Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca2+ signals otherwise obscured during large Ca2+ elevations. We find the rising phase of global Ca2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca2+ depletion. The continuing rise in Ca2+, and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca2+ signaling. Puffs make only small, transient contributions to global Ca2+ signals, which are sustained by diffuse release of Ca2+ through a functionally distinct process. These two modes of IP3-mediated Ca2+ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca2+-dependent effector functions and Ca2+ transport.The ‘building-block’ model of inositol trisphosphate (IP Ca2+ diffusing from one open channel may thus trigger the opening of adjacent channels, with self-reinforcing CICR countered by Ca2+ feedback inhibition. The clustered distribution of IP3Rs further shapes the extent of this regenerative process. CICR may remain locally restricted to a single cluster, containing from a few to a few tens of functional IP3Rs, to produce a puff; whereas it is proposed that a global response is generated by successive cycles of CICR and Ca2+ diffusion acting over longer spatial ranges to recruit successive puff sites  during even large amplitude global Ca2+ elevations we developed image processing and analysis routines to analyze local fluctuations in Ca2+ fluorescence signals  microscopy to resolve signals arising near the plasma membrane, and by lattice light-sheet (LLS) microscopy to acquire optical sections through the cell interior. We find that rapid flurries of Ca2+ puffs accompany the rising phase of global Ca2+ signals evoked by photoreleased IP3 and by agonist stimulation of the IP3 signaling pathway, but these rapidly terminate before the peak of the response through a mechanism regulated by ER Ca2+ store content. The punctate liberation of Ca2+ via transient, localized Ca2+ puffs contributes only a small fraction of the total Ca2+ liberated during global Ca2+ signals, which are instead sustained by diffuse Ca2+ liberation through a functionally distinct mode of release. These two modes of IP3-mediated Ca2+ release will likely selectively activate different populations of effectors; those positioned close to the IP3R clusters that mediate puffs and which respond to brief, repetitive transients of [Ca2+], and others that respond to a more sustained, spatially diffuse elevation of bulk cytosolic [Ca2+].Here, we examined the nature of Cak’ model  in which signals , a chall signals . We appl3-evoked Ca2+ liberation during cell-wide Ca2+ signals arises through coordinated activation of pulsatile, spatially -localized events, analogous to the local Ca2+ puffs observed with weaker IP3 stimulation or after loading cells with EGTA to suppress global signals , and a band-pass temporal Butterworth filter (3–20 Hz). The resulting image stack was then processed by a running boxcar window (160 ms) that, for each pixel, calculated the standard deviation (SD) of the fluorescence signal at that pixel throughout the duration of the window. These parameters were chosen to optimally ‘tune’ the algorithm to reject slow changes in baseline fluorescence and attenuate high-frequency photon shot noise while retaining frequencies resulting from puff activity . Lastly,e source .2+ elevation (panels ii-iv), but ceased at the time of the peak response (panel v). This behavior is further illustrated by the black traces in 2+ response to photoreleased i-IP3 but had largely subsided by the time of the maximal global Ca2+ elevation. Even though the global Ca2+ level then stayed elevated for many seconds the mean SD signals at these regions remained low. Measurement of the SD signal derived from a ROI encompassing the entire cell  and kinetics to that evoked by photoreleased i-IP3 (6.9 ΔF/F0) in To further validate the fluctuation analysis algorithm, we examined a situation where cytosolic  is not the primary mechanism causing puff activity to terminate during whole-cell Ca2+ responses. They further buttress other evidence that the decline in SD signal during the rising phase of the response does not arise because the indicator dye becomes saturated, but faithfully reflects a physiological termination of puff activity.Taken together, these results demonstrate that inhibition of IP2+ elevations because of falling luminal ER [Ca2+], rather than rising cytosolic [Ca2+]. We tested this idea by imaging i-IP3 evoked global Ca2+ signals after partially depleting ER Ca2+ stores while minimizing changes in cytosolic free [Ca2+].We next considered the possibility that puff activity may terminate during the rising phase of global Ca2+ from the ER and a small elevation of cytosolic Ca2+. Following wash-out of CPA, the cell was maintained in Ca2+-free medium so that the cellular Ca2+ content (including that of the ER) gradually depleted owing to passive and active extrusion across the plasma membrane. After about 4 min the resting cytosolic Ca2+ level had returned close to the original baseline, and we delivered a photolysis flash to photorelease i-IP3. This evoked a substantial elevation in global Ca2+, yet the SD signal showed almost no transient puff activity during this response. Similar results were obtained in a further seven cells, as shown by the mean ΔF/F0 and SD traces in 2+ depletion, we repeated this experiment, now making a paired comparison of i-IP3-evoked responses between cells that were bathed for 30 min after washing out CPA either in Ca2+-containing medium to allow ER store refilling  as a function of the global Ca2+ level (ΔF/F0) during the rising phase of the global response further highlighted these differences  rose to roughly 1.5, 6, and 3 for types 1, 2, and 3 IP3Rs, respectively; and similarly large differences were evident in the global Ca2+ level attained when puff activity terminated.We evoked Can of CCH . All thrlevation . Neverthd longer . In contng phase . Ca2+ sing IP3R3  were simferences . Ca2+ fl3R isoforms  of puff sites in WT HEK cells are located  microscopy to record Ca2+ signals within diagonal optical ‘slices’ through the cell volume  and SD signals from ROIs encompassing peripheral (red traces) and central (blue traces) regions of the cells, as indicated in the leftmost lower panels of 2+ activity monitored by SD fluctuations started within a few hundred ms of the photolysis flash and was maximal during the early portion of the rising phase. The SD signal then declined, returning close to baseline as the global Ca2+ signal approached a peak. For the cell illustrated in 2+ was slightly smaller. In contrast, the cell illustrated in 2+ level as peripheral sites  that were determined from the tail-end of the global Ca2+ decay for that particular cell.We then calculated the instantaneous Ca2+ liberation during Ca2+ signals under normal conditions, and when puff activity had been inhibited by partial depletion of ER Ca2+ store content. 2+ responses of comparable peak amplitudes (black traces). However, whereas the SD signals (grey traces) exhibited the normal flurry of puff activity in the control cell  and CPA-treated cells (n = 6). Colored areas indicate the relative cumulative amounts of Ca2+ entering the cytosol  in CPA-treated cells where puff activity was substantially abolished (blue shading), and the additional Ca2+ flux (pink shading) in control cells showing flurries of puffs. From these respective areas, we estimate that, in normal conditions, the punctate liberation of Ca2+ through puff activity contributes about 41% of the total Ca2+ release responsible for the initial rise of Ca2+ toward its peak. 2+ (black), rate of Ca2+ release into the cytosol (red), and cumulative amount of Ca2+ released (blue) during the entire time course of global Ca2+ signals evoked by photoreleased i-IP3  we used for most experiments may have approached saturation, thus ‘clipping’ the signals to artifactually suppress the temporal SD signal and giving a false impression that puff activity terminates as the Ca2+ level and fluorescence rise during global signals. Several lines of evidence convincingly argue that this is not the case. Notably: (i) maximal, saturating signals evoked by ionomycin (~19 ΔF/F0) were much higher than mean peak IP3-evoked fluorescence signals (~7 ΔF/F0), and puff activity began to decline as fluorescence rose above ~2 ΔF/F0; (ii) we observed patterns of puff activity using the low affinity indicator fluo-8L (Kd1.86 µM) that closely matched those obtained with Cal520 (Kd320 nM)  ; (iii) wevations ; (iv) th signals ; (v) theelevated  or, convlic EGTA . Finallydepleted  strongly3-mediated global Ca2+ signals. However, although puff activity was evident during the initial foot of the response and peaked early during the rising phase, it then subsided during the later portion of the rising phase, with few or no transient, local Ca2+ signals evident by the time of the peak. Notably, overall Ca2+ levels continued to rise after puffs had largely ceased, and cytosolic Ca2+ remained elevated for several seconds in the face of rapid removal from the cytosol, during which time Ca2+ fluctuations were largely suppressed.In agreement with previous findings  our fluc2+ liberated as puffs to fill in spaces between release sites. Diffusion would be rapid (e.g. mean time of ~300 ms to diffuse 5 μm assuming an effective diffusion coefficient of 20 μm2 s−1). In any case, the average fluorescence signal would not be expected to increase appreciably if the total amount of Ca2+ in the imaging volume remained constant. Utilizing lightsheet imaging we further excluded the possibility that the continuing rise in near-plasmalemmal Ca2+ observed by TIRF imaging might arise through diffusion of Ca2+ over longer distances after liberation at sites in the cell interior. Finally, as noted previously, the observation of large global Ca2+ signals in the absence of detectable fluorescence fluctuations  of the cumulative flux during its entire time course. Thus, a second component of continuous, spatially diffuse release of intracellular Ca2+ is responsible for generating and sustaining a large part of whole-cell Ca2+ signals. These two components are further discriminated by procedures that selectively promoted either puff activity (2+ buffers), or global Ca2+ elevations in the absence of localized Ca2+ transients . We term these two modes of IP3-mediated Ca2+ release as ‘punctate’ (discontinuous in time and space) and ‘diffuse’ (smoothly varying in time and space).By deriving the time course of cumulative Caactivity   fraction of the IP3Rs in a cell are stationary   , it has unctions . On the ions  and cell lines natively expressing exclusively type 1 , type 2  and type 3  isoforms generated from that parental WT HEK293 cell line by CRISPR/Cas9 genetic engineering in the Yule lab were purchased from Kerafast . HEK cell lines were characterized as described in 3R isoform-expressing cell lines were cultured in Eagle’s Minimum Essential Medium , and HeLa cells were cultured in Dulbecco’s modified Eagle Medium  from Thermo Fisher Scientific . Both EMEM and DMEM were supplemented with 10% fetal bovine serum (#FB-11) from Omega Scientific . All cell lines were cultured in plastic 75 cm flasks and maintained at 37°C in a humidified incubator gassed with 95% air and 5% CO2. For imaging, cells were collected using 0.25% Trypsin-EDTA (Thermo Fisher Scientific #25200–056) and grown on poly-D-lysine  coated (1 mg/ml) 35 mm glass bottom imaging dishes (#P35-1.5–14 C) from MatTek  or 12 mm glass coverslips for 2–3 days.HEK293 wild-type (WT) cells were kindly provided by Dr. David Yule (University of Rochester). An IP2+ indicator Cal520/AM  for 1 hr at room temperature (RT) in a Ca2+-containing HEPES buffered salt solution (Ca2+-HBSS). Where indicated cells were additionally loaded with membrane permeant esters of either the caged IP3 analogue ci-IP3/PM   or the caged Ca2+ buffer NP-EGTA [o-nitrophenyl EGTA/AM]  in conjunction with Cal520. For the experiments in 2+-HBSS to attenuate global Ca2+ elevations. To address possible saturation of Cal520 at high Ca2+ levels, cells were alternatively loaded with the lower affinity Ca2+ indicator fluo8L/AM  together with 1 µM ci-IP3/PM for 1 hr at RT in Ca2+-HBSS. Following incubation with AM esters, cells were incubated for 30 min at room temperature in Ca2+-HBSS. Cal520/AM, ci-IP3/PM, and NP-EGTA/AM, EGTA/AM, and fluo8L/AM were all solubilized with DMSO/20% pluronic F127 (Thermo Fisher Scientific #P3000MP).Immediately before imaging, cells were incubated with the membrane-permeant fluorescent Ca2O. Ca2+-HBSS contained (in mM) 135 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH = 7.4). Nominal Ca2+-free HBSS consisted of the same formulation as Ca2+-HBSS except that CaCl2 was omitted; for zero Ca2+-HBSS, 300 µM EGTA was added to nominal Ca2+-free HBSS. For lattice light-sheet imaging, the plasma membrane was stained by adding Cell Mask Deep Red (Thermo Fisher Scientific #C10046) to the bathing solution in the imaging chamber at 1/50,000 dilution.FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone) and CPA (cyclopiazonic acid), were purchased from Millipore Sigma , and solubilized in 100% ethanol. Carbachol (#C4832) and histamine (#H7125), also from Millipore Sigma, were reconstituted in deionized H2+ signals was accomplished using a home-built system, based around an Olympus  IX50 microscope equipped with an Olympus 60X oil immersion TIRF objective (NA 1.45). Fluorescence images were acquired with an Evolve EMCCD camera  with a bit depth of 16 bits, using 2 × 2 binning for a final field at the specimen of 128 × 128 binned pixels (one binned pixel = 0.53 µm) at a rate of ~125 frames s−1. Image data were streamed to computer memory using Metamorph v7.7  and stored on hard disk for offline analysis.Total internal reflection fluorescence (TIRF) imaging of Ca−1 and 16 bit depth. Ca2+ signals were imaged in Cal520-loaded cells for several seconds following photorelease of i-IP3 by a flash from a 405 nm laser diode, utilizing 473 nm laser fluorescence excitation and a 510–560 nm bandpass emission filter. A 562 nm laser and 590 nm long-pass filter were used to image the plasma membrane stained with Cell Mask Deep Red. Images were streamed to disk using MicroManager .Lattice light-sheet imaging was performed using a home-built system as previously described . Images 3 was evoked by UV light from a xenon arc lamp filtered through a 350–400 nm bandpass filter and introduced by a UV-reflecting dichroic in the light path to uniformly illuminate the field of view. The amount of i-IP3 released was controlled by varying the flash duration, set by an electronically controlled shutter . The same system was used for photolysis of NP-EGTA (i.e. caged Ca2+). For the local delivery of solutions to cells during imaging, glass micropipettes were prepared from borosilicate glass capillary filaments  using a micropipette puller  to produce tip diameters of ~1–2 µm. Micropipettes were positioned above the cell understudy with local delivery controlled using a pneumatic picospritzer. The delivery of micropipette contents and the duration and intensity of the UV-flash were empirically adjusted to evoke rapid rises in whole-cell cytosolic Ca2+ levels.Photorelease of i-IP2+ or zero Ca2+-HBSS containing 0.3 mM EGTA and no added Ca2+.All imaging was performed while cells were bathed in HBSS containing 2 mM Cahttp://flika-org.github.io), a freely available open-source image processing and analysis software in the Python programming language  of eviously . In briespatial fluctuations in Ca2+ image stacks. For this, black-level subtracted image stacks were first temporally band-pass filtered as before, and then new image stacks were derived by taking the difference of weak (sigma two pixel) and stronger (sigma eight pixel) Gaussian blur functions. Essentially, this functioned as a spatial bandpass filter, attenuating high frequencies caused by pixel-to-pixel shot noise variations and low frequency variations resulting from spread of Ca2+ waves across the cell, while retaining spatial frequencies corresponding to the spread of local Ca2+ puffs. Next, for each frame, we calculated the spatial variance among pixels within an imaging field, took the square root to obtain a measure of the spatial SD signal and subtracted the predicted increase in spatial shot noise with increasing fluorescence. Finally, we derived traces showing the average spatial fluctuations across a region encompassing the entire cell within a moving boxcar window of 160 ms  to give global Ca2+ signals. It has, however, been difficult to examine this hypothesis because as Ca2+ puffs become more frequent, it becomes impossible to resolve them from background signal. The present work applies an elegant noise analysis method  to assess the subcellular heterogeneity of Ca2+ signals evoked by IP3. The results are both unexpected and important in that they suggest that most cytosolic Ca2+ signals are not associated with the noise expected from Ca2+ puffs. The results convincingly challenge prevailing dogma by suggesting that Ca2+ puffs contribute only a small fraction the cytosolic Ca2+ signals evoked by IP3, with the remainder likely arising from openings of single IP3Rs. The authors have done an excellent job of revising the manuscript to address the technical concerns of the reviewers, and overall this is a rigorous, well done and significant body of work that justifies publication in eLife.Parker's pioneering work established that the initial responses to low concentrations of IP3 comprise CaDecision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Colin W Taylor (Reviewer #1); Grant Hennig (Reviewer #3).Thank you for submitting your work entitled "Inositol trisphosphate mediated Ca2+ spikes arise through two temporally and spatially distinct modes of Ca2+ release" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in 2+ indicators and the spatial and temporal Ca2+ release patterns.The reviewers agreed that your work is interesting and has the potential to be quite important, but there were major concerns with the SD approach, on which most of the presented findings hinge, involving the dynamic range of the Ca2+ indicators have become saturated at peak cytoplasmic background Ca2+ levels . This would make it essentially impossible to resolve punctate events, even though punctate release events may still be occurring – akin to "signal clipping". While SD analysis may provide some additional resolution in situations with narrow dynamic fluorescence ranges, it still depends on the indicator having some dynamic range. This concern needs to be carefully addressed so that readers are confident that the presented findings are accurate and biological.The first concern is that after uncaging or after the addition of agonists, the cytoplasmic background fluorescence in cells increases to a seemingly very high level. As punctate release events are only observed when background levels are low to moderate, it brings into question whether the CaSome ways that could be used to examine the potential dynamic range issue of the indicators include:2+ levels during uncaging/agonists which could be compared to Kd/saturation mM of Cal-520 and GCamP etc.• use a ratiometric indicator (Fura) to estimate actual cytoplasmic Ca2+ using low/moderate levels of EGTA (to preserve the dynamic range of the indicator) and examine whether punctate release patterns are still reduced after uncaging etc.• pharmacologically limit the increase in cytoplasmic Ca2+ levels to near maximum with ionomycin then initiate an uncaging event to see puncate release sites can still be resolved .• purposefully increase cytoplasmic Ca2+ concentrations. At these levels where there is extremely limited dynamic range and greater shot noise, noise aggregates and punctate release sites will appear similar – but they can be separated using perimeter:area ratios (high for noise aggregates), or Gaussian fitting (better for release sites). This will require no Gaussian filtering of the data and some form of deconvolution to reduce the smoothing of objects due to Z-smear (where appropriate).• use quantitative shape analysis (not ROIs) of transient bright regions at high cytoplasmic Ca2+ behaviors are included or filtered by varying the space/time variance parameters.The second concern is discussed adequately in the reviews below, but questions whether the SD approach can detect synchronous, widespread puffs/ release events, or high frequency puffs/release events that appear and behave like changes in cytoplasmic background. It would be useful to the reader to see how different duration timespans affect the variance signal , to assess what range of CaeLife.Without additional information to address the major concerns of the reviewers, it is not possible to assess whether the work represents a sufficient advance to warrant publication in Reviewer #1:2+ puffs, wherein a few IP3Rs within an immobile cluster almost simultaneously open to evoke a local and short-lived cytosolic Ca2+ signal. It has been widely assumed that Ca2+ released by these events recruits further Ca2+ puffs (by Ca2+-induced Ca2+ release) to give global Ca2+ signals. It has, however, been difficult to examine this hypothesis because as Ca2+ puffs become more frequent, it becomes impossible to resolve them from background signal. The present work applies an elegant noise analysis method  to assess the subcellular heterogeneity of Ca2+ signals evoked by IP3. The results are both unexpected and important in that they suggest that most cytosolic Ca2+ signals are not associated with the noise expected from Ca2+ puffs. The results convincingly challenge prevailing dogma by suggesting that Ca2+ puffs contribute only a small fraction the cytosolic Ca2+ signals evoked by IP3, with the remainder likely arising from openings of single IP3Rs. The rigour and significance of the work justify publication in eLife; none of my comments should detract from that recommendation.Parker's pioneering work established that the initial responses to low concentrations of IP3 comprise Ca2+ signals develop and that a comparable increase in global cytosolic [Ca2+] evoked by ionomycin is “noise-free”, as expected for homogenous Ca2+ release. My understanding is that after some filtering and spatial blurring, most analyses calculate the SD for each pixel as a 160-ms (ie 20 frames) boxcar average. The original view posits that a global Ca2+ signal reflects most Ca2+ puff sites firing very frequently (perhaps 20 or so sites active at each instant), each spreading a µm or more from its source. Might this cause elevations in cytosolic [Ca2+] that are less noisy than the infrequent Ca2+ puffs observed initially, because each pixel would be influenced by Ca2+ reaching it from several puffs? It's perhaps too tall an order to require quantitative simulation of this on SD measurements, but it would be helpful to have the authors consider the issue explicitly.1) Figure 1 provides compelling evidence that noise decreases as the IP3-evoked Ca2+ from the ER causes puffs to terminate without appreciably abrogating the global increase in cytosolic [Ca2+]. That proposal would align with a long-standing, but contentious, suggestion that luminal Ca2+ supports IP3R activity, but it seems difficult to align with other work from this lab:2) From results in Figure 5, the authors propose that loss of CaeLife  proposes that luminal Ca2+ inhibits IP3R activity, such that loss of ER Ca2+ would be expected to enhance Ca2+ puffs.A paper from this lab and under review with 2+ puffs do not detectably affect ER luminal [Ca2+], since steps during the falling phase of a Ca2+ puff (reflecting channel closures) are of fixed amplitude. It is not thereby clear that the flurry of puff activity would cause the loss of ER Ca2+ proposed to terminate Ca2+ puffs.Substantial evidence, much of it form this lab, indicates that individual CaThe authors should discuss both issues.Reviewer #2:2+ release by the inositol trisphosphate receptor (IP3R) Ca2+ release channel. They use HEK and HeLa cells to examine by TIRF microscopy  and light sheet microscopy, and a heavy dose of image processing abd fluctuation aanlyses, to address the issue of the sources of Ca2+ release and their mechanisms of transitioning from discrete Ca2+ signals, referred to as puffs, to global ones.In this manuscript, Lock and Parker expand the ongoing work in the Parker lab to examine the temporal and spatial properties and their regulation of Ca2+ release is associated with a flurry of noisy events during the rising phase of the global Ca2+ signal that terminates before the peak of the global Ca2+ signal is achieved. They suggest that the noisy events are Ca2+-release events from IP3R clusters, i.e. Ca2+ puffs, on a background of IP3R-mediated more diffuse release. Using procedures to partially deplete Ca2+ stores, they conclude also that the Ca2+ puffs terminate because of depletion of the ER stores, although they cannot determine whether it represents local or global store depletion, whereas the diffuse release, from what they consider to be a separate population of IP3R from those in the Ca2+-puff inducing clusters, continue to release Ca2+. By measuring the rate of cytoplasmic Ca2+ concentration relaxation following a pulse of cytoplasmic Ca2+, they calculate the relative contributions of Ca2+ puffs (punctate release) and diffuse release during the rising phase and during the entire Ca2+ transient.They find that IP3-mediated Ca2+ signaling (mostly developed by Parker); and because they address a conundrum in the field regarding the role and existence of "silent" IP3Rs that are localized throughout the cell but were previously  considered to not participate. Here, the suggestion is that in addition to active IP3R in clusters near the plasma-membrane, these other IP3R channels might indeed be active and contribute to the diffuse release that continues after puffs have stopped firing.This is an elegant analysis and an interesting set of studies, and an not least of all because they address and challenge a major model of IP3-mediated local and global CaI have a few comments that might suggest that the authors could clarify a few points.1) Why aren't local release events observed in the raw ratio images, as Parker has described many times?2+ puffs. Rather their size suggests to me that these are entire regions of the endoplasmic reticulum (ER) that are near the plasma membrane in the TIRF evanescent field. If this is the case, are the authors selecting small sites within the large "contact" sites for the SD analyses? And, I would think that there is a "global"/homogenous signal at these sites that is contributed by Ca2+ that has diffused into these "contact" sites. This might also be consistent with the observation that the TIRF-detected high-SD signal terminates before the peak signal is achieved, because the peak is contributed by slow Ca2+ diffusion that persists after release has terminated.2) Figure 3—figure supplement 1 shows the sites with high standard deviation (SD) to be rather large, ~10 uM, a size that is inconsistent with the size of clusters of ~15 IP3R that generate the Ca3) The authors describe an inverted U relationship between the amplitude of the SD signal and the amplitude of the global response. What does it mean?2+ doesn't inhibit the IP3R as a primary mechanism to terminate Ca puffs. Why doesn't it inhibit?4) The authors conclude that elevated cytoplasmic Ca2+ concentration after an elevation. They refer to this as sequestration. I'm not sure of their meaning. Although they suggest that the kinetics can be described by a single exponential, there must be at least two processes involved: sequestration by the ER, and extrusion by the plasm membrane Ca2+ pump. Indeed, in the discussion they use the phrase "efflux rate from the cytoplasm". I think what the authors mean to suggest in the sequestration away from the cytoplasm. If so, this should be stated more clearly.5) The authors examine the decay in cytoplasmic CaReviewer #3:2+ events in the context of global cytoplasmic Ca2+ levels. While previous studies used pharmacological tools to largely prevent global changes in Ca2+ in order to better reveal small discrete Ca2+ release events, the current study uses a variety of software routines that utilize fluorescence variance changes – from which punctate and global Ca2+ event behaviors are derived from. The authors surmise that punctate and global Ca2+ events are likely produced by 2 separate populations of IP3Rs. The experimental manipulations to investigate depletion of stores, Ca2+ entry, IP3R channel KO etc are well thought out and appropriate – but some concerns remain as to the ability of the authors to resolve puffs once background/cytoplasmic Ca2+ levels reach a certain intensity.In this manuscript, the authors present a method that enables some resolution and measurement of small, punctate Ca2+ release events reach a steady state, then the variance technique becomes essentially useless. This is my main concern with some of the conclusions drawn from the analysis.The use of signal variance to isolate different components of mixed fluorescence signals relies on those components having distinct (and variable) spatio-temporal signatures. However, if Ca2+ levels increase, the ability to detect puffs is hampered by i) closing of the max difference between background and puff sites and ii) an increase in the amplitude of shot noise that may start to mimick fast and small puff events. To resolve between genuine puff events, that are inherently Gaussian (1-2 µm) and random noise aggregates that occur during high background levels, is difficult – but is made worse by applying Gaussian filters to the data that reshape noise aggregates into more Gaussian shapes. Employing shape analysis before smoothing (perimeter:area ratios) is an effective way of resolving puffs (low P:A – smooth circles) even when surrounded by noise aggregates of equal intensity and size (high P:A – leafy looking). This may assuage some doubts as to the authors ability to resolve punctate events once background/global levels have reached moderate to high levels.The ability to detect puffs is greatest during low background conditions, but the as global CaSimilarly, the use of user-placed (or randomly positioned) ROIs based on visual inspection of bright spots in the videos is subjective and can heavily bias results to what the user wants to show. Many "hot spots" don't appear to have ROIs drawn over them and some ROIs appear to be sampling the same site. Small ROIs are more sensitive to spatial shifts in the position of the underlying fluorescent sites. The "flickering" variance may be a function of out-of-sync release events from a number of channels/clusters covered by each ROI. When these channels syncronize (in the open position?) or fluorescence sites increase in size, this will dramatically reduce the signal variance, even though puffs may still be occurring.The shape of the cell and the distance and size of the cytoplasm located away from the evanescent field can dramatically affect the blurring of the background . How do the authors discriminate between "global" cytoplasmic changes and large puff sites that are heavily blurred being out of focus . This effect still remains using lightsheet imaging.The pixel resolution used during TIRF imaging was 0.5µm and is unlikely to fully resolve individual puffs, especially after smoothing. This may bias analysis to release events that are comprised of multiple channel clusters.The use of black level subtraction can normalize absolute intensity levels, but does not compensate for the higher shot noise in areas with high static background. Was this taken into account?Gaussian blur is reported as 2 pixels (in text) but as an SD of 2 pixels in the Materials and methods. What was the actual kernel size in pixels  and the SD.Random ROIs were used for HEK cells devoid of IP3Rs. What does the data look like if the authors select the brightest spots in the 3KO video – or use random ROIs in controls?Figure 3—figure supplement 3C appears to show a stochastic build up of high frequency events correlated to baseline increases – until background fluorescence levels may start to swamp out the ability to resolve small punctate events.eLife. Your letter of appeal has been considered by a Senior Editor and the reviewers, and we are prepared to consider a revised submission.Thank you for choosing to send your work entitled "Inositol trisphosphate mediated Ca2+ spikes arise through two temporally and spatially distinct modes of Ca2+ release" for consideration at The reviewers have also provided additional feedback below after reading your rebuttal to hopefully better convey the technical concerns.Specific points:1) Indicator Saturation/Clipping concerns:In your rebuttal, you state (i) that "…maximal signal evoked by ionomycin is>2 fold higher than peak IP3-evoked fluorescence signals"2+ release may be reaching levels that saturate the dye. Similarly, the large areas that show uniform white intensity  are either due to: i) over zealous contrast/brightness adjustment, ii) indicator saturation @ max or iii) camera chip saturation (clipping). Assuming reasonable contrast adjustment and digitizing in 16-bits? , this suggests that saturation is still occurring. As none of the results figures presented data in absolute intensity values and used relative a.u. or F/F0 ratios which can give different "max levels" depending on the F0, it is hard for a reviewer to determine what the actual peak/max levels are and if indicator saturation was reached.However, in Figure 1, the maximum raw signal during uncaging (1C) and after ionomycin (1F) looks exactly the same – both topping out at 8500 a.u – and suggests that IP3-stimulated Ca2+ release uniformity is magnified in using variance as the primary measure to resolve Ca2+ release events. Large, saturated areas have essentially no variance (after subtracting shot noise), and could dramatically underestimate SD values and obscure Ca2+ events. In a similar fashion, simultaneous firing of multiple puffs throughout a cell could also be difficult to interpret, as one would lose the ability to spatially discriminate individual events. This, also, would reduce SD values and make it hard to distinguish between cytoplasmic and "punctate" events.The importance of indicator saturation and/or Ca2+ concentrations goes some way to assuage concerns over indicator saturation, Ca2+ levels may still be saturating this dye given the nature of the abrupt IP3-induced Ca2+ release. Out of curiosity, what does an IP3-uncaging event look like in a cell treated with ionomycin?While the use of Fluo8L, which has a much larger dynamic range in response to CaIf the saturated/clipped regions from your recordings were dynamically filtered out using image processing and SD/intensity recalculated in parts of the cell in which the indicator is presumably unsaturated, this would help to allay saturation concerns on SD measurements.2) Figure 1, ROIs2+ release events, such as an apparent reduction in intensity variability in traces that reach near-maximal intensity (see first point).Towards the end of the rebuttal ("Summarizing key points…"), where you indicate the attention drawn to subjectively-placed ROIs was based on a "misconception", then one has to question ROIs were used at all. ROIs do illustrate important features of the Ca3) 2 populations of IP3Rs…2+ liberation implicate a second population of IP3Rs with properties distinct from those clustered at puff sites" is too strong a statement given the evidence presented.Concerning point v) Figure 4 , if potential methodological concerns are addressed – and indeed the punctate responses do drop out as global levels rise – the concluding statement that: "Our findings of a diffusive mode of Ca2+ responses can be unrelated for this to be the case. In most examples presented the pattern of punctate release events appear to correlate with changes in cytoplasmic/global Ca2+.We think there needs to be experiments/analysis to show that punctate and global Ca2+ from baseline. In F (blue) the ramping up of SD events is reflected as a more gradual, sloping increase in global Ca2+. These relationships are illustrated nicely in G.For example, in Figure 2, the rate and amplitude of the SD events appears to be mirrored in the shape of the global responses. In E (puce color), the abrupt increase in SD is reflected as a sharp transition in global Ca2+ increases seems to occur at the point of maximum SD events. See Figure 4, Figure 5G and H , Figure 6, Figure 8 .The maximum slope of global Ca2+ mass / SD mass) followed by regression, cross-correlation (with ∆ time offsets) and/or inflection analysis would be appropriate to examine those potential relationships. Performing this analysis on specific regions in cells to elucidate any potential zone-of-influence between punctate and global Ca2+ characteristics.To disprove that there is just one population of IP3Rs, comparisons of the rate change of SD and global fluorescence should show little (or highly variable) correlation strength. Cumulative integration of SD and cumulative global fluorescence signals . This would make it essentially impossible to resolve punctate events, even though punctate release events may still be occurring – akin to "signal clipping". While SD analysis may provide some additional resolution in situations with narrow dynamic fluorescence ranges, it still depends on the indicator having some dynamic range. This concern needs to be carefully addressed so that readers are confident that the presented findings are accurate and biological.The first concern is that after uncaging or after the addition of agonists, the cytoplasmic background fluorescence in cells increases to a seemingly very high level. As punctate release events are only observed when background levels are low to moderate, it brings into question whether the Ca3-evoked responses. However, these data may not have been presented with sufficient clarity. We describe below our arguments and call out specific figures and places in the text where they are presented in our revised manuscript.We agree that this is a crucial issue. Our original submission already included several lines of evidence indicating that fluorescence signals were well below the saturating level, even at the peak of large IP3-evoked fluorescence signals, and suppression of puff activity occurred at yet lower levels . See also the sample traces we show in i) The maximal, saturating signals evoked by ionomycin were substantially higher than peak IP2+ liberation using the low affinity indicator fluo-8L (Kd 1.86 µM) as compared to Cal520 (Kd 320 nM) which we used for the majority of our experiments .ii) We observed the same patterns of Ca3-evoked global fluorescence increases .iii) We were able to resolve instances of local puff activity even at the peak of IP3R was suppressed during the rising phase of global Ca2+ signals and followed a similar pattern to WT cells, even though the magnitude of the peak fluorescence increase was <40% of that in WT cells .iv) Puff activity  in cells exclusively expressing type 1 IP3-mediated global Ca2+ signals were little altered when the initial basal Ca2+ level was elevated  or, conversely, when the global Ca2+ rise was attenuated by buffering with cytosolic EGTA .v) The onset and termination of puff activity during the rise of IP2+ liberation throughout all phases of IP3-evoked Ca2+ responses evoked when ER Ca2+ stores are partially depleted  strongly supports our proposal that Ca2+ liberation can arise in a diffuse manner that sustains Ca2+ signals after puff activity terminates during the rising phase of the response.vi) The suppression of punctate Ca3-evoked signals reflects the physiological functioning of IP3Rs and is not an artifact of indicator saturation.We thus conclude that the termination of puff activity during IPSome ways that could be used to examine the potential dynamic range issue of the indicators include:2+ levels during uncaging/agonists which could be compared to Kd/saturation mM of Cal-520 and GCamP etc.• use a ratiometric indicator (Fura) to estimate actual cytoplasmic Cad of both Fura-2 and Cal520 in the cytosolic environment. Further, accurate calibration of Fura-2 signals could be impaired because Fura-2 itself may be nearly saturated; especially given that the affinity of Fura-2 is higher than Cal520. Instead, we believe our approach of comparing the peak amplitude of IP3-evoked Cal520 signals with the maximal (saturating) amplitude evoked by ionomycin provides a more direct and convincing evidence that IP3-evoked Cal520 signals were well below saturation.We do not feel this approach is appropriate, with problems including uncertainties in determining the K2+ using low/moderate levels of EGTA (to preserve the dynamic range of the indicator) and examine whether punctate release patterns are still reduced after uncaging etc.• pharmacologically limit the increase in cytoplasmic Ca3-evoked Cal520 signal even though the peak amplitude of the global signal was much (~40%) lower than in control, non-EGTA-loaded cells (paragraph two subsection “Ca2+ puffs do not terminate because of rising cytosolic Ca2+ during cell-wide elevations.”).This is a good suggestion. Thank you! We performed this experiment (luckily before our lab was shut down in response to the Covid19 crisis!) and incorporate the new data as Figure 4C,D. Our results show the same pattern of SD activity in EGTA loaded cells as in non-loaded cells – i.e. puff activity ceased before the peak of the global IP2+ levels to near maximum with ionomycin then initiate an uncaging event to see puncate release sites can still be resolved .• purposefully increase cytoplasmic Ca2+ (rather than ionomycin) to elevate cytosolic [Ca2+] prior to evoking an IP3-mediated response already address this issue. Our results showed that puff activity was still triggered even with prior [Ca2+] elevations to levels  at which puffs are suppressed during the rising phase of IP3-evoked responses beginning from baseline cytosolic [Ca2+].Although performed for a different purpose, our experiments  using photorelease from caged Ca2+ concentrations. At these levels where there is extremely limited dynamic range and greater shot noise, noise aggregates and punctate release sites will appear similar – but they can be separated using perimeter:area ratios (high for noise aggregates), or Gaussian fitting (better for release sites). This will require no Gaussian filtering of the data and some form of deconvolution to reduce the smoothing of objects due to Z-smear (where appropriate).• use quantitative shape analysis (not ROIs) of transient bright regions at high cytoplasmic Ca3 were <40% of the maximal response evoked by ionomycin .This is an interesting suggestion, but developing, verifying and applying appropriate algorithms and software would incur a substantial time delay. We further contest the statement that dynamic range would be extremely limited. Mean peak Cal520 fluorescence signals evoked by i-IP2+ behaviors are included or filtered by varying the space/time variance parameters.The second concern is discussed adequately in the reviews below, but questions whether the SD approach can detect synchronous, widespread puffs/ release events, or high frequency puffs/release events that appear and behave like changes in cytoplasmic background. It would be useful to the reader to see how different duration timespans affect the variance signal , to assess what range of CaWe now include  an evaluation of how varying the space/time variance parameters of the algorithm affect the resulting SD fluctuation signal. Other concerns are dealt with below in our responses to specific comments by the reviewers.Without additional information to address the major concerns of the reviewers, it is not possible to assess whether the work represents a sufficient advance to warrant publication in eLife.2+ elevations could be attributed to an artifact of indicator saturation. We feel that this issue, and other concerns of the reviewers, are fully addressed by our responses detailed here, and that our findings provide convincing support for our proposal of two different functional modes of IP3-mediated signaling involving both punctate and diffuse Ca2+ liberation. Our model represents a major revision of the widely accepted “fire-diffuse-fire” notion by which global Ca 2+ signals are generated by coordination of transient, punctate release events. This represents an important advance and re-evaluation of current dogma in the field of Ca2+ signaling that we hope would warrant publication in eLife.A major concern of the reviewers was that the decline of the SD signal (termination of puff activity) during the rise of global CaReviewer #1:2+ signals develop and that a comparable increase in global cytosolic [Ca2+] evoked by ionomycin is “noise-free”, as expected for homogenous Ca2+ release. My understanding is that after some filtering and spatial blurring, most analyses calculate the SD for each pixel as a 160-ms (ie 20 frames) boxcar average.1) Figure 1 provides compelling evidence that noise decreases as the IP3-evoked CaWe now include data  illustrating how the SD signal is affected by varying parameters for spatial filtering and boxcar averaging.2+ signal reflects most Ca2+ puff sites firing very frequently (perhaps 20 or so sites active at each instant), each spreading a µm or more from its source. Might this cause elevations in cytosolic [Ca2+] that are less noisy than the infrequent Ca2+ puffs observed initially, because each pixel would be influenced by Ca2+ reaching it from several puffs? It's perhaps too tall an order to require quantitative simulation of this on SD measurements, but it would be helpful to have the authors consider the issue explicitly.The original view posits that a global Ca2+ after puff activity ceases might arise as Ca2+ released during puffs diffuses into spaces between puff sites – i.e. that all the Ca2+ released during global responses originally derives from puffs. Perhaps the strongest argument supporting a distinct, “diffuse” mode of release comes from our experiments with partial depletion of ER Ca2+ content , where we show Ca2+ responses that arise in the absence of detectable puff activity. Moreover, a simple calculation indicates that Ca2+ diffusion would be too fast to account for the continuing smooth rise in Ca2+. Assuming a value of D = 20 mm2 s-1 for the effective diffusion coefficient of Ca2+, the mean time t for a Ca2+ ion to diffuse midway between puff sites separated by 10 mm ( d =10/2 mm) would be about 300 ms (t = d2/4D). In contrast, global Ca2+ signals typically continued to rise and were maintained for several seconds in the face of SERCA uptake after puff activity ceases . We had further addressed this issue in the context of whether Ca2+ diffusing from release sites deep in the cell interior might underlie the noise-free rise observed by TIRF imaging, but our lightsheet imaging indicated this was not the case .We interpret the essence of this concern as to whether the continued, “noise-free” rise in cytosolic Ca2+ signals”).We now explicitly discuss this issue in the revised manuscript  From results in Figure 5, the authors propose that loss of Ca2+ inhibits IP3R activity, such that loss of ER Ca2+ would be expected to enhance Ca2+ puffs.A paper from this lab and under review with eLife  proposes that luminal Ca3R activity would create a positive feedback loop, such that any local depletion of ER Ca2+ would speed Ca2+ liberation through puffs and more rapidly terminate that liberation if local stores at puff sites became more completely emptied. We prefer not to include discussion of this point at present, as the Mak et al. paper is not yet published.The proposal in the Mak et al. paper does not appear to be incompatible with our model. The proposed luminal regulation of IP2+ puffs do not detectably affect ER luminal [Ca2+], since steps during the falling phase of a Ca2+ puff (reflecting channel closures) are of fixed amplitude. It is not thereby clear that the flurry of puff activity would cause the loss of ER Ca2+ proposed to terminate Ca2+ puffs.Substantial evidence, much of it form this lab, indicates that individual CaThe authors should discuss both issues.2+ during the falling phase of puffs do suggest that there is little decline of ER Ca2+ during individual puffs evoked at relatively low frequency. However, it is plausible that ER depletion may occur during the rapid flurries of puffs evoked at multiple sites during the rising phase of global signals. We now incorporate discussion of this point .Findings of uniform step decrements of CaReviewer #2:[…]I have a few comments that might suggest that the authors could clarify a few points.1) Why aren't local release events observed in the raw ratio images, as Parker has described many times?Local release events are indeed present in the raw ratio images. They are not readily visible in the figures because the grey scale range was adjusted to encompass the much higher fluorescence levels during global responses, and because puffs in the HEK cells we used for most experiments are smaller than in cell lines such as SHSY-5Y and HeLa. We now include a note in the text (Results paragraph two) to clarify the point for readers who may also have this question.2+ puffs. Rather their size suggests to me that these are entire regions of the endoplasmic reticulum (ER) that are near the plasma membrane in the TIRF evanescent field. If this is the case, are the authors selecting small sites within the large "contact" sites for the SD analyses? And, I would think that there is a "global"/homogenous signal at these sites that is contributed by Ca2+ that has diffused into these "contact" sites. This might also be consistent with the observation that the TIRF-detected high-SD signal terminates before the peak signal is achieved, because the peak is contributed by slow Ca2+ diffusion that persists after release has terminated.2) Figure 1—figure supplement 1 shows the sites with high standard deviation (SD) to be rather large, ~10 uM, a size that is inconsistent with the size of clusters of ~15 IP3R that generate the Ca2+ signals”).All of our SD analyses  were done using regions of interest encompassing the entire cell. There is thus no issue of “selecting small sites … for the SD analyses”. We now emphasize this point more clearly in the manuscript  The authors describe an inverted U relationship between the amplitude of the SD signal and the amplitude of the global response. What does it mean?2+ dependence of activation of IP3Rs. However, further experiments  indicated that is not the case. Our interpretation  is that the cytosolic Ca2+ signal is an inverse mirror of the local ER Ca2+ store level, and that the decline in SD signal arises as puff activity declines owing to falling ER store content.We show plots of amplitude of the SD signal and the amplitude of the global response as a convenient means of summarizing and depicting the relationship between these parameters in a time-independent manner. As described in the manuscript we originally thought the inverted U relationship might correspond to the “bell-shaped curve” of Ca2+ doesn't inhibit the IP3R as a primary mechanism to terminate Ca puffs. Why doesn't it inhibit?4) The authors conclude that elevated cytoplasmic Ca2+] would be expected to inhibit opening of IP3R channels, a likely explanation is simply that depletion of ER Ca2+ stores is the more dominant mechanism to terminate puff activity during global signals. This interpretation is buttressed by new data we present , demonstrating that puffs still terminate even when the global cytosolic Ca2+ elevation is substantially reduced by loading with EGTA.Although high cytosolic  The experimental manipulations to investigate depletion of stores, Ca2+ release events reach a steady state, then the variance technique becomes essentially useless. This is my main concern with some of the conclusions drawn from the analysis.The use of signal variance to isolate different components of mixed fluorescence signals relies on those components having distinct (and variable) spatio-temporal signatures. However, if Ca2+ release events reach a steady state” we interpret the reviewer to mean that Ca2+ release through the IP3Rs that mediate puffs becomes continuous; i.e. that some or all of the IP3R channels remain open all the time, resulting in a “noise-free” signal. If this indeed occurs, we believe it is valid and useful to regard such continuous release as a mode distinct from the transient puffs. The issue, then, is whether the same IP3Rs underlie puffs and continuous release, or whether these modes arise from separate populations of IP3Rs. Although we favor the notion that distinct populations of IP3Rs mediate the two modes of Ca2+, we now explicitly discuss the possibility the two modes may instead arise through a switch of the same IP3Rs from one form of regulation to another (Discussion and see response to similar query of reviewer #1).By the statement “Ca2+ levels increase, the ability to detect puffs is hampered by i) closing of the max difference between background and puff sites and ii) an increase in the amplitude of shot noise that may start to mimick fast and small puff events. To resolve between genuine puff events, that are inherently Gaussian (1-2 µm) and random noise aggregates that occur during high background levels, is difficult – but is made worse by applying Gaussian filters to the data that reshape noise aggregates into more Gaussian shapes. Employing shape analysis before smoothing (perimeter:area ratios) is an effective way of resolving puffs (low P:A – smooth circles) even when surrounded by noise aggregates of equal intensity and size (high P:A – leafy looking). This may assuage some doubts as to the authors ability to resolve punctate events once background/global levels have reached moderate to high levels.The ability to detect puffs is greatest during low background conditions, but the as global Ca2+ responses truly represents a decline in puff activity, not a failure to detect puffs in the face of elevated Ca2+levels. Notably; (i) Puff activity  was still observed in response to IP3 activation when the basal cytosolic Ca2+ was substantially elevated by prior photorelease of Ca2+ . (ii) We now include new data showing that puff termination follows similar kinetics when peak global Ca2+ elevations were reduced by EGTA buffering . Moreover, we present additional evidence in our rebuttals to concerns of the other reviewers that our ability to detect puffs at elevated Ca2+ levels may have been impaired by limited dynamic range of the indicator.Use of shape analysis is an interesting suggestion; but developing, verifying and applying appropriate algorithms and software would incur a substantial time delay. We feel that the data we already present convincingly demonstrate that the fall in SD signal during the rising phase of global CaSimilarly, the use of user-placed (or randomly positioned) ROIs based on visual inspection of bright spots in the videos is subjective and can heavily bias results to what the user wants to show. Many "hot spots" don't appear to have ROIs drawn over them and some ROIs appear to be sampling the same site. Small ROIs are more sensitive to spatial shifts in the position of the underlying fluorescent sites. The "flickering" variance may be a function of out-of-sync release events from a number of channels/clusters covered by each ROI. When these channels syncronize (in the open position?) or fluorescence sites increase in size, this will dramatically reduce the signal variance, even though puffs may still be occurring.2+ signals”).We show SD traces from small, subcellular ROIs only in Figure 1, for the purpose of better illustrating the basis of our technique. For all the other experiments – from which our conclusions are drawn – we used a single ROI encompassing the entire cell. There is thus no issue of subjective bias resulting from user-placed ROIs. The reviewer appears to have missed this methodological point, which we now emphasize more clearly in the text . How do the authors discriminate between "global" cytoplasmic changes and large puff sites that are heavily blurred being out of focus . This effect still remains using lightsheet imaging.2+ level across the entire TIRF footprint of the cell. We do not attempt to discriminate on the basis of spatial patterns between global signals and large puffs.Again, all our analyses are based on SD and DF/F0 traces derived from the same, single ROI encompassing each cell. They represent an average of the temporal fluctuations and CaThe pixel resolution used during TIRF imaging was 0.5µm and is unlikely to fully resolve individual puffs, especially after smoothing. This may bias analysis to release events that are comprised of multiple channel clusters.The camera pixel resolution, subsequent Gaussian smoothing, and boxcar calculation of SD were chosen to optimally “tune” for the spatio-temporal characteristic of puffs while minimizing stochastic photon shot noise. The SD signal simply reports an average of pixel-by-pixel temporal fluctuations across the entire cell. We do not attempt to discriminate between individual or multi-cluster events.In response to the editor’s comment we now include a supplementary figure  illustrating how different parameters in our algorithm affect the SD signal. The spatial smoothing we applied (σ corresponding to ~1 mm at the specimen) effectively reduced photon shot noise see without smoothing, but did not otherwise appreciably alter the SD signal arising from puff activity.The use of black level subtraction can normalize absolute intensity levels, but does not compensate for the higher shot noise in areas with high static background. Was this taken into account?2+ images highlights transient signals” paragraph three).Our algorithm subtracts the mean shot noise level predicted for the intensity level on a pixel-by-pixel basis (subsection “Fluctuation processing of CaGaussian blur is reported as 2 pixels (in text) but as an SD of 2 pixels in the Materials and methods. What was the actual kernel size in pixels  and the SD.Spatial smoothing was accomplished using a Python routine (skimage) that applies a two-dimensional Gaussian blur with specified standard deviation (σ) out to a range of 4 σ. We now clarify this description, and state σ in terms of both pixels and corresponding micrometers at the specimen . See new Figure 1—figure supplement 1 for an illustration of how the SD signal is affected by different values of Gaussian blur.Random ROIs were used for HEK cells devoid of IP3Rs. What does the data look like if the authors select the brightest spots in the 3KO video – or use random ROIs in controls?As noted above, we show traces from small, subcellular ROIs only in Figure 1, for the purpose of helping explain the basis of our SD approach. Use of random ROIs for the 3KO cell would seem appropriate for that purpose. Excepting the introductory Figure 1, all data were acquired from single regions of interest encompassing the entire cell .Figure 3—figure supplement 3C appears to show a stochastic build up of high frequency events correlated to baseline increases – until background fluorescence levels may start to swamp out the ability to resolve small punctate events.As described in responses to several of the reviewers’ comments, we present convincing evidence that fluctuations from punctate events do not become “swamped out” at higher background fluorescence levels, but rather that the punctate release activity itself declines.[Editors’ note: what follows is the authors’ response to the second round of review.]The reviewers have also provided additional feedback below after reading your rebuttal to hopefully better convey the technical concerns.Specific points:1) Indicator Saturation/Clipping concerns:In your rebuttal, you state (i) that "…maximal signal evoked by ionomycin is>2 fold higher than peak IP3-evoked fluorescence signals"2+ release may be reaching levels that saturate the dye.However, in Figure 1, the maximum raw signal during uncaging (1C) and after ionomycin (1F) looks exactly the same – both topping out at 8500 a.u – and suggests that IP3-stimulated Ca2+ fluorescence signal matching the amplitude and kinetics of the IP3-evoked signal in Figure 1A,C. Our intent was to compare the output of our SD analysis routine with signals of similar size where punctate release was either present or absent. For this purpose, we delivered a small aliquot of ionomycin at a distance  from cells imaged in zero Ca2+ solution, so that the slow diffusion and dilution of ionomycin evoked gradual release of Ca2+ from intracellular stores.We apologize that we had not fully described the procedure used to evoke the ionomycin response in Figure. 1, leading to the reviewer’s confusion. The experiment in Figure 1F,H was done under conditions deliberately chosen to evoke a Ca3-evoked evoked response in Figure 1 as ΔF/F0 values as well as in fluorescence units.Separately, we used ionomycin to determine the saturating fluorescence of the indicator by complete bath exchange of medium containing high (10 mM) calcium with ionomycin. We now clarify these points in the text, and also state the peak amplitudes of the ionomycin-and i-IPSimilarly, the large areas that show uniform white intensity  are either due to: i) over zealous contrast/brightness adjustment, ii) indicator saturation @ max or iii) camera chip saturation (clipping). Assuming reasonable contrast adjustment and digitizing in 16-bits? , this suggests that saturation is still occurring.Camera digitization was 16 bit; the camera was operated far below full-well capacity; and image processing was done using 64 bit floating point arithmetic. No clipping occurred anywhere through the entire imaging pipeline. This is now stated in Materials and methods.Areas of uniform white in Figure 1A,F resulted because we chose a grey scale that would still display some data at low fluorescence levels, and this inadvertently clipped the higher intensity values. We have now re-made this figure with a more extended grey scale so that the Cal520 fluorescence image panels at the peak of the response are no longer clipped.As none of the results figures presented data in absolute intensity values and used relative a.u. or F/F0 ratios which can give different "max levels" depending on the F0, it is hard for a reviewer to determine what the actual peak/max levels are and if indicator saturation was reached.2+ level (setting F0) is consistent between cells prepared under the same conditions. From many years’ experience, including comparison measurements with fura-2, we find this to be the case.The term “absolute intensity value” (even if expressed as photons/pixel/unit time) has little meaning in this context. The fluorescence intensity will depend on factors including excitation laser intensity, concentration of indicator loaded in the cytosol, TIRF angle setting, etc.; factors that will vary between cells and between experiments. Instead, we follow the widely used practice of expressing data as ΔF/F0 values to normalize for these factors. The only crucial underlying assumption is that the basal free Ca3 in 3 cells black), and maximal responses to ionomycin in 3 cells (red). These data are displayed as both “raw” fluorescence in arbitrary camera units (left) and as F/F0 values (right).For reference, we show in 2+ release uniformity is magnified in using variance as the primary measure to resolve Ca2+ release events. Large, saturated areas have essentially no variance (after subtracting shot noise), and could dramatically underestimate SD values and obscure Ca2+ events.The importance of indicator saturation and/or Ca3-evoked Ca2+ signals were well below the level of indicator saturation, and that our key conclusion that puff activity terminates before the peak of global signals is not an artifact of indicator saturation.While we agree that our data and conclusions would be dramatically affected if the indicator became saturated, we present numerous lines of evidence  clearly demonstrating that IPIn a similar fashion, simultaneous firing of multiple puffs throughout a cell could also be difficult to interpret, as one would lose the ability to spatially discriminate individual events. This, also, would reduce SD values and make it hard to distinguish between cytoplasmic and "punctate" events.Our SD signal reflects the cell-wide aggregate of fluctuations at each pixel, and is independent of any requirement to spatially discriminate individual events.2+ concentrations goes some way to assuage concerns over indicator saturation, Ca2+ levels may still be saturating this dye given the nature of the abrupt IP3-induced Ca2+ release.While the use of Fluo8L, which has a much larger dynamic range in response to Ca2+ binding to the indicator (and its fluorescence) will equilibrate with free [Ca2+] on a timescale vastly more rapidly than the timescale of seconds for the IP3-evoked signal.We do not understand this point. CaOut of curiosity, what does an IP3-uncaging event look like in a cell treated with ionomycin?2+ from the ER, we would not expect any response to uncaged IP3 if no Ca2+ remained to be released. In Figure 4A,B we show i-IP3-evoked responses after cytosolic [Ca2+] was elevated using caged calcium.Because ionomycin will cause uncontrolled loss of CaIf the saturated/clipped regions from your recordings were dynamically filtered out using image processing and SD/intensity recalculated in parts of the cell in which the indicator is presumably unsaturated, this would help to allay saturation concerns on SD measurements.3-evoked signals are well below the level of indicator saturation. Our revised manuscript includes new experimental data, and a new section in the Discussion summarizing and evaluating this evidence.Again, we present compelling evidence that our IP3-evoked signals is quite homogeneous throughout the cell (TIRF footprint), without evidence of saturated/clipped regions.As shown in (revised) Figure 1A and Video 1, the fluorescence at the peak of IP2) Figure 1, ROIs2+ release events, such as an apparent reduction in intensity variability in traces that reach near-maximal intensity (see first point).Towards the end of the rebuttal ("Summarizing key points…"), where you indicate the attention drawn to subjectively-placed ROIs was based on a "misconception", then one has to question ROIs were used at all. ROIs do illustrate important features of the CaWe show traces from subcellular ROIs in Figure 1 as a way to illustrate the behavior of puff sites (particularly to provide helpful background for readers who may not be familiar with this literature), and to show how the aggregate behavior of multiple sites results in the whole-cell ROI traces we present in the rest of the manuscript. We have reworded the text to better communicate this intent. We agree that there is valuable information to be derived from analysis of individual release events, but leave that analysis for a future publication.3) 2 populations of IP3Rs…2+ liberation implicate a second population of IP3Rs with properties distinct from those clustered at puff sites" is too strong a statement given the evidence presented.Concerning point v) Figure 4 , if potential methodological concerns are addressed – and indeed the punctate responses do drop out as global levels rise – the concluding statement that: "Our findings of a diffusive mode of Ca2+ release, and in the Discussion speculate as to whether these modes may arise from two separate populations of IP3R, or through a modulation of the functional properties of a single population. We further address this point in our detailed responses to the original comments of the reviewers.We agree that our statement was too strong. In our revised manuscript we now refer to two “modes” of Ca2+ responses can be unrelated for this to be the case.We think there needs to be experiments/analysis to show that punctate and global Ca2+ signal is evoked without detectable puff activity ; i.e. in this situation  punctate and diffuse modes of Ca2+ release are indeed unrelated.Figure 5A,B,D,F,H,I present clear-cut cases where a global Ca2+.In most examples presented the pattern of punctate release events appear to correlate with changes in cytoplasmic/global Ca2+ from baseline. In F (blue) the ramping up of SD events is reflected as a more gradual, sloping increase in global Ca2+. These relationships are illustrated nicely in G.For example, in Figure 2, the rate and amplitude of the SD events appears to be mirrored in the shape of the global responses. In E (puce color), the abrupt increase in SD is reflected as a sharp transition in global Ca2+ increases seems to occur at the point of maximum SD events. See Figure 4, Figure 5G and H , Figure 6, Figure 8 .The maximum slope of global Ca2+ release though puffs contributes a substantial  fraction of the total Ca2+ liberation during the rising phase of global Ca2+ signals. We thus expect that the rising phase of the global signal will reflect both punctate and diffuse modes of Ca2+ liberation.This is consistent with, and predicted by, our proposal that punctate Ca2+ mass / SD mass) followed by regression, cross-correlation (with ∆ time offsets) and/or inflection analysis would be appropriate to examine those potential relationships.To disprove that there is just one population of IP3Rs, comparisons of the rate change of SD and global fluorescence should show little (or highly variable) correlation strength. Cumulative integration of SD and cumulative global fluorescence signals (akin to Ca2+ release, and we discuss whether these may arise from two populations of IP3Rs or a single population. Because we conclude that punctate Ca2+ release makes up an appreciable part of the total Ca2+ released during the rising phase of the cell-wide response, we do expect correlation between the SD signal and the global fluorescence.As noted above, our revised manuscript refers to two modes of Ca2+ characteristics.Performing this analysis on specific regions in cells to elucidate any potential zone-of-influence between punctate and global Ca2+ and diffuse Ca2+ release arise at different sites in the cell. An interesting question, but one we feel is beyond the scope of the present manuscript, and a topic for future experiments once our lab reopens after the Covid-19 pandemic.This suggestion appears predicated on a proposal that punctate Ca


We crystallized and determined the X-ray crystal structure of the LT1009 Fab fragment in 10 mM CaCl2 and found that it binds two Ca2+ in a manner similar to its antigen-bound state. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds Ca2+ in solution and inductively-coupled plasma-mass spectrometry (ICP-MS) revealed that, although Ca2+ is preferred, LT1002 can bind Mg2+ and, to much lesser extent, Ba2+. Isothermal titration calorimetry (ITC) indicated that LT1002 binds two Ca2+ ions endothermically with a measured dissociation constant (KD) of 171 μM. Protein and genome sequence analyses suggested that LT1002 is representative of a small class of confirmed and potential metalloantibodies and that Ca2+ binding is likely encoded for in germline variable chain genes. To test this hypothesis, we engineered, expressed, and purified a Fab fragment consisting of naïve murine germline-encoded light and heavy chain genes from which LT1002 is derived and observed that it binds Ca2+ in solution. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved across diverse mammalian genomes.LT1009 is a humanized version of murine LT1002 IgG1 that employs two bridging Ca The two Ca2+ are partially coordinated within close proximity (<4 Å) of one another by three aspartic acid residues (Asp30-Asp31-Asp32) from light chain complementarity-determining region loop 1 (CDR-L1) and a fourth aspartic acid (Asp92) from CDR-L3. This arrangement allows for one oxygen atom from the phosphate head group of S1P to complete a μ bridging bond to both bound Ca2+. Subsequent in vitro analysis confirmed that the bound ions are indeed Ca2+ and characterization of the antibody/antigen complex by surface plasmon resonance (SPR) spectroscopy revealed the binding to be extremely favorable with a dissociation equilibrium constant (KD) in the low nM range. Removal of Ca2+, by addition of chelators such as EDTA or EGTA, as well as mutation of Ca2+-coordinating amino acid residues completely disrupted the complex to the same or even greater extent than did mutation of residues that contact the antigen directly.At least one-third, and perhaps as many as one-half, of all functioning proteins are predicted to be metalloproteins ,2,3. Inc2+ interactions. For example, can the antibody bind Ca2+ independently of the antigen or is prior interaction with S1P a requirement for Ca2+ binding? Does the antibody bind to metal ions other than Ca2+? With what affinity is the Ca2+ bound? It also raised questions about the biology underlying the anti-S1P antibody such as, did the metalloantibody develop its ability to bind to Ca2+ during the processes of somatic recombination and/or affinity maturation or is the potential to coordinate Ca2+ evolutionarily conserved within germline antibody gene sequences? In addition, are there other examples of antibodies that employ Ca2+ or other metal ions in this fashion to recognize different antigens?This direct observation of an antibody that was raised by immunization in mice employing interfacial bridging metal ions as a required component of its antigen recognition site called to mind several questions concerning the chemical nature of the anti-S1P antibody:Ca2+-mediated antigen binding is an evolutionarily conserved component of a complete and robust antibody repertoire. Our results suggest that the anti-S1P antibody does not require antigen in order to bind Ca2+ and that the default Ca2+ binding mode is similar to that observed in the LT1009:Ca2+:S1P complex X-ray crystal structure. We observe that the antibody binds Ca2+ selectively, with an appreciable, but significantly lower capacity for binding Mg2+ and even lower affinity toward Ba2+. We measure the Ca2+ binding affinity and find that its dissociation constant (KD) is an order of magnitude lower than the typical concentration of Ca2+ in plasma, suggesting that the anti-S1P antibody is preloaded with Ca2+ in vivo. We identify the Ca2+ binding sequence in previously characterized antibodies, some, but not all, of which also require Ca2+ for binding to their respective antigens. Finally, we observe that the amino acid sequence signature for Ca2+ binding is encoded for within the germline sequences of select antibody kappa light chain gene variable regions across diverse species and that an antibody Fab fragment expressing the mouse germline-encoded sequences from which the anti-S1P antibody is likely derived also binds to Ca2+ in solution. These results strongly suggest that the Ca2+-dependent antigen binding observed in the anti-S1P antibody is an evolutionarily conserved and diversely applied component of a functional adaptive immune system.In this study, we report results from a series of structural, in vitro biochemical, and protein engineering experiments that were performed in order to characterize the metal ion binding properties of the anti-S1P antibody and to test the hypothesis that CaPlasmids encoding murine (LT1002) and humanized (LT1009) anti-S1P antibodies have been described previously . For cong for 5 min and vacuum filtrated through 0.2 μm nitrocellulose membrane (Millipore) and passed by gravity through 1 mL of Ni Sepharose Fast Flow resin  at 4 °C. The column was washed with 25 mM TRIS-HCl pH 8.0, 150 mM NaCl, and 25 mM imidazole, and protein was eluted with wash buffer containing 150 mM imidazole. The eluted protein was treated with 0.1 M EDTA pH 8.0, filtered through a 0.2 μm syringe tip filter (Millipore), and purified on a SuperDex75 16/60 size exclusion chromatography column  in 25 mM TRIS-HCl pH 8.0 and 150 mM NaCl. Peak fractions were combined and concentrated to 5 mg/mL in a 10 kDa molecular weight cutoff (MWCO) centrifugal concentrator (Millipore), flash frozen in liquid nitrogen, and stored at −80 °C.Full length murine (LT1002) and humanized (LT1009) anti-S1P antibodies were produced in stable CD-CHO cell lines as previously described . Briefly2. For optimal crystal growth, 1 μL of 12 mg/mL LT1009 Fab was mixed with 1 μL reservoir solution comprised of 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 1.5 M Li2SO4, and 10 mM CaCl2 on a siliconized coverslip and sealed with high vacuum grease over 1 mL of reservoir solution. Plate-like crystals of dimensions 0.3 × 0.3 × 0.02 mm grew for 63 days at room temperature. Crystals were harvested with nylon loops and immersed into 0.1 M TRIS-HCl pH 8.5, 0.2 M Li2SO4, 30% w/v polyethylene glycol 4000, 10 mM CaCl2, and 10% glycerol for approximately ten seconds prior to flash cooling in liquid nitrogen.Crystals were grown by the hanging drop-vapor diffusion method at room temperature. Initial screening for conditions that promote crystallization of antigen-free LT1009 Fab were identified by testing Crystal Screen I (Hampton Research) reagents supplemented with 10 mM CaCl2+ binding sites were carried out using REFMAC5 and FFT, respectively, in CCP4 [Synchrotron data were collected at 100 K on a NOIR-1 CCD detector at the Advanced Light Source Beamline 4.2.2, Lawrence Berkeley National Laboratory. Diffraction data indexing and scaling of intensities were carried out with HKL2000 . Data co in CCP4 . Model b in CCP4 . All fur in CCP4 . Final a in CCP4 . Refinem in CCP4 .2·2H2O (EMD® #401800) prepared in 20 mM Na-HEPES pH 7.2 and 20 mM NaCl was added to 1.8 mL of 1 mg/mL LT1002, 1 mg/mL LT3015, 1 mg/mL naïve Fab, or Na-HEPES buffer. The resulting solutions at 10 mM CaCl2 were applied to 3.5 kDa MWCO dialysis units . In order to monitor the rate of equilibration, samples were dialyzed against 0.5 L of 20 mM Na-HEPES pH 7.2 and 20 mM NaCl and analyzed at time points 0, 1.5, 3.5, 7.5, and 22.5 h. In subsequent studies, samples were dialyzed against Na-HEPES buffer for 24 h with four changes of buffer. Addition metals were tested by substituting with 100 mM stock solutions of MgCl2·6H2O (VWR® #VW1483-01), SrCl2·6H2O , BaCl2·2H2O (EMScience #43129930), Zn(C2H3O2)2·2H2O (Fisher Scientific® #053087), CdCl2 , MnCl2·4H2O (Fisher Scientific #041398), CoCl2·6H2O (Fisher Scientific® #040819), NiSO4·6H2O (EMDTM 35044548), CuSO4·5H2O (CAS 7758-99-8), YbCl3·6H2O (CAS 10035-01-05), GdCl3 (CAS 10138-52-0), and EuCl3·6H2O (CAS# 13758-92-7) in 20 mM Na-HEPES pH 7.2 and 20 mM NaCl.Prior to testing, proteins were treated with 0.1 M EDTA pH 8.0 and purified either by dialysis (LT1002/LT3015 FAAS and ICP-MS experiments) or by size exclusion chromatography (naïve germline-encoded Fab antibody ICP-MS). Then, 200 μL of 100 mM stock solution of CaClg for 5 min. Analysis was carried out on a 240 AA instrument (Agilent), which was calibrated by a 6-point dilution of 1.0 ppm CaCO3 standard. Instrument settings: lamp current, 70% of 10 mA; compressed air, 9.0 psi; burner height, 15 mm; burner position, 90 °C; fuel flow, 1.1 L/min.; Ca2+ absorbance, 422.7 nm; slit width, 0.5 nm. Between samples, the injection port was washed with 5.0% (v/v) HNO3 solution.To begin, 20 μL of sample solution was removed from the dialysis unit, treated nitric acid , heated at 98 °C for 30 min, and centrifuged at 14,000× Samples were prepared as for FAAS and analyzed on an Agilent 4500 Series Inductively Coupled Plasma Mass Spectrometer (ICP-MS) through the Institute for Integrated Research Materials, Environments and Society at California State University, Long Beach.2 solution was titrated at 2.0 μL increments into solutions of 300 μM LT1002, 300 μM LT3015, or no antibody in 50 mM Na-HEPES pH 7.2 and the binding isotherms were measured on a MicroCal™ iTC200 instrument  available through Protein Production and Analysis at the Sanford Burnham Prebys Medical Discovery Institute. Data were analyzed by Origin™ software.A 20 mM CaClhttp://www.ncbi.nlm.nih.gov/igblast) to identify naïve V, D, and J genes from which the LT1002 anti-S1P antibody is most likely derived [http://blast.ncbi.nlm.nih.gov) search with a murine Igkv17-121 gene sequence [Heavy and light chain variable domain light chain sequences were analyzed on the IgBlast server  with S1P antigen and all nonbonded atoms  removed. After rigid-body and restrained refinement of the model against diffraction data to 3.0 Å resolution, FO-FC difference Fourier maps were calculated and analyzed for the presence of unaccounted for peaks of electron density. Two spheres of positive electron density were visible at the CDR-L1/CDR-L3 binding sites at a contour level of 3.5 σ  of 20.9% and Rfree of 26.0% and with very good stereochemistry . By this approach, buffered solutions of LT1002 anti-S1P antibody that had been rendered metal ion free by pretreatment with EDTA were incubated in solution containing Ca2+ and dialyzed against excess amounts of Ca2+-free buffer. In order to monitor the rate of dialysis under the conditions tested, we first carried out an experiment in which the amount of Ca2+ retained within the dialysate fraction was monitored over time relative to similarly prepared and buffered whole LT3015, an antibody specific for lysophosphatidic acid (LPA) that we had previously demonstrated does not involve Ca2+ in binding to its lipid antigen [2+ could be detected relative to LT3015 anti-LPA antibody by FAAS after 24 h, and the amount of Ca2+ remaining within the dialyzed antibody samples did not change significantly during the final 15 h of dialysis  detection.We next wished to determine whether metal ions other than Ca2+ to a significantly greater degree than does the control LT3015 antibody .We did not observe significant binding to Sr spheres . Finally2+ and the anti-S1P antibody in solution, we next carried out isothermal titration calorimetry (ITC). By this approach, controlled amounts of Ca2+ were introduced to a HEPES buffered solution of LT1002 antibody and the change in electrical current required to maintain a steady solution temperature was carefully measured. This allows for direct determination of the enthalpy associated with binding. Analysis of the titration endpoint can be fit to determine binding association constants, through which enthalpic components of the binding free energy can be deduced. Through this method, full thermodynamic profiles of metals for proteins, including binding affinities, have been determined [In order to thermodynamically characterize the interaction of Catermined ,27.2+ immediately revealed that binding of the metal is an endothermic (heat requiring) process . This represents a relatively low affinity interaction. However, when one considers the energy associated with shielding the charges on the two Ca2+ as they bind within close proximity of one another as well as the fact that only three coordination sites on each of the two bound ions are filled by the carboxylate oxygens of the critical four Asp residues, it seems reasonable that the binding affinity would not be too strong. The measured value is certainly consistent with our observation that at 10 mM CaCl2, there appears to be full occupancy of Ca2+ bound throughout the crystal. From the perspective of physiological relevance, 171 μM is roughly an order of magnitude lower than the typical plasma concentration of Ca2+. This suggests that under physiological conditions, the anti-S1P antibody exists in it predominantly Ca2+-bound state. As indicated from our previous antigen binding and X-ray crystallographic studies, subsequent binding to S1P antigen significantly increases the Ca2+ binding affinity. Titration of Ca2+ into buffered LT3015 anti-LPA antibody or HEPES buffer alone revealed no interaction between Ca2+ and HEPES or the control antibody  in CDR-L1 and a fourth Asp residue equivalent to Asp92 in CDR-L3 [KD) in the absence of Ca2+ and 1.6 nM affinity in the presence of 25 mM Ca2+, clearly identifying it as a bona fide metalloantibody [2+ for Q425 indirectly, they arrived at a value of 187 μM, which agrees surprisingly well with what we measured by ITC for Ca2+ binding to LT1002. This is despite the fact that X-ray crystallography of the Q425 Fab fragment in 10 mM CaCl2 suggests that only one Ca2+ binds to Q425 at a site that differs completely from what is observed in the anti-S1P antibody crystal structures [Upon confirming that LT1002 binds Can CDR-L3 . Among tantibody . It is wructures .2+-coordinating Asp residues is murine 2C10, which was originally identified as an anti-double-stranded DNA (anti-dsDNA) antibody expressed in an MRL/1 mouse model of the autoimmune disease systemic lupus erythematosus (SLE) [A second antibody identified by virtue of its close homology to LT1002 that includes each of the four Caus (SLE) . Subsequus (SLE) . Recentlus (SLE) . No dire2+-coordinating amino acid residues is the MR1 antibody that binds to a polypeptide epitope near the N-terminus of a mutant epidermal growth factor receptor (EGFRvIII). X-ray crystallography of the complex between an MR1 single chain variable domain (scFv) and a 13-mer antigen peptide reveals that none of the CDR-L1 amino acids are involved in antigen binding and that no metal ions are observed or suspected in the complex [2+-binding potential do not necessarily require metals for antigen recognition and binding.A third antibody that shares all of the LT1002 Ca complex . The CDR2+ in antigen binding by LT1002 is in regard to the manner by which the anti-S1P antibody developed its ability to bind Ca2+. Individual antibodies arise in developing B lymphocytes through a series of well characterized yet complicated molecular biological reactions that include somatic hypermutation and recombination of naïve germline-encoded starting sequences throughout the processes of B cell receptor (BCR) gene assembly and antibody affinity maturation [2+ binding developed during these processes or if it can be found encoded within the germline DNA sequences of antibody light chain genes. Lacking access to the cells in which the anti-S1P antibody originally developed, we relied upon analysis by the IgBlast server to identify the likely murine variable and joining genes from which LT1002 is derived [2+-coordinating residues, Asp30, Asp31, Asp32, and Asp92, are present in the naïve germline variable gene sequence , macaque (Vk5.4), and human (Igkv5-2) genomes that bear each of the four Asp residues in positions that correspond to murine LT1002  for binding to two Ca2+ at 171 μM. This is significant on several counts. First, this is roughly ten times lower than the typical concentration of Ca2+ in blood plasma, which suggests that LT1002 is generally preloaded with Ca2+ as it circulates in the blood stream. We had previously speculated that the antibody might bind to Ca2+ and S1P simultaneously through a concerted binding mechanism or that initial antigen binding might induce subsequent affinity for Ca2+. In light of the present study, we are confident that circulating anti-S1P antibodies associate with Ca2+ independent of antigen. Second, the low affinity correlates with the significant increase in bond distances to the two Ca2+ ions observed in the antigen-free X-ray crystal structure relative to its S1P antigen-bound structure. This suggests that the Ca2+ are bound with significantly higher affinity upon antigen binding and explains why Ca2+ remained bound even at 100 mM Mg2+ concentration used for crystallization of the S1P antigen-bound complex [2+ binding agrees remarkably well with that deduced in an elegant SPR study on the closely related anti-CD4 Q425 antibody, which also showed that Ca2+ affinity was increased by a factor of greater than 50,000 in the presence of its CD4 protein antigen [Our characterization of ion binding by LT1002 in solution reveals that Caavors it ,40. ITC  complex . Finally antigen .2+-coordination signature  in other antibodies immediately raises the question of whether implementation of bridging Ca2+ ions is a common mechanism employed by antibodies. Other than Q425, neither of the other two antibodies identified as containing the Ca2+-coordination signature, the 2C10 anti-dsDNA antibody, and the MR1 anti-EGFRvIII antibody are known or suspected to rely upon bridging metal ions to bind their respective antigens. By expressing the murine Igkv17-121 germline sequence as part of a naïve antibody Fab fragment and confirming its ability to bind Ca2+ in solution, we have proven that Ca2+ binding potential is inherent to these sequences and preserved within the mouse germline repertoire. Our observation of antibody light chain genes that contain the Ca2+-coordinating residues within germline sequences of diverse mammals makes a strong case for interfacial Ca2+-dependent antigen recognition as an evolutionarily conserved component of the mammalian antibody repertoire.Identification of the interfacial Ca