We have previously shown increased cardiac stanniocalcin-1 (STC1) in patients with idiopathic dilated cardiomyopathy. STC1 localizes to the inner mitochondrial membrane and transgenic over-expression of STC1 is associated with increased energy utilization.
Cardiolipin (CL) is a unique phospholipid found in mitochondrial inner membrane. It is a key component for mitochondrial function in both respiration and apoptosis. The level of CL is an important parameter for investigating these intracellular events and is a critical indicator of a number of diseases associated with mitochondrial respiratory functions. 10-Nonyl acridine orange (NAO) is the only fluorescent dye currently available for CL detection. However, the performance of NAO is far from satisfactory in terms of selectivity and sensitivity. In this work, we report an aggregation-induced emission-active fluorogen, TTAPE-Me, for CL detection and quantification. With improved sensitivity and excellent selectivity to CL over other major mitochondrial membrane lipids, TTAPE-Me could serve as a valuable fluorescent sensor for CL quantification. The use of TTAPE-Me for the quantification of isolated mitochondria is also demonstrated.
Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER-mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.
The mitochondrial contact site and cristae organizing system (MICOS) is crucial for the formation of crista junctions and mitochondrial inner membrane architecture. MICOS contains two core components. Mic10 shows membrane-bending activity, whereas Mic60 (mitofilin) forms contact sites between inner and outer membranes. Here we report that Mic60 deforms liposomes into thin membrane tubules and thus displays membrane-shaping activity. We identify a membrane-binding site in the soluble intermembrane space-exposed part of Mic60. This membrane-binding site is formed by a predicted amphipathic helix between the conserved coiled-coil and mitofilin domains. The mitofilin domain negatively regulates the membrane-shaping activity of Mic60. Binding of Mic19 to the mitofilin domain modulates this activity. Membrane binding and shaping by the conserved Mic60-Mic19 complex is crucial for crista junction formation, mitochondrial membrane architecture and efficient respiratory activity. Mic60 thus plays a dual role by shaping inner membrane crista junctions and forming contact sites with the outer membrane.
Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function.
Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle-specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF-κB activation, and enhanced expression of pro-inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF-κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell-autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF-κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.
The conserved MICOS complex functions as a primary determinant of mitochondrial inner membrane structure. We address the organization and functional roles of MICOS and identify two independent MICOS subcomplexes: Mic27/Mic10/Mic12, whose assembly is dependent on respiratory complexes and the mitochondrial lipid cardiolipin, and Mic60/Mic19, which assembles independent of these factors. Our data suggest that MICOS subcomplexes independently localize to cristae junctions and are connected via Mic19, which functions to regulate subcomplex distribution, and thus, potentially also cristae junction copy number. MICOS subunits have non-redundant functions as the absence of MICOS subcomplexes results in more severe morphological and respiratory growth defects than deletion of single MICOS subunits or subcomplexes. Mitochondrial defects resulting from MICOS loss are caused by misdistribution of respiratory complexes in the inner membrane. Together, our data are consistent with a model where MICOS, mitochondrial lipids and respiratory complexes coordinately build a functional and correctly shaped mitochondrial inner membrane.
The Mitochondrial-Targeted Compound SS-31 Re-Energizes Ischemic Mitochondria by Interacting with Cardiolipin
- Journal of the American Society of Nephrology : JASN
- Published over 5 years ago
Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.
Mitochondrial crista structure partitions vital cellular reactions and is precisely regulated by diverse cellular signals. Here, we show that, in Drosophila, mitochondrial cristae undergo dynamic remodeling among distinct subcellular regions and the Parkinson’s disease (PD)-linked Ser/Thr kinase PINK1 participates in their regulation. Mitochondria increase crista junctions and numbers in selective subcellular areas, and this remodeling requires PINK1 to phosphorylate the inner mitochondrial membrane protein MIC60/mitofilin, which stabilizes MIC60 oligomerization. Expression of MIC60 restores crista structure and ATP levels of PINK1-null flies and remarkably rescues their behavioral defects and dopaminergic neurodegeneration. In an extension to human relevance, we discover that the PINK1-MIC60 pathway is conserved in human neurons, and expression of several MIC60 coding variants in the mitochondrial targeting sequence found in PD patients in Drosophila impairs crista junction formation and causes locomotion deficits. These findings highlight the importance of maintenance and plasticity of crista junctions to cellular homeostasis in vivo.
Mitochondria exist as a highly interconnected network that is exquisitely sensitive to variations in nutrient availability, as well as a large array of cellular stresses. Changes in length and connectivity of this network, as well as alterations in the mitochondrial inner membrane (cristae), regulate cell fate by controlling metabolism, proliferation, differentiation, and cell death. Given the key roles of mitochondrial dynamics, the process by which mitochondria constantly fuse and fragment, the measure of mitochondrial length and connectivity provides crucial information on the health and activity of various cell populations. However, despite the importance of accurately measuring mitochondrial networks, the tools required to rapidly and accurately provide this information are lacking. Here, we developed a novel probabilistic approach to automatically measure mitochondrial length distribution and connectivity from confocal images. This method accurately identified mitochondrial changes caused by starvation or the inhibition of mitochondrial function. In addition, we successfully used the algorithm to measure changes in mitochondrial inner membrane/matrix occurring in response to Complex III inhibitors. As cristae rearrangements play a critical role in metabolic regulation and cell survival, this provides a rapid method to screen for proteins or compounds affecting this process. The algorithm will thus provide a robust tool to dissect the molecular mechanisms underlying the key roles of mitochondria in the regulation of cell fate.