OPA1 functionally interacts with MIC60 but is dispensable for crista junction formation.

Remodeling of crista junctions (CJs) is observed in numerous human disorders and during apoptosis. The functional interplay of OPA1 and MIC60, two key players in this context, is unclear. We show that OPA1 modulates cristae morphology but is dispensable for CJ formation. MIC60 is strongly enriched at CJs, whereas OPA1 is distributed evenly across the inner membrane. MIC60 levels are increased in OPA1-/- cells which show increased cellular resistance to apoptosis induction. Endogenous OPA1 and MIC60 show a physical interaction. Overall, we suggest that the regulation of CJ remodeling during apoptosis is mediated via an interplay between OPA1 and MIC60.

Inner membrane dynamics in mitochondria.

Combining the use of cells with sparse cristae marked with IMP-EGFP and short pulsed sub-saturating fluorescence excitation (non-saturation fluorescence microscopy/NSFM) revealed inhomogeneous fluorescence distribution along mitochondria in living cells. Also the matrix located TMRE was distributed non-uniformly and at least in part filling the gaps between the IMP-EGFP fluorescence: fluorescence intensities are modulated in space and time in part in an antidromic manner. The spatial modulations can be interpreted to represent cristae/matrix distributions. The temporal fluctuations of fluorescence vary within 0.3-3s. Because most peak positions of IMP fluorescence remain stationary up to at least several minutes, temporal intensity modulations may result from varying emissions related to the degree of excitation and/or represent wobbling of cristae, i.e. lateral movements, bending or size changes. Modulations by noise and non-saturated excitation have been reduced by 3 steps of deconvolution followed by averaging 4 images. This allowed a final temporal resolution of 150ms. Disappearance of cristae or formation of new ones takes place within a few seconds, but these are rare events. Thus position of cristae seems to be rather stable, but they regularly disassemble close to fission sites. Treatment with oligomycin strongly reduces "wobbling" activity.

Emergence of the mitochondrial reticulum from fission and fusion dynamics.

Mitochondria form a dynamic tubular reticulum within eukaryotic cells. Currently, quantitative understanding of its morphological characteristics is largely absent, despite major progress in deciphering the molecular fission and fusion machineries shaping its structure. Here we address the principles of formation and the large-scale organization of the cell-wide network of mitochondria. On the basis of experimentally determined structural features we establish the tip-to-tip and tip-to-side fission and fusion events as dominant reactions in the motility of this organelle. Subsequently, we introduce a graph-based model of the chondriome able to encompass its inherent variability in a single framework. Using both mean-field deterministic and explicit stochastic mathematical methods we establish a relationship between the chondriome structural network characteristics and underlying kinetic rate parameters. The computational analysis indicates that mitochondrial networks exhibit a percolation threshold. Intrinsic morphological instability of the mitochondrial reticulum resulting from its vicinity to the percolation transition is proposed as a novel mechanism that can be utilized by cells for optimizing their functional competence via dynamic remodeling of the chondriome. The detailed size distribution of the network components predicted by the dynamic graph representation introduces a relationship between chondriome characteristics and cell function. It forms a basis for understanding the architecture of mitochondria as a cell-wide but inhomogeneous organelle. Analysis of the reticulum adaptive configuration offers a direct clarification for its impact on numerous physiological processes strongly dependent on mitochondrial dynamics and organization, such as efficiency of cellular metabolism, tissue differentiation and aging.

Determination of protein mobility in mitochondrial membranes of living cells.

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F(1)F(0) ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D=0.7µm(2)/s and 0.6µm(2)/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F(1)F(0) ATPase: 0.4µm(2)/s and 0.0005µm(2)/s; COX: 0.3µm(2)/s and 0.007µm(2)/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.

Do UCP2 and mild uncoupling improve longevity?

Mild uncoupling of mitochondrial respiration is considered to prolong life span of organisms by reducing the production of reactive oxygen species (ROS). Experimental evidence against this hypothesis has been brought forward by premature senescence in cell cultures treated with uncouplers. Exposing HUVEC to a mixture of nutritionally important fatty acids (oil extract of chicken yolk) mild uncoupling with "naturally acting substances" was performed. This treatment also resulted in premature senescence although ROS production did not increase. Fatty acids activate uncoupling proteins (UCP) in the inner mitochondrial membrane. UCP2 expression proved to be sensitive to the presence of fatty acids but remains unchanged during the ageing process. UCP3 expression in senescent HUVEC and avUCP expression in senescent CEF were considerably less than in young cultures. No indication for protonophoric reduction of mitochondrial membrane potential was found in UCP2 overexpressing HeLa cells and only little in HUVEC. ROS levels increased instead of being reduced in these cells. Stable transfection with UCP2-GFP was possible only in chick embryo fibroblasts and HeLa cells and resulted in decreased proliferation. Stable transfection of HUVEC with UCP2-GFP resulted in death of cultures within one or two weeks. The reason for this behaviour most probably is apoptosis preceded by mitochondrial fragmentation and loss of membrane potential.