Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent

Wolf, Dane M.; Segawa, Mayuko; Kondadi, Arun Kumar; Anand, Ruchika; Bailey, Sean T.; Reichert, Andreas S.; Bliek, Alexander M. van der; Shackelford, David B.; Liesa, Marc; Shirihai, Orian S.

doi:10.15252/embj.2018101056

Cited by 235 publications

(257 citation statements)

References 52 publications

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“…This does not exclude that the MICOS complex can dynamically change its composition or assembly state; (iii) CJs can dynamically merge and split; (iv) cristae are mainly, but not necessarily always, attached to CJs; and (v) cristae can undergo transient pinching-off from CJs and remerge at the same CJ or another one (transverse or neighbouring CJ; Fig 9Di). The formation of separated cristae subcompartments is consistent with another study showing that, within the same mitochondrion, individual cristae can maintain a stable membrane potential over time and that neighbouring cristae can maintain disparate DΨ [60]. This is also consistent with our FRAP data showing that TIMM23-GFP shows a reduced mobility upon loss of CJs, presumably by enhanced trapping in the CM.…”

Section: Discussionsupporting

confidence: 92%

Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

et al. 2020

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The mitochondrial inner membrane can reshape under different physiological conditions. How, at which frequency this occurs in living cells, and the molecular players involved are unknown. Here, we show using state‐of‐the‐art live‐cell stimulated emission depletion (STED) super‐resolution nanoscopy that neighbouring crista junctions (CJs) dynamically appose and separate from each other in a reversible and balanced manner in human cells. Staining of cristae membranes (CM), using various protein markers or two lipophilic inner membrane‐specific dyes, further revealed that cristae undergo continuous cycles of membrane remodelling. These events are accompanied by fluctuations of the membrane potential within distinct cristae over time. Both CJ and CM dynamics depended on MIC13 and occurred at similar timescales in the range of seconds. Our data further suggest that MIC60 acts as a docking platform promoting CJ and contact site formation. Overall, by employing advanced imaging techniques including fluorescence recovery after photobleaching (FRAP), single‐particle tracking (SPT), live‐cell STED and high‐resolution Airyscan microscopy, we propose a model of CJ dynamics being mechanistically linked to CM remodelling representing cristae membrane fission and fusion events occurring within individual mitochondria.

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Section: Discussionsupporting

confidence: 92%

Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

et al. 2020

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“…Third, it has been argued that the CJs make a natural diffusion barrier in the inner membrane, effectively separating the IBM from the CMs (40,41). Indeed, recent high resolution studies of live cells have shown that cristae can be biophysically distinct to the IBM as a consequence of CJs providing a form of physical insulation (42). Therefore, it is unclear how easy it would be for complexes made in the inner boundary membranes to diffuse through the constricted membranes around the MICOS complexes at the cristae junction.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, another current limitation is that mitochondrial FUNCAT cannot allow synthesis to be followed in real time. Considering the superb nanoscopy images that are being generated to allow us to visualise the ultrastructure of the mitochondrion in real time (4,42,44,45), we hope to soon adapt our protocol to follow mitochondrial protein synthesis in live cells.…”

Section: Discussionmentioning

confidence: 99%

High resolution imaging of nascent mitochondrial protein synthesis in cultured human cells

Zorkau

Albus

Berlinguer‐Palmini

et al. 2020

Preprint

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Human mitochondria contain their own genome, mtDNA, that is expressed in the mitochondrial matrix. This genome encodes thirteen vital polypeptides that are components of the multi-subunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially-encoded components are also integral members of these complexes, where does nascent protein synthesis occur? Transcription, mRNA processing, maturation and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites ? We have adapted a click chemistry based method, coupled with STED nanoscopy to address these questions. We report that in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis occurs at the cristae membranes and is spatially separated from the sites of RNA processing and maturation. the architecture of the membranes (11) and iii) large (10 -40nm) complexes termed cristae junctions (CJ), composed of the Mitochondrial contact site and Cristae Organising System (MICOS; (12-14)), which forms the cristae and separates them from the IBM.There has also been much focus on the mitochondrial genome, how and where it is expressed. This has led to the identity and characterisation of the nucleoid (15-18) and more recently the mitochondrial RNA granule, a complex formed by phase transition where transcripts are processed and matured (19)(20)(21). Translation occurs on membraneassociated mitochondrial ribosomes, which are also at least partially assembled in the RNA granule (22,23). Exactly where, however, protein synthesis is performed in human mitochondria, whether at the CM, CJ or IBM, within, close to, or distal to the RNA granules, is unknown. To follow this process in yeast mitochondria, Jakobs and colleagues harnessed an elegant series of experimental approaches (24). The yeast S. cerevisiae express a battery of translational activators that show specificity to mt-mRNAs, which encode individual components of OXPHOS complexes III, IV and V (25). Using immuno-labelling super resolution and electron microscopy, data were produced that were consistent with mtDNA-encoded complex V subunits being synthesised predominantly on the cristae membranes, whilst complex III and IV components were synthesised both at the IBM and CM. This approach is not feasible in human cells as with the exception of TAC...

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“…Recently, studies showed great improvements in live-cell imaging using STED or Airyscan microscopy. These methods were used in combination with fluorescent dyes or inner membrane markers for analysis of the mitochondrial ultrastructure 21, 22 , measurement of the membrane potential of individual cristae 24 or even cristae dynamics 40 . Although these live-cell imaging methods enabled following of cristae dynamics in real time, they also exhibited very fast damaging of mitochondrial vitality and are also not suitable for protein localization studies.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the possibility to investigate cristae morphology and the localization of mitochondrial proteins is of broad interest. Up to now, most light microscopy approaches have been performed using STED 21, 22, 23 or Airyscan microscopy 24 . Although very successful in cristae visualization, the limitation is the restricted availability of super-resolution microscopes in standard cell biology laboratories as tools for investigating the mitochondrial ultrastructure.…”

Section: Introductionmentioning

confidence: 99%

Using Expansion Microscopy to visualize and characterize the morphology of mitochondrial cristae

Kunz

Goetz

Gao

et al. 2020

Preprint

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15Mitochondria are double membrane bound organelles indispensable for biological processes such as 16 apoptosis, cell signalling, and the production of many important metabolites, which includes ATP that 17 is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane 18 contains folds called cristae, which increase the membrane surface and thus the amount of membrane-19 bound proteins necessary for the OXPHOS. These folds have been of great interest not only because 20 of their importance for energy conversion, but also because changes in morphology have been linked 21 to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to ageing and 22 infection. With a distance between opposing cristae membranes often below 100 nm, conventional 23 fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this 24 reason, various highly specialized super-resolution methods including dSTORM, PALM, STED and SIM 25 have been applied for cristae visualisation. 26Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on 27 conventional confocal microscopes by embedding the sample into a swellable hydrogel that is 28 isotropically expanded by a factor of 4-4.5, improving the resolution to 60-70 nm on conventional 29 confocal microscopes, which can be further increased to ~ 30 nm laterally using SIM. Here, we 30 demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein 31 GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial 32 membrane, can be used as a cristae marker. Applying ExM on mitochondria labelled with this construct 33 enables visualization of morphological changes of cristae and localization studies of mitochondrial 34 proteins relative to cristae without the need for specialized setups. For the first time we present the 35 combination of specific mitochondrial intermembrane space labelling and ExM as a tool for studying 36 internal structure of mitochondria. 37

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Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent

Cited by 235 publications

References 52 publications

Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

Cristae undergo continuous cycles of membrane remodelling in a MICOS ‐dependent manner

High resolution imaging of nascent mitochondrial protein synthesis in cultured human cells

Using Expansion Microscopy to visualize and characterize the morphology of mitochondrial cristae

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