In parallel to the ultrastructure of the IM, the morphology of mitochondria was determined by fluorescence microscopy. To analyze the structure of these cristae we used electron tomography.
These results show that the disappearance and reappearance of cristae are rapid and depend on functional Mgm1. We conclude that the formation of lamellar and tubular cristae relies on two different pathways. Moreover, Mgm1 plays a direct role in the formation and maintenance of lamellar but not of tubular cristae. This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing.
To further study the role of Mgm1 in cristae formation we first explored where Mgm1 is exactly located in mitochondria. Mgm1 is present in two isoforms Shepard and Yaffe, ; Wong et al. Importantly, both l-Mgm1 and s-Mgm1 forms are required for Mgm1 function in mitochondrial fusion DeVay et al. To test where s-Mgm1 is located, we generated sub-mitochondrial vesicles by sonication and separated them by gradient density centrifugation.
Remarkably, s-Mgm1 co-fractionated with the outer membrane marker Tom70, indicating that it is associated with the outer membrane Figure 3B. Left panel, four mitochondrial profiles M showing gold particles at the IBM.
Scale bar, 0. Right panel, quantification of gold particles. Mitochondrial vesicles generated by sonication were subjected to sucrose density gradient centrifugation followed by fractionation, SDS-PAGE and immunoblotting. Arrow, s-Mgm1. The membrane anchor of l-Mgm1 is notorious for being clipped off during isolation of mitochondria c.
Figures 5C and 7C. Therefore, l-Mgm1 was not detected after mitochondrial subfractionation. Cor2, subunit two of respiratory chain complex III. Dimeric F 1 F O provides positive curvature to crista membranes Dudkina et al. Deletion of Su e or Su g leads to the formation of membrane sheets that are crossing the matrix space completely and to onion-like structures.
Examination of a large number of EM sections showed that these membrane sheets are not cristae as previously assumed, but septa. Onions represent cross sections of curved septa Figure 1. This is also apparent upon tomographic reconstruction of isolated mitochondria from cells lacking dimeric F 1 F O Davies et al. A fundamental difference between cristae and septa is that crista membranes are strongly bent to generate crista rims and thus the sac-like crista structure, whereas septa are not closed but divide the matrix into membrane-limited subcompartments.
In spite of this drastic alteration of mitochondrial architecture, growth is only moderately retarded under respiratory conditions Paumard et al. It is presently unknown how and when during crista formation this bending is taking place. This observation is compatible with the assumption that the distribution of F 1 F O in cristae and IBM correlates with its oligomeric state.
Thus, the assembly of dimeric F 1 F O is likely to take place during the formation of cristae when the bending of membranes has to occur. Mitochondria lacking Mic60 contain characteristic stacked membranes in the matrix that have no connections to the IBM Alkhaja et al.
The protein composition of these mitochondria is very similar to that of WT cells Harner et al. Importantly F 1 F O is present in the dimeric form Rabl et al.
Arrow head, l-Mgm1; arrow, s-Mgm1; asterisk, cross-reaction of the Mic60 antibody. Mitochondria were isolated and lysed using digitonin. The result of one of four independently performed experiments is shown. SM, septum membrane; SJ, septum junction. This observation is in line with the finding that this MICOS subunit has an important role in the formation of the narrow ring or slot like structure of the crista junctions Barbot et al.
Taken together, this suggests that cristae and stacks are functionally similar if not identical. These findings raise the questions of how the internal membrane stacks originate and how proteins reach them in view of the virtually complete absence of connections with the IBM.
Its deletion leads to the formation of a highly branched and interconnected mitochondrial network Bleazard et al. Loss of Mgm1 in cells depleted of Dnm1 does not lead to loss of cristae Sesaki et al. This surprising observation appeared to contradict our suggestion that Mgm1 is directly required for crista formation and maintenance. We observed that mitochondrial architecture differed significantly from that in WT. These mitochondria are considerably enlarged, densely filled with vesicular profiles and short tubular cristae, while CJs are abundant along the IBM Figure 6A.
In comparison to WT, the number of cristae with lamellar structure was found to be much lower as highlighted by electron tomography. These tubules seem to have the ability to fuse and to branch. Interestingly, we identified tubular cristae also in WT cells by tomography, albeit much less frequently Figure 2E ; Video 1.
This is consistent with the higher number of cristae and CJs in this mutant. The necessity for the accommodation of OXPHOS and other membrane proteins likely requires an increase of the number of cristae since the surface area of tubular cristae compared to lamellar cristae is much lower. In summary, we reasoned that the mitochondrial network is in an almost completely fused state in the absence of Dnm1, and thus the rate of fusion is strongly decreased.
The diminished Mgm1 activity might then result in a drastically reduced formation of lamellar cristae and massive production of tubular cristae. Analysis as in Figure 4D.
Thus, Mgm1 appears to be essential for the formation of lamellar cristae, but is not required for the formation of tubular cristae and CJs. The strains were cultured on YPG medium to logarithmic phase followed by growth analysis on the indicated media by drop dilution assay. Mean values of three independent experiments.
Error bars, standard deviation. Arrow head, l-Mgm1; arrow, s-Mgm1. Green, IBM; other colors, perforated tubular-sheet like membrane structures in the matrix without connections to the IBM. To be able to discriminate between the different tubular elements they are shown in different colors.
H Quantitative evaluation of the EM analysis of the indicated mutant strains. Indeed, we observed mitochondria with numerous tubular cristae Figure 7—figure supplement 1 , supporting the idea that tubular cristae are the predominant type in the absence of fusion activity. Since it has been repeatedly suggested that a separate Dnm1-independent fission mechanism exists for the IM Gorsich and Shaw, ; Ishihara et al.
Together, these observations suggest that mitochondrial fusion is important for generating crista structure. Vesicular-tubular profiles, often located close to the IBM and associated with sheet-like membrane structures, were present in the interior of the mitochondria.
Tomographic reconstructions revealed a rather corrugated and perforated appearance, in contrast to the planar structure of WT lamellar cristae. Therefore, these membranes are most likely formed by association of extended tubules, which partly fuse with each other Figure 7F ; Video 5. MICOS components were an exception, as they were reduced to different degrees.
The level of Cox2, a mitochondrial gene product, was also reduced Figure 7C. This results in irregular networks which, as indicated by the biochemical and the growth phenotype, lack the characteristic properties of cristae. Green, IBM and cristae connected to the IBM; blue and red, fused tubular structures and perforated tubular-sheet like membrane structures in the matrix without connections to the IBM; yellow, ER-type tubules.
Deletion of both Dnm1 and Su e resulted in cells that grew extremely slowly on non-fermentable carbon source Figure 7A. Mitochondria contained many highly branched septa and multi-layered onion-like profiles Figure 7G.
Furthermore, the drastically altered membrane structures present in cells lacking Dnm1 and at the same time MICOS core components or Su e cannot substitute for lamellar and tubular cristae to maintain mtDNA and respiratory growth. Major advances have been made in our understanding of the biogenesis of mitochondrial protein complexes and supercomplexes, but the biogenesis of mitochondrial architecture, the next higher level of organization, has been investigated to a much lesser degree.
In particular, organization and formation of the cristae are poorly understood. We report here on novel and basic insights into the molecular mechanisms underlying the formation of lamellar and of tubular cristae, and the key factors involved in these pathways.
Based on our findings and the literature about these three factors and their interactors, we developed a hypothesis that postulates the existence of two distinct mechanisms for the generation of lamellar and tubular cristae. Regarding the formation of lamellar cristae, a first important issue is how Mgm1 performs fusion of the mitochondrial IM Figure 8A.
It is known that dysfunction of the components that mediate mitochondrial membrane fusion, Fzo1, Ugo1, and Mgm1, leads to loss of mtDNA and loss of cristae, concomitant with septa formation and inhibition of IM fusion Hermann et al. Fusion of the OM by Fzo1 would first generate a mitochondrion with a planar septum consisting of non-fused IM. According to our hypothesis, the fusion of the IM is then initiated by Mgm1 and proceeds along the IM-OM contacts, thereby generating a membrane sac protruding into the matrix.
Shifting the temperature sensitive mgm mutant to non-permissive temperature leads to rapid fragmentation of mitochondria Meeusen et al. Quantitative EM analysis of this process reveals that, in addition to fragmentation, a rapid and extensive loss of cristae takes place, while septa are formed. These findings strongly suggest that the activity of Mgm1 not only is required for fusion but also for maintenance of cristae. Importantly, cristae are rapidly regenerated upon reactivation of Mgm1.
Equally important, mtDNA is retained during inactivation of Mgm1 and remains functional. Thus, it is not the loss of mtDNA that causes loss of cristae and formation of septa, emphasizing a role of Mgm1 in crista maintenance. A Formation of lamellar cristae by the fusion dependent pathway. Steps in the conversion of the septa membranes of two fusing mitochondria into a crista membrane. B Formation of tubular cristae by the fusion independent pathway. C Formation of bizarre membrane assemblies in mitochondria of MICOS deficient cells by the fusion independent pathway.
At the same time these findings raise the question how this rapid reformation of cristae can be explained. The fusion rate of mitochondria in wild type cells has been measured by live cell microscopy Jakobs et al. Under steady-state conditions at least one fusion and one fission event was observed per minute and cell. This rate is apparently determined by regulatory processes that ensure a balance of fusion and fission.
In contrast, fusion of the IM in mgm mutant cells by reactivation of Mgm1 is likely to reflect an entirely different situation. Here, the conversion of a septum to a crista requires only the time of IM fusion by Mgm1 which upon its reactivation is immediately ready to function. The overall process of mitochondrial fusion requires a multitude of steps, including fusion of the OM. Fusion of the IM likely represents one of the fastest steps. Notably in this context, fusion intermediates have not been identified convincingly in intact cells.
These conclusions are in good agreement with observations that acute ablation of Opa1 in mouse embryonic fibroblasts leads to disorganized cristae and reduced respiratory function, but maintenance of mtDNA Cogliati et al.
The conversion of two membranes into a single one by Mgm1 entails the necessity to bend the membrane.
Very interestingly, recent experiments with reconstituted vesicles have revealed that Mgm1 mediates membrane bending Rujiviphat et al.
Therefore, it is reasonable to assume that bending of crista membranes takes place concurrently with the fusion process, and dimeric F 1 F O -ATP synthase is required to stabilize the bending of the membrane.
Rather, OM fusion still occurs in the absence of IM fusion and septa are generated. The continual import of proteins and lipids then leads to expansion of the septa membranes.
This expansion may cause the curved structures of septa that in EM sections appear as onion-like profiles Figure 1. Our hypothesis proposes that fusion of IM is halted by the assembly of MICOS complexes, leading to the generation of a CJ and thereby represents the final step in the formation of lamellar cristae Figure 8A. Since membrane proteins and protein complexes can shuttle between cristae and the IBM Vogel et al.
In addition, our hypothesis provides a rational explanation for the generation of the stacked closed membrane sheets lacking connection with the IBM that are formed in the absence of functional MICOS complex.
Interestingly, a very similar mechanism was suggested for the fusion of vacuoles, the yeast homolog of lysosomes Wickner, Upon fusion of vacuoles an internal vesicle is generated Wang et al. It might well be that these vesicles are generated like the internal stacks in Mic10 or Mic60 deletion mutants.
According to our hypothesis, during fusion septa membranes and the nascent crista membrane form a continuum. This allows free distribution of proteins between the IBM and the nascent crista membrane. This would also explain the similarity of the composition of intramitochondrial membrane sheets, made in the absence of MICOS, to crista membranes as well as their full functionality in mediating OXPHOS.
After completion of fusion and formation of CJs, cristae may grow by uptake of newly synthesized proteins, along with assembly of subunits of the F 1 F O -ATP synthase, which extend the crista rims. Thus, our hypothesis also offers an explanation of how the characteristic shape of lamellar cristae is generated and how cristae can expand. Our results also suggest the existence of a second pathway of crista biogenesis Figure 8B. In this pathway, import of newly synthesized proteins and lipids into the IBM and passage through CJs leads to the formation of tubular cristae.
Dimerization of F 1 F O would stabilize bending of the tubular cristae, very much like it stabilizes the rims of lamellar cristae. Interestingly, regular helical zipper-like structures of presumably dimeric F 1 F O were observed at the surface of the tubular cristae of the protist Paramecium multimicronucleatum by scanning EM Allen et al.
We suggest that they are generated by the continual influx of proteins and lipids and shaped by dimeric F 1 F O. This points to a correlation between presence of cristae and mtDNA.
In fact, mtDNA, condensed in nucleoids, was reported to be bound to crista membranes, possibly to crista rims Kopek et al. This second pathway of crista biogenesis suggested in our hypothesis becomes apparent when fission is compromised. In this situation, the mitochondrial network is virtually completely fused and Mgm1-dependent fusion activity should be drastically reduced. The lack of requirement of Mgm1 in this pathway provides a rational explanation for the most surprising observation that Mgm1 can be deleted in the Dnm1 deletion background without loss of cristae.
Residual formation of lamellar cristae in the absence of Dnm1 can be explained by residual fusion and fission activity of mitochondria Gorsich and Shaw, ; Ishihara et al. The virtually complete lack of lamellar cristae in cells deficient in both Dnm1 and Mgm1 further supports this assumption.
There is no fixed shape for mitochondria and they can often be seen changing shape as the cell moves through different degrees of energy requirement. However, when viewed as a thin, cross section under the electron microscope, the most common shape for a mitochondrion is that of a hot dog. Each mitochondrion has two membranes: an inner membrane which is highly convoluted and folded into finger-like projections called cristae , and a smooth outer membrane that is a selective barrier to molecules from the cytoplasm.
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Download references. We thank Jaydev Jethwa for carefully reading the manuscript and Peter Ilgen for help with graphics. You can also search for this author in PubMed Google Scholar.
Correspondence to Stefan Jakobs. Reprints and Permissions. Stephan, T. Live-cell STED nanoscopy of mitochondrial cristae. Sci Rep 9, Download citation. Received : 10 June Accepted : 13 August Published : 27 August Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.
Nature Communications Nature Methods Nature BIOspektrum Scientific Reports By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Fluorescence imaging Mitochondria Super-resolution microscopy. Abstract Mitochondria are highly dynamic organelles that exhibit a complex inner architecture.
Introduction Mitochondria form tubular and highly dynamic networks in mammalian cells that constantly undergo fusion and fission events 1 , 2. Results Visualization of the cristae architecture in living cells We have generated a human HeLa cell line that stably expresses the full length cytochrome c oxidase subunit 8A COX8A , an integral protein of the mitochondrial inner membrane, fused to the SNAP-tag Figure 1.
Full size image. Figure 2. Discussion The intricate mitochondrial membrane architecture is vital for the functioning of mitochondria as cellular powerhouses. Image processing No deconvolution was used. Data Availability All raw data used to create the figures and videos in this paper are available from the corresponding author upon reasonable request. References 1. Article Google Scholar Acknowledgements We thank Jaydev Jethwa for carefully reading the manuscript and Peter Ilgen for help with graphics.
View author publications. Ethics declarations Competing Interests The authors declare no competing interests. Supplementary information. Supplementary Movie S1. Supplementary Movie S2. Supplementary Movie S3. Supplementary Movie S4.
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