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 both 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.
Mitochondria are highly mobile and dynamic organelles that continually fuse and divide. These processes allow mitochondria to exchange contents, including mitochondrial DNA (mtDNA). Here we examine ...the functions of mitochondrial fusion in differentiated skeletal muscle through conditional deletion of the mitofusins Mfn1 and Mfn2, mitochondrial GTPases essential for fusion. Loss of the mitofusins causes severe mitochondrial dysfunction, compensatory mitochondrial proliferation, and muscle atrophy. Mutant mice have severe mtDNA depletion in muscle that precedes physiological abnormalities. Moreover, the mitochondrial genomes of the mutant muscle rapidly accumulate point mutations and deletions. In a related experiment, we find that disruption of mitochondrial fusion strongly increases mitochondrial dysfunction and lethality in a mouse model with high levels of mtDNA mutations. With its dual function in safeguarding mtDNA integrity and preserving mtDNA function in the face of mutations, mitochondrial fusion is likely to be a protective factor in human disorders associated with mtDNA mutations.
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► Mice lacking mitochondrial fusion in skeletal muscle exhibit mitochondrial myopathy ► Fusion is essential for maintenance of mitochondrial DNA (mtDNA) levels ► Loss of mitochondrial fusion results in increased mtDNA mutations ► Fusion protects mitochondrial respiration in cells with high loads of mutated mtDNA
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the ...most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.
Mutations in the mitochondrial fusion gene
Mfn2 cause the human neurodegenerative disease Charcot-Marie-Tooth type 2A. However, the cellular basis underlying this relationship is poorly understood. ...By removing
Mfn2 from the cerebellum, we established a model for neurodegeneration caused by loss of mitochondrial fusion. During development and after maturity, Purkinje cells require Mfn2 but not Mfn1 for dendritic outgrowth, spine formation, and cell survival. In vivo, cell culture, and electron microscopy studies indicate that mutant Purkinje cells have aberrant mitochondrial distribution, ultrastructure, and electron transport chain activity. In fibroblasts lacking mitochondrial fusion, the majority of mitochondria lack mitochondrial DNA nucleoids. This deficiency provides a molecular mechanism for the dependence of respiratory activity on mitochondrial fusion. Our results show that exchange of mitochondrial contents is important for mitochondrial function as well as organelle distribution in neurons and have important implications for understanding the mechanisms of neurodegeneration due to perturbations in mitochondrial fusion.
The heteroaggregation rates of negatively charged multiwalled carbon nanotubes (CNTs) and positively charged hematite nanoparticles (HemNPs) were obtained over a broad range of nanoparticle ...distributions using time-resolved dynamic light scattering (DLS). Binary systems comprising CNTs and HemNPs were prepared using low ionic strength solutions to minimize the concurrent occurrence of homoaggregation. To elucidate the mechanisms of heteroaggregation, the structures of CNT–HemNP aggregates were observed using cryogenic transmission electron microscopy (cryo-TEM). An initial increase in the CNT concentration, while keeping the HemNP concentration constant, resulted in a corresponding increase in the rate of heteroaggregation, which occurred through the bridging of HemNPs by CNT strands. At the optimal CNT/HemNP mass concentration ratio (CNT/HemNP ratio) of 0.0316, the heteroaggregation rate reached 3.3 times of the HemNP homoaggregation rate in the diffusion-limited regime. Increasing the CNT/HemNP ratio above the optimal value, however, led to a dramatic decrease in the growth rate of heteroaggregates, likely through a blocking mechanism. In the presence of humic acid, the trends in the variation of the heteroaggregation rate with CNT/HemNP ratio were similar to that in the absence of humic acid. However, as the humic acid concentration was increased, the maximum aggregate growth rate decreased due to the lessening in the available surface of the HemNPs that CNTs can attach to through favorable electrostatic interaction.
The events that occur during the fusion of double-membraned mitochondria are unknown. As an essential step toward determining the mechanism of mitochondrial fusion, we have captured this event in ...vitro. Mitochondrial outer and inner membrane fusion events were separable and mechanistically distinct, but both required guanosine 5'-triphosphate hydrolysis. Homotypic trans interactions of the ancient outer transmembrane guanosine triphosphatase, Fzo1, were required to promote the fusion of mitochondrial outer membranes, whereas electrical potential was also required for fusion of inner membranes. Our conclusions provide fundamental insights into the molecular events driving mitochondrial fusion and advance our understanding of the evolution of mitochondrial fusion in eukaryotic cells.
Defects in mitochondrial fusion or fission are associated with many pathologies, raising the hope that pharmacological manipulation of mitochondrial dynamics may have therapeutic benefit. This ...approach assumes that organ physiology can be restored by rebalancing mitochondrial dynamics, but this concept remains to be validated. We addressed this issue by analyzing mice deficient in Mff, a protein important for mitochondrial fission. Mff mutant mice die at 13 wk as a result of severe dilated cardiomyopathy leading to heart failure. Mutant tissue showed reduced mitochondrial density and respiratory chain activity along with increased mitophagy. Remarkably, concomitant deletion of the mitochondrial fusion gene Mfn1 completely rescued heart dysfunction, life span, and respiratory chain function. Our results show for the first time that retuning the balance of mitochondrial fusion and fission can restore tissue integrity and mitochondrial physiology at the whole-organ level. Examination of liver, testis, and cerebellum suggest, however, that the precise balance point of fusion and fission is cell type specific.
The immediate responses to inhibition of phosphatidylcholine (PC) biosynthesis in yeast are altered phospholipid levels, slow growth, and defects in the morphology and localization of ER and ...mitochondria. With chronic lipid imbalance, yeast adapt. Lipid droplet (LD) biogenesis and conversion of phospholipids to triacylglycerol are required for restoring some phospholipids to near-wild-type levels. We confirmed that the unfolded protein response is activated by this lipid stress and find that Hsp104p is recruited to ER aggregates. We also find that LDs form at ER aggregates, contain polyubiquitinated proteins and an ER chaperone, and are degraded in the vacuole by a process resembling microautophagy. This process, microlipophagy, is required for restoration of organelle morphology and cell growth during adaptation to lipid stress. Microlipophagy does not require ATG7 but does requires ESCRT components and a newly identified class E VPS protein that localizes to ER and is upregulated by lipid imbalance.
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•Phospholipid imbalance results in morphology defects of ER and mitochondria•Excess lipids stimulate lipid droplet (LD) biogenesis at ER aggregates•LDs are associated with damaged proteins and are degraded in the vacuole•LD delivery to vacuole requires ESCRT proteins and a regulator of ESCRT Ylr312p
Phosphatidylcholine biosynthesis defects produce phospholipid imbalance and ER stress. Vevea et al. find they also cause aberrant organelle morphology and distribution of mitochondria and ER in yeast. Lipid droplets form at ER aggregates and remove excess lipids and potentially unfolded proteins from the ER for degradation by microautophagy-like, ATG7-independent microlipophagy.
Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many ...of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies.
Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases--OPA1 and the mitofusins Mfn1 and Mfn2--are essential for the fusion of mammalian ...mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast.
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