We thank Ganley et al. for their thoughtful comment on our recent report [Citation1]. We agree with Ganley et al. that both mito-Keima and mt-QC detect mitophagy. Our report demonstrates, however, that mito-Keima is more sensitive than mt-QC in cell culture and in vivo. This difference in sensitivity may lead investigators to different conclusions depending on which reporter they use.
Specifically, we found we could detect significantly increased mitophagy in the heart following exhaustive exercise with mt-Keima but not with mito-QC. The mt-Keima mice were backcrossed twice on BL/6 (the same background as the mito-QC mice), and the remaining background difference is unlikely to account for dramatic difference in response for the two reporters. The difference is more likely due to the increased sensitivity of mt-Keima relative to mito-QC, as this was also observed in primary cells from the reporter mice and HeLa cells into which the reporters were introduced by lentiviral transduction.
The difference in reporter sensitivity may also account for some contradictory findings in the literature, such as those concerning whether or not Pink1-park/PRKN mediate basal mitophagy in the indirect flight muscle of adult Drosophila. Using the mt-Keima reporter, Cornelissen et al. found it does [Citation2]; whereas, using the mito-QC reporter, Lee et al. found it does not [Citation3]. If this difference is due to the reporters used, then the correct interpretation is likely that Pink1-park/PRKN mediate basal mitophagy in adult Drosophila, as the mt-Keima reporter is more sensitive than mito-QC. The detection of Pink1-park/PRKN-dependent basal mitophagy in adult Drosophila using the mt-Keima reporter is also consistent with recent data from the Pallanck lab [Citation4,Citation5]. They found that the Pink1-park/PRKN pathway is responsible for approximately 25% of the basal turnover of mitochondrial proteins in Drosophila [Citation5].
Our findings point to both the targeting of the fluorescent protein (outer mitochondrial membrane vs. matrix) and the fluorescent protein used (Keima vs. tandem EGFP-mCherry) as important for the superior sensitivity of mt-Keima. These findings are consistent with a recent report from Katayama et al., which we discuss in our report, and are additionally consistent with the report from the Goessling lab, which Ganley et al. cite in their comment and which was published while our report was in revision [Citation6,Citation7]. In the latter report, the authors used a tandem GFP-mCherry construct (mito-GR) targeted to the mitochondrial matrix (and so, distinct from mito-QC, which is targeted to the outer membrane) and mt-Keima [Citation7]. Consistent with our findings regarding the relative sensitivity of the reporters, they note that “at greater imaging depths the ubi:mito-Keima signal decayed more consistently and provided higher signal-to-noise than [mito-GR].” Thus, there appears to be a growing consensus that for a mitophagy reporter (1) mKeima is more sensitive than tandem GFP-mCherry and (2) targeting to the matrix is superior to targeting to the outer mitochondrial membrane.
As we noted in our discussion and Ganley et al. noted in their comment, mito-Keima, although more sensitive, has important limitations, such as the need to image the reporter live. A fixable mitophagy reporter such as mito-QC has clear advantages. Our results and those of Katayama et al. suggest how the mito-QC may be improved as a fixable reporter by directing it to the mitochondrial matrix rather than the outer membrane and substituting mCherry with TOLLES, which has superior resistance to acidity [Citation1,Citation6]. Katayama et al. implemented these and other changes in mt-SRAI, which they showed is fixable and performs better than mito-QC at least in cell culture [Citation6]. We hope our findings will help guide investigators in their choice of reporter and spur the development of even better reporters for mitophagy research.
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References
- Liu Y-T, Sliter DA, Shammas MK, et al. Mt-Keima detects PINK1-PRKN mitophagy in vivo with greater sensitivity than mito-QC. Autophagy. 2021;1–10. DOI:https://doi.org/10.1080/15548627.2021.1896924
- Cornelissen T, Vilain S, Vints K, et al. Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila. Elife. 2018;7. DOI:https://doi.org/10.7554/eLife.35878
- Lee JJ, Sanchez-Martinez A, Zarate AM, et al. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. J Cell Biol. 2018;217:1613–1622.
- Vincow ES, Merrihew G, Thomas RE, et al. The PINK1–Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo. Proc Nat Acad Sci. 2013;110:6400–6405.
- Vincow ES, Thomas RE, Merrihew GE, et al. Autophagy accounts for approximately one–third of mitochondrial protein turnover and is protein selective. Autophagy. 2019;1–14.
- Katayama H, Hama H, Nagasawa K, et al. Visualizing and modulating mitophagy for therapeutic studies of neurodegeneration. Cell. 2020;181:1176–1187.e16.
- Wrighton PJ, Shwartz A, Heo J-M, et al. Quantitative intravital imaging in zebrafish reveals in vivo dynamics of physiological-stress-induced mitophagy. J Cell Sci. 2021;134. DOI:https://doi.org/10.1242/jcs.256255