolonged the peak duration of [Ca2+]i. Whole-cell patch-clamp experiments revealed that NF increased Ca2+ influx via L-type Ca2+ channels  and prolonged the action potential duration (APD). Further analysis revealed that NF shifted the relaxation phase of sarcomere lengthening vs. [Ca2+]i trajectory to the right and increased [Ca2+]i for 50 % of sarcomere relengthening (EC50), suggesting myofilament Ca2+ desensitization. Butanedione monoxime (BDM), a myosin ATPase inhibitor that reduces myofilament Ca2+ sensitivity, abolished the NF-induced enhancement of [Ca2+]i amplitude and the tau of [Ca2+]i decay, indicating the association of myofilament Ca2+ desensitization with the changes in [Ca2+]i profile in NF. NF reduced intracellular pH ([pHi]). Increasing [pH]i buffer capacity with HCO3/CO2 attenuated Δ [pH]i and reversed myofilament Ca2+ desensitization and Ca2+ handling in NF. Collectively, greater Ca2+ influx through LTCCs and myofilament Ca2+ desensitization, via reducing [pH]i, are likely responsible for the positive inotropic and lusitropic effects of NF. Computer simulation recapitulated the effects of NF.Fatty acid (FA)-dependent oxidation is the predominant process for energy supply in normal heart. Impaired FA metabolism and metabolic insufficiency underlie the failing of the myocardium. So far, FA metabolism in normal cardiac physiology and heart failure remains undetermined. Here, we evaluate the mechanisms of FA and major metabolic substrates (termed NF) on the contraction, relaxation, and CaThe online version of this article (doi:10.1007/s00424-016-1892-8) contains supplementary material, which is available to authorized users. Constant supply of ATP through cardiac metabolism is essential to maintain the normal functions of the heart i) binds to troponin C (TnC) in the thin filament (troponin complex), prompts actin association with myosin, and initiates contraction. Reuptake of Ca2+ into the SR via Ca2+-dependent ATPase (SERCA) and Ca2+ extrusion via Na+-Ca2+ exchanger (NCX) decrease intracellular Ca2+, lead to the dissociation of Ca2+ from TnC and myosin from actin, myofibril relaxes. As such, factors that increase or decrease the amount of intracellular Ca2+ plays important roles in myocardial contractile function. In addition, myofilament Ca2+ sensitivity is pivotal in determining myocyte contraction and relaxation in the functioning heart. On the other hand, recent consensus is that changes in the myofilament Ca2+ sensitivity, per se, regulates intracellular Ca2+ homeostasis and therefore myocyte contractility in normal and diseased hearts i. As shown in Fig. 2+]i . Analysis of [Ca2+]i profile showed that the time constant of [Ca2+]i decay  was abbreviated in NF  and TR50 of [Ca2+]i were prolonged . These results suggest that NF increased [Ca2+]i transients and decay kinetics of [Ca2+]i but prolonged peak time of [Ca2+]i during systole.Next, we tested whether NF increased [Ca2+]i and contraction in NF. Figure Ca in LV myocytes before and after NF. And the inset in Fig. Ca at 0 mV for Ca2+ influx analysis. NF reduced the amplitude of ICa at 0 mV  but slowed the inactivation of ICa at 0 mV  was greater in NF  but not at +20 mV.We aimed to test the key elements of excitation-contraction coupling responsible for increased myocyte [CaCa influx in NF may be responsible for the prolongations of action potential (AP) plateau (and therefore APD) and [Ca2+]i duration. Accordingly, we examined the APD duration in NF. As shown in Fig. 20, APD50, and APD90 were significantly prolonged in NF . Collectively, these results suggest that Ca2+ influx via LTCC is greater and APD is prolonged, which may be responsible for increased [Ca2+]i amplitudes and prolonged peak time of [Ca2+]i in NF.Greater I2+ sensitivity is regulated by NF and contributes to greater contraction and faster relaxation, we recorded sarcomere shortening and [Ca2+]i simultaneously in Fura-2-loaded LV myocytes and plotted a phase-plane loop of the changes in sarcomere length vs. [Ca2+]i transient with and without NF. As shown in Fig. 2+]i transient relationship shifted to the right in NF; [Ca2+]i at 50 % sarcomere relengthening (EC50) was significantly increased . These results suggest that myofilament Ca2+ sensitivity was significantly reduced by NF.To test whether myofilament Ca2+ and changing myofilament Ca2+ sensitivity affects [Ca2+]i homeostasis i and facilitated tau of [Ca2+]i in NF. As shown in Fig. 2+]i and faster tau . These results suggest that reduced myofilament Ca2+sensitivity in NF may, at least in part, increase [Ca2+]i and facilitate [Ca2+]i decline in rat LV myocytes. The duration of [Ca2+]i peak , however, remain prolonged in NF in the presence of BDM  is reduced by NF and contributes to reduced myofilament Ca2+ sensitivity, we detected changes of pHi in SNARF-1/AM-loaded LV myocytes. As shown in Fig. i was gradually decreased with NF (3–5 min). Averaged results showed that NF significantly reduced pHi  weaken the changes in pHi by NF , which is in line with the experimental results.Next, we employed the computer simulation to further confirm whether reduced pHged Fig. , in linesed Fig. . In addi2+ influx via LTCC and increasing [Ca2+]i transients, despite that myofilament Ca2+ sensitivity was reduced. Importantly, both experimental and computer simulation results suggest that myofilament Ca2+ desensitization, at least in part, contributes to increased [Ca2+]i , greater contraction, abbreviated tau, and faster relaxation by NF. Intracellular pH, which is reduced by NF, is important in mediating the effects of NF on myocyte contraction, relaxation, and [Ca2+]i. These results suggest that supplementation of metabolic substrates (including fatty acids) potentiates myocyte inotropy and lusitropy by regulating key elements of Ca2+ handling processes and myofilament Ca2+ sensitivity, in addition to their effects on cardiac metabolism.The present study showed that supplementation of metabolic substrates  increased LV myocyte contraction and facilitated relaxation from rat heart by increasing Ca2+]i, facilitated Ca2+ reuptake into the SR via SERCA, and prolonged peak time duration of [Ca2+]i. Since LTCC and Ca2+ release from the SR are important in shaping AP and [Ca2+]i profiles and myofilament Ca2+ sensitivity and myofilament mechanics dominate myocyte contraction, we analyzed these parameters in the presence of NF. As shown in both experimental and computer simulation results, peak ICa density was reduced by NF; however, the integral of ICa  was increased due to slower inactivation of LTCC , suggesting more Ca2+ is fluxed into the myocyte under these conditions. Accordingly, APD was prolonged remarkably with NF. These results suggest that [Ca2+]i elevation with NF may be due to increased Ca2+ influx via LTCC and the changes in LTCC are likely responsible for the prolongations of APD and the peak duration of [Ca2+]i.The main theme of the study was that supplementation of metabolic substrates essential for cardiac metabolism  influenced myocyte function from normal rat hearts. That is, NF enhanced LV myocyte contraction and promoted myocyte relaxation in almost all the myocytes studied. Furthermore, NF increased diastolic and systolic i buffering capacity i [2+ desensitization with BDM increased [Ca2+]i in sham but not in hypertension (where myofilament Ca2+ sensitivity was reduced). Furthermore, myofilament Ca2+ desensitization in hypertension, with BDM or with high stimulation frequency, prompted greater ICa inhibition due to [Ca2+]i increment [2+ desensitization with BDM prevented NF-induced increase in the amplitude of [Ca2+]i are consistent to our previous results. Furthermore, computer simulation with reduced Ca2+ binding and myofilament Ca2+ desensitization recapitulated greater [Ca2+]i and the prolongation of the peak duration of [Ca2+]i, supporting the experimental results with NF. These results strongly suggest that both LTCC and myofilament contribute to increased [Ca2+]i and myocyte contraction/relaxation with NF in rat heart.NF regulation of myofilament Cacapacity , 19. Foruffering . In fact [Ca2+]i  and myofncrement . Here, t2+ sensitivity and LTCC by NF. It is possible that enhanced mitochondrial activity secondary to the supplementation of metabolic substrates may produce greater ROS via oxidative phosphorylation i. To confirm, when intracellular pH buffering was increased with NaHCO3 + CO2, the effects of NF on myofilament Ca2+ sensitivity and [Ca2+]i were diminished. Alternatively, reduced pH may increase [Ca2+]i via enhancing the activity of Na+-H+ exchanger and the reverse mode of cardiac Na+-Ca2+ exchanger [Our results showed that intracellular pH was reduced by NF. Since low pH is a potent modulator of myofilament Cal muscle , 16, 24,xchanger . It is pxchanger  and consxchanger . Mechani2+ desensitization is intracellular ATP. This is because ATP desensitizes myofilament response to Ca2+ in muscle and myocardium [2+ desensitization and myofilament buffering of Ca2+ with metabolic substrates need further investigation.Another possible mechanism for myofilament Caocardium , 10. Pre2+ transients, and ICa were successfully reproduced by computer simulation which assumed that the effects of NF are all mediated by lowered intracellular pH. Saegusa et al. [+ concentration at low intracellular pH affects ICa by modulating charge screening/binding of surface charge and permeability of LTCC. As NF was found to reduce intracellular pH ~0.1 U .Most of experimentally observed effects of NF on AP, Caa et al.  proposed1 U Fig.  in our eignaling ). In our2+ handling processes and myofilament play in concert to potentiate intracellular Ca2+ level and induce greater contraction. Our results provide useful evidence for better understanding of cardiac excitation–contraction coupling with comprehensive metabolic supplies.In conclusion, our results clearly show that supplementation of metabolic substrates, including fatty acids, is important in strengthening cardiac metabolism and contractile function. Under these conditions, CaSupplementary Figure 1(PDF 17.5 kb)Supplementary Figure 2(PDF 161 kb)


However, the lifetime of individual membrane-bound activated cPKCs is an order of magnitude shorter than the average time needed for target-protein phosphorylation. Here, we employed intermolecular Förster resonance energy transfer (FRET) in living cells combined with computational analysis to study the spatial organization of cPKCs bound to the plasma membrane. We discovered Ca2+-dependent cPKC nano-clusters that significantly extend cPKC’s plasma-membrane residence time. These protein patterns resulted from self-assembly mediated by Ca2+-binding C2-domains, which are widely used for membrane-targeting of Ca2+-sensing proteins. We also established clustering of other unrelated C2-domain containing proteins, suggesting that nano-cluster formation is a key step for efficient cellular Ca2+-signaling.Conventional protein kinase Cs (cPKCs) are key signaling proteins for transducing intracellular Ca In the context of bacterial chemotaxis, large long-lived receptor clusters were shown to enhance signaling sensitivity92+-signaling evidence for the existence and contribution of nano-clusters to downstream signaling is indirect at best. In vitro studies reported evidence for interactions between different domains of the conventional protein kinase Cα (PKCα) that lead to dimerization1112in vitroin vivo for the existence, let alone for a functional role of PKCα oligomerization or nano-clustering. However, the aforementioned discrepancy between membrane residence times (<75 ms) and the time required for one phosphorylation event (>150 ms) requires the existence of some mechanism that either extends the activation lifetime of PKCα or decreases the time necessary for phosphorylation. In light of the observed roles of nano-clusters in signaling, in this work we study the existence of PKCα-nano-clusters and their possible role in signaling.In the context of Ca2+ from internal stores2+ to the C2-domain of PKCα increases its affinity for the inner leaflet of the plasma membrane, notably to negatively charged phospholipids such as phosphatidylserine (PS)  that resulted in accumulation of the fusion-protein on the plasma membrane within a few seconds . The undine (PS) 1617. We ansients . Notablyansients , which iR77A) with substantially reduced DAG binding18R77A compared to wt, but it remained significantly slower than the underlying Ca2+ decay . In the cytosol, they are occupied independently at a rate 2+ that we take to be spatially homogenous. Unbinding of Ca2+ from the two sites occurs independently at rate ωa.In order to further explore the mechanistic consequences of intimate PKCα interactions, we performed a computational analysis of PKCα dynamics accounting for the process of PKCα aggregation . In the ωt and are irreversible. The unbinding rate of Ca2+ depends on the state of PKCα: In state 1, the rate depends on the number n of neighboring molecules 2+ at a free Ca2+-binding site at rate 2+ it detaches from the membrane at rate ωd.From the observation that the FRET signal decayed in presence of a constant fluorescence signal, we inferred that membrane-bound PKCα can be in two states: in state 1 it can form a cluster, such that it is tightly bound to the membrane and neighboring molecules. In state 2, it cannot form a cluster. In our model, transitions from state 1 to state 2 take place within the cluster at rate ωit. These transitions spread rapidly through the clusters and destabilize them. The lifetime of a PKCα cluster is thus determined by the rate of transitions from state 1 to state 2 and by the total number of molecules in the cluster.Motivated by the rapid dissociation of PKCα clusters observed for local translocation events2+ oscillations depicted in app, app transients showed the same prolonged decay as the measured FRET signal, when compared to the Ca2+ transient  C2-eCFP) and eYFP and investigated possible FRET changes upon a Ca2+ increase  well-known to cause maximal PKCα translocationTo scrutinize our findings, we studied the fluorescence lifetime of eCFP fusion proteins at the plasma membrane in the absence and presence of appropriate FRET partners, i.e. eYFP fusion proteins. In order to maximize accumulation of PKCα molecules on the plasma membrane in the absence of Ca2+ induced translocation to the plasma membrane  according to the vendor´s recommendations. Cells were investigated 48 hours after transfection.HEK293 cells were cultured as described previously2, 1.8 mM CaCl2, 10 mM glucose, 10 mM HEPES adjusted to pH 7.35 with NaOH unless stated otherwise. All compounds used were of research grade. Changes in the extracellular solution were achieved by a gravity-driven custom-made local perfusion system or by manually exchanging the bath solution.All experiments were conducted at room temperature (20–22 °C) and used an extracellular solution (Tyrode) comprising: 135 mM NaCl, 5.4 mM KCl, 2 mM MgCl2+ concentration, we made use of the Ca2+ -ionophore ionomycin  and adapted the extracellular Ca2+ concentration as detailed in the figures. Extracellular Ca2+ concentrations were modulated simultaneously with the application of the ionophore. Please note that this experimental design could result in varying speeds of Ca2+ changes due to the varying efficiency, with which ionomycin incorporated into the plasma membrane. The resulting changes in the intracellular Ca2+ concentration were quantified after pre-loading the cells with Indo-1 or Mag-Indo-1 (see below).For long-term changes in the intracellular CaThe wild-type human PKCα protein, C1 domain mutated (R77A) PKCα protein and full length of human PKCβII protein were fused with eYFP or eCFP at the C-terminus in the pCDNA3 plasmid as described previouslyThe C2 domain of PKCα was cloned from human PKCα by PCR with the following primers, 5′-AGAATTCATGGATCACACTGAGAAGAGG and 5′-ATCTCGAGCGGTCCGTGAGTTTCACTCG, and fused with eYFP or eCFP at the C-terminus respectively in the pCDNA3 plasmid.The truncated form of synaptotagmin-1 (SYT1-C2AB) was cloned from the full length of synaptotagmin-1 (EMBL) with the following pair of primers, 5′-ATCTCGAGAGAAATGTTTGTTCAAAA and 5′- TGGATCCTACTTCTTGACGGCCAG, and fused with eYFP or eCFP respectively at the N-terminus in the pCDNA3 plasmid.All plasmids were confirmed by sequencing.2+-imaging employing Indo-1 or MagIndo-1 we utilized an automated inverted microscope  and a monochromator  for generating the required excitation light . The resulting pairs of images were projected on the two halves of a fast CCD camera  after passing through appropriate dichroic mirrors . Imaging was performed through a 20x multiimmersion lens . The entire setup was controlled by LA software . Before the experiments with combined FRET and Ca2+ measurements were started the HEK293 cells on the coverslip were loaded with either Indo-1 AM (0.5 μM) or MagIndo-1 AM (0.5 μM) in Tyrode for 30 minutes. Thereafter the cells were bathed in normal Tyrode without the dye for de-esterification (20 minutes). To perform the calculation of the FRET efficiency and the Ca2+ concentration, we acquired three pairs of images; two image pairs CFP/YFP at the CFP excitation and YFP excitation wavelengths and one image pair at the Indo-1, MagIndo-1 excitation wavelength. Images acquired in a frequency of 0.5 fps with image size 200 × 300 pixels.For combined FRET and CaWe performed confocal imaging on an inverted microscope  using an oil immersion objective . The microscope was attached to a fast 2D-kilobeam array scanner  that simultaneously scans 2500 parallel laser beams across the specimen and projects the resulting fluorescence images on two spectrally separated EMCCD-cameras . For alternating excitation of eCFP and eYFP (see below) we employed two solid-state lasers; eCFP excitation with a 445 nm laser , eYFP excitation with a 514 nm laser . The two emission channels were separated through a dichroic mirror (491 nm). The entire setup was integrated and controlled through VoxCellScan software . To perform the calculation of the FRET efficiency, we acquired two pairs of images CFP/YFP at the CFP excitation and YFP excitation wavelengths. Images (256 × 256 pixels) were recorded at 0.5 fps.After the experiments, the resulting image series were transferred into a large-scale image database system running OMERO 5.02  for long-term storage. Images were processed either in MatLab (see below) or in ImageJ. To obtain fluorescence over time plots, the fluorescence information from regions-of-interests were averaged, saved and imported into Igor software .0), for which the fluorescence at a given time point (F) was divided by the resting fluorescence (F0) to account for different dye loading and/or expression of the fluorescent proteins and their distribution in subcellular compartments .et al.EfD and EfA, which are the absolute FRET efficiencies scaled by the respective fractions of donors and acceptors in the FRET state and the donor mole fraction (xD = [Dt]/([Dt] + [At])), were determined for each pixel for each FRET measurement. Here [Dt] and [At] denote total donor and acceptor concentrations, respectively. Data stacks acquired at alternating excitation wavelengths (445 nm and 514 nm) were obtained with two emission images each , corrected for background, inhomogeneous illumination, and further processed according to Prasad et al.xD values, we calculated the predicted apparent FRET efficiency xD = 0.5), assuming a standard dimerization modelTo investigate cPKC nano-clusters we performed quantitative lux-FRET experiments according to Wlodarczyk Fluorescence lifetime microscopy (FLIM) images were acquired with a custom made time-correlated single photon counting (TCSPC) system comprising an inverted microscope (Nikon TE-2000-U), a super-continuum laser  with a repetition rate of 40 MHz coupled into a laser scanning unit . The setup was controlled by image acquisition software . The sample was scanned using a 40x, 1.4 NA Plan-Fluor objective, creating images of the size 384 × 384 pixel. The start and stop signals for each line of the image were transferred from the scanning unit into the TCSPC system  for constructing the resulting image. Emitted photons were detected by an avalanche photodiode (APD), acquired, time-stamped and analyzed by the TCSPC Software . Excitation of eCFP was carried out through a bandpass filter (434/17 nm) and the emission was detected through a bandpass filter .In our computational model, PKCα-molecules are represented by particles with a diameter of 30 nm moving on a cubic lattice with an edge of length 7.2 μm × 7.2 μm × 1.8 μm representing the cytosol. Sites have a lateral extension equaling a particle diameter. The membrane is represented by a square lattice that coincides with one of the faces of the cubic lattice introduced before. We apply periodic boundary conditions in the directions lateral to the membrane.2+-free PKCα molecules, PKCα bound to one and to two Ca2+ ions. Unbound Ca2+ ions are assumed to form a reservoir. Binding of a Ca2+ ion to PKCα occurs at rate ωb,c[Ca2+]i, where [Ca2+]i is the (intracellular) Ca2+ concentration. PKCα release Ca2+ ions at rate ωd,c. Particles can hop to empty neighboring sites at constant rate corresponding to a diffusion constant Dc.We consider three states of PKCα: Ca2+ ions can attach to the membrane at rate ωa . Particles on the membrane exist in one of three states corresponding to PKCα that is not part of a cluster and to two conformations of PKCα when it is part of a cluster. The detachment of Ca2+ from an isolated PKCα occurs at rate ωd,m. Ca2+ binds to membrane-bound PKCα at rate ωb,m[Ca2+]i. PKCα free of Ca2+ detaches from the membrane at rate ωd.The membrane is described by a 7.2 μm × 7.2 μm square lattice that coincides with one face of the cube. PKCα bound to two Ca2+ are stabilized. The rate of Ca2+ release then depends on the number n of adjacent PKCα in the cluster and equals ωd,mc(n) = ωd,m exp{−αn}.Two adjacent PKCα molecules on the lattice are assumed to strongly interact and to immediately form a cluster. A particle that has not been part of a cluster before changes its state to the stable cluster state. For PKCα in this state, the interactions between PKCα and Ca2+-ions bind to membrane-bound b,m [Ca2+]i. t. Proteins in the new conformation are denoted as t to ωit. d,m.CaThe particles obey the following reactions occurring stochastically. The actual values for the rates of the reactions are given in c and the diffusion constant of membrane-bound particles Dm differ by a factor of 10. At the system’s boundaries we apply periodic boundary conditions. Only particles which diffuse in the bottom layer of the bulk can bind to membrane. At the top of the geometry, we apply no flux boundary conditions. The clusters are formed when membrane-bound We analyze the dynamic behavior of In the simulations we do not model the fluorescence explicitly. Fifty percent of the particles are marked with the label ‘YFP’, all the rest is marked with the label ‘CFP’. ‘CFP’- and ‘YFP’-labelled particles have exactly the same molecular properties. The total FRET signal is defined as the number of ‘CFP’-labelled particles in a complex with at least one adjacent ‘YFP’-labelled particle.Statistical analysis of the data was performed in Prism 6 software . After testing for Gaussian distribution  data were analyzed with an unpaired t-test. Bar graphs depict mean ± SEM. Statistical significance was defined as follows; *p < 0.05, **p < 0.01, ***p < 0.001. N numbers give the number of cells analyzed, whereas the number of experiments depict independent experiments (different passage or cover slip).How to cite this article: Mike, B. et al. C2-domain mediated nano-cluster formation increases calcium signaling efficiency. Sci. Rep.6, 36028; doi: 10.1038/srep36028 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


However, it is unclear whether short Ca2+ peaks influence glomerular biology and cause podocyte injury. Here we generated a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) knock-in mouse line to manipulate intracellular Ca2+ levels. By mating to a podocyte-specific Cre driver we are able to investigate the impact of Ca2+ peaks on podocyte biology in living animals. Activation of the engineered G-protein coupled receptor with the synthetic compound clozapine-N-oxide (CNO) evoked a short and transient Ca2+ peak in podocytes immediately after CNO administration in vivo. Interestingly, this Ca2+ peak did neither affect glomerular perfusion nor filtration in the animals. Moreover, no obvious alterations in the glomerular morphology could be observed. Taken together, these in vivo findings suggest that chronic alterations and calcium overload rather than an induction of transient Ca2+ peaks contribute to podocyte disease.Chronic alterations in calcium (Ca Overexpression of the G-protein-coupled angiotensin II type 1 receptor, which results in an increased Ca2+ flux, causes glomerulosclerosis in rats82+ signallingIn vivo, expression of either constitutively active Rac1 or RhoA both caused albuminuria and foot process effacement212+ dependent degradation of the actin-binding protein synaptopodin via the calcineurin/cathepsin-L pathway further emphasise the importance of Ca2+ in podocytes23Diseases of the glomerular filter are a leading cause of end stage renal failure. Podocyte injury is the common final pathway of any glomerular disease. Podocytes are highly specialized epithelial cells and part of the three-layered kidney filtration barrier as they enwrap the glomerular capillaries with their primary and secondary foot processes completely23567871012132+ dependent signalling in podocytes. However, most studies only provide indirect evidence that indeed Ca2+ signalling is required to regulate podocyte function in vivo as they focus on e.g. receptor function and do not study Ca2+ levels in vivo. Due to technical limitations most experiments performed to investigate the role of Ca2+ have been performed on cultured podocytes ex vivo. To further study the role of Ca2+ dependent signalling events in podocytes in vivo and its implication on podocyte function we applied the DREADD (Designer Receptor Exclusively Activated by a Designer Drug) concept and generated a novel podocyte-specific transgenic mouse model in which we were able to induce increased intracellular Ca2+ levels by administration of a specific chemical compound. To this end, we use a previously well characterized mutant human muscarinic type 3 receptor (hM3D), which leads to an exclusive activation by binding of the inert compound Clozapine-N-Oxide (CNO)25in vivo approach we demonstrate that single transient Ca2+ peaks do not affect glomerular function.Taken together, numerous reports highlight the role of Ca3D construct in murine immortalized mouse podocytes and generated cell lines either expressing FLAG-tagged hM3D or GFP as control. Immunofluorescence stainings revealed a clear membrane localization of the receptor . We also excluded receptor desensitization or internalization by repetitive Ca2+ imaging experiments at 0 hrs and 24 hrs after the initial stimulation  as published by Schreiber et al.3D-expressing animals in comparison to their littermate controls . However, we could not observe any signs of glomerular disease based on histological analyses as well as urinary albumin-to-creatinine ratios . Due to two point mutations (Y149C/A239G) this receptor can solely be activated by the chemical compound CNO but no longer by its native ligand acetylcholine253 receptors, which makes CNO a potent agonist of the mutated M3 receptor253 to the IP3-receptor at the ER resulting in a strong Ca2+ release from the ER into the cytoplasm. This allows studying effects of increasing intracellular Ca2+ levels in vivo in a novel, highly specific manner. As we inserted a LoxP-STOP-LoxP cassette in the ROSA26 locus upstream of the hM3D sequence, this mouse model may be used to study effects of Gq-coupled receptor activation in a variety of tissues and cell types. Here, we mated the ROSAhM3D/wt mice to a podocyte-specific Cre recombinase driver line (NPHS2.Cretg/wt)2+ after CNO administration solely occurred in podocytes.In this study, we present a novel transgenic mouse model to study Ca3D receptor in podocytes as demonstrated in vitro, ex vivo and in vivo, activation of the PLC pathway did not affect glomerular filtration or perfusion nor caused glomerular disease even after a prolonged stimulation for 18 consecutive days. Apparently, short and transient Ca2+ peaks as visualized by calcium imaging in vivo in this study are not sufficient to cause overt morphological alterations or functional changes at the renal filtration barrier. We did not observe any effect on glomerular perfusion, filtration or the onset of albuminuria. Our findings are in line with previously reported effects of G-protein coupled receptors in regulating glomerular biology. Expression of a constitutively active Gq-coupled receptor mutant in podocytes did also not result in any glomerular dysfunction under physiological conditionsSurprisingly, despite the proven functionality of the hM2+ signalling events are dispensable for podocyte function. As we activated the PLC pathway, increased Ca2+ levels primarily derive from the ER. This might be of particular importance in podocytes with their delicate cytoskeletal architecture. Increased Ca2+ levels close to the ER, which is primarily found in perinuclear regions in the podocyte cell body, might not resemble the intracellular Ca2+ concentration as observed in patients with TRPC6 gain-of-function mutations2+ levels as they harbor several homeostasis mechanisms including the Na+-Ca2+-exchanger, the ATP-dependent plasma membrane Ca2+ pump (PMCA) as well as Ca2+-buffers like calbindin and parvalbumin2+ levels due to the CNO administration might be compensated by these homeostasis mechanisms. In addition, the slit diaphragm might serve as a Ca2+ microdomain orchestrating Ca2+-dependent proteins like the TRPC channels, GTPases and synaptopodin. Furthermore, administration of CNO did only result in a transient Ca2+ peak in podocytes.These findings do not allow the assumption that Ca3D-transgene gets rapidly internalized or undergoes desensitization upon stimulation. However, studies performed in hippocampal neurons expressing a similar hM3D-transgene showed repetitive activation of the receptor 24 hrs after the initial CNO treatment2+ levels by stimulating podocytes with CNO at 0 and 24 hrs. It is therefore easily conceivable, that a sustained Ca2+ influx rather than a single Ca2+ peak is required to induce cytoskeletal changes to cause foot process effacement in podocytes.One might also speculate, whether the hMDREADD mouse model to study Ca2+ signalling events in vivo. As the DREADD sequence is integrated into the ROSA26 locus it can be easily applied for all murine tissues by mating the loxP-STOP-loxP hM3D mice with tissue specific Cre recombinase mouse lines. In podocytes, a transient Ca2+ peak alone is not sufficient to impair podocyte function challenging the current view of transient Ca2+ peak as regulators of glomerular biology.In conclusion, we present a novel 3D or GFP we used the commercially available pLenti6/V5-dest vector  as previously published3D or GFP construct respectively.Immortalized mouse podocytes were cultured with RPMI-1640 medium  with 10% fetal bovine serum, 5% sodium pyruvate solution 100 mM  and 5% HEPES buffer solution 1 M  as previously reported2HPO4 and 2 mM KH2PO4) containing 1 mM CaCl2 and 0.5 mM MgCl2 for three times. The cells were stained with an anti-FLAG antibody over night at 4 °C. The primary antibody was diluted in 1 x PBS with 1 mM CaCl2, 0.5 mM MgCl2 and 0.1% Triton-X and with 5% NDS. Prior to incubation with the secondary antibody, cells were washed again three times with 1 x PBS with 1 mM CaCl2 and 0.5 mM MgCl2. The respective secondary antibody was diluted in 1 x PBS with 1 mM CaCl2, 0.5 mM MgCl2 and 0.1% Triton-X. Mounting was performed with Prolong Gold with DAPI . Antibody dilution and commercial source are listed in Immortalized mouse podocytes were grown at 37 °C for 14 days. The cells were fixed with 4% paraformaldehyde, further blocking was performed with 5% normal donkey serum for 30 min. The cells were washed with 1 x PBS  and 1 mM of the serine protease inhibitor PMSF Phenylmethanesulfonyl fluoride  and 2 mM of the phosphatase inhibitor Na3VO4 . Following incubation on ice for 15 min and subsequent centrifugation  the supernatant was used for further analyses. The diluted lysates (with 2x SDS-PAGE buffer) were separated by SDS-PAGE. For gel electrophoresis the XCell SureLockTM Mini-Cell System was used. Electrophoresis was performed with 70 V for 30 min, followed by 25 mA/gel for 1:45 h. Blotting was performed on PVDF membranes  for 1 h at 12 V. After the transfer, membranes were blocked with 5% bovine serum albumin (BSA)  for 30 min, followed by three washing steps. The primary antibody was diluted in washing buffer  Tween20, pH 7.5) and incubation was performed over night at 4 °C. The respective secondary antibody was diluted in washing buffer and incubated 45 min at room temperature, followed by visualization with chemiluminescence with a Fusion Solo S . Antibody dilution and commercial source are listed in Immortalized mouse podocytes were stimulated with 1 μM CNO  diluted in medium for 10 min. Afterwards the cells were harvested using a cell scraper and lysed in 100 μl 1% Trition-X 100 buffer  for 30 min at 37 °C. Afterwards they were kept in imaging buffer for 20 min at room temperature. Imaging was performed with a Confocal microscope LSM710/Axiobserver Z1 using an 40x objective. Pictures were taken every 0.78 secs. 2 μM CNO was added after 10 pictures for the immortalized mouse podocytes and 100 μM after 20 pictures for the primary glomerular cells. 3 μM Ionomycin was used as positive control and added to the immortalized cells after 280 pictures and after 130 pictures to the primary cells.Immortalized mouse podocytes were grown at 33 °C and cultured on 8 chamber slides . The cells were incubated with 5 μM Fluo-8  in imaging buffer  were performed according to manufacturer’s instructions.hM3D/wt mice is described in the results section in detail. Briefly, to generate mice expressing the hM3D transgene in podocytes, we mated ROSA26hM3D/wt mice to NPHS2.Cretg/wt mice2+ reporter mouse GCaMP3tg/wt hM3D/wt; NPHS2.Cretg/wt mice were mated with GCaMP3tg/wt to achieve a simultaneous hM3D and Ca2+ reporter expression in podocytes exclusively. All animals were on a pure C57/Bl6 background. The mouse holding was done in the University of Cologne animal facility according to standardized specific pathogen-free conditions. All animal experiments were performed in accordance with the guidelines provided by the LANUV NRW . The experimental protocol was examined and approved by the LANUV NRW.Generation of the ROSA26To isolate primary cells we perfused the kidneys via the renal arteries with magnetic beads  and isolated glomeruli as previously published41Mice were injected intra-peritoneally with 5 mg/kg bodyweight CNO  dissolved in NaCl either 5 or 18 consecutive days as published previouslyQuantification of the urinary albumin levels was performed with a specific mouse albumin ELISA kit according to the manufactor’s instructions . Creatinine levels were quantified with a urinary creatinine kit according to the manufactor’s instructions .® O.C.T.™ compound . Paraformaldehyde fixed kidneys were embedded in paraffin and used for cutting 2 μm thick sections. To study morphological changes we performed a periodic-acid Schiff staining. To this end, sections were stained with 0.9% periodic acid  for 10 min, followed by incubation in Schiff’s reagent for 10 min . After washing with tap water for 2 min sections were incubated for 10 min in Mayer’s hematoxylin solution . Mounting was performed with histomount. For immunofluorescence analysis frozen kidney sections (5 μm) were used. After fixation with paraformaldehyde sections were blocked with 5% normal donkey serum in phosphate buffered saline with 0, 1% Triton-X (PBS-T) and subsequently incubated with primary antibody diluted in PBS-T with 5% NDS overnight at 4 °C. The respective secondary antibody was diluted in PBS-T and incubation was performed for 45 min at room temperature. Mounting was done with Prolong Gold antifade with DAPI . All images were taken with an Axiovert 200 M microscope/C-Apochromat 63x/1.20 W . Images were further processed with ImageJ/Fiji Software and Photoshop CS4 version 11.0. Antibody dilution and commercial source are listed in Mice were perfused with phosphate-buffered saline via the heart and kidneys were either fixed in 4% paraformaldehyde overnight or frozen in Tissue-TekhM3D/wt; NPHS2.Cretg/wt; GCaMP3tg/wt mice were anaesthetized using isoflurane and buprenorphine (0.1 mg/kg). A tube was placed into the trachea to facilitate breathing, and the right carotid artery was cannulated for dye infusion and injection of CNO. 70 kDa Texas red dextrane was injected i.a. to label the vasculature. The left kidney was exteriorized via a small incision at the left flank of the animal. The mouse was placed onto a heated mouse holder and the kidney was stabilized and covered with a coverslip for imaging. Body temperature was maintained during the imaging time. Calcium imaging was performed with a TSC SP8 upright Multi-Photon microscope (Leica) using an IR Apo L25x/0.95 W objective and a Coherent Chameleon Vision II laser at a wavelength of 940 nm. For acquisition two external hybrid detectors were used. The time courses of calcium signals were acquired at 1 frame per second over 90 seconds. After 10 seconds of baseline recording, 5 mg/kg bodyweight CNO was injected. The images were further processed with Leica Application Suite X, Version 1.1.0.4 weeks old ROSAhM3D/wt; NPHS2.Cretg/wt mice were anaesthetized with isofluran and a sensor was taped directly on their skin. After injecting 5 mg/kg CNO i.p., we injected 15 mg/100 g FITC-Sinistrin i.v. and measured the GFR for 60 min in the freely moving mice. Calculation of the GFR was performed according to Schreiber et al.To study the glomerular filtration rate we made use of a transcutaneous measurement approachhM3D/wt; NPHS2.Cretg/wt mice and their littermate controls were perfused ex-situ at a constant perfusion pressure (100 mmHg) as described in detail previouslyKidneys from ROSAP-value < 0.05 was considered significant. For two groups with two independent variables 2 way ANOVA combined with Bonferroni’s multiple comparison test was applied and a P-value < 0.05 was considered significant.All results are expressed as means ± SEM, except for the GFR measurement, which is depicted as mean. Statistical significance was evaluated using GraphPad Prism version 6 for Windows . For two groups t-test was applied and a How to cite this article: Koehler, S. et al. Single and Transient Ca2+ Peaks in Podocytes do not induce Changes in Glomerular Filtration and Perfusion. Sci. Rep.6, 35400; doi: 10.1038/srep35400 (2016).


CalQuo , offers some automatic features in calcium analysis. Here, we present an advanced version of the software package: CalQuo2. CalQuo2 analyzes the calcium response in the Fourier-domain, allowing the number of user-defined filtering parameters to be reduced to one and a greater diversity of calcium responses to be recognized, compared to CalQuo that directly interprets the calcium intensity signal. CalQuo2 differentiates cells that release a single calcium response and those that release oscillatory calcium fluxes. We have demonstrated the use of CalQuo2 by measuring the calcium response in genetically modified Jurkat T-cells under varying ligand conditions, in which we show that peptide:MHCs and anti-CD3 antibodies trigger a fraction of T cells to release oscillatory calcium fluxes that increase with increasing koff rates. These results show that CalQuo2 is a robust and user-friendly tool for characterizing global, single cell calcium responses.Intracellular calcium acts as a secondary messenger in a wide variety of crucial biological signaling processes. Advances in fluorescence microscopy and calcium sensitive dyes has led to the routine quantification of calcium responses in non-excitable cells. However, the automatization of global intracellular calcium analysis at the single-cell level within a large population simultaneously remains challenging. One software,  In cell-mediated immunity, Ca2+ signaling is crucial for T-cell development, tolerance, and homeostasis3. In particular, Ca2+ signaling plays a key role in T-cell activation5, in which 75% of genes derived from the cDNA of stimulated T cells were found to be Ca2+ influx dependent6. Specifically, triggering of the T-cell receptor (TCR) following engagement with peptide:MHC (pMHC) leads to the release of Ca2+ from the endoplasmic reticulum (ER) by the opening of the inositol 1,4,5-triphosphate receptor (IP3R), an ER transmembrane Ca2+ channel8. Subsequent Ca2+ fluxes result from store operated channels (SOC), known as Ca2+ release-activation Ca2+ (CRAC) channels9. Various Ca2+ responses have been found in cells with correlatively different downstream effects on the activity of transcription factors, e.g. Nuclear Factor of Activated T cell (NFAT), and Nuclear Factor (NF)-κB11.Calcium  within each cell needed to then quantify the Ca2+ response22.Considering the importance of CaCalQuo is a MATLAB-based software that attempts to overcome these limitations by automatically reading in and segmenting images, allowing for the classification of the global Ca2+ intensity of a few thousand cells simultaneously over time and the subsequent quantification of cells with a particular type of calcium response20. CalQuo analyzes the changes in Ca2+ intensity of each cell over time to determine whether a cell has triggered, allowing the fraction of cells that have triggered in the sample population to be quantified20. CalQuo is compatible with images acquired using a fluorescence spinning-disk confocal microscope, which can image Ca2+ responses on the sub-second time-scale20. In order to use CalQuo, however, several parameters regarding the curve of the Ca2+ intensity must be manually input to achieve successful filtering of the data20. User-dependent parameters describing the landing event where the cell first makes contact with the activating surface, the peak fluorescent intensity, the time between landing and peak events, and the time-scale of the signal decay are required to characterize a typical curve. Despite the success of this approach, input of these parameters limits the flexibility in which a triggering cell may be recognized and does not differentiate between the various Ca2+ responses a cell may have20. In addition, determining each parameter is time-consuming and opens the possibility of user error and bias.CalQuo, called CalQuo2, where new algorithms to analyze and characterize Ca2+ responses have been developed and implemented. Using antigen functionalized coverslips to trigger T cells and, thus, induce Ca2+ release  evolution to segment each individual cell and integrate its Ca2+ intensity over time. Building upon CalQuo, CalQuo2 implements the Bio-Formats 5.3.3 toolbox (http://www.openmicroscopy.org/site/products/bio-formats) to accelerate the rate at which an image stack is read into MATLAB . Once the Ca2+ response curve has been extracted a Fourier transform of the Ca2+ based intensity signal is calculated, allowing filtering of the signal to be carried out based on the phase and frequency of the Ca2+ response (see Supplementary Information). Critically, triggering and non-triggering cells have distinct signatures in Fourier space, and choosing a radius in the imaginary (|Im|) or real (|Re|) part on the complex plane allows the two signatures to be separated  above baseline in a normalized intensity curve, will hence have near zero values for their imaginary and real components .To address these concerns, we developed an updated version of ase Fig. , time-coell Fig. . As descnts Fig. . Likewisdes Fig. . Thus, a2+ pattern elicited from a triggering T cell is important in determining the nuclear localization of the transcription factors and thus the type of T cell response11, CalQuo2 further builds upon the capabilities of CalQuo by separating cells with a single Ca2+ flux from cells with oscillatory Ca2+ fluxes. By setting a minimum threshold, defining a peaked response in the derivative of the intensity curve, the program can separate between a cell that displays only a single peak and those that display multiple intensity peaks above the minimum threshold. Cells that show two or more peaks above the minimum threshold are defined as oscillatory. The difference between singular peak, oscillatory, sustained, and no Ca2+ flux can be seen in the normalized intensity plots and Fourier domain plots , suggesting that oscillatory Ca2+ flux responses specify weak stimulation, whereas singular responses signify fully activating T cells. We find that CalQuo2 was able to successfully demonstrate that pMHCs and anti-CD3 Abs trigger a fraction of T cells to release oscillatory Ca2+ fluxes that increase with increasing koff rates.In the following, we first validate the performance of et al.1. The inserted sequence was then sub-cloned into the pHR vector, according to the protocol by Aleksic et al.2. By over-expressing the 1G4 β chain, the original β chains in the E61_57 cells were removed. Using UCHT1, the transfected cells were stained and sorted to keep TCR negative cells. The same process was repeated twice before a final cell line, E61_57_1G4-β+ cell line, was produced. The 1G4-α chains were then added to achieve the E61_57_1G4-TCR cell line, which we refer to as the 1G4-TCR Jurkat T cells.Wild-type Jurkat E61_57 T-cells were transduced to express 1G4-TCRs and CD8 coreceptors to produce 1G4-TCR Jurkat T-cells, given by Veronica Chang, a former member of the Simon J. Davis laboratory . The Jurkat clones given by Takamasa Ueno , were devoid of the TCR α chain, low in the TCR β chain expression, and low in CD4 expression. To transduce these Jurkats, cDNA of the 1G4-TCR’s α and β chains, given by Vincenzo Cerundolo , were amplified via polymerase chain reaction (PCR). Using the AgeI and KpnI restriction sites, the amplified PCR products were ligated into the region following the gene that encodes the chicken RPTPσ signal peptide of the pHLsec vector, according to Li 2. Cells were maintained at ~5–10 × 105 cells/mL in 25 mL of RPMI media . The media was supplemented with 1% of 10,000 units of penicillin and 10 mg/mL of streptomycin , 1% of 200 mM L-glutamine , 1% of 100 mM sodium pyruvate , 1% of 1 M of HEPES , and 10% of fetal bovine serum (FBS) .1G4-TCR Jurkat T-cells were incubated in a T-75 flask at 37 °C in 5% CO5 cells/mL and a cell viability of 80%, were taken from a T-75 flask and centrifuged at 1500 rpm for 3 minutes to form a pellet. After discarding the supernatant, cells were resuspended in 50 µL of L15 medium , 50 µL of HBS  buffer containing 2.5 µM of probenecid , and 1 µL of 1 mM Fluo-4 AM . Cells were incubated for 5 minutes at 37 °C followed by washing twice in HBS buffer. The HBS buffer contained 150 mM of NaCl and 10 mM of HEPES in distilled water with a final pH of 7.05 and was then supplemented with probenecid to contain 2.5 µM of probenecid in HBS.2 mL of the 1G4-TCR Jurkat T-cells, with a cell count of 5–10 × 10All experiments were conducted on 18 mm # 1.5 circular, glass coverslips . Each coverslip was first coated with 20 µL of a biotin-BSA:BSA mix, containing biotinylated BSA at a concentration of 200 µg/ml and BSA at a concentration of 800 µg/ml in distilled water. After 1.5 hrs. of incubation with the biotin-BSA:BSA at room temperature, the coverslips were washed three times in 2 mL of PBS. Coverslips were then coated with 20 µL of streptavidin at a concentration of 10 µg/ml and incubated at room temperature for 1.5 hrs. Coverslips were washed three times with 2 mL of PBS. Coverslips were then coated with 20 µL of either biotinylated pMHCs, biotinylated OKT3, biotinylated UCHT1 at a concentration of 10 µg/ml. For negative control coverslips, the addition of antigen at the final step was omitted. After a further 1.5 hrs. of incubating at room temperature the coverslips were washed with 2 mL of PBS three times and stored at room temperature until required for imaging. Coverslips were prepared fresh on the day of the experiment.157–165 peptide23. Two variants of the NY-ESO-1157–165 peptide were presented on the MHC class I complexes used: one with a mutation to valine at the 165 position (referred now on as 9V) and one with a mutation to aspartate at the 160 position (referred now on as 4D)23. Peptides were synthesized by Generon (UK).The 1G4-TCR binds specifically to the NY-ESO-1Protein biotinylation was required to specifically functionalize glass surfaces. To achieve biotinylation, an N-Hydroxysuccinimidet (NHS)-ester labelling method was used that targets amine groups typically found on free lysines within the protein. All proteins to be biotinylated were first passed through a desalting column  to remove any buffer components that may interfere with the biotinylation reaction (e.g. Tris). The reaction was conducted in a volume of 200 μL and a protein concentration of 1 mg/ml. The pH of the solution was adjusted to pH 8 using a 1 M solution of NaOH. Biotin-NHS  was added to the protein solution at a molar ratio of 10:1 and incubated for 1 h at 37 °C. Following incubation, the solution was passed through a further desalting column to remove any unbound biotin.2+ flux. All imaging was conducted at 37 °C and at 5% CO2. The Ca2+ flux was visualized by the excitation of the calcium sensitive dye Fluo-4 AM with 488 nm laser light set to 70% of the maximum output power (measure at the objective to be 0.52 mW). The movies were captured using an AxioCam MRm camera. Each experimental acquisition was 1000 frames long, with a frame time of 820 milliseconds, set to maximize signal to noise whilst capturing sufficient temporal resolution. Ionomycin was added during the final 100 frames (82 s). Frame rates and laser powers were chosen to reduce photo-bleaching while maintaining sufficient temporal resolution to capture the dynamics of the Ca2+ response. By integrating the intensity of the whole acquisition frame over time, it is possible to appreciate how on the time scale of the Ca2+ response, the level of photo-bleaching is minimal .2+ responses in Jurkat T-cells24 but manual analysis makes large scale quantification of these differing and complex responses challenging. We exemplified the use of CalQuo2 by analyzing the Ca2+ patterns in Jurkat T-cells interacting with glass coverslips functionalized with antigens of differing binding affinities  esterified Fluo-4 that allows the dye to permeate the cell membrane, upon which a de-esterified Fluo-4 acts as a fluorescent indicator with a 100-fold increase in fluorescence intensity when bound to Ca2+28. A Zeiss spinning disk confocal scanning microscope was used to visualize the Ca2+ response with a numerical aperture (NA) of 0.45 and a frame rate of 1.2 frames per second (s). After 900 frames (738 s), ionomycin was added to ensure that each cell could release Ca2+ from its ER stores. Furthermore, the cell(s) showing the greatest fluorescence intensity on addition of ionomycin was used to normalize the fluorescence intensity across the population of cells to account for possible inhomogeneous loading of Fluo-4 AM in each acquisition .To first characterize the accuracy of ime Fig. . The difCalQuo2, we quantified its ability to distinguish single and oscillating responses. Compared to visual inspection and manual sorting of Ca2+ responses, CalQuo2 accurately separated oscillatory and single peak Ca2+ responses on average of 90 ± 4% of the time in the 100 cells picked in a random sample in each of the three replicates in which the T cells had landed on the 9V:MHC functionalized coverslips .To further asses the performance of ips Fig. . The dif2+ response classification, CalQuo2 is able to quantify the time between the landing of the cell and increase in Ca2+ intensity which is referred to as the triggering time. Using the derivative of the Ca2+ intensity to find peaks, using the MATLAB ‘findpeaks’ function , the triggering time was determined by subtracting the time of the lowest peak from the highest peak. The triggering time, period of time that passed between cell landing and the first flux of Ca2+; triggering fraction, the fraction of cells that triggered; single peak fraction, fraction of triggered cells with only one Ca2+ influx; and oscillatory peak fraction, fraction of triggered cells with multiple Ca2+ influxes, were measured to characterize the Ca2+ response for each antigen coated surface (see Supplementary Information).In addition to CaCalQuo2 to quantify differences in the Ca2+ response to differing peptides, Jurkat T-cell were systematically exposed to different pMHCs and their Ca2+ responses quantified and classified by CalQuo2 . Additionally, the mean single peak fraction of the triggered cells that landed on the 9V:MHC coverslip was significantly greater than that of triggered cells that landed on the 4D:MHC coverslip by 0.217 a.u. with p = 0.039. The reverse was seen in the oscillatory peak fraction of triggered cells . For example, despite UCHT1 having the lowest koff rate among the four antigens used, T cells exposed to 9V:MHC had the highest single peak fraction, which was significantly higher than T cells exposed to 4D:MHC. Additionally, despite OKT3 having a similar koff rate to 9V:MHC, which are 0.39 s−1 and 0.33 s−1, respectively30, T cells contacting OKT3 did not have a significantly higher single peak fraction than 4D:MHC, which has a koff rate of 2.59 s−1 23. However, when evaluating cells stimulated by only pMHCs or by only Abs, the mean single peak fraction of triggered cells monotonically decreases with increasing koff rates, especially in regards to cells stimulated by pMHCs . By analyzing the response function in Fourier space, CalQuo2 is robust against noise in the raw data, offering improved performance over algorithms based on a single threshold21.From the raw fluorescent images, 2+ responses both manually and by CalQuo2, we demonstrate the capacity of CalQuo2 to differentiate between triggering and non-triggering cells and between single and oscillatory cells without biasing the data towards one category over another. Thus, CalQuo2 successfully employs the Fourier transform and just one user-defined parameter, the triggering radius, to categorize calcium responses as either triggering or non-triggering. Analyzing calcium responses transformed into the Fourier space removes the need to specify each of the five parameters defining a single, specified model of a calcium curve, as was required for the previous version of CalQuo. Reduction in these user input parameters increases the flexibility in which a triggering calcium curve can be identified and lowers the introduction of user bias and error. Moreover, users with limited programming and computational ability can more easily use and access this quantitative Ca2+ analysis tool, broadening the application of such a software. On top of enhanced performance, CalQuo2 offers the ability to separate cells with single and oscillatory calcium responses.Comparing the quantifications of CaCalQuo2 to analyze the triggering time, we were able to show that T cells interacting with uncoated negative control coverslips took longer to release Ca2+ than T cells interacting with antigen functionalized coverslips. The triggering fraction, however, did not show a significant difference in the Ca2+ response between T cells landing on antigen functionalized and on non-antigen functionalized glass coverslips. Nonetheless, when separating the cells into those that express a single Ca2+ flux and those that express oscillatory Ca2+ fluxes, statistically significant differences were found between the T cells placed on the UCHT1, 9V:MHC, OKT3, or 4D:MHC functionalized coverslip and the T cells placed on the negative control coverslips. And, although a monotonic negatively correlated trend could not be seen between the koff rates and the single peak fraction of the triggered cells, when separating these T cells into those that were stimulated by pMHCs and those by anti-CD3 Ab, the single peak fraction were seen to decrease with increasing koff rates. These results indicate that while the koff rate of an antigen is important in determining the type of Ca2+ responses induced in T cell, the type of antigen and where that antigen binds/stimulates the TCR-CD3 complex are also significant factors in the type of Ca2+ response.Using CalQuo2 is a software capable of quantifying large populations of cells displaying diverse Ca2+ responses. This will be important when links are to be drawn between the nature of the calcium signaling and the wider biological function. Single, oscillatory, and sustained Ca2+ fluxes have been observed in various studies28. Wülfing et al.21 propose that the different Ca2+ patterns may arise from different transformations of the T cells used, with hybridomas displaying greater oscillatory Ca2+ responses. In the presented experiments, one type of T cell is used, and different Ca2+ responses are seen under different ligand conditions, suggesting that the strength and type of stimulus affect the pattern of the Ca2+ response. A single Ca2+ flux is consistent with the type of response seen in wild type Jurkat T - cells20, while oscillatory Ca2+ fluxes may be indicative of a T cell trying to increase the sensitivity to weaker stimuli, since there is not a linear correlation between Ca2+ levels and activation of transcription factors28. Therefore, it is believed that single Ca2+ flux on a twelve-minute time-scale is indicative of a stronger triggering response than an oscillatory Ca2+ response. Without CalQuo2, we would not be able to demonstrate the differences between single and oscillatory Ca2+ responses in a quantitative manner, thereby forfeiting the ability to understand the biological importance of Ca2+ in T-cell triggering and activation. Crucially, CalQuo2 addresses the need within the field, providing a software capable of quantifying diverse Ca2+ responses necessary to understand the broader functional role of such responses.CalQuo2 is an improved MATLAB-based version of CalQuo, capable of automatizing the analysis of Ca2+ responses found in single cells and within the whole sample population. With only one manual parameter that can optionally become automatic, there are less degrees of freedom with CalQuo2, increasing the accuracy in which cells are considered to be triggering or non-triggering based on their Ca2+ response. The automatization of CalQuo2 without any complex mathematical computation required of the user makes CalQuo2 easy to use and reduces user bias and error. CalQuo2 is suitable for analyzing Ca2+ responses in non-excitable cells at sub-second time-scales.In conclusion, Supplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5