The final stage of eukaryotic cellular respiration occurs in mitochondria. The terminal respiratory reactions are catalyzed by the oxidative phosphorylation (OXPHOS) electron transport chain (ETC) – a series of large membrane protein complexes that reside in the inner mitochondrial membrane (IMM). Several ETC complexes are redox-coupled H⁺ pumps that connect oxygen consumption to adenosine triphosphate (ATP) synthesis by the generation of a proton motive force (pmf) across the IMM that is used to power the ATP synthase complex. The generation of the pmf is driven by the transfer of electrons from reduced substrates (NADH and succinate) to O₂ via four ETC complexes (Complexes I-IV, CI-IV) and electron carriers ubiquinone (coenzyme Q, CoQ) and cytochrome c (cyt c). In addition to their independent existence, ETC complexes can form higher-order structures known as supercomplexes (SCs) (Letts and Sazanov, 2017; Schägger and Pfeiffer, 2000). Across species, the most common SCs are formed between CI, a dimer of CIII (CIII₂) and CIV (SC I+III₂+IV) Letts et al., 2016b; CI and CIII₂ alone (SC I+III₂) Letts et al., 2019; and CIII₂ and CIV (SC III₂ +IV) (Hartley et al., 2018; Rathore et al., 2018; Vercellino and Sazanov, 2021). The advantage of SC formation remains undefined, but they are found across eukaryotes, often as the most abundant form of the ETC complexes (Davies et al., 2018; Schägger and Pfeiffer, 2001; Zhou et al., 2022).

CI couples the transfer of electrons from NADH to CoQ to the pumping of four H⁺ across the IMM (Galkin et al., 1999; Jones et al., 2017). It has an ‘L’ shaped structure consisting of two arms: a peripheral arm (PA) that extends into the matrix and a membrane arm (MA) that is embedded in the IMM. CI accepts electrons from NADH onto an FMN co-factor near the distal tip of the PA and transfers them to CoQ via a series of seven iron-sulfur (FeS) clusters. With few exceptions, the catalytic core of 14 subunits is conserved across species and contains all the redox cofactors and active sites required for catalysis (Hirst, 2013; Sazanov, 2015). In addition to the core subunits, eukaryotic CI has varying numbers of accessory subunits, e.g., 29 in the yeast Yarrowia lipolytica and 31 in mammals (Carroll et al., 2003; Letts and Sazanov, 2015; Padavannil et al., 2021; Parey et al., 2019). The accessory subunits are needed for the assembly and stability of the complex (Garcia et al., 2017; Stroud et al., 2016) and in some cases may play an active role in regulating turnover (Padavannil et al., 2021).

The molecular mechanism of the coupling between electron transfer and proton pumping has been the target of much research and debate (Chung et al., 2022a; Kampjut and Sazanov, 2022). Nonetheless, thanks to a plethora of high-resolution structures, a framework for the coupling mechanism is emerging in which specific conformational changes in the CoQ binding site loops at the interface of the PA and MA initiate a wave of conformational changes and electrostatic interactions upon CoQ reduction that propagate along the MA via a hydrophilic axis of amino acid residues resulting in H⁺ pumping (Kampjut and Sazanov, 2020; Kravchuk et al., 2022; Zickermann et al., 2015). Although this model is consistent with most mutagenesis and structural data, more experiments are needed to confirm the predictions of the model and examine possible variations in coupling and regulation across organisms (Klusch et al., 2021; Maldonado et al., 2020; Zhou et al., 2022).

Complicating the structural elucidation of the coupling mechanism is the fact that resting CI from yeast and mammals used for detailed structural studies have been shown to exist in two distinct biochemical states: the catalytically competent active (A) state and the off-pathway deactive (D) state (Gavrikova and Vinogradov, 1999; Maklashina et al., 2003). In these species, CI spontaneously undergoes an A-to-D transition when exposed to physiological temperatures in the absence of reduced substrates (Babot et al., 2014). Biochemically, the D state is characterized by a solvent-exposed cysteine residue on the transmembrane helix 1–2 loop of the ND3 core subunit (TMH1-2^ND3) that can be modified by thiol-reactive agents such as N-ethyl maleimide (NEM) (Galkin et al., 2008). Modification of the cysteine residue traps CI in the D state. The presence of the D state is thought to protect cells from ROS-mediated damage due to CI reverse electron transport (Chouchani et al., 2013). It remains unclear whether conformations of CI in the presence (Kampjut and Sazanov, 2020) or absence (Agip et al., 2018; Blaza et al., 2018) of added substrates, which differ in the structure of ND3’s cysteine-containing loop and in the relative positions of the PA and MA, correspond to the A and D states of CI or if they are part of CI’s catalytic cycle (Chung et al., 2022a; Kampjut and Sazanov, 2022).

Nonetheless, the A-to-D transition is not universally conserved across species (Maklashina et al., 2003). Biochemical characterization of CI from bacteria and more recently the ciliate Tetrahymena thermophila has failed to detect a deactive state using the standard NEM approach (Maklashina et al., 2003; Zhou et al., 2022). Structural analysis of the T. thermophila CI demonstrated that its PA/MA interface is more extensive than in other species (Zhou et al., 2022), likely precluding the opening of the complex in the same manner observed in mammals (Agip et al., 2018; Kampjut and Sazanov, 2020) and E. coli (Kolata and Efremov, 2021; Kravchuk et al., 2022). Although fungi and metazoans diverged approximately 1300 million years ago they form a broad clade of eukaryotes known as the opisthokonts and are thus more closely related to each other than either is to the ciliate T. thermophila. This suggests that the A-to-D transition may be a biochemical feature of CI that evolved in early opisthokonts. However, even within the opisthokonts the A-to-D transition is not universally detected. An NEM-sensitive D-state is seen in the CI of all characterized fungi but within metazoans, it is observed in deuterostomes but absent in protostomes (Figure 1A; Maklashina et al., 2003). Deuterostomes and protostomes diverged after the evolution of bilaterians approximately 600 million years ago (Dunn et al., 2014). This indicates either the loss of an ancestral NEM-sensitive D-state in Protostomia or the evolution of distinct, though biochemically similar, D-states in Fungi and Deuterostomia. It is important to note that across opisthokonts, and eukaryotes more broadly, CI from very few species have been biochemically characterized in detail. Thus, the evolution of this biochemical feature of the complex remains poorly understood.

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Further structural and biochemical analyses of CI in organisms without an A-to-D transition are needed to determine whether the observed conformational changes are catalytic or part of the off-pathway deactive state. Specifically, structural and functional characterization of a protostomian CI, like that from the model insect D. melanogaster, would be an important target due to its relatively close evolutionary relationship to the most well-characterized mammalian complexes. D. melanogaster is a genetically tractable system, has a similar ETC to humans, and is an emerging model for CI assembly and regulation (Garcia et al., 2017; Murari et al., 2022; Murari et al., 2021; Murari et al., 2020; Rhooms et al., 2019; Xu et al., 2019). Furthermore, CI is an established target for insecticides and agricultural pesticides (Murai and Miyoshi, 2016). Particularly with the emergence of Drosophila suzukii as a major pest for soft summer fruits (Tait et al., 2021), it is important to elucidate the structure of CI from a Drosophila species to identify unique aspects of CI in this genus. Here, we report the functional and structural characterization of thoracic muscle Dm-CI.

We present here the structure of mitochondrial CI from the model organism D. melanogaster (Dm-CI), providing a representative structure for insects and Protostomes more generally – a broad group of animals that split from mammals and other Deuterostomes approximately 600 million years ago. During the evolution of metazoans, a split in the biochemical behavior of mitochondrial CI occurred corresponding to the Protostomia/Deuterostomia divide (Figure 1A). According to established biochemical assays (Babot et al., 2014), all characterized CIs from Deuterostomes can enter a NEM sensitive deactive ‘D’ state, a property that they share with fungal CIs, whereas this is not the case for CI from Protostomes (Figure 1A; Babot et al., 2014; Maklashina et al., 2003). However, it is important to note that although all current data are consistent with a Protostomia/Deuterostomia split in the biochemical behavior of CI, only a small subset of species from each group have been investigated (Figure 1A). Characterization of additional species across both groups will provide a better understanding of the evolution of this functional split. We show here that although Dm-CI is insensitive to NEM after incubation at elevated temperatures in the absence of substrate (Figure 1B, E and G, and Figure 1—figure supplement 1D, F) it does shows an activation lag phase that is consistent with the presence of an off-pathway resting state (Figure 1E and G and Figure 1—figure supplement 1D, F). Recently the structural basis of the A-to-D transition has become central to the debate over the CI coupling mechanism (Chung et al., 2022a; Kampjut and Sazanov, 2022).

In fungi and mammals, the state-dependent accessibility of the ND3 cysteine in the active ‘A’ and D states is clearly understood from CI structures. The mammalian CI structure has been solved in multiple states that broadly fall into two categories, either ‘open’ or ‘closed,’ originally defined by the angle between the PA and the MA (Agip et al., 2018; Kampjut and Sazanov, 2020; Parey et al., 2021). In the open state the TMH1-2^ND3 loop, which harbors the reactive cysteine, is disordered indicating flexibility and accessibility, whereas in the closed state, the TMH1-2^ND3 loop is well ordered and the reactive cysteine is buried and inaccessible. This led to the proposal that the open state of CI corresponds to the D state and the closed state of the complex corresponds to the A state (Agip et al., 2018; Zhu et al., 2016). This proposal is supported by the structure of deactivated bovine CI which was found to contain 87.5% open-state particles (Blaza et al., 2018). However, it was also found that under turnover conditions ovine CI adopts open states, leading Kampjut and Sazanov to propose that open states are part of the catalytic cycle and that the deactive state is a particular ‘deep’ open state (Kampjut and Sazanov, 2020). They tested their model by deactivating the ovine complex prior to structure determination and reporting a large conformational shift in TMH4^ND6 (Figure 5—figure supplement 2; Kampjut and Sazanov, 2020). Thus, they conclude that the deactive state is a specific open state characterized by a large displacement of TMH4^ND6 (Kampjut and Sazanov, 2022; Kampjut and Sazanov, 2020; Kravchuk et al., 2022). However, all deposited maps of the deactivated ovine complex (EMDB-11260, EMDB-11261, EMDB-11262, and EMDB-11263) show very weak or no density for ND5-HL, TMH16^ND5, NDUFA11 or TMH4^ND6 (Kampjut and Sazanov, 2020). Poor density in these regions has been associated with so-called ‘state 3’ particles which are proposed to correspond to CI in the first stages of dissociation (Chung et al., 2022b; Zhu et al., 2016). This leads to the possibility that, despite being able to measure activity, the deactivation treatment of Kampjut and Sazanov may have partially denatured the ovine complex or sensitized it to disruption during the cryoEM grid preparation (Kampjut and Sazanov, 2020). If these structures represent the D state, then it follows that disorder of ND5-HL, TMH16^ND5, and NDUFA11 in addition to the movement of TMH4^ND6 would also be features of the D state. Although that is possible, despite lower resolution, these features were not observed in the deactivated bovine complex (EMDB-3731) (Blaza et al., 2018). Altogether, it is clear that CI’s deactive state is an open state. However, given the discrepancies between mammalian structures, it remains unclear how the deactive open state differs from other open states that may be part of CI’s catalytic cycle.

In the ‘open-and-closed-states’ model of CI turnover, the proposed function of open states during catalysis is twofold. Firstly, the disordering of the CoQ-site loops, in particular the TMH1-2^ND3 and β1-2^S2 loops, would disrupt the CoQ binding site and open the CoQ-binding cavity to solvent after the formation of ubiquinol (CoQH₂), thereby ‘washing out’ the highly hydrophobic substrate that may otherwise remain ‘stuck’ in the active site tunnel. Second, an open state during turnover would disrupt the hydrophilic axis via the formation of a π-bulge in TMH3^ND6, rotating hydrophobic residues into the axis and preventing a futile cycle caused by the ‘back flow’ of protons to the solvent-accessible CoQ site (Kampjut and Sazanov, 2022; Kampjut and Sazanov, 2020; Kravchuk et al., 2022).

Alternatively, the ‘closed-states-only’ model proposes that catalysis only occurs through a series of closed states and that all observed open states correspond to the D state (Chung et al., 2022a; Chung et al., 2022b). In this model, the multiple observed open states would stem from greater flexibility between the PA and MA caused by the angle between them increasing and the release of the TMH1-2^ND3 loop from the PA/MA interface and all open states observed from structures under turnover conditions are artifacts of sample preparation or the harsh conditions of the cryoEM grid. In the closed-states-only model, CoQ entry and exit would occur though standard diffusion in and out of the active site tunnel with more subtle conformational changes resulting in changing affinities for CoQ and CoQH₂ (Chung et al., 2022b; Chung et al., 2022a). Also, this model of turnover proposes that the α-helix-to-π-bulge transition of TMH3^ND6 does not occur during catalysis and that the TMH3^ND6 π-bulge is only a feature of the D state. Both models are consistent with conformational changes in the CoQ site loops being important during turnover, which has been demonstrated through site-specific cross-linking of the TMH1-2^ND3 loop with NDUFS7 (Cabrera-Orefice et al., 2018), but differs in the degree of conformational change needed.

The structures of resting Dm-CI reported here inform this debate. First, in the helix-locked state, the angle between the PA and MA is not consistent with either the open states of Y. lipolytica or mammalian CIs but the active site loops are fully ordered and buried by the NDUFA9 latch (Figure 5C). This state also lacks the TMH3^ND6 π-bulge indicating that the water wire of the hydrophilic access is intact, though a higher resolution structure is needed to confirm this. These features are most consistent with the closed/active state of CI. However, the NDUFA9 latch binding on top of the TMH1-2^ND3 loop and pinning down Arg50^ND3 (Figure 5C) is a feature not seen in mammalian closed/active states to date. Second, in the flexible class 1 state in which the PA and MA twist to bring NDUFAB1-α and NDUFA10 into close proximity, we see increased flexibility in the TMH1-2^ND3 loop, and the formation of the TMH3^ND6 π-bulge is more consistent with the open/deactive state of CI. However, it is important to note that the range of disordered residues in the TMH1-2^ND3 loop is distinct and shorter than that seen in other open/deactive states (Figure 5—figure supplement 1) and the conserved Cys41^ND3 remains well-ordered and hydrogen-bonded to Tyr134^ND1 (Figure 5E and F). Additionally, unlike what is seen in other species, no differences are seen between the β1-2^S2 loop, the α1-2^S7 loop, and the TMH5-6^ND1 loop between the Dm-CI states (Figure 5—figure supplement 1A, B, C).

Given that both the helix-locked and flexible class 1 states are resting-state structures, i.e., no substrate was provided to the complex prior to flash freezing, we cannot make strong conclusions regarding the catalytic relevance of these states. Nonetheless, four scenarios are possible: (1) both states are catalytically relevant on-pathway states, (2) the flexible state is on the pathway and the helix-locked state is an off-pathway resting state, (3) the flexible state is an off-pathway resting state and the helix-locked state is on the pathway, or (4) both are off-pathway resting states. From the current structures, we are unable to fully differentiate between these scenarios. However, scenario 1 is unlikely given that we were able to detect the presence of an off-pathway resting state (Figure 1E and G and Figure 1—figure supplement 1D, F) and the unique features of the Dm-CI states. The presence and absence of the NDUFS4 lock-helix, which is not adjacent to the active site nor conserved in other CIs of known structure (Figure 4—figure supplement 3), suggests that the NDUFS4 lock-helix is a regulatory element that has evolved in a specific branch of eukaryotes to either stabilize the resting (scenario 2) or active (scenario 3) states. If the NDUFS4 lock-helix evolved to regulate the transition between the off-pathway resting state and on-pathway states this would also argue against scenario 4 in which all the observed states are off-pathway. Further, scenario 1 would necessitate the binding and release of the lock-helix during turnover, which may not be rate limiting on the timescale of CI turnover but seems unlikely given the absence of the lock-helix in other species. Therefore, scenarios 2 and 3 seem most likely, though further work is needed to establish which possible scenario is correct.

The ‘opening-and-closing’ model of CI turnover would favor scenario 2. Large conformational changes in the TMH1-2^ND3 loop, such as an order-to-disorder transition, during CI turnover (Cabrera-Orefice et al., 2018) would also support scenario 2. Flexibility and opening of the TMH1-2^ND3 loop during turnover are supported by the enhanced reactivity of the ND3 cysteine seen in Bos taurus CI and our 2 mM NEM pre-activation conditions (Figure 1B; Burger et al., 2022). The presence of the NDUFA9 latch holding the TMH1-2^ND3 loop down in the helix-locked state may block conformational changes in TMH1-2^ND3, suggesting that it is a resting state incapable of turnover. The NDUFA9 latch would also explain the insensitivity of this potential off-pathway resting state to NEM (Figure 1B). If the Dm-CI flexible state is a catalytically competent state, the lack of the NDUFA9 latch would allow for conformational changes in the TMH1-2^ND3 loop and it would support the hypothesis that the TMH3^ND6π-bulge forms as part of the catalytic cycle.

Given that the proposed physiological function of the D state is to prevent reactive oxygen species production by reverse electron transport (Chouchani et al., 2013), it is possible that multiple distinct mechanisms have evolved to achieve this end. If the helix-locked state is a D-like resting state, it suggests that reverse electron transport by CI can be blocked in two distinct ways: (1) opening the CoQ site to solvent and thereby preventing CoQH₂ binding as seen in the standard D state; or (2) blocking conformational changes needed for coupling between CoQ reduction/CoQH₂ oxidation and H⁺-pumping as seen in the NDUFA9-latched-helix-locked-state. If the helix-locked state is an auto-inhibited state, these structures represent a novel regulatory mechanism that may be exploited to inhibit CI turnover in other species.

The ‘closed-states-only’ model of CI turnover would favor scenario 3. According to this model, only the helix-locked state could be the active state as it contains the fully α-helical TMH3^ND6 which is proposed to only be found in the π-bulge conformation in D-like resting states. If so, given the presence of the NDUFA9 latch this would suggest that either the TMH1-2^ND3 loop does not undergo conformational changes during turnover or the conformational changes are concerted across multiple subunits and would require additional open-like-states to explain the evidence that the cysteine on this loop becomes exposed during turnover (Figure 1B; Burger et al., 2022). Nonetheless, this interpretation is consistent with the structure of T. thermophila CI in which the TMH1-2^ND3 loop is also buried (Zhou et al., 2022). Given the presence of the lock-helix, in this scenario, CI would turnover without large changes in the angle between the PA and MA. This would be consistent with species whose CI have additional bridging interactions between the PA and MA that may limit changes in the PA/MA angle, such as plants, Tetrahymena, and the thermophilic yeast Chaetomium thermophilum (Klusch et al., 2021; Laube et al., 2022b; Zhou et al., 2022).

Recently, structures of E. coli CI, which does not deactivate (Maklashina et al., 2003) but does have an uncoupled resting-state (Belevich et al., 2017), revealed a variety of ‘open’ states in which the TMH1-2^ND3 loop was disordered and TMH3^ND6 featured a π-bulge (Kravchuk et al., 2022). In this study, a mixture of open and ‘closed’ states, in which the TMH1-2^ND3 loop is well ordered and TMH3^ND6 is full α-helical, was only observed under turnover conditions (Kravchuk et al., 2022). This confirms that the closed state is a catalytically relevant active state and supports the hypothesis that open states are not solely a consequence of deactivation. However, to demonstrate that the open states are catalytically relevant other possible explanations for the observation of open states must be ruled out. Thus, it is important to note that another recent structural study of E. coli CI found that intact biochemically active preparations contained a significant fraction of broken particles on the cryoEM grids (Kolata and Efremov, 2021). After classification Kolata and Efremov found that most E. coli CI particles used for the reconstruction of the PA (151,357 vs. 134,976 particles) were particles that had completely dissociated from the MA (Kolata and Efremov, 2021). The biochemical preparations for the Kravchuk et al., 2022, and Kolata and Efremov studies are different and Kravchuk et al., used gentler conditions and do not report any disrupted particles. Nonetheless, the results of Kolata and Efremov are a stark reminder that structural studies on extracted membrane protein complexes exist within a spectrum of biochemical stability and that complexes that are intact according to size exclusion chromatography, mass photometry, and activity assays, can still end up as broken particles on the cryoEM grid (Han et al., 2022; Kolata and Efremov, 2021). Therefore, it is not unreasonable to consider that the interaction with the air-water interface during grid preparation may act to convert closed-state CI to the lower energy (higher entropy due to disorder and flexibility) open states.

Therefore, interactions with the air-water interface should be noted as an alternate explanation as to why most structures of CI across species appear to have open states, and complexes in which the PA and MA are more stably associated should be sought out for additional corroborative functional work. Although Dm-CI adopts an open-like flexible state, it may be more resistant to disruption during grid preparation due to the expanded interaction between NADUFA5 and NDUFA10 (Figure 3B). However, Dm-CI needs to be further characterized in the presence of substrates before general conclusions can be drawn. Due to the presence of the lock-helix, Dm-CI is an ideal system for further structural and functional work as the presence of the NDUFS4 helix can act as a strong signal for the different states under different turnover and grid preparation conditions.

In conclusion, the structure of Dm-CI from thoracic muscle reveals unique features of CI from Protostomia that do not share the standard A-to-D transition as defined biochemically. Overall, with a few notable differences, the structure of Dm-CI is like that of mammals, validating its use as a genetically tractable model for the study of metazoan CI physiology. Given that inhibitors of CI have been developed as potential agricultural pesticides (Murai and Miyoshi, 2016), this structure will be a valuable resource for the development of more selective inhibitors. Due to its close relatedness to D. melanogaster (Table 2), our structure is of particular value to develop targeted pesticides against spotted-wing drosophila (D. suzukii), a major invasive agricultural pest of the berry and wine industry in Southeast Asia, Europe, and America (Tait et al., 2021). More broadly, the unique features of Dm-CI revealed here suggest strategies for the development of insecticides that could help control insect vectors of human disease. This study highlights the utility of diverse model organisms in the study of important biochemical processes as we learn as much from the differences as we do from the similarities. In addition, our structures reveal unanticipated mechanisms that have evolved to regulate the assembly and activity of mitochondrial CI that may be exploited to modulate assembly or activity in other organisms. Additional studies on Dm-CI as well as other species are needed to fully understand the different mechanisms which have evolved to regulate the assembly and activity of this important enzyme.

Single-particle cryogenic electron micrograph movies are available on the Electron Microscopy Public Image Archive, accession code EMPIAR-11272. The maps and models are available on the Electron Microscopy Database (EMDB) and Protein Data Bank (PDB). The accession codes for the Helix-locked state are EMDB-28582, PDB-8ESZ and for the flexible class 1 state are EMDB-28581, PDB-8ESW.

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   ID 11272. Mitochondrial complex I from Drosophila melanogaster.

   https://www.ebi.ac.uk/empiar/
2. 1. Letts JA
   2. Padavannil A

   (2022) Electron Microscopy Data Bank

   ID EMD-28582. Structure of mitochondrial complex I from Drosophila melanogaster, Helix-locked state.

   https://www.ebi.ac.uk/emdb/search/EMDB-28582
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   ID EMD-28581. Structure of mitochondrial complex I from Drosophila melanogaster, Flexible class 1.

   https://www.ebi.ac.uk/emdb/search/EMDB-28581
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   ID 8ESZ. Structure of mitochondrial complex I from Drosophila melanogaster, Helix-locked state.

   https://www.rcsb.org/structure/unreleased/8ESZ
5. 1. Letts JA
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   (2022) RCSB Protein Data Bank

   ID 8ESW. Structure of mitochondrial complex I from Drosophila melanogaster, Flexible class 1.

   https://www.rcsb.org/structure/unreleased/8ESW

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Author details

1. Abhilash Padavannil

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9949-6776

2. Anjaneyulu Murari

   Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7532-964X

3. Shauna-Kay Rhooms

   Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

4. Edward Owusu-Ansah

   1. Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, United States
   2. The Robert N. Butler Columbia Aging Center, Columbia University Irving Medical Center, New York, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3451-1752

5. James A Letts

   Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   jaletts@ucdavis.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9864-3586

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