Mitochondrial Inner Membrane Fusion Research Articles

Inner‐membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

Fussy mitochondria fuse in response to stress

Fussy mitochondria fuse in response to stress

Computer-assisted live cell analysis of mitochondrial membrane potential, morphology and calcium handling

Mitochondria are crucial for many aspects of cellular homeostasis and a sufficiently negative membrane potential (Δ ψ) across the mitochondrial inner membrane (MIM) is required to sustain most mitochondrial functions including ATP generation, MIM fusion, and calcium uptake and release. Here, we present a microscopy approach for automated quantification of Δ ψ and mitochondrial position, shape and calcium handling in individual living cells. In the base protocol, cells are stained with tetramethyl rhodamine methyl ester (TMRM), a fluorescent cation that accumulates in the mitochondrial matrix according to Δ ψ, and visualized using video-microscopy. Next, the acquired images are processed to generate a mitochondria-specific binary image (mask) allowing simultaneous quantification of mitochondrial TMRM fluorescence intensity, shape and position. In a more advanced version of this protocol a mitochondria-targeted variant of green fluorescent protein (mitoAcGFP1) is expressed to allow mask making in TMRM-stained cells. The latter approach allows quantification of Δ ψ in cells with a substantially depolarized Δ ψ. For automated quantification of mitochondrial calcium handling in space and time mitoAcGFP1-expressing cells are stained with rhod-2, a fluorescent calcium indicator that accumulates in the mitochondrial matrix. In this paper, a detailed step-by-step description of the above approaches and its pitfalls is provided.

4] Lipid enrichment and fusion of mitochondrial inner membranes

Publisher Summary This chapter discusses the details of methods developed in the laboratory over the past few years which permit a significant incorporation or bulk enrichment of the mitochondrial inner membrane with a variety of membrane lipids. Related techniques, which permit the conversion of the typically small mitochondrial inner membranes to ultralarge inner membranes through a fusion process, are also reviewed. One method provides osmotically active, ultralarge (>200/μm) individual, spherical inner membranes. The lipid-enrichment procedure gives enriched inner membranes in bulk suspension whereas the fusion procedures are best suited for microanalysis of individual, enlarged inner membranes on microscope slides. Lipid-enriched inner membranes and fused inner membranes have been used in studies on the nature of the interaction of oxidation-reduction components utilizing routine reaction-kinetic approaches, fluorescence recovery after photobleaching, and flash-induced absorption anisotropy. Such studies reveal that the major oxidation-reduction components of the inner membrane diffuse laterally and independently in the membrane plane and rotate on an axis normal to the membrane plane. Lipid-enrichment and fusion methods are applied successfully on other membrane systems, such as microsomes and red blood cell ghosts and it is believed that most membrane systems can be treated similarly.

Fusion of bacteriorhodopsin with submitochondrial particles yields a new system with retention of energy coupling and acquisition of photophosphorylation activity

Submitochondrial particles were fused with purple membranes of Halobacterium halobium cells by means of a freeze-thawe sonication procedure. It is reported that fusion of inner mitochondrial membranes with a bacterial membrane yields a new particle which shows not only retention of redox- and photon-linked energy-coupling activities, but also creation of an additional energy-coupling process, light-driven ATP synthesis.

Membrane fusion in mitochondria. II. An ultrastructural interpretation of cristae-enriched megamitochondria induced by cuprizone.

Ultrastructure of megamitochondria in mouse hepatocytes induced by cuprizone was analyzed in the light of the fusion of inner mitochondrial membranes. Hepatic parenchymal cells of mice fed with a 0.5% cuprizone-diet for 7-8 days were characterized by megamitochondria with numerous cristae including disoriented, shorter ones than those of the control and those in vesicular configuration. After feeding the animals with cuprizone for 15-16 days, megamitochondria increased their sizes further and the number of cristae decreased drastically. Numbers of cristae based on a unit-mitochondrial area were 41.3 +/- 10.8 for the former type of cristae-enriched megamitochondria and 3.8 +/- 1.8 for the latter type of megamitochondria with the value of 23.9 +/- 6.2 for the control. Considering the content of cytochromes in megamitochondria previously reported, these results might suggest that the fusion and defusion of inner mitochondrial membrane take place along with the fusion of outer membranes during the megamitochondrial formation.

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles: Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.

Serial section analysis of mitochondrial form and membrane relationships in the neonatal rat liver cell

Serial sections of mitochondria from osmium-fixed neonatal rat liver cells were examined by electron microscopy. In every case, dumbbell-shaped mitochondria in single sections were found to be part of approximately disk-shaped mitochondria with flattened centers. The endoplasmic reticulum closely approximated the flat site, in one case giving rise to a nipple-like protrusion into a fold of the mitochondrion. Electron dense lines could be traced between endoplasmic reticulum and mitochondria, but were not considered to be real connections because they occurred at sites of obvious membrane slope. Connections between inner and outer mitochondrial membranes (membrane reversal) were examined, and all could be explained on the basis of small slopes of mitochondrial membranes through the extent of the section rather than real continuity. Interlacing or fusion of inner mitochondrial membranes in the flat zone could also be explained on the basis of sloped membranes. Apparent location of inner and outer mitochondrial membranes within the mitochondrial matrix was termed the staircase effect; it could be explained by membranes which were alternately sloped and perpendicular to the incident electron beam through the extent of a section.