Despite intense efforts, these important steps in the synaptic vesicles’ cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca2+-sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca2+ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca2+ sensor for vesicle priming. Ca2+ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca2+ concentration up to 50 μM. The rate of vesicle replenishment during sustained elevated intracellular Ca2+ concentration exhibited little Ca2+-dependence. Finally, quantitative mechanistic release schemes with five Ca2+ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca2+ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.The Ca While the basal free intracellular Ca2+ concentration is ~50 nM, thousandfold higher local microdomains of Ca2+ build and decay very fast around the Ca2+ channels . Here, we investigate the Ca2+-dependence of priming and replenishment, which increases the number of release-ready vesicles. Previous work provided evidence that priming and replenishment are strongly Ca2+-dependent . To finally measure the Ca2+-dependence of vesicle replenishment, we focused on the sustained component of release occurring during 100 ms of flash-evoked Ca2+ increase.The discrepant findings of the Caferences . The ratferences . The aim2+ concentrations between 30 and 180 nM, a significant release below 5 µM, an apparent shallow dose-response curve in the studied Ca2+ concentration range of 1 to 50 µM, and little Ca2+-dependence of vesicle replenishment during sustained elevated intracellular Ca2+ concentrations. Computational simulations incorporating mechanistic release schemes with five Ca2+ binding steps and fast vesicle replenishment via sequential or parallel pools of vesicles could explain our data. Our results show the co-existence of Ca2+ sensors with high- and low-affinities that cover a large range of intracellular Ca2+ concentrations and mediate fast signaling at this synapse.Our data revealed a strong dependence of the number of release-ready vesicles on basal Ca2+ concentration on synaptic release, we performed simultaneous patch-clamp recordings from presynaptic cerebellar mossy fiber boutons (cMFB) and postsynaptic granule cells (GC) of 5- to 6-week-old mice at physiological temperatures . Two-photon quantitative Ca2+ imaging with the dual-indicator method using Fluo-5F as the Ca2+ indicator , respectively . The EPSC rise and decay kinetics were not significantly different . Subtraction of the remaining artefact in the background region of the two-photon line scan resulted in artefact-free fluorescence signals  and the intensity (10–100%) and the duration (100 or 200 µs) of the UV laser pulse (2+ elevation was assessed by UV illumination of caged fluorescein mixed with glycerol (2+ concentration was quantified with either high- or low-affinity Ca2+ indicator (Fluo-5F or OGB-5N). To measure the kinetics of neurotransmitter release independent of dendritic filtering or postsynaptic receptor saturation, vesicular fusion was quantified by measuring the presynaptic capacitance with a 5 kHz-sinusoidal stimulation .In addition to the speed of vesicle fusion, we analyzed the delay from the onset of the UV-illumination to the onset of the rise of membrane capacitance, which was a free parameter in our fitting functions see . The deltrations , which i2+ uncaging in the presynaptic terminal evoked EPSCs with kinetics, which strongly depended on the post-flash Ca2+ concentration. The cumulative release obtained from deconvolution analysis of the recorded EPSCs was fitted as previously done for capacitance traces (2+ concentrations (<5 µM), a significant amount of neurotransmitter release could be measured, which is consistent with previous reports from central synapses  and no significant overshoots were observed (see below). Secondly, we experimentally compared strong and short UV illumination  with weak and long UV illumination , because a Ca2+ overshoot is expected to primarily occur with strong and short UV illumination. Comparison of these two groups of UV illumination resulted in similar post-flash concentrations but did not reveal a significant difference in the corresponding release rate indicating that undetectable Ca2+ overshoots did not affect the measured release rate  of the merged capacitance and deconvolution data showed no signs of saturation consistent with our previous analyses of each data set separately. Fitting a Hill equation to the merged data indicated a KD >50 μM and a Hill coefficient of 1.6  was on average more than 10 times smaller  released between 10 and 100 ms after flash onset  and the other pool represents a replacement pool (V1), which can undergo rapid docking and fusion  with different Ca2+-sensitivity exist, where both types of vesicles can fuse with different Ca2+ affinity  on flash-evoked release. The pre-flash Ca2+ concentration can only be reliably determined with the Ca2+ indicator Fluo5F used in the experiments with weak flashes . Due to the presence of the Ca2+ loaded DMn cage, the pre-flash Ca2+ concentrations were on average higher than the resting Ca2+ concentration in physiological conditions of around 50 nM  these vesicles will fuse very rapidly and the further increase in the release kinetics (causing the non-saturating dose-response curve) can be explained by rapid vesicle replenishment from a sequential pool of vesicles exhibiting use-dependent lowering of the Ca2+-affinity , therefore, these questions remain to be investigated at the cMFBs. Third, currently available techniques to estimate fast release rates at near-physiological temperatures in the Ca2+ range above 50 µM are limited by the sampling frequency of capacitance measurements and dendritic filtering, which could prevent the detection of saturation at the upper end of the dose-response curve.The here obtained dose-response curve has the following three caveats. First, the cMFB to GC synapses in lobule IX are functionally distinct based on the origin of the mossy fibers . Therefo2+-dependence of vesicle priming and replenishment at cMFBs was analyzed more indirectly with the Ca2+ chelator EGTA .In previous reports, the Cator EGTA  and the t models . Furtherat cMFBs . Our paiat cMFBs  and 7 cl Munc13s . Another2+ concentration of ~3 µM; 2+-dependent. This interpretation would also provide an explanation why in a recent report, triggering an action potential in the range of 10–50 ms before another action potential restored the synchronicity of synaptic vesicle fusion in mutant synapses which had an impaired synchronous release  was initially interpreted as a sub-pool of release-ready vesicles that fuse with slower kinetics . Thus, during sustained activity at cMFBs, vesicle replenishment is mediated by either an apparently Ca2+-independent process because of a saturated high-affinity Ca2+ sensor or a Ca2+-independent process.The upstream steps of vesicle priming, referred to as replenishment, recruitment, refilling, or reloading, remain controversial in particular with respect to their speed. The slow component of release . Therefore, at the cMFB, the coupling distance of the vesicles released by a single action potential is if anything even smaller than the previous estimate of 20 nm.The Caproaches . Our preproaches  was base2+ hypothesis’ explaining synaptic facilitation  O2/95% (vol/vol) CO2]). All recordings were restricted to lobules IV/V of the cerebellar vermis to reduce potential functional heterogeneity among different lobules  0.001, equilibrated with 95% O2 and 5% CO2. Cerebellar mossy fiber boutons (cMFBs) were visualized with oblique illumination and infrared optics  and CaCl2 were added depending on the aimed post-flash Ca2+ concentration, such that either 0.5, 2, or 10 mM was used for low, middle, or high target range of post-flash Ca2+ concentration, respectively  until the temperature in the center of the recording chamber with immersed objective was between 36.0°C and 36.3°C. This process was repeated before using a new brain slice. During recordings, the thermometer was put at the side of the recording chamber and the readout was monitored to avoid potential drifts in temperature . The room temperature was controlled using an air conditioner set to 24°C. Presynaptic patch-pipettes were from pulled borosilicate glass  to open-tip resistances of 3–5 MΩ (when filled with intracellular solution) using a DMZ Puller . Slices were superfused with artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 105, NaHCOd optics . Whole-cectively . To quan2O with 200 mM CsOH in H2O. The purity of each DMn batch was determined in the intracellular solution used for patching through titration with sequential addition of Ca2+ as previously described , we used consecutive flashes on the same cell .After waiting for at least one minute in whole-cell mode to homogenously load the terminal with intracellular solution, capacitance measurements were performed at a holding potential of −100 mV with sine-wave stimulation  were whole-cell voltage-clamped with intracellular solution containing the following (in mM): K-gluconate 150, NaCl 10, K-HEPES 10, MgATP three and Na-GTP 0.3 . 10 μM Atto594 was included to visualize the dendrites of the GCs . After w2 (5 mM EGTA / 0.412 mM CaCl2 or 6.24 mM EGTA / 1.65 mM CaCl2), we aimed to clamp the free Ca2+ concentration to low and high resting Ca2+ concentrations of ~50 or ~200 nM, respectively, while maintaining a free EGTA concentration constant at 4.47 mM. The underlying calculations were based on a Ca2+ affinity of EGTA of 543 nM  and was found to be to ~30 or ~180 nM, respectively  and divided it by the fluorescence of the Ca2+-insensitive Atto594 . The ratio (R) of green-over-red fluorescence was translated into a Ca2+ concentration through the following calculation  and maximum (Rmax) fluorescence ratios, respectively. We performed these measurements in cMFBs and GCs as well as in cuvettes. Consistent with a previous report .To avoid pipetting irregularities, which might influence the quantification of the fluorescence signals, pre-stocks of Cas report , both Rm boutons . This ad2+ sensitive and insensitive dyes can differentially bleach during UV flash  using weak flashes, deconvolution was first performed with a set of trial parameters for each cell pair. The mini-parameters of the deconvolution were optimized in each individual recording to yield low (but non-negative) step-like elevations in the cumulative release corresponding to small EPSCs measured from the postsynaptic terminal . Next, using the average of the mini-parameters obtained from weak flashes, the deconvolution parameters for the residual current (β and d) were optimized in each recording with strong flashes until no drops occurred in the cumulative release in the range of 5–50 ms after the stimulus . Finally, we averaged the values of each parameter and the deconvolution analysis of all recordings was re-done using the average parameters values. To test the validity of this approach, cumulative release from deconvolution of EPSCs and presynaptic capacitance recordings were compared in a subset of paired recordings (n = 9 pairs) similarly as done in previous investigations  and a single junction silver chloride reference electrode  was read out with a pH meter in voltage mode. A series of standard solutions, with defined Ca2+ concentration (Thermo Fisher Scientific) covering the whole range of our samples, were used to plot a calibration curve of the potential (mV) versus Ca2+ concentration (μM). Then, the potential of several sample solutions containing the same intracellular solution used for patching, but with different Ca2+ concentrations buffered with EGTA, was determined. This way, we got a direct measure of the free Ca2+ concentration of several sample solutions, which were later used after the addition of Ca2+-sensitive fluorometric indicators to plot the fluorescence signal of each solution versus the corresponding free Ca2+ concentration verified by the Ca2+-sensitive electrode, and accordingly the DK of the Ca2+ indicators were obtained from fits with a Hill equation. The estimated DK was two-fold higher than the estimate obtained using only the Ca2+ Calibration Buffer Kit (Thermo Fisher Scientific) without including intracellular patching solution (2+ concentration using Maxchelator software (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/). Therefore, we used two independent approaches to confirm the DK of OGB-5N. We found that TEA increased the potential of the solutions measured through the Ca2+-sensitive electrode, which is consistent with a previous report showing a similar effect of quaternary ammonium ions on potassium sensitive microelectrodes . Caged fluorescein (2 mM) was mixed with glycerol (5% caged fluorescein/ 95% glycerol) to limit the mobility of the released dye . We did The increase in membrane capacitance and in cumulative release based on deconvolution analysis was fitted with the following single or bi-exponential functions using Igor Pro (WaveMetrics) including a baseline and a variable onset.2mono/χ2bi > 1.04), (2) the time constants of the fast and the slow components differed by a factor >3, and (3) the relative contribution of each component was >10%  at least 4% decrease in the sum of squared differences between the experimental trace and the fit compared with a mono-exponential fit  was fit on the decadic logarithm of the release rates and x was the decadic logarithm of the intracellular Ca2+ concentration.Hill equations were used to fit the release rate versus intracellular Ca2+ dynamics using a single compartment model. The kinetic reaction schemes for Ca2+ and Mg2+-uncaging and -binding  includes the free form ([DMn]), the Ca2+-bound form ([CaDMn]), and the Mg2+-bound form ([MgDMn]). Each of these forms is subdivided into an uncaging fraction (α) and a non-uncaging fraction (1-α). The uncaging fractions were further subdivided into a fast (af) and a slow (1-af) uncaging fraction:We simulated the intra-bouton Ca-binding  were con-binding . The iniT = [DMn]f + [DMn]s + [CaDMn]f + [CaDMn]s+ [MgDMn]f + [MgDMn]s[DMn]f + [DMn]s[DMn] = [DMn]f = α af [DMn][DMn]s = α (1-af) [DMn][DMn]f + [CaDMn]s[CaDMn] = [CaDMn]f = α af [CaDMn][CaDMn]s = α (1-af) [CaDMn][CaDMn]f + [MgDMn]s[MgDMn] = [MgDMn]f = α af [MgDMn][MgDMn]s = α (1-af) [MgDMn][MgDMn]f) or slow (τs) time constants, respectively. Free Ca2+ or Mg2+-bound DMn decomposed into two photoproducts  differing with respect to their binding kinetics. The binding kinetics of all species were governed by the corresponding forward (kon) and backward (koff) rate constantsH is the Heaviside step function and flasht the time of the UV flash. Ca2+ and Mg2+-binding to the dye, ATP, and an endogenous buffer (EB) were simulated by second-order kinetics:The suffixes ‘T’, ‘f’, and ‘s’ indicate total, fast or slow, respectively. The transition of fast and slow uncaging fractions into low-affinity photoproducts (PP) occurred with fast  prim/kunprim= k and V1Ca(0), V2Ca(0), and Vfused(0) was zeroprim. k was the sum of a Ca2+-dependent and Ca2+-independent rate constants. The Ca2+-dependence was implemented as a Michaelis-Menten kinetic with a maximum rate constant of 30 s−1  = 1 for the default pre-flash resting Ca2+ concentration of 227 nM , was obtained from the simulations as described in the previous paragraph. 2+ concentration (i.e. simulated Ca2+(t) transient) the sampling, addition of noise, and exponential fitting were repeated 50 times. The median of these values represents the prediction of the model for each peak post flash Ca2+ concentration. The parameters of the r were manually adjusted to obtain best-fit results.The differential equations were solved with the NDSolve function of Mathematica. The Caase rate , 6D, Vfuunctions . The sel2+ binding steps mediating fusion and two Ca2+-dependent priming steps defined according to the following differential equationsModel 2 was a sequential two-pool model based on , …, and2+ ions, respectively, and To implement the use-dependent slowing of the release rate constants of this model  in a detonk and offk were linearly interpolated between two values depending on 2+-dependence of the rate constants in these differential equations.The reserve pool 1(0) prim1/kunprim1= k and V2,0Ca(0) = (prim1/kunprim1k)*(prim2/kunprim2k). The initial condition of the other state V2,1Ca(0) to V5,0Ca(0), Vfused(0), and prim1. k and prim2k were the sum of a Ca2+-dependent and Ca2+-independent rate constant defined similarly as described in unprim1k and unprim2k were defined such that the occupancy V1(0) = 1 and V2,0Ca(0) = 1 for the default pre-flash resting Ca2+ concentration of 227 nM  prim1/kunprim1= k and V2,0Ca(0) = (prim1/kunprim1k)*(prim2/kunprim2k). The initial condition of the other state V1,1Ca(0) to V1,0Ca(0), V1,fused(0), and V2,1Ca(0) to V2,0Ca(0), V2,fused(0) were zeroprim1. k was a Ca2+-independent rate constant and prim2k was the sum of a Ca2+-dependent and Ca2+-independent rate constants defined similarly as described in unprim1k and unprim2k were defined such that the occupancy V1,0Ca(0) = one and V2,0Ca(0) = one for the default pre-flash resting Ca2+ concentration of 227 nM  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Acceptance summary:The authors examined the calcium dependence of exocytosis at cerebellar mossy fiber boutons using multiple experimental approaches - electrophysiology, calcium imaging, calcium uncaging and capacitance measurements. These specific approaches have been used at other synapses over many years, and the authors show intriguing differences among different types of synapses that are likely functionally significant. A strength of this work is the masterful implementation and explanation of the techniques that combine to lend the paper reliability and give it lasting value.Decision letter after peer review:Thank you for submitting your article "Calcium dependence of neurotransmitter release at a high fidelity synapse" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Gary Westbrook as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted the below summary to help you prepare a revised submission. For the most part, the revisions can be addressed by more better discussion of the issues enumerated below and by addressing limitations of the approach.Essential revisions:1. The introduction could be improved. The authors do a good job on the calcium dependence of release, but the presentation of priming and replenishment is confusing. They should set up the idea that the time course of release will be used to provide insight into replenishment. The issues of basal calcium on the amplitude of evoked responses is unlikely to be the same thing as replenishment . Part of the problem is that they ignore a long and rich history of studies describing how residual calcium leads to enhancement of neurotransmitter release, even though the experiments examining how basal free calcium levels influence neurotransmitter release are very closely related to those papers. They finally (and briefly) discuss a small fraction of that literature in the discussion (and they could do a much better job of it).2. MFs come from a variety of sources and are likely to have highly heterogeneous properties. This is not adequately dealt with in the current study. Chabrol et al  previously studied the properties of MF synapses and found tremendous diversity in their release properties. The conclusion was that that the origin of MFs strongly influenced their release properties. Based on the properties they describe it would be reasonable to assume that the calcium dependence of release could be very different for MFs from different sources. That study was performed in lobule X, where there may be more diversity. The current study was performed in lobules IV and V, which reduces interlobule variability but not intralobule variability. I do not think that there has been a comparable study characterizing the diversity of MF inputs in these regions. They need to deal with this issue and the complications it would introduce into this study.3. Comment not requiring additional experiments: Stefan Hallermann has previously published a beautiful paper describing the role of Bassoon in vesicle reloading at mossy fiber synapses . If they had studied knockout animals in the current study, they could make a much stronger case about vesicle replenishment, and it would have the potential to be a much more convincing paper.4. 36C is not really a physiological temperature. Although there seems to be some variability in the physiological temperature it seems as if 37.5-38.5 is a better approximation of physiological temperature. See: Kiyatkin EA (2019) Brain temperature and its role in physiology and pathophysiology: Lessons from 20 years of thermo-recording. Also, they state that experiments were done at 36C. I think many experimenters find that there is some fluctuation in the actual temperature of the slice. The authors should provide an accurate estimate of the range of temperatures used in their experiment.5. Does the elevation of basal calcium affect mEPSC frequency? It would be useful to know the number of claws on each recorded granule cell and scale mEPSC frequency effects accordingly in order to estimate the effects on release from the recorded mossy fiber. It is important to know whether the same elevation of basal calcium is sufficient to influence release in other ways.6. Organization of the results . One reviewer wondered why the authors started with the basal calcium experiment? " I like it. I think it is interesting. But it seems slightly unrelated to much of the rest of the paper and is of a very different flavor. It also seems almost unrelated to the introduction. It is not very well integrated into the rest of the paper. It seems as if it would be better to move it to near Figure 7."7. Distant mossy fibers on the same axon could complicate capacitance measurements. Have the authors considered how this could affect their capacitance measurements?8. How might endocytosis influence the capacitance measurements?9. On line 245-247 the authors say that they did simultaneous capacitance and deconvolution measurements. Please present these data in a supplementary figure to show the extent to which they agree and disagree. It would be useful to have a quantitative comparison of these two approaches. This is particularly important because it seems as if there is a big difference between the two approaches in 5B.10. One reviewer raised a concern with Fig 5B. It seems as if the capacitance is measuring from the entire presynaptic bouton (release onto many granule cells) and the granule cell recording is extremely complicated because it has a direct component and a scaled down spillover component that is difficult to convert into vesicle number.11. Please add additional discussion of the direct release and the spillover release components at mossy fiber synapses and how it could affect their measurements.12. On page 32, it would seem that if you did simulations, you would expect an overshoot. Why isn't there an overshoot for brief uncaging?13. The paper would great benefit from a more complete Ca2+ dose response curve. Because the authors goal is to completely characterize how release is regulated at the cerebellar mossy fiber bouton, it would significantly strengthen the conclusions of the manuscript to provide data on release rates at Ca2+ concentrations under 1 µM and higher than 50 µM in the dose response curve. We recognize that these experiments may not be completely feasible, but the issue should be openly discussed in the manuscript. The authors demonstrate that the release machinery is not saturated up to 50 µM Ca2+. It would be ideal to determine if the release machinery can be saturated.Thank you for resubmitting your work entitled "Calcium dependence of neurotransmitter release at a high fidelity synapse" for further consideration by eLife. Your revised article has been evaluated by Gary Westbrook (Senior Editor) . The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Reviewer #1:As you will see the authors are pleased with the quality and rigor of the experiments. However, we really want you to address the issue raised by Reviewer 1 concerning the intellectual and experimental background that are critical to the eventual standing of this work.This is an impressive paper. Its great strength is that it addresses an important question so carefully and thoroughly that it makes a substantial contribution to the field.My one quibble it that I still think they have done a woefully inadequate job of discussing a great deal of previous literature centered around the residual calcium hypothesis. They have added a new section and an eclectic set of new references (55-80), but they disregard many classic papers and relevant reviews in the field. The residual calcium hypothesis has been most extensively discussed in terms of forms of short-term synaptic plasticity, such as facilitation, augmentation and post-tetanic potentiation. The literature goes about 70 years. I will leave it up to the authors to fix the issue to their satisfaction. Even though it is a minor point, I think this reflects badly on the authors. I leave it up to them to fix it to their own satisfaction.Reviewer #2:The authors have done a good job responding to the reviews. Their response to point 7 should have led to a comment  somewhere in the methods, rather than just a comment to the reviewer. Otherwise, I have no further concerns with the work and feel it is ready for publication. It is a fine paper. Essential revisions:1. The introduction could be improved. The authors do a good job on the calcium dependence of release, but the presentation of priming and replenishment is confusing. They should set up the idea that the time course of release will be used to provide insight into replenishment. The issues of basal calcium on the amplitude of evoked responses is unlikely to be the same thing as replenishment . Part of the problem is that they ignore a long and rich history of studies describing how residual calcium leads to enhancement of neurotransmitter release, even though the experiments examining how basal free calcium levels influence neurotransmitter release are very closely related to those papers. They finally (and briefly) discuss a small fraction of that literature in the discussion (and they could do a much better job of it).2+ on neurotransmitter release in the introduction and discussion (for example line 55 to 80 of the introduction). Furthermore, we now explain already in the introduction how priming, fusion, and replenishment were dissected (line 93 to 99):We thank the reviewers for this comment. We re-wrote the introduction and now discussed more literature on the impact of residual Ca2+-dependence of vesicle priming, we first directly manipulated the free basal intracellular Ca2+ concentration and measured the amount of action potential-evoked release. To determine the Ca2+-sensitivity of vesicle fusion, we measured the initial release kinetics of the fusion of the primed vesicles upon Ca2+ uncaging (with time constants mostly << 10 ms). To finally address the Ca2+-dependence of vesicle replenishment, we focused on the sustained component of release occurring during 100 ms of flash-evoked Ca2+ increase.”“To dissect the Ca2. MFs come from a variety of sources and are likely to have highly heterogeneous properties. This is not adequately dealt with in the current study. Chabrol et al  previously studied the properties of MF synapses and found tremendous diversity in their release properties. The conclusion was that that the origin of MFs strongly influenced their release properties. Based on the properties they describe it would be reasonable to assume that the calcium dependence of release could be very different for MFs from different sources. That study was performed in lobule X, where there may be more diversity. The current study was performed in lobules IV and V, which reduces interlobule variability but not intralobule variability. I do not think that there has been a comparable study characterizing the diversity of MF inputs in these regions. They need to deal with this issue and the complications it would introduce into this study.We thank the reviewer for this important comment. We agree that cMFBs could be functionally and molecularly distinct in lobule IV/V, similar to what has been reported in lobule IX in Chabrol et al. . We now discussed this further as general caveat of this study in lines 604-612:2+ dependence of release at the calyx of Held synapse could in part also be due to the recently revealed heterogeneity between different classes of functionally and structurally distinct calyces .”“The here obtained dose-response curve has the following three caveats. First, the cMFB to GC synapses in lobule IX are functionally distinct based on the origin of the mossy fibers . Therefore, the here-recorded boutons in lobule IV/V could be molecularly and functionally distinct leading to the observed scatter in the dose-response curve, which could cause an apparent shallowing. Yet, the scatter in the dose-response curve of comparable studies measuring the Ca3. Comment not requiring additional experiments: Stefan Hallermann has previously published a beautiful paper describing the role of Bassoon in vesicle reloading at mossy fiber synapses . If they had studied knockout animals in the current study, they could make a much stronger case about vesicle replenishment, and it would have the potential to be a much more convincing paper.2+ dependence of release at the cerebellar mossy fiber to granule cell synapse and combining UV Ca2+ uncaging and quantitative two-photon Ca2+ imaging with presynaptic capacitance measurements and deconvolution techniques during paired pre- and postsynaptic recordings. It was a great effort to establish the methods used in this manuscript, and to provide a detailed analysis which has been done at this level of detail only at the calyx of Held. We believe that this manuscript focusing only on wild-type cerebellar mossy fiber synapses provides relevant novel findings.We are relieved that the editor and the reviewers agreed that repeating the analysis on knockout mice would be beyond the scope of this manuscript. We would like to point out that this is the first study measuring the Ca4. 36C is not really a physiological temperature. Although there seems to be some variability in the physiological temperature it seems as if 37.5-38.5 is a better approximation of physiological temperature. See: Kiyatkin EA (2019) Brain temperature and its role in physiology and pathophysiology: Lessons from 20 years of thermorecording. Also, they state that experiments were done at 36C. I think many experimenters find that there is some fluctuation in the actual temperature of the slice. The authors should provide an accurate estimate of the range of temperatures used in their experiment.We thank the reviewer for pointing this out. We now explained in more details how we controlled the recording temperature in the methods section lines 742-750:“All recordings were performed at near-physiological temperature by adjusting the set temperature of the TC-324B perfusion heat controller  until the temperature in the center of the recording chamber with immersed objective was between 36.0 and 36.3°C. This process was repeated before using a new brain slice. During recordings, the thermometer was put at the side of the recording chamber and the readout was monitored to avoid potential drifts in temperature . The room temperature was controlled using an air conditioner set to 24°C.”5. Does the elevation of basal calcium affect mEPSC frequency? It would be useful to know the number of claws on each recorded granule cell and scale mEPSC frequency effects accordingly in order to estimate the effects on release from the recorded mossy fiber. It is important to know whether the same elevation of basal calcium is sufficient to influence release in other ways.2+ experiments .”“Interestingly, the frequency of miniature currents in-between the current injections used to elicit action potentials had a tendency to increase with elevated basal Ca6. Organization of the results . One reviewer wondered why the authors started with the basal calcium experiment? " I like it. I think it is interesting. But it seems slightly unrelated to much of the rest of the paper and is of a very different flavor. It also seems almost unrelated to the introduction. It is not very well integrated into the rest of the paper. It seems as if it would be better to move it to near Figure 7."2+ experiments could be better introduced and integrated in the manuscript. We would like to start the manuscript with physiological stimuli  and proceed with unphysiological UV uncaging for a mechanistic analysis. We therefore favor this order of presentation. However, we now introduced priming and recruitment better, and revised the introduction as mentioned above to better motivate the story flow.We agree with the reviewer that the basal Ca7. Distant mossy fibers on the same axon could complicate capacitance measurements. Have the authors considered how this could affect their capacitance measurements?2+ currents from neighboring boutons during depolarizations for 1 to 30 ms can indeed contaminate the Ca2+ current recordings, in which case the experiments were excluded from the analysis as previously described .This is an interesting point, which was addressed previously at hippocampal mossy fiber boutons . In that study, multi-compartment models of the bouton, filopodia and adjacent axons were used to judge how the capacitance measurements could be influenced by neighboring cellular structures. The results indicate that high-frequency sine-wave techniques as used in our study (5 and 10 kHz) probe changes in capacitance of the bouton accurately (<10 % error) and are hardly affected by release from neighboring boutons. Note, that more low frequency signals such as Ca8. How might endocytosis influence the capacitance measurements?Endocytosis might indeed complicate the measurements of the rates of vesicle fusion. This is an unavoidable error in capacitance measurements in general. For the dose response curve of vesicle fusion, we focused on the initial fast component of release occurring within the first 10 ms, which we assume is less affected by endocytosis, since it is still earlier than the fastest reported form of endocytosis . However, the sustained release between 10 and 100 ms  could well be contaminated by endocytosis. We therefore separated the sustained release rate measured with capacitance measurements and deconvolution techniques . Both techniques have different limitations and indeed we found some differences between these two techniques (see response to the following comments).9. On line 245-247 the authors say that they did simultaneous capacitance and deconvolution measurements. Please present these data in a supplementary figure to show the extent to which they agree and disagree. It would be useful to have a quantitative comparison of these two approaches. This is particularly important because it seems as if there is a big difference between the two approaches in 5B.We thank the reviewer for raising this point. We have added a supplementary figure  showing a correlation between the time constants obtained from capacitance measurements versus the time constants obtained from deconvolution analysis for the recordings where we have both measurements simultaneously. Please note that our results are consistent with a previous comparison of capacitance measurements and this type of deconvolution analysis .2+ concentration for deconvolution and capacitance measurements. However, we thank the reviewer for realizing that there is indeed a difference between these two data sets. The release rate observed with deconvolution analysis is about two times higher compared with a release rate observed with capacitance measurements . However, the difference is small compared with the large scatter in the data with a 100-fold difference in the rate of release between individual cerebellar mossy fiber boutons. Furthermore, there is a weak but significant correlation in the data set observed with deconvolution analysis compared with the capacitance data. We therefore split the data in 5B into two separate figures based on the method used to estimate the number of vesicles . In the manuscript we now carefully discuss the limitations of each of these technique (line 676-681):The sustained release rate differs mostly in the used Ca“However, it should be mentioned that measuring the sustained release rate is technically very challenging with both presynaptic capacitance and postsynaptic current recordings, because the former cannot differentiate between exo- and endocytosis occurring simultaneously, and the latter can fail to dissect direct release from spill-over current, which is prominent at this synapse . Therefore, these results must be considered cautiously”.10. One reviewer raised a concern with Fig 5B. It seems as if the capacitance is measuring from the entire presynaptic bouton (release onto many granule cells) and the granule cell recording is extremely complicated because it has a direct component and a scaled down spillover component that is difficult to convert into vesicle number.We used the deconvolution technique that was established by Neher and Sakaba , which we adjusted for the cerebellar mossy fiber to granule cell synapse . This deconvolution method takes into account the glutamate spill-over and the delayed clearance of the neurotransmitter. Despite our efforts to establish deconvolution techniques and capacitance measurements, we agree that both techniques have different intrinsic limitations when investigating strong long-lasting release. In order to address the reviewer’s concern, we split the data in 5B into two separate figures based on the method used to estimate the number of vesicles released . As mentioned above, we now carefully discuss the limitations of these approaches.11. Please add additional discussion of the direct release and the spillover release components at mossy fiber synapses and how it could affect their measurements.We have added additional discussion to address this point. Please see response to the above comments.12. On page 32, it would seem that if you did simulations, you would expect an overshoot. Why isn't there an overshoot for brief uncaging?2+ and Mg2+ binding and unbinding kinetics in particular of the photoproducts . In addition, in our simulations ATP and endogenous buffers were taken into account, which further dampened the initial Ca2+ spike, as already anticipated in Bollmann et al. .There is no overshoot due to the recent improvements in the quantification of Ca13. The paper would great benefit from a more complete Ca2+ dose response curve. Because the authors goal is to completely characterize how release is regulated at the cerebellar mossy fiber bouton, it would significantly strengthen the conclusions of the manuscript to provide data on release rates at Ca2+ concentrations under 1 µM and higher than 50 µM in the dose response curve. We recognize that these experiments may not be completely feasible, but the issue should be openly discussed in the manuscript. The authors demonstrate that the release machinery is not saturated up to 50 µM Ca2+. It would be ideal to determine if the release machinery can be saturated.2+ dependence of release in the Ca2+ range below 1 µM could well form the basis of a separate manuscript. Regarding the upper end of our dose-response curve, we reached the limit of resolving the release rates at 30 to 50 µM Ca2+ concentration, although we increased the sampling frequency of our capacitance recordings to a high frequency (10 kHz). Therefore, with the current techniques, it seems impossible to measure the release rate at this synapse at Ca2+ levels higher than 50 µM. To explain the limitations of our study we added the following sentences in the discussion:We agree with the reviewer that it would be interesting to extend the dose response curve to values below 1 µM. To our knowledge this has only been achieved in a few studies performed at the calyx of Held synapse. As the reviewers know, direct presynaptic recordings and simultaneous pre- and postsynaptic recordings at the cerebellar mossy fiber to granule cell synapse are technically extremely challenging, and the number of experiments in the present paper is already at 139 (80 presynaptic and 59 paired recordings). Therefore, we think that a detailed analysis of the Ca2+ range (<1 µM), therefore, these questions remain to be investigated at the cMFBs. Finally, currently available techniques to estimate fast release rates in the Ca2+ range above 50 µM are limited by the sampling frequency of capacitance measurements and dendritic filtering which could prevent the detection of saturation at the upper end of the dose-response curve.”“Second, we could not investigate allosteric or two-sensor models  because we did not address the release rates in the low CaReviewer #1:This is an impressive paper. Its great strength is that it addresses an important question so carefully and thoroughly that it makes a substantial contribution to the field.My one quibble it that I still think they have done a woefully inadequate job of discussing a great deal of previous literature centered around the residual calcium hypothesis. They have added a new section and an eclectic set of new references (55-80), but they disregard many classic papers and relevant reviews in the field. The residual calcium hypothesis has been most extensively discussed in terms of forms of short-term synaptic plasticity, such as facilitation, augmentation and post-tetanic potentiation. The literature goes about 70 years. I will leave it up to the authors to fix the issue to their satisfaction. Even though it is a minor point, I think this reflects badly on the authors. I leave it up to them to fix it to their own satisfaction.We thank the reviewer for pointing this out. We fully agree that the manuscript profits from further discussion of our results on vesicle priming in the context of the ‘residual calcium hypothesis’ because these two mechanisms are very likely to converge into a mechanism explaining synaptic facilitation. Therefore, we followed the reviewer’s suggestion and explained in more details the potential interplay between residual calcium, vesicle priming, and synaptic facilitation in a new section in the discussion:“Implications for synaptic facilitation2+ hypothesis’ explaining synaptic facilitation . The strong dependence of the action potential-evoked release on basal Ca2+  supports the critical effect of residual Ca2+ on synaptic strength. Our mechanistic analysis  indicates that the number of release-ready vesicles rather than the vesicular release probability is regulated by residual Ca2+. The high-affinity Ca2+ sensor Synaptotagmin-7  could be a sensor for the changes in basal Ca2+ levels and mediate the here-reported three-fold increase in synaptic strength . Synaptotagmin‑7 has been shown to mediate vesicle recruitment , asynchronous release , and synaptic facilitation . If the recruitment and priming steps are fast enough, they could provide a powerful mechanism for synaptic facilitation. Indeed, there is increasing evidence for ultra-fast Ca2+-dependent recruitment and priming (reviewed in Neher and Brose 2018) as well as facilitation mediated by an increase in the number of release-ready vesicles rather than the vesicular release probability . Our data are therefore consistent with the emerging view that facilitation is mediated by rapid Synaptotagmin‑7/Ca2+-dependent recruitment and priming of vesicles.”Our data might contribute to a better understanding of the mechanisms of the ‘residual CaFurthermore, we more precisely defined the aim of this study and introduced the relation with facilitation in the introduction:2+ channels . According to Imig et al. (2014) molecular priming is the functional correlate of vesicle docking . Vesicle replenishment refers to the delivery of new vesicles during sustained activity. The effect of the residual Ca2+ on the strength of synapses particularly during synaptic facilitation has been studied for decades with a particular focus on the release probability of vesicles (see discussion). Here we investigate the Ca2+-dependence of priming and replenishment, which increases the number of release-ready vesicles.”“We refer to vesicle priming as the molecular and positional preparation of vesicles for fusion near CaReviewer #2:The authors have done a good job responding to the reviews. Their response to point 7 should have led to a comment  somewhere in the methods, rather than just a comment to the reviewer. Otherwise, I have no further concerns with the work and feel it is ready for publication. It is a fine paper.We followed the reviewer’s suggestion and included this in the methods section:“Distant mossy fiber boutons on the same axon are unlikely to contaminate capacitance measurements because investigations at hippocampal mossy fiber buttons indicate that the high-frequency sine-wave techniques as used in our study are hardly affected by release from neighboring boutons on the same axon .”


Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca2+ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17β-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca2+ entry via SOCE without affecting other ion channels and pumps involved in Ca2+ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.T cell activation starts with formation of second messengers that release Ca •2-Ethyl-3-sulfamoyloxy-17β-cyanomethylestra-1,3,5(10)-triene is a SOCE inhibitor.•novel inhibitor antagonizes SOCE and subsequent signaling events in different T cells•3 receptor, KV channels or SERCA pumpsno antagonist effects on IP Althoiewed in . Furtheriewed in . Therebyiewed in . Recentliewed in . In the 2+ entry in T cells, screened a library of 2ME2 analogues, and characterized STX564 as novel, potent and specific SOCE inhibitor, similar in efficacy to the known SOCE inhibitor Synta66 i) from the ratio data:Finally, every measurement was calibrated using 0.1% . The fluorescence intensities of deleted data points were interpolated from those just before and after the deleted time points.s 224 nM . R is thv/v) for all conditions.Estradiol, 2ME2 and 2ME2 derivatives were dissolved in DMSO at a concentration of 50 mM and stored at −20 °C for up to 6 months. For concentration-response curves, stock solution was freshly diluted with DMSO to obtain the same DMSO concentration of 0.2%  were calculated and subtracted from the basal Ca2+ concentration that represents the mean Ca2+ concentration before the first compound addition. These plateau Ca2+ concentrations were normalized to 100% (DMSO condition of every data set) and 0% . The compound concentrations were then log transformed. Finally, the data were fitted to a corresponding nonlinear fit of variable slope using Prism 6 .For calculating IC2.62+ quenching of Fura2 was used to analyze influx of divalent cations into Jurkat T cells. Like for Ca2+ measurements, 106 loaded Jurkat T-lymphocytes were pelleted and Ca2+ buffer was exchanged for nominal Ca2+ free buffer (see above). Instead of Ca2+, Mn2+ was added 1220 s after the start of the measurement. To investigate Mn2+ quenching independently of any effects of Ca2+, Fura2 was excited at its isosbestic point, which was experimentally determined to be 357 nm. The emission wavelength was set to 495 nm. In contrast to the Ca2+ measurement experiments, these measurements were not calibrated.Mn2+ addition, there was a very fast drop in Fura2 fluorescence in all experiments. This drop lasted for about 10 s and was probably due to quenching of extracellular Fura2. This phase was not considered in the calculation of the initial slope. The data after this rapid drop were fitted to a one phase exponential decay with the following equation:For quantitative analysis, the velocity of quenching was calculated as the initial slope. After Mny0 is the fluorescence at the start of the decay, P is the theoretical fluorescence at the end of the decay at infinite times, K is the rate constant, and x is the time.The first derivative of this equation was used to calculate the slope at 1230 s:2+ addition due to bleaching. The fluorescence before Mn2+ addition was fitted to a straight line using the following equation:In all experiments, fluorescence gradually decreased before Mnm is the slope, x is the time and c is the y-intercept. To not overestimate the initial slope, the slope of this linear fit was subtracted from the initial slope of the exponential decay. So the initial slope (si) after Mn2+ addition was calculated using the following equation:2.76 Jurkat T–lymphocytes were pelleted at 400 to 450 ×g for 5 min at room temperature and pellets were dissolved in 500 μl RPMI 1640 medium (Gibco) without phenol red containing l-glutamine, supplemented with 7.5% (v/v) NCS. 30 μg plasmid-DNA (G-GECO1.2-ORAI1) was transferred into a 4 mm electroporation cuvette  and the Jurkat T-lymphocytes were added. G-GECO1.2-ORAI1 was a gift from Michael Cahalan, USA (Addgene plasmid # 73562)  after IP3 addition was subtracted from the mean [Ca2+] between 25 and 75 s before IP3 addition (dPeak). For analyzing SERCA inhibition, the data after compound addition, namely thapsigargin, STX564, Synta66 or DMSO as a vehicle control, and the decay after ATP addition was fitted to a ‘plateau followed by one phase exponential decay’ using GraphPad Prism. The rate constant K of the fitted data was used as a quantitative read-out for SERCA pump activity and its inhibition.As a quantitative read-out for inhibition of IP2.9w/v) PLL solution  diluted 1:50 with high-purity water  for 30 min. Coated culture dishes were rinsed twice with high-purity water, dried at room temperature under sterile conditions and then stored at 4 °C for up to 1 week. Just before starting the experiments, coated culture dishes were pre-warmed for 5 min at room temperature. A recording chamber  that was preliminary coated with a thin layer of baysilone-paste  of medium viscosity was placed into the coated cell culture dish. About 105 Jurkat T-lymphocytes were placed into this recording chamber and were allowed to settle down for 5–10 min. Afterwards the cells were rinsed twice with 200 μl extracellular buffer (composition: see below next paragraph) and finally taken up in 200 μl extracellular buffer containing 200 nM thapsigargin as well as either K+ channel inhibitors, STX compounds or 0.2% (v/v) DMSO as a vehicle control. The cell culture dish was placed in a custom-made holder placed on an inverted microscope (Zeiss AxioVert 100). Microelectrodes were manually pulled from 1.5 mm diameter borosilicate glass capillaries  and firepolished via a Flaming Brown micropipette puller , resulting in pipette resistances between 2.7 and 5.1 MΩ (mean: 3.9 MΩ). The microelectrodes were filled with intracellular solution (composition: see below next paragraph). The holding potential was set to −60 mV. Extracellular buffer for KV measurement contained 128 mM CH3SO3Na, 4 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose; pH 7.4, NaOH .V channels contained 125 mM aspartic acid, 5 mM MgCl2, 2 mM MgSO4, 15 mM HEPES, 12 mM BAPTA  were compensated by Patchmaster . C-slow and series resistance was compensated before every sweep. The voltage was applied to the cells and the currents were recorded via an EPC9/10 amplifier at a sampling rate of 20 kHz.Intracellular buffer for measuring Kied from ). For me2.10+ T cells retrovirally transduced to express enhanced GFP (TMBP cells) were established as previously reported i is essential for downstream signaling events of T cell activation, inhibition of Ca2+ entry is likely the mechanism by which 2ME2 decreases NFAT translocation, T cell proliferation and cytokine production [2+]i upon exposure to STX564, we excluded experimentally (i) inhibition of IP3 evoked Ca2+ release, (ii) inhibition of SERCA activity, and (iii) inhibition of KV as discussed above [We clearly demonstrated that the hormone estradiol, its non-estrogenic metabolite 2ME2, and its synthetic analogue STX564 inhibit Caoduction  as well oduction . Among ted above .+/K+ ATPases in human and mouse cardiac myocytes [+ channels were described, e.g. activation [V channels nor SERCA pumps.While it was shown that estradiol activates (i) Namyocytes ,43 (ii) myocytes , and . Actionl target ,54, modul target ,54, whicl target . Finallyl target . Howevereceptors .2+ release channels, SERCA pumps nor KV channels. This compound should serve as an attractive new chemical biology tool for future studies on SOCE, but also in autoimmune disease models.In conclusion, we characterized the 2ME2 derivative STX564 as a novel potent and specific SOCE inhibitor affecting neither CaSource data are available from the corresponding author upon request.10.13039/501100001659Deutsche Forschungsgemeinschaft (DFG), Germany,  and by the Hertie Stiftung, Germany . BVLP is a 10.13039/100010269Wellcome Trust Senior Investigator .This work was supported by the Anke Löhndorf: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration. Leon Hosang: Methodology, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Wolfgang Dohle: Investigation, Resources, Writing – review & editing. Francesca Odoardi: Conceptualization, Methodology, Validation, Investigation, Writing – original draft, Writing – review & editing, Supervision, Project administration. Sissy-Alina Waschkowski: Formal analysis, Investigation, Data curation, Writing – review & editing. Anette Rosche: Formal analysis, Investigation, Data curation. Andreas Bauche: Formal analysis, Investigation, Data curation. Riekje Winzer: Investigation. Eva Tolosa: Investigation. Sabine Windhorst: Investigation. Stephen Marry: Methodology, Formal analysis, Investigation, Data curation, Writing – review & editing. Alexander Flügel: Conceptualization, Validation, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition. Barry V.L. Potter: Conceptualization, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition. Björn-Philipp Diercks: Methodology, Formal analysis, Investigation, Data curation, Writing – review & editing. Andreas H. Guse: Conceptualization, Validation, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition.The authors declare no competing interest.


However, as shown in the literature reviewed here, many continuous trafficking steps can be up- or down-regulated by Ca2+, including several steps associated with human pathologies. Notably, we describe a series of Ca2+ pumps, channels, Ca2+-binding effector proteins, and their trafficking machinery targets that together regulate the flux of cargo in response to genetic alterations as well as baseline and agonist-dependent Ca2+ signals. Here, we review the most recent advances, organized by organellar location, that establish the importance of these components in trafficking steps. Ultimately, we conclude that Ca2+ regulates an expanding series of distinct mechanistic steps. Furthermore, the involvement of Ca2+ in trafficking is complex. For example, in some cases, the same Ca2+ effectors regulate surprisingly distinct trafficking steps, or even the same trafficking step with opposing influences, through binding to different target proteins.Constitutive vesicle trafficking is the default pathway used by all cells for movement of intracellular cargoes between subcellular compartments and in and out of the cell. Classically, constitutive trafficking was thought to be continuous and unregulated, in contrast to regulated secretion, wherein vesicles are stored intracellularly until undergoing synchronous membrane fusion following a Ca Alternatively, the differentiation of B cells to plasma cells requires a marked increase in secretory capacity, driven in part by sequential waves of protein expression2. However, there is still much to learn about the regulation of constitutive secretion. The milieu of ions, lipids, and proteins that constitutive trafficking externalizes, degrades, or delivers to various organelles affects nearly every aspect of cell metabolism, making dynamic regulation of these pathways of great significance to biomedical research.Eukaryotic cells are in a constant state of membrane maintenance, renewal, and expansion, accomplished by an interwoven web of constitutive vesicle trafficking pathways connecting the endoplasmic reticulum (ER), Golgi, plasma membrane (PM), and the endolysosomal system. Classically, constitutive secretion is thought to be continuous and unregulated. This contrasts with the highly controlled, regulated secretory pathway in which proteins stored in intracellular granules are released en masse in response to external factors or signals. However, constitutive secretion is also regulated. By way of example, during mitosis in mammalian cells, the Golgi is fragmented for partitioning into progeny cells. This transition from S-to-M phase necessitates that most, if not all, membrane trafficking pathways are temporarily shut down, only to be resumed later during cytokinesis2+ is actively sequestered into several organelles for use in a myriad of signaling processes3. Many of these Ca2+ stores, such as the ER, Golgi, and lysosome, are prominent in constitutive vesicle trafficking pathways -to-Golgi and Golgi-to-ER retrograde trafficking is inhibited by the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) but not the slower chelator of comparable Ca2+ affinity, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)4. In addition to illustrating a role for calcium in constitutive secretion, that study implied tight spatial coupling of the vesicular fusion machinery and Ca2+, wherein the putative fusion site is close to the Ca2+ release site. Since then, further evidence continued to mount and various groups have implicated Ca2+ in the fusion of yeast vacuoles5, the heterotypic fusion of late endosomes with lysosomes, or the reformation of lysosomes from hybrid compartments6.Intracellular Capathways . Interes2+, focusing mainly on studies after 2007, when we published a review on the same topic7. We discuss a variety of secretory effectors, organized by the organelle wherein the primary secretory effects spatially occur, that modulate secretion via their capacity to bind Ca2+. In addition to these Ca2+ effectors, what has been most obvious in recent years is the rise in the number of calcium pumps or channels that have been found to affect constitutive trafficking. Channels mobilize Ca2+ and create a dynamic Ca2+ landscape in the cytoplasm8 but still must act upstream of Ca2+ sensors and trafficking machinery effectors to influence trafficking. Pumps, on the other hand, tend to affect trafficking on the luminal side by providing sufficiently high Ca2+ environments for Ca2+-dependent sorting or retention processes. Prominent example studies from the text are briefly summarized in 2+ sensors that could translate a Ca2+ flux into changes in secretion. This may mean that the critical Ca2+-binding effectors or channel-binding partners have not been defined yet.In this review, we discuss multiple proteins that appear to regulate constitutive trafficking and have direct or indirect ties to Ca12 but not covered in this review except regarding Ca2+-specific observations. In addition, the ER acts as a principal Ca2+ store within cells facilitating Ca2+ signals in the cytosol or multiple organelles via ER-organelle contact sites15. In this section, we describe effects on secretion via ER-localized Ca2+ channels or their cytosolic effectors; finally, we note ways in which luminal calcium has been described to affect secretion.The ER folds and assembles newly synthesized proteins destined for trafficking via the secretory pathway, dysregulation of which leads to the unfolded protein response (UPR), reviewed elsewhere16. TRPP2, like all TRP family members, functions as a cation channel, principally directing the flow of Ca2+ into the cytosol. TRPP2 knockdown (KD) caused enhanced production of type II collagen in the zebrafish notochord sheath16, a trait reversed by expression of an ER-retained mutant of TRPP217. This implied that TRPP2 activity in the ER could directly or indirectly affect collagen translation or vesicle trafficking. Indeed, TRPP2 depletion led to an increase in the mRNAs of inner COPII coat components Sec23B, Sec24C, and Sec24D16, factors predicted to increase rates of ER-to-Golgi trafficking. Given that the increased coat expression could also be a compensation mechanism for loss of TRPP2, it is unclear whether TRPP2 depletion itself increased secretion. However, COPII coat protein KD or chemical inhibition of ER-to-Golgi transport via low-level brefeldin A (BFA) treatment was sufficient to reverse the enhanced collagen and aberrant morphology in the notochord sheath. This implies that the secretory pathway was an important target for manifesting the ECM abnormalities. Ultimately, in that study, a specific effector mechanism was not implicated nor were Ca2+ dynamics monitored, but previous work had shown that TRPP2 significantly decreased ER Ca2+18 and increased ER agonist-stimulated release of Ca2+19 or reduced it in the case of mutant TRPP220. These findings are reminiscent of a study21 focusing on a separate Ca2+ channel: inositol trisphosphate receptor-3 (IP3R-3). IP3Rs are broadly localized; they are present at the Golgi22), nucleus25, and the PM27 but are by far most abundant at the cells’ largest Ca2+ store: the ER28. Held et al. showed that IP3R-3 depletion increased spontaneous Ca2+ oscillations and produced an increase in ER-to-Golgi trafficking of a VSV-G transmembrane cargo21. It is possible that the two channels—IP3R and TRPP2—regulate trafficking via the same mechanism, possibly mediated in part by the demonstrated ability of TRPP2 to bind to ER IP3R and potentiate its activity19.Originally studied for its role in polycystic kidney disease, TRPP2 was suggested to have a role in altered extracellular matrix (ECM) integrity. TRPP2 localizes to the ER membrane, although it is also present at the PM and primary cilia2+ concentrations, mediated by changes in channel expression (as described above) or signaling events, likely affect secretion via direct binding of Ca2+ to effectors. At the ER, no effectors are more prominent than the cytosolic penta-EF-hand (PEF) proteins. Common features of this family include (1) five serial Ca2+-binding EF-hand motifs, (2) capacity to dimerize via EF-hand domains, and (3) Ca2+-dependent effector binding, often at membrane sites. Of this family, the most studied is apoptosis-linked gene-2 (ALG-2). ALG-2 has more than 20 Ca2+-dependent-binding partners and possesses several binding pockets for effectors, implying that one ALG-2 homodimer can bind two or more target proteins simultaneously in response to Ca2+-induced conformational changes29. Furthermore, in mammalian cells, ALG-2 is present in two splice forms that have a different spectrum of interaction partners30. Early research on ALG-2 in secretion indicated that it localizes principally with Sec31 at ER exit sites (ERES) in a manner concomitant with intracellular calcium oscillations33, suggesting a role for ALG-2 in affecting ERES function in response to a Ca2+ signal. Other early work reported that recombinant ALG-2 inhibited COPII vesicle budding and/or fusion in vitro35, implying an inhibitory role for ALG-2. Other cellular studies showed that recombinant ALG-2 bound to Sec31 was able to stabilize ERES36, and dominant-negative ALG-237 and depletion38 studies also indicated a positive regulatory role. Recently, our lab helped reconcile some of these apparent contradictions by demonstrating that ALG-2 can either stimulate or inhibit ER-to-Golgi transport in the same system39. This duality in the regulatory role of ALG-2 may come down to multiple partners it can bind under different circumstances. In this review, we will discuss multiple ALG-2 protein complexes that can or do affect secretion.Changes in local Ca2+ oscillations, ALG-2 binds to the outer COPII coat subunit Sec3132, where it colocalizes with another PEF protein: peflin40. Likely, the colocalized peflin and ALG-2 at ERES are comprised of the earlier reported 1:1 heterodimer, observed in vitro to dissociate in response to Ca2+41. Interestingly, depletion of peflin in NRK cells produced increased ALG-2 at ERES38 and an increase in ER-to-Golgi trafficking of multiple COPII cargoes39, a situation reminiscent of ALG-2-binding to ERES in response to intracellular Ca2+ oscillations33, indicating a straightforward positive and negative role for ALG-2 and peflin at ERES, respectively. Furthermore, double KD of ALG-2 and peflin did not decrease transport below control levels, indicating that neither is actually required for secretion and both are purely regulatory in this context. Importantly, this result is in partial contrast to that of another study. McGourty et al. showed that the ubiquitin ligase CUL3 and its adaptor KLHL12 required both peflin and ALG-2 together for recognition and ubiquitination of Sec31 and further that both PEF proteins were positively required for collagen I secretion40. In that study, treatment of cells with histamine or ionomycin caused enlargement of COPII membrane sites or vesicles in an ALG-2- and peflin-dependent manner, suggesting activation of collagen export in response to surges in cytoplasmic Ca2+. The reason for the opposite effect of peflin depletion in Sargeant et al.39 and McGourty et al.40 is not known.In the basal state, in the absence of significant cytosolic Ca2+- and lipid-binding proteins in trafficking is long and complex; a 2017 review discussed the potential involvement of annexins in a variety of plant membrane trafficking steps42. Briefly, there is very strong evidence that annexin A2 is involved in regulated exocytosis of chromaffin granules43 as well as several annexins being involved in Ca2+-dependent PM repair45, but the role of Anxa11 in ER-to-Golgi transport seems to be a unique example of an annexin playing a regulatory role at a constitutive trafficking step. At ERES, Anxa11, like ALG-2, has a role in stabilizing Sec31A46. There, Anxa11 likely anchors the ALG-2-Sec31A complex directly to the phospholipid bilayer, stabilizing the Sec31A interaction with ALG-2 rather than competing with it. Additionally, studies testing specific mutants that disrupt ALG-2-Anxa11-binding but not Sec31A-binding suggest that one ALG-2 molecule can bind Sec31A and Anxa11 at the same time, potentially through different binding sites. If this is true, then Anxa11 would not exactly be an additional ALG-2 target but rather an ALG-2 “co-effector” of Ca2+ since Anxa11 is itself a Ca2+-binding protein whose interactions with phospholipids could be independently modulated by Ca2+, separately from the Ca2+ modulation of ALG-2-binding to Anxa11 and Sec31A. This dual Ca2+-sensing unit could create complex functional responses, as has been observed in the Ca2+-dependent changes of ALG-2 in ER-to-Golgi transport. Also in support of more complex functions of ALG-2, ALG-2 has been reported to bind trk-fused gene (TFG) Ca2+-dependently at ERES in a Sec31A-independent manner47. GFP-MAPK1-interacting and spindle-stabilizing like (GFP-MISSL) also colocalized with ALG-2 in response to increased Ca2+48. The effector complex responsible for this targeting phenomenon appeared to be distinct from the Sec31A-ALG-2-Anxa11 or ALG-2-TFG complexes and was comprised of ALG-2 bound to both MISSL and MAP1B (microtubule-associated protein 1B) but not Sec31A. MISSL itself appeared to have a positive role in secretion as its KD attenuated secretion of SEAP , possibly via sequestration of MAP1B, KD of which reversed the secretion defect. Furthermore, since the MISSL-ALG-2-MAP1B effector complex does not contain a member of the transport machinery, its effects on transport could be indirect . Collectively, these studies indicate that ALG-2 has the capacity to serve multiple roles in secretion through binding to an array of target proteins.Other binding partners for ALG-2—for example, annexin A11 (Anxa11)—continue to support a regulatory role for ALG-2. The role of the annexin family of Ca39. In NRK cells, exposure to histamine or ATP for up to 2.5 hours caused an ALG-2-dependent depression of ER-to-Golgi trafficking, but in neuroendocrine PC12 cells, the same treatment caused ALG-2-dependent enhancement of transport. Another recent report found a stimulatory effect of ER Ca2+ release in the ER-to-Golgi transport of a bulk flow cargo in HeLa cells49. We noted that the important distinction between a stimulatory or inhibitory effect of Ca2+ is not the cell type per se but the pattern and longevity of the Ca2+ signal produced, since by adjusting the pattern of signaling using different protocols, it was found that NRK cells could either down-regulate or up-regulate ER export in response to Ca2+ signaling in an ALG-2-dependent manner39. Mechanistically, the Ca2+-dependent decrease of ER export co-occurred with less COPII outer shell and more peflin at ERES but the opposite was true for the Ca2+-dependent enhancement of ER export. We proposed that different Ca2+ signals were able to elicit different effects via ALG-2, likely via other ALG-2- binding partners that may bind in response to different intensities and durations of Ca2+ signals. The ability of ALG-2 to influence Sec31A targeting may relate to the earlier observation that ALG-2 binding to the Sec31 subunit of the outer coat heterodimer Sec13/31 seems to dramatically potentiate its interaction with the inner coat heterodimer sec23/2435; the inner-outer coat interaction is thought to be critical for cargo sorting and vesicle formation.Recently, our lab showed that distinct calcium signals and patterns can cause opposite effects on ER export in an ALG-2-dependent manner2+ —and also relates to ALG-2. This work examined the effect of Ca2+ dysregulation on the O-GlcNAcylation and membrane targeting of Sec31A50. O-GlcNAc transferase (OGT) is an enzyme responsible for this newly appreciated cytosolic post-translational modification. OGT was found in a previous article to interact directly with Sec31A and mediate O-GlcNAcylation at position S96451. In the more recent article, extremely high Ca2+ for 24 hours (brought about with A23187 or A-beta peptide) caused disruption of Sec31A targeting to membranes whereas 24-hour exposure to EGTA caused enhanced Sec31A targeting. The low-Ca2+, Sec31A targeting-promoting condition was accompanied by increased O-GlcNACylation of Sec31A and less ALG-2 association whereas the high-Ca2+, Sec31A targeting-inhibiting condition was accompanied by decreased O-GlcNACylation and increased ALG-2 association. An S694A mutation of Sec31A that cannot be O-GlcNACylated was not subject to the targeting regulation by Ca2+ flux, suggesting that O-GlcNACylation is a powerful determinant of Sec31A targeting. One interesting speculation is that since the ALG-2 binding site on Sec31A (residues 839–85136) is relatively close to S694, ALG-2-binding and OGT-binding could be mutually exclusive, in which case Ca2+ could regulate Sec31A O-GlcNACylation through Ca2+-dependent ALG-2-binding.A completely different line of research has also focused on modification of the COPII subunit Sec31A in response to Ca2+ in the preceding examples is presumed to be the cytoplasm, where released Ca2+ regulates the trafficking machinery, such as vesicle coats. However, Ca2+ inside the lumen of organelles can also affect trafficking by altering the function of receptors or other vesicle components that influence whether luminal cargo is recruited to a budding vesicle. Substantial depletion of ER Ca2+ has been associated with the abnormal secretion of ER-resident proteins normally retained in the ER54. This abnormal ER exit has been demonstrated to affect proteins containing a C-terminal KDEL retrieval sequence, and their exit can be suppressed by over-expression (OE) of KDEL receptors55. Although the mechanism of retrieval via the KDEL receptor is well established, it is not known how luminal Ca2+ contributes to the retention of ER proteins, a phenomenon first noted for the ER chaperone calreticulin in 199456. Several putative mechanisms have been proposed to explain this departure, including (i) proteins with KDEL sequences such as ER chaperones BiP or GRP94 bind Ca2+ with low affinity and interact with both each other and misfolded proteins, possibly forming a kind of retention matrix. A decrease in Ca2+ could reduce their interactions and cause them to be secreted. Or (ii) the ER environment may exist as a dynamic hydrogel because of a high concentration of Ca2+ and Ca2+-interacting proteins, and the hydrogel properties may limit access of resident proteins to ERES. A decrease in the Ca2+ concentration could thereby decrease the hydrogel viscosity, allowing ER resident proteins to escape and overwhelm the KDEL retrieval pathway. The precise mechanism for Ca2+-dependent ER protein retention remains to be explored and will have medical significance to the many diseases whose pathologies involve severe disruption of homeostasis 57.The locus of action of Ca2+. ERGIC-53 Ca2+ dependently binds soluble glycoprotein cargoes in the ER lumen59. Example cargoes include cathepsins C and Z60, α1-antitrypsin61, and serotonin neuroreceptors62. In addition, ERGIC-53 forms a 1:1 Ca2+-dependent complex with a small Ca2+-binding protein MCFD2, both of which are necessary for the trafficking of coagulation factor V (FV) and factor VIII (FVIII)63. Mutations in ERGIC-53 or MCFD2 induce the autosomal recessive disorder combined deficiency of factor V and factor VIII (F5F8D)64. Previously, ERGIC-53 bound to cargo was thought to assemble in the ER lumen before dissociating in the ERGIC/Golgi in a manner dependent upon Ca2+ release facilitated by decreasing pH between organelle compartments65. However, a more recent study found that the carbohydrate-binding domain (CRD) of ERGIC-53 bound to Ca2+ was insensitive to the changes in pH predicted to occur between the ER and Golgi66. Importantly, that study also showed that CRD-ERGIC-53 had very little binding to a glycan-like moiety below 0.4 mM CaCl2. It remains unresolved whether differences in pH between the ER and Golgi, or differences in Ca2+ concentration, play a more significant role in regulating ERGIC-53 cargo loading and release.The cargo receptor ERGIC-53  is another protein modulated by luminal Ca2+-binding protein from the CREC  protein family. In striated muscle, CALU expression changes are coupled to Ca2+ cycling likely via Ca2+-dependent interaction with the SERCA pump or ryanodine receptors67, whereas CALU in thrombocytes may act as a cargo receptor for thrombospondin-1 (TSP1)—an ECM adhesive glycoprotein. In this system, CALU and TSP1 form a Ca2+-dependent complex and are released together in response to thrombin68. Whether CALU regulates cargo secretion in the same cells in which it regulates ER Ca2+ entry/exit remains to be seen. If such a role were observed, this could be an interesting example of a protein that regulates both the Ca2+ environment necessary for secretion and the actual secretion event itself.Other ER luminal proteins also act as cargo receptors. Calumenin (CALU) is a CaHighlights of this section are summarized in 2+ and is similarly endowed with Ca2+ pumps, Ca2+ release channels, and Ca2+-binding proteins69. The Ca2+ concentration at the Golgi diminishes from cis to trans (300 –> 150 μM), reaching the lowest concentration at the trans-Golgi network (TGN) (50–100 μM)9  and ryanodine receptors71. Similarly, three Golgi proteins considered Ca2+ regulators, two of which have direct roles in secretion (discussed below), are localized to distinct Golgi compartments: nucleobindin-1 in the cis-Golgi74, DNA-binding EF-hand acidic amino acid-rich non-glycosylated Ca2+-binding protein (NEFA/p54) in the medial-Golgi75, and Cab45 in the trans-Golgi and TGN77. It is possible that just as ERGIC-53 may use Ca2+ to regulate cargo loading and release, distinctive Ca2+ levels in the Golgi could aid cargo progression . In this section, we define specific instances of Ca2+ regulating secretion via its flux near the cytosolic surface or within luminal Golgi compartments.The Golgi apparatus plays a central role in the trafficking and sorting of secretory cargo. Like the ER, the Golgi holds a vast reservoir of Ca2+ channels and Ca2+ effectors in regulating trafficking. To begin, we discuss Sly41, an SLC-family solute transporter that constitutively cycles between the ER and Golgi in Saccharomyces cerevisiae. Although the substrate for the transporter remains unknown, genetic interactions with the PMR1 Golgi Ca2+ pump led to the discovery that Sly41 OE generated elevated cytosolic Ca2+78. This was an important trafficking discovery since Sly41 was originally identified in a screen for genes whose OE suppressed COPII-dependent membrane tethering deficiencies . Mechanistically, this meant that raising the cytosolic Ca2+ concentration through Sly41 OE compensated for the loss of an ER-to-Golgi vesicle tethering factor  that is essential for intracellular transport pathways and efficient SNARE assembly79. These findings, on their own, suggest that Ca2+ promotes membrane fusion between COPII vesicles and the Golgi. Curiously, although the addition of Ca2+ in the absence of maximal vesicle tethering factors did indeed stimulate membrane fusion, it did nothing when saturating levels of vesicle tethering factors were present. A Ca2+ effector protein such as ALG-2 or calmodulin (CaM) that, for example, interacts with SNAREs or tethers and mediates the stimulation of transport was not identified. However, some Ca2+ effects on membrane fusion could instead be due to the fusion-promoting properties of Ca2+ itself without the requirement for a Ca2+ effector (to be discussed below).Many studies have implicated Golgi Ca2+ concentrations on Golgi structure80. The authors exposed HeLa cells to the Ca2+ agonist histamine or the SERCA inhibitor thapsigargin for 0.5 to 2 hours. They noted morphological fragmentation of Golgi stacks via a mechanism that involved PKCα-mediated phosphorylation of Golgi reassembly stacking protein 55 (GRASP-55). The fragmentation response appeared to be independent of ER stress and UPR activation. Satisfyingly, in this example, we have both an established Ca2+ effector (PKCα) and the trafficking machinery impacted (GRASP-55). However, the trafficking purpose for the effect is less clear. The authors suggested that it was part of a Golgi stress adaptive mechanism or signaling pathway. Cargo trafficking effects through the Golgi were regarded as mild; however, there was measurable acceleration that could have contributed to increased secretion. It would be interesting to know whether the PKCα-mediated response to Ca2+ signaling in the Golgi works in concert with changes in ER export mediated by ALG-2 that were discussed above.A recent study investigated the effect of high cytosolic Ca81. Human TRPC3 and 7 are localized on the PM but also are found in intracellular pools at the TGN and Golgi stack82. Furthermore, OE of either of these channels was found to increase constitutive secretion of SEAP by two- to four-fold82. The precise locus of the secretion effects observed in that study was not elucidated, and it cannot be eliminated that the over-expressed channels mobilized ER Ca2+ in addition to Golgi Ca2+ and that ER Ca2+ was the functional site, possibly via the effects of ALG-2 on COPII targeting, as discussed above. However, it is tempting to speculate that what occurred was local stimulation of intra-Golgi cargo transport, a process conventionally thought to be facilitated, through the process of cisternal maturation, by COPI-coated vesicles . Interestingly, two groups have presented evidence that COPI targeting to membranes, like COPII targeting, is Ca2+-dependent83 but that ALG-2 did not affect COPI targeting34. It would be very exciting if a separate Ca2+ effector regulated COPI; furthermore, this putative effector could be the key to understanding TRPC effects on secretion.As with the ER possessing TRPP2, the Golgi also possesses resident TRP channels that appear to affect secretion. The transient receptor potential canonical (TRPC) sub-family of TRP channels are non-selective cation channels that are known to open in response to phospholipase C activation, although their precise ligands  vary2+ effector is the cytosolic phospholipase A2 alpha (cPLA2α). cPLA2α is a member of the superfamily of phospholipase A2 enzymes (PLA2) reviewed extensively elsewhere84. In general, PLA2 hydrolyze the fatty acid from the sn-2 position of membrane phospholipids creating a lysophospholipid and a free fatty acid. An inverted-cone-shaped lipid, the lysophospholipid favors positive curvature of lipid membranes and thereby promotes membrane tubule formation. Tubule-mediated (as opposed to vesicle-mediated) trafficking is gaining recognition for its contributions to intra-Golgi transport as well as Golgi-to-PM transport of certain cargoes85. Three specific PLA2 enzymes have been implicated in tubule regulation at the Golgi87, including cPLA2α88. Whereas cPLA2α is Ca2+ activated, the other two Golgi-associated PLAs are not but could instead be activated by G protein βγ complexes89. Notably, cPLA2α is specifically recruited to the Golgi complex, likely via its C2 domain90, in response to transient increases in Ca2+ apparently initiated by the arrival of secretory cargo88. There it was required for intra-Golgi transport of VSV-G through the Golgi, apparently via induction of small inter-stack tubules. In the study by San Pietro et al., depletion of cPLA2α specifically inhibited transport through the Golgi and did not impair ER-to-Golgi transport of VSV-G or delivery of VSV-G from the TGN to the PM or retrograde Golgi-to-ER transport of the KDEL receptor88. Another article showed that cPLA2α contributes to the delivery of transmembrane proteins to junction complexes in endothelial cells91. This point was demonstrated by noting that the inhibition or RNA interference-mediated KD of cPLA2α prevented delivery of VE-cadherin, occludin, and claudin-5 to cell-to-cell contacts. Instead, these proteins accumulated in the Golgi, indicating that either transport through the Golgi or transport from it was inhibited91. The evidence for cPLA2α in the regulation of intra-Golgi transport is thus quite strong, but the Ca2+ signaling that activates cPLA2α on the Golgi is less clear. One report suggested that Ca2+ is released from the Golgi itself92 when a bolus of secretory cargo enters the Golgi, although specific channels or pumps and their mode of activation were not identified.A prominent Golgi Ca2+-ATPase (SPCA1) is a primarily trans-Golgi-localized Ca2+ ATPase70, which, along with the ER SERCA pump, is responsible for the maintenance of Golgi Ca2+ levels. Mutations in SPCA1 (gene name: ATP2C1) usually manifest as a decrease in expression and elicit the autosomal dominant skin disorder Hailey-Hailey disease93. As we will see in the following examples, the role of SPCA1 in secretion seems to be mainly in providing the lumen of the Golgi and associated organelles with high Ca2+ that is used in sorting and processing of secretory cargoes en route to their final destinations. The role of SPCA1 in trafficking was first demonstrated in yeast, where its medial Golgi-localized ortholog PMR1 transports both Ca2+ and Mn2+ into secretory compartments94. In that early study, pmr1 mutants are both unable to sort carboxypeptidase properly to the vacuole and unable to degrade misfolded carboxypeptidase, a luminal ER protein—a trait that was later found to be reversible via addition of external calcium95. This suggested that low luminal calcium in secretory organelles is the primary cause of these sorting defects. Further studies reinforce this idea. For example, SPCA1 inhibition also produced a decrease in insulin-like growth factor receptor (IGF1R) at the cell surface, a phenotype caused by defective proteolytic processing and accumulation of previously undetectable TGN-localized pro-IGF1R96. One putative effector for the sorting role of SPCA1 in secretion arose later when it was discovered that the luminal, secreted Ca2+-binding protein Cab45, of the CREC family, preferentially accumulates near SPCA1, where it oligomerizes with secretory cargoes in response to the presumably high local concentration of Ca2+97. Cab45 does not play a general role in secretion but rather binds a few select secretory cargoes, including lysozyme C and cartilage oligomeric matrix protein (COMP), and presumably assists their secretion by forming condensates98. Curiously, Cab45 is secreted along with its client cargoes. This could imply that Cab45 has additional functions in the extracellular environment.Secretory pathway Ca2+ effector protein. NUCB1 is widely distributed within the cell. The protein contains both an ER signal sequence and an ER export signal and displays prominent localization to the cis Golgi74 prior to being constitutively secreted99. Surprisingly, NUCB1 is also present in the cytoplasm100. In the cytoplasm, it has been suggested to, among other things, undergo Ca2+-dependent interactions with G⍺i subunits on Golgi membranes102. Importantly, cytosolic NUCB1 acts as a regulator of endosomal recycling of lysosomal receptors such as the mannose-6-phosphate receptor and sortilin, which capture cargo at the TGN and ferry it to late endosomes for ultimate deposition in the lysosome103. In these studies, NUCB1 appeared to act in the cytosol and as a regulator of rab7 activation to bring about recruitment of the retrograde coat, retromer, that mediates return of the lysosomal receptors to the TGN. The role of Ca2+ in NUCB1 function here was not investigated.Nucleobindin-1  appears to be a multi-functional soluble EF-hand-containing Ca105. In the Golgi, NUCB1 may function as a regulator of Golgi Ca2+ homeostasis since its OE increased Ca2+ uptake into the Golgi73 while NUCB1 KO induced a loss in Golgi luminal Ca2+106. Furthermore, NUCB1 KO and NUCB1 with mutant EF hands produced a delay in the trafficking of ECM constituents MMP2 and MT1-MMP at the cis-to-trans Golgi stage. Again, like Cab45, NUCB1 appeared to regulate the trafficking of only a subset of cargoes; for example, NUCB1 KO had no effect on the trafficking of lysozyme C106. This trafficking defect was a delay and not a block, possibly due to compensatory mechanisms mediated by other Ca2+-binding proteins. The trafficking function of NUCB1 was presumed to be mediated by direct interactions with the cargo, but a more detailed mechanism of how NUC1B facilitates anterograde transport while remaining in the cis-Golgi has not yet been established. Finally, NUCB1 is reported to, after a long period in the Golgi, be constitutively secreted into the extracellular space99, where it may have signaling functions via its capacity to bind Ca2+, such as in bone matrix maturation107.In regard to NUCB1 that is targeted to the luminal cellular domain, some reports have suggested a chaperone-like activity due to its ability to bind Alzheimer’s amyloid precursor protein (APP) and assist in its folding and biogenesisHighlights of this section are summarized in 2+, reaching concentrations that approach that of the ER lumen108. We have known for quite some time that LEL trafficking requires luminal Ca2+ and that CaM is an important Ca2+ effector for this109. Resident in the lysosome is the TRPML1 channel that is critical in Ca2+-dependent lysosome trafficking; lack of TRPML1 activity causes the genetic disorder mucolipidosis and enlarged vacuole-like lysosomes110. TRPML1 belongs to the mucolipin subgroup of the TRP ion channel family whose members we have discussed in the sections on trafficking in the ER and the Golgi. Like other TRP channels, TRPML1 acts as a non-selective ion channel permeable to Ca2+. Some work has indicated that TRPML1 is an activator of lysosome-MVB fusion. In this context, TRPML1 was suggested to regulate exosome release since this process is inhibited when lysosomes fuse with MVBs, the source of exosomes112. Other work, however, has implicated TRPML1 in LEL fission113. TRPML1 has been shown to be regulated by pH108 and activated by the rare LEL phospholipid PIP2. Deficiency of either TRPML1 or PIP2 produced enlarged LEL, while OE of TRMPL1 both attenuated the defect observed with PIP2 deficiency and enhanced vacuolar calcium release114. This local increase in juxta-organellar calcium could serve to recruit cytosolic complexes necessary for membrane fusion and fission. In fact, CaM was implicated as the specific Ca2+ effector for the role of TRPML1 in LEL fission113, and the mammalian target of rapamycin 1 (mTORC1) served as a required downstream target115. ALG-2 (discussed above in the ER section) has also been implicated as an effector of Ca2+ released by TRPML1 since it binds to TRPML1 in a Ca2+-dependent manner116. Furthermore, activation of TRPML1 via PIP2 caused ALG-2 to Ca2+-dependently couple dynein-dynactin with the N-terminus of TRPML110. This ALG-2-mediated adaptor system ultimately permitted migration of lysosomes to the perinuclear region in response to Ca2+ signals. Distinct from the proposed role of CaM in LEL fission, this dynein-mediated trafficking implicates Ca2+ and ALG-2 in autolysosome formation, which occurs when the migrated lysosomes fuse with autophagosomes.The late endosomes/lysosomes (LELs) are central organelles responsible for macromolecule recycling. Importantly, they are also intracellular stores for Ca2+ release channel activated by luminal ATP in a pH-dependent manner. In a 2015 article, it was shown that P2X4 promotes LEL fusion in a cell-free assay and that this effect was blocked by various inhibitions of P2X4-mediated Ca2+ release117. Although inhibition of P2X4 activity by the low pH of LEL was overcome pharmacologically in these experiments, it is unknown whether and how this inhibition is overcome in cells. That study also showed that P2X4 activation recruits CaM and that together they form a complex at the LEL membrane. Finally, the fusion effect of P2X4 was shown to be suppressed by inhibiting CaM117. The authors do not specifically identify the downstream targets for CaM in this instance, although it may be relevant that CaM interacts with LEL SNARE proteins in a Ca2+-dependent manner118. It is interesting to note that the same research group also implicated CaM as the effector for TRPML1-mediated LEL fission113, but how two distinct channels could mediate opposing trafficking phenomena (fusion vs. fission) via the same Ca2+ effector was not resolved.Also localized to LEL, purinergic receptor P2X4 is a CaHighlights of this section are summarized in 2+ concentrations an order of magnitude lower than agonist-stimulated release119, can exceed stimulated release over long periods120. A high-affinity Ca2+ sensor for baseline mucin secretion is proposed to be K+ channel-interacting protein 3 (KChIP3), a member of the neuronal Ca2+ sensor (NCS) family of EF-hand-containing proteins121. KChIP3 inhibits baseline exocytosis, which was shown by an increase in baseline mucin secretion during KChIP3 KD and a decrease in baseline secretion during KChIP3 OE. This effect was demonstrated to be entirely dependent on intracellular Ca2+ oscillations, as increased intracellular calcium oscillations abrogated the inhibitory effect of KChIP3 OE. Finally, this Ca2+-dependent control of KChIP3 activity was discovered to be dependent on the ryanodine receptors, activity of which caused KChIP3 to dissociate from mucin granules121. Although this process sounds more akin to regulated secretion, it was able to proceed without stimulation by external agonists and did not require synaptotagmin, thus blurring the distinction between the regulated and constitutive secretory pathways. In vivo, the Ca2+ oscillations may be driven by fluid flow over the epithelium, as goblet cells were found to increase Ca2+ oscillations and mucin secretion in response to cell perfusion120.Specialized epithelial goblet cells secrete gel-forming mucins, the first line of defense against pathogens or allergens. Following biogenesis at the Golgi, mucin granules undergo maturation and eventually release their contents at the PM. Although mucin granules undergo stimulated exocytosis in response to agonists such as ATP, it was discovered in 2015 that baseline secretion of mucins, which requires intracellular Ca2+ of up to 2 mM, equivalent to the extracellular Ca2+ concentration, whereas its free Ca2+ store is about 500 μM8. Meanwhile, the Golgi apparatus sits at an average of 250 μM free Ca2+8, although this number varies; Ca2+ steadily diminishes from the cis- to the trans-Golgi compartments122. Finally, the average free Ca2+ of lysosomes is comparable to the ER at about 500 μM10. Although so far we have discussed the influence of these stores primarily in the context of their capacity to regulate Ca2+-binding proteins that in turn regulate vesicle trafficking, here we discuss the possibility that Ca2+ directly modulates trafficking by influencing the structural and functional properties of membrane lipids. Indeed, experimental and theoretical work has shown that Ca2+ can quickly and concentration-dependently tighten and order lipid bilayers, primarily via coordination with anionic oxygens of acidic phospholipids such as phosphatidylinositol-4,5-bisphosphate [PIP2]124 and phosphatidylserine (PS)127. Together these studies suggested a role for cytosolic Ca2+ in promoting membrane fusion, possibly by masking the negative charge of anionic lipids and more readily allowing close association of membranes. Ca2+ concentrations on the order of 200 to 400 μM overcame DOPS (dioleoyl phosphatidylserine) inhibition of lipid or content mixing in prepared vesicles128. Importantly, this Ca2+ concentration is on the order one might expect for focal Ca2+ signals near release sites130. We note that, in a previously discussed study, elevations in cytosolic Ca2+ were observed to overcome a block in membrane tethering to allow fusion78. Since no Ca2+ effector was evident in that study, it could indicate that Ca2+ alone may have promoted membrane fusion in a physiological system.Secretory pathway organelles harbor significant stores of calcium. The ER is reported to be the highest with a total Ca2+ may have additional roles in vesicular trafficking. Studies have shown, for example, induction of membrane tubulation in lipid vesicles in response to local Ca2+ addition at concentrations above 1 mM131. The authors of that study note that the observed inward spontaneous curvature and membrane bending are likely due to a large, Ca2+-facilitated reduction in the surface charge density of one membrane leaflet causing stress asymmetry and ultimately bending of the membrane. A study using a giant unilamellar vesicle transfer assay demonstrated that Ca2+ can trigger deformation of membranes containing PS or PIP2 in a direction that points away from the ion source132, in a manner reminiscent of vesicle budding. It is important to note that the concentration of Ca2+ required to manifest the induced curvature of membranes appears to be on the order of a few hundred micromolar. Although this number falls well within the bounds of free luminal Ca2+ for secretory pathway organelles, it is highly unlikely that high levels of free Ca2+ alone could lead to a membrane budding event. Instead, it is much more plausible that luminal Ca2+ stores support induction of membrane curvature by working in concert with other proteins such as the annexins133. Finally, in support of a system wherein Ca2+ can facilitate membrane budding, we note that the membranes of organelles most enriched in the Ca2+-binding lipid PS are the luminal leaflets of the ER, Golgi, and mitochondria135, two of which are critical organelles for constitutive secretion.In addition to putative direct effects on fusion, Ca7, Ca2+ regulation of constitutive trafficking has expanded from examples employing mostly in vitro systems using Ca2+ addition or chelation into complex systems wherein specific Ca2+-related gene products, many implicated in human pathologies, fundamentally alter trafficking steps throughout secretory and LEL trafficking. Furthermore, whereas prior to this it was difficult to distinguish whether Ca2+ played only permissive, co-factor roles, now we know that Ca2+ signaling can play dynamic roles, altering trafficking rates or patterns in response to natural ligands. Furthermore, we note that Ca2+ signaling can have unexpectedly complex roles; for example, in the case of ER export, ALG-2, together with its multiple targets, can act to either restrict or expand entry of cargo into the secretory pathway, depending upon the intensity and duration of a Ca2+ signal. It is now clear that Ca2+ is a fundamental regulator of the cell’s trafficking toolkit but that each case is also mechanistically unique rather than following a universal pattern. Overall, constitutive trafficking is looking a lot more Ca2+-regulated and therefore less “constitutive” than once believed.Since our last review of this topic