On 2017 Mar 22, Paola Pizzo commented:

We thank the Authors for their Reply to our Letter. However, given the importance of this topic, we have to add an additional comment to further clarify some criticisms. First, the Authors reason that the average mitochondrial surface in contact with ER can be extracted from the data presented in table S1, S2, S3 of their paper (Naon D, 2016). However, these data have not the same relevance that presenting the total number of ER-mitochondria contacts in the two situations. Indeed, the percentage of mitochondrial surface in contact with ER substantially varies whenever the analysis is restricted only to mitochondria that display contacts with the ER, or includes also contact-deprived mitochondria. In their analysis, the Authors considered only those mitochondria that are engaged in the interaction with the ER (and not the total mitochondrial population), as they stated in the Results section (“we devised an ER-mitochondria contact coefficient (ERMICC) that computes [....] the perimeter of the mitochondria involved in the interaction”). This approach could be misleading: considering that in Mfn2-depleted cells a higher percentage of mitochondria endowed with contacts has been found (Filadi R, 2016), the fraction of contact-deprived mitochondria should be taken into account to calculate the real average OMM juxtaposition surface. Second, the Authors argue that the fluorescent organelle proximity probes they used (both ddGFP and FRET-based FEMP probe) do not artificially juxtapose organelles: we did not claim this in our Letter, and we apologize if, for space limitations, this was not clear enough. Nevertheless, as to the FEMP probe, its propensity to artificially force ER-mitochondria juxtaposition, already after few minutes from rapamycin treatment, has been clearly shown by EM analysis in the original paper describing this tool (Csordás G, 2010). Additionally, it is worth to mention that comparison of FRET values between different conditions is possible only when the dynamic range (i.e., the difference between minimal and maximal FRET values) of a given FRET probe is similar in these different conditions. The new data provided by the Authors in their Reply (Tables 1 and 2) show that the average rapamycin-induced FRETmaximal values are dramatically different between wt and Mfn2-depleted cells. Thus, we believe that at least some cautions should be adopted to claim the use of this probe as a reliable tool for the comparison of ER-mitochondria tethering in such different conditions. Recently, we suggested how the fragmented/altered mitochondrial and ER morphology, present in Mfn2-depleted cells, may impair the rapamycin-induced assembly of this probe (Filadi R, 2017), thus severely complicating the interpretation of any result. Regarding the other fluorescent probe (the ddGFP) used by Naon et al. (Naon D, 2016), it is unclear why only ~10 % of the transfected wt/control cells (mt-RFP positive cells; Fig. 1G and 2E) are positive for the ddGFP signal (claimed as organelles tethering indicator). Should not ER-mitochondria juxtaposition be a feature of every cell? Concerning the Ca²⁺ experiments, we are forced to discuss additional criticisms present in the Authors’ Reply. Our observation that, in Naon et al. (Naon D, 2016), mitochondrial Ca²⁺ peaks in control cells (mt-YFP traces), presented in Fig. 3F, are ~ 100-fold higher than those in Fig. 3B was rejected by the Authors, because in Fig. 3F “Mfn2^flx/flx cells were preincubated in Ca²⁺ -free media to equalize cytosolic Ca²⁺ peaks”. However, in our Letter, we clearly referred to control, mt-YFP expressing cells, and not to Cre-infected Mfn2^flx/flx cells. Nevertheless, even if a “preincubation in a Ca²⁺ -free media to equalize cytosolic Ca²⁺ peaks” (i.e., a treatment that decreases the ER Ca²⁺ content) was applied to both cell types, the prediction is that, in Fig. 3F, the ATP-induced mitochondrial Ca²⁺ peaks would be lower for both Mfn2^flx/flx and control (mt-YFP) cells, and not higher than those presented in Fig. 3B. Lastly, as members of a lab where mitochondrial Ca²⁺ homeostasis has been studied over the last three decades, we have here to point out that, in our opinion, the reported values for ATP-induced mitochondrial [Ca²⁺ ] peaks (i.e., 160 nM and 390 nM in Fig. 3B and 3C, respectively) are unusually very low and hardly considerable to be over the basal mitochondrial matrix [Ca²⁺ ] (~ 100 nM). Furthermore, these low [Ca²⁺ ] values cannot be reliably measured by the mitochondrial aequorin probe (Brini M, 2008) used by Naon et al. (Naon D, 2016). Finally, concerning the speed of Ca²⁺ accumulation in isolated mitochondria, we clearly stated in our Letter that at 50 uM CaCl2 in the medium and no Mg^2+, the rate of Ca²⁺ accumulation is limited by the activity of the respiratory chain (Heaton GM, 1976), and thus does not offer any information on the MCU content. We did not refer to problems in respiratory chain activity in Mfn2-depleted cells, as interpreted by Naon et al. in their Reply.<br> Overall, while we appreciate the attempt of the Authors to highlight some aspects of the controversy, we renew all the concerns we discussed in our Letter.

On 2017 Mar 22, Paola Pizzo commented:

We thank the Authors for their Reply to our Letter. However, given the importance of this topic, we have to add an additional comment to further clarify some criticisms. First, the Authors reason that the average mitochondrial surface in contact with ER can be extracted from the data presented in table S1, S2, S3 of their paper (Naon D, 2016). However, these data have not the same relevance that presenting the total number of ER-mitochondria contacts in the two situations. Indeed, the percentage of mitochondrial surface in contact with ER substantially varies whenever the analysis is restricted only to mitochondria that display contacts with the ER, or includes also contact-deprived mitochondria. In their analysis, the Authors considered only those mitochondria that are engaged in the interaction with the ER (and not the total mitochondrial population), as they stated in the Results section (“we devised an ER-mitochondria contact coefficient (ERMICC) that computes [....] the perimeter of the mitochondria involved in the interaction”). This approach could be misleading: considering that in Mfn2-depleted cells a higher percentage of mitochondria endowed with contacts has been found (Filadi R, 2016), the fraction of contact-deprived mitochondria should be taken into account to calculate the real average OMM juxtaposition surface. Second, the Authors argue that the fluorescent organelle proximity probes they used (both ddGFP and FRET-based FEMP probe) do not artificially juxtapose organelles: we did not claim this in our Letter, and we apologize if, for space limitations, this was not clear enough. Nevertheless, as to the FEMP probe, its propensity to artificially force ER-mitochondria juxtaposition, already after few minutes from rapamycin treatment, has been clearly shown by EM analysis in the original paper describing this tool (Csordás G, 2010). Additionally, it is worth to mention that comparison of FRET values between different conditions is possible only when the dynamic range (i.e., the difference between minimal and maximal FRET values) of a given FRET probe is similar in these different conditions. The new data provided by the Authors in their Reply (Tables 1 and 2) show that the average rapamycin-induced FRETmaximal values are dramatically different between wt and Mfn2-depleted cells. Thus, we believe that at least some cautions should be adopted to claim the use of this probe as a reliable tool for the comparison of ER-mitochondria tethering in such different conditions. Recently, we suggested how the fragmented/altered mitochondrial and ER morphology, present in Mfn2-depleted cells, may impair the rapamycin-induced assembly of this probe (Filadi R, 2017), thus severely complicating the interpretation of any result. Regarding the other fluorescent probe (the ddGFP) used by Naon et al. (Naon D, 2016), it is unclear why only ~10 % of the transfected wt/control cells (mt-RFP positive cells; Fig. 1G and 2E) are positive for the ddGFP signal (claimed as organelles tethering indicator). Should not ER-mitochondria juxtaposition be a feature of every cell? Concerning the Ca²⁺ experiments, we are forced to discuss additional criticisms present in the Authors’ Reply. Our observation that, in Naon et al. (Naon D, 2016), mitochondrial Ca²⁺ peaks in control cells (mt-YFP traces), presented in Fig. 3F, are ~ 100-fold higher than those in Fig. 3B was rejected by the Authors, because in Fig. 3F “Mfn2^flx/flx cells were preincubated in Ca²⁺ -free media to equalize cytosolic Ca²⁺ peaks”. However, in our Letter, we clearly referred to control, mt-YFP expressing cells, and not to Cre-infected Mfn2^flx/flx cells. Nevertheless, even if a “preincubation in a Ca²⁺ -free media to equalize cytosolic Ca²⁺ peaks” (i.e., a treatment that decreases the ER Ca²⁺ content) was applied to both cell types, the prediction is that, in Fig. 3F, the ATP-induced mitochondrial Ca²⁺ peaks would be lower for both Mfn2^flx/flx and control (mt-YFP) cells, and not higher than those presented in Fig. 3B. Lastly, as members of a lab where mitochondrial Ca²⁺ homeostasis has been studied over the last three decades, we have here to point out that, in our opinion, the reported values for ATP-induced mitochondrial [Ca²⁺ ] peaks (i.e., 160 nM and 390 nM in Fig. 3B and 3C, respectively) are unusually very low and hardly considerable to be over the basal mitochondrial matrix [Ca²⁺ ] (~ 100 nM). Furthermore, these low [Ca²⁺ ] values cannot be reliably measured by the mitochondrial aequorin probe (Brini M, 2008) used by Naon et al. (Naon D, 2016). Finally, concerning the speed of Ca²⁺ accumulation in isolated mitochondria, we clearly stated in our Letter that at 50 uM CaCl2 in the medium and no Mg^2+, the rate of Ca²⁺ accumulation is limited by the activity of the respiratory chain (Heaton GM, 1976), and thus does not offer any information on the MCU content. We did not refer to problems in respiratory chain activity in Mfn2-depleted cells, as interpreted by Naon et al. in their Reply.<br> Overall, while we appreciate the attempt of the Authors to highlight some aspects of the controversy, we renew all the concerns we discussed in our Letter.