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. 2020 Jan 1;31(1):18-26.
doi: 10.1091/mbc.E19-08-0450. Epub 2019 Nov 20.

Coordinated Organization of Mitochondrial Lamellar Cristae and Gain of COX Function During Mitochondrial Maturation in Drosophila

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Free PMC article

Coordinated Organization of Mitochondrial Lamellar Cristae and Gain of COX Function During Mitochondrial Maturation in Drosophila

Yi-Fan Jiang et al. Mol Biol Cell. .
Free PMC article

Abstract

Mitochondrial cristae contain electron transport chain complexes and are distinct from the inner boundary membrane (IBM). While many details regarding the regulation of mitochondrial structure are known, the relationship between cristae structure and function during organelle development is not fully described. Here, we used serial-section tomography to characterize the formation of lamellar cristae in immature mitochondria during a period of dramatic mitochondrial development that occurs after Drosophila emergence as an adult. We found that the formation of lamellar cristae was associated with the gain of cytochrome c oxidase (COX) function, and the COX subunit, COX4, was localized predominantly to organized lamellar cristae. Interestingly, 3D tomography showed some COX-positive lamellar cristae were not connected to IBM. We hypothesize that some lamellar cristae may be organized by a vesicle germination process in the matrix, in addition to invagination of IBM. OXA1 protein, which mediates membrane insertion of COX proteins, was also localized to cristae and reticular structures isolated in the matrix additional to the IBM, suggesting that it may participate in the formation of vesicle germination-derived cristae. Overall, our study elaborates on how cristae morphogenesis and functional maturation are intricately associated. Our data support the vesicle germination and membrane invagination models of cristae formation.

Figures

FIGURE 1:
FIGURE 1:
Mitochondria undergo development upon Drosophila eclosion. Thin-section EM micrographs of Drosophila IFM at day 1 (a), week 1 (b), and week 4 (c) showing the development of mitochondrial cristae. Red arrows indicate ribosome/polysomelike densities. Orange arrows indicate the cristae. Western blot analysis of mitochondrial proteins, ATP5A, PDHA1, SOD2, and CytC, and ribosomal protein, RPS6, in day 1, week 1, and week 4 flies (d, e). The relative protein abundance was quantified by densitometry and normalized to α-tubulin. The ratios were subsequently normalized to week 4 flies (f).
FIGURE 2:
FIGURE 2:
Serial-section tomography of immature mitochondria reveals cristae organization. Serial-section tomography slice (a) and segmentation (b) of Drosophila IFM at day 1. A few organized lamellar cristae are labeled in blue, cytoplasmic ribosomallike densities are green, and mitochondrial ribosomallike densities are red. (c) 3D segmentation of arbitrarily colored mitochondria showing the polymorphic shapes of immature mitochondria rather than the consistent ovoid shape of mature mitochondria.
FIGURE 3:
FIGURE 3:
Lamellar cristae formation in immature mitochondria was coincident with the gain of COX activity. Thin-section EM micrographs of Drosophila IFM at day 1 (a) and week 4 (b) stained for COX activity.
FIGURE 4:
FIGURE 4:
Cristae morphogenesis and functional maturation were associated during mitochondrial development. Serial-section tomography of Drosophila IFM at day 1 stained for COX activity. Tomography slices across the z-axis and the corresponding segmentation are shown in the top and bottom panels, respectively (a). At day 1, a few lamellar cristae show organization and strong COX staining, top panels. COX-positive structures are colored red and COX-negative structures are colored yellow in the bottom panels. The red arrows indicate a single lamellar crista across z-sections. 3D representations of the tomographic segmentation are shown in b and c to visualize the distribution of the COX-positive and COX-negative structures in a mitochondrion. (d) 3D representation of the tomographic segmentation showing COX-positive lamellar and reticular structures. (e) 3D representation of the segmentation of the whole serial-tomograms showing COX-positive lamellar cristae in or without connection to the IBM.
FIGURE 5:
FIGURE 5:
COX4 localizes predominantly to organized lamellar cristae. (a) Apex2 staining of the IFM of COX4-Apex2 knock-in flies at day 1. Positive Apex2 signals appeared darkly stained and were localized mainly in organized lamellar cristae. Red arrows indicate positive Apex2 staining in lamellar cristae. (b) Apex2 staining of WT flies at day 1 as a negative control. (c) The Western blot analysis of COX4-Apex2 expression at day 1, week 1, and week 4 by anti-flag tag. The relative protein abundance was quantified by densitometry and normalized to α-tubulin. The ratios were subsequently normalized to week 4 flies. (d) Thin-section EM of the IFM from COX4-Apex2 knock-in flies at week 4 stained for COX activity.
FIGURE 6:
FIGURE 6:
OXA1 localization in immature mitochondria supported COX-positive cristae formation by a process additional to the invagination of the IBM. Apex2 staining of the IFM of OXA1-Apex2 knock-in flies (a) and the WT control (b) at day 1. Positive OXA1-Apex2 staining appeared as granular densities on the matrix side of cristae and IBM where the Apex2 tag is expected to be localized. Apex2 staining of S2 cells transfected with (c) and without (d) plasmids expressing D. melanogaster OXA1-Apex2. High OXA1-Apex2 expression yielded strong staining on the matrix side of the cristae and IBM where the Apex2 tag is expected to be found. Red arrows in a and c indicate positive Apex2 staining on the matrix side of the membranes.
FIGURE 7:
FIGURE 7:
Hypothetical model of cristae formation. Lamellar cristae (1) organize at the cristae junction and extend from the IBM as the membranes acquire COX function, or (2) extend from the cristae from the matrix side by organizing reticular structures in coordination with OXA1-mediated COX assembly. (3) Lamellar cristae form in the matrix as organized reticular structures, which later contact the IBM and establish cristae junctions via MICOS.

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