Functional Ca2+ Channels between Channel Clusters are Necessary for the Propagation of IP3R-Mediated Ca2+ Waves
Round 1
Reviewer 1 Report
This manuscript undertakes a computational analysis of the CICR via IP3 receptors. They utilize prior experimental results to model the calcium waves, taking advantage of two dyes with different kinetics. The experimental setup is clever and the analysis is thoughtful and clearly describes the required assumptions. Overall, the authors conclude that the propagation of the wave in their model system most likely requires the presence of single IP3Rs interspersed between the clusters of receptors. While this is not a definitive conclusion (in part due to the model system chosen and the required assumptions) it is an intriguing and important result.
Major comments:
1. The authors run their computations using two different values of d and V, however little else is done to determine how sensitive the model is to changes in key parameters. It would be useful to include some sensitivity analysis to determine how changing key parameters affect the model output. This is particularly important for d. The authors should give some indication of the optimal value for this parameter based on their computation.
2. Do oocytes have any endogenous sources of calcium release other than IP3Rs (such as RyR) which might contribute to the observed calcium?
Minor comments:
1. The authors should include a table listing the parameter values and definitions.
2. A cartoon of the model and proposed clustering might make reading the paper and understanding the assumptions easier.
Author Response
We included a sensitivity analysis to observe how changes the probability of opening one IP3R as a function ot the distance d from the calcium source (Fig. 3(a,b)). In these figures it can be observed that the probability decays to the basal value at the typical inter-cluster distance (1.4 um). At the half distance between clusters, 0.6-0.7 um, the clusters can remain coupled. As we are performing an approximation of the different terms in Eq. (8) we cannot estimate an optimal value of d quantitatively.
The immature oocyte of Xenopus laevis has long been a favoured cell preparation in which to study IP3Rs-mediated Ca2+ signalling (Berridge1993). It has the advantage that there are no other sources of calcium release such as ryanodine receptors which simplifies the analysis of Ca2+ signals (Parker1991, DeLisle1992).
We included two tables: one in the Material and Methods Section including the numerical simulations parameters (Table 2), and another in the Results Section that summarizes the parameters varied to compute P0 (Table 3).
The Figure 3 included in this new version better explains the assumpions of Ca2+ propagation.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The present study is an extension of previous published work by the authors. In previous work, they found the existence of inter-clusters IP3Rs that may contribute to the propagation of Ca2+ signals by experiment and simulations. The present work performaces a more quantative analysis on experiments and simulation of previous work, which provides further insight into the functional role of inter-clusters IP3Rs on the global propagation of Ca2+ signals.
Major Comments:
1. As their simulation, the non-cluster IP3Rs (0.6 um) is easier to be activated than IP3Rs in second cluster (1.4 um) by the CICR mechanism. However, in a ca2+ waves, the ca2+ signal needs to continue propagating to the second IP3R cluster. So the authors need to compare the Po of second clustered IP3Rs between these modes: (1)Ca2+ puff in 1st IP3Rs cluster --> 2nd IP3Rs cluster (1.4 um), and 2) Ca2+ puff in 1st IP3Rs cluster --> non-cluster IP3Rs (0.6 um)-->2nd IP3Rs cluster (1.4 um). Especially when the number of non-cluster IP3Rs is much fewer than that of clustered IP3Rs.
2. In their simulation, the authors use a simply linear relatinship between the release current and opening number of IP3Rs. However, considering the ER depletion during Ca2+ puff, the relationship between Nc (opening IP3Rs number) and Ca2+ release flux will be not linear. Will there be any affect on the results?
minor suggestions:
1)in line 309, "The emitted fluorescence was detected in the 500-600 nm and the 600-630 ranges, respectively, with PMT detectors." The Rhod-2 emission peak is ~570 nm, so the emitted fluorescences should be 500-550 nm and the 550-630 ranges.
Author Response
We included the comparison of the probability when an IP3R is added in between clusters with the case in which no IP3R is added (Figure 3(c)).
We included the comparison of the probability when an IP3R is added in between clusters with the case in which no IP3R is added (Figure 3(c)).
It is supposed that the ER depletion rapidly recovers (Lopez2016). However, if the relationship between nc and Ca2+ release is not linear it can be reduced to a problem with a smaller Ca2+ current and, hence, accompanies our results since it will be more difficult to have the first and the second clusters coupled.
Lopez, Lucía Fernanda; Ponce Dawson, Silvina Martha; Luminal Ca 2+ dynamics during IP 3 R mediated signals; IOP Publishing; Physical Biology; 13; 3; 5-2016; 036006,1-12
The Rhod-2 emission effectively is in the 500-550 nm and the 550-630 ranges but. As we simultaneously acquired the fluorescence coming from two dyes (Fluo-4 and Rhod-2) in two channels, we removed the Fluo-4 emission, (500-600)nm, in the Rhod-2 channel to avoid bleeding.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
My questions got clarified by the authors in the revised version.
