Peroxisomes contribute to intracellular calcium dynamics

  1. Yelena Sargsyan
  2. Uta Bickmeyer
  3. Katrin Streckfuss-Bömeke
  4. Ivan Bogeski
  5. Sven Thoms

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Abstract

Peroxisomes communicate with other cellular compartments by transfer of various metabolites. However, whether peroxisomes are sites for calcium handling and exchange has remained contentious. Here we generated sensors for assessment of peroxisomal calcium and applied them for single cell-based calcium imaging in HeLa cells and cardiomyocytes. We found that peroxisomes in HeLa cells take up calcium upon depletion of intracellular calcium stores and upon calcium influx across the plasma membrane. Further, we show that peroxisomes of neonatal rat cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes can take up calcium in a controlled manner. Our results indicate that peroxisomal and cytosolic calcium signals are tightly interconnected. Hence, peroxisomes may play an important role in shaping cellular calcium dynamics by serving as buffers or sources of intracellular calcium.

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  1. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    This study outlines calcium probes for assessing the poorly understood role of peroxisomes in calcium signaling. The authors suggest that these organelles sequester calcium from either calcium influx across the plasma membrane or from release from the ER/SR. This is important since we need to know more about the roles of these organelles in calcium homeostasis and signaling. However, it needs to be robustly demonstrated that the probes are targeted to the right organelle without confounding contamination from other organelles which can be very significant even for a small degree of mis-targeting.

    Major

    1. The difference between the signals seen between the peroxisome and cytosolic D3 versions are not compelling, other than a dampened spike with the former (higher resting levels, smaller peak). See below for pH concerns.
    2. How clean is the peroxisome distribution? Prove that D3 spillover from its being partially in (or on) other compartments (e.g. cyto, ER) is not contributing to the changes. Selective manipulation of Ca2+ in these other compartments should not affect the peroxisome signal.
      • a. For example, the small changes in the D3-px could be explained by peroxisome not changing at all but rather the other compartments (where larger responses are observed) signal(s) contaminating the response.
      • b. e.g. if in the ER lumen, the signal should be eliminated with SERCA inhibitors (thapsigargin, CPA). They used Thapsigargin in cardiac myocytes, why not in HeLa during characterization)?
    3. Any Ca2+ reporter will pH-sensitive to an extent, even D3 (Ca2+ binding, inherent fluorescent proteins).
      • a. It is essential to prove that the signal changes are not due changes perox pH. Target pH-sensitive proteins to the perox lumen by the same strategy and show that the same Ca2+ interventions do not cause pH changes.
      • b. The authors claim different resting levels of [Ca2+] in cytosol/mitochondria/peroxisome. The resting FRET level also depends on the resting pH of the compartments which may also be different. Certainly, mitochondria are more alkaline than the cytosol. Again, to interpret these are real Ca2+ differences requires the pH to be accounted for.
    4. I am puzzled by the model, in particular in view of Fig 3. The genetically-encoded calcium indicator (GECI) is allegedly in on the cytosolic face of the peroxisome and measuring peri-peroxisomal Ca2+.
      • a. The changes with this reporter look pretty similar to the luminal reporter (save that the resting ratio may be lower). I don't understand how the lumen [Ca2+] > cytosolic [Ca2+] without a higher local [Ca2+] (unless there is an energy-driven uptake mechanism, but then how does this fit in with ER-driven Ca2+ release?).
    5. The claim that resting peroxisome [Ca2+] is higher than cytosol is questionable. Is this a calibration artifact (e.g. compartment pH-differences or the reporter behaves differently in the lumen)? Such a gradient could not be sustained without energy-dependent Ca2+ uptake. The authors make no discussion of this.

    Minor

    1. Quantitate localization. Pearson's coefficients for GECIs and Peroxisomes.
    2. Different upstroke rates of D3 with His vs Cao. Quantify.
    3. Page 5. Line 161. 'Different sites', do the authors mean different sides? Similarly, the Legend of Fig 3.

    Significance

    Good peroxisome calcium probes is important to the genral calcium signaling field. This is fundamental science of interst to all cell biologists.

    There has been little published on peroxisome calcium, although for example, the Pozzan lab published a paper in JBC in 2008 on a GFP-based lumenally targeted peroxisome probe. There is contradictory data in the field and reliable new approaches are needed.

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Sargsyan et al describes an unappreciated role for peroxisomes in Calcium dynamics. Specifically, the authors propose that GPCR/VDCC/SOCE-mediated cytosolic Ca2+ elevation is rapidly sensed by peroxisomes and sequestered. The authors used/generated a peroxisome-targeted genetically encoded Ca2+ indicators which is elegant and powerful tool to monitor the luminal Ca2+ dynamics. While the results and conclusions are novel, there are some important gaps that need to be addressed for consideration for publication in EMBO J.

    Comments:

    Peroxisomes are single membrane bound organelles which are conserved across species spanning from yeast to humans. While housing only -100 proteins, they are responsible for essential steps in lipid metabolism, amino acid metabolism and ROS homeostasis. Unlike other organelles, peroxisomes import fully folded and cofactor-bound proteins into their matrix. Though peroxisomes house specific metabolic functions, there is extensive crosstalk with other organelles, including mitochondria. It is essential to test and define whether silencing/knockdown of mitochondrial Ca2+ transport components like MCU will impact peroxisome Ca2+ uptake upon stimulation with histamine or electrical stimulation.

    Since peroxisomes buffer significant amount of Ca2+, it is worth testing whether blockade of mitochondrial Ca2+ uptake would not alter peroxisome mediated Ca2+ influx. This analysis will provide Ca2+ uptake rate of mitochondria vs peroxisomes (mallilankaraman K. et al CELL 2012 and Nemani N. et al Science Signaling 2020).

    Peroxisomal synthesis of plasmalogens is Ca2+ and oxygen tension dependent, it is essential to show that altering Ca2+ controls plasmalogen synthesis.

    In the introduction authors have stated that "Elevated mitochondrial uptake increases 39 mitochondrial reactive oxygen species (ROS) production and is associated with heart falure and ischemic 40 brain injury (Starkov et al., 2004; Santulli et al., 2015)." These cited articles remotely links MCU and ROS elevation. It is important to point out that Tomar et al 2016 Cell Reports clearly demonstrated that genetic ablation of MCU suppresses mROS production that is mitochondrial Ca2+ dependent.

    Significance

    The significance of the work is very high. The authors employ a variety of complementary techniques and experimental systems to demonstrate that peroxisomes indeed buffer a large quantity of Ca2+ upon stimulation.

  3. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    These are straight forward studies aimed to develop probes to asses peroxisomal Ca2+ in rest and in response to receptor stimulation. The probes were designed to measure intraperoxisomal Ca2+ and the Ca2+ the peroxisome experience when cytoplasmic Ca2+ is increased. The pobes fill a need in understanding peroxisomal Ca2+ and Ca2+ signaling in general and should be very useful to investigators in the field.

    The comments are aimed to help in improving the studies and taking them to the next stage.

    The grammar needs improvement and the introduction needs sharpening. It is long and, in many places, not to the point. The results and discussion sections are also quite verbose.

    The sidedness of the probes need to be validated further, especially since the peroxisomal Ca2+ increase follows the cytoplasmic and the slower reduction rate may results from the environment experienced by the probe. Simple experiments: how the probes respond to Ca2+ ionophore; does Ca2+ reduced rapidly when removed from the media of the digitonin permeabilized cells; how the cytoplasmic and peroxisomal thapsigargin responses compare using the protocols in 2A and 4A? Sidedness of PEX13-D3cpV was not examined.

    Calculation of peroxisomal Ca2+ are based on Kd reported in the literature. The Kds of D3cpV-px and PEX13-D3cpV should be determined when in the peroxisome in permeabilized cells for the numbers to have any meaning.

    How the localization of the probes look in the differentiated cardiomyocytes? How it compares to RyRs, VACC, etc..

    The major weakness of the study is that the probes are used only as a tool. The enhance the study and bring it beyond an excellent technical achievement, the authors should use them to study a significant Ca2+-dependent peroxisomal function and show how the use of the tools eliminate the role of Ca2+ in such a function.

    Significance

    These are straight forward studies aimed to develop probes to asses peroxisomal Ca2+ in rest and in response to receptor stimulation. The probes were designed to measure intraperoxisomal Ca2+ and the Ca2+ the peroxisome experience when cytoplasmic Ca2+ is increased. The pobes fill a need in understanding peroxisomal Ca2+ and Ca2+ signaling in general and should be very useful to investigators in the field.

    The major weakness of the study is that the probes are used only as a tool. The enhance the study and bring it beyond an excellent technical achievement, the authors should use them to study a significant Ca2+-dependent peroxisomal function and show how the use of the tools eliminate the role of Ca2+ in such a function.

  4. Version published to 10.1101/2020.09.02.279174 on bioRxiv