Structural implications of mitochondrial dynamics

Mitochondrial components are continuously distributed throughout the whole chondriome of a cell by fusion and fission. Thus, a single mitochondrion represents a transient fraction of the chondriome. Mitochondrial dynamics are responsible for intracellular distribution and reaction of mitochondria to functional requirements. Dynamics occur on different levels: overall morphology, inner membrane-matrix compartment, turnover and rearrangements of mitochondrial proteins and DNA. Electron micrographs of serial sections of human umbilical vein endothelial cells reveal perinuclear mitochondria of extreme length and with branches in those cells that also have short peripheral mitochondria. Interactions of mitochondria with cytoskeletal elements are revealed in cells treated with cytochalasin D to destroy actin fibrillar structures or after disassembling microtubule by nocodazole. In the latter case mitochondria not only become immobilized, they also acquire a multiple ring structure. In F-actin-disturbed cells, motility (shape changes in particular) is increased and the mitochondria become elongated. Mechanisms of how F-actin might render mitochondria immobile may involve dynamin-related protein 1 (DRP1) or interaction with anion channels. This may be responsible for the lack of mitochondrial motility in senescent cells. Fusion between mitochondria revealed local fluctuations of mitochondrial red fluorescent protein (mtRFP), indicating novel fast inner membrane reorganizations. Mitochondrial dynamics result from a complex interplay between the molecular organization of the inner membrane-matrix complex and cytoskeletal elements outside.     

Ca-dependent regulation of mitochondrial dynamics by the Miro–Milton complex

Calcium oscillations control mitochondrial motility along the microtubules and in turn, support on-demand distribution of mitochondria. However, the mechanism mediating the Ca²⁺ effect remained a mystery. Recently, several papers reported on the Ca²⁺-dependent regulation of mitochondrial dynamics by a Miro–Milton complex linking mitochondria to kinesin motors. Both mitochondrial motility and fusion–fission dynamics seem to be sensitive to a Ca²⁺-dependent switch by this complex. Evidence is emerging that calcium signaling through Miro–Milton is central to coordination of the local oxidative metabolism with the energy demands and protection against Ca²⁺-induced cell injury.     

Opening the doors to cytochrome c: Changes in mitochondrial shape and apoptosis

Mitochondria are key organelles in the regulation of apoptosis induced by intrinsic stimuli. This is accomplished by the release in the cytoplasm of cytochrome c and of other cofactors that ensure the activation of effector caspases. Multiple changes in the shape of the organelle occur around the time of the release of these factors, including fragmentation of the mitochondrial network and the activation of the so-called “cristae remodeling” pathway. However, contrasting evidence exist on the functional role of these changes. Here we review the molecular mechanisms that control mitochondrial shape, their changes during apoptosis and the role that these changes might play in the amplification of the apoptotic cascade.     

The regulation of mitochondrial morphology: intricate mechanisms and dynamic machinery

Mitochondria typically form a reticular network radiating from the nucleus, creating an interconnected system that supplies the cell with essential energy and metabolites. These mitochondrial networks are regulated through the complex coordination of fission, fusion and distribution events. While a number of key mitochondrial morphology proteins have been identified, the precise mechanisms which govern their activity remain elusive. Moreover, post translational modifications including ubiquitination, phosphorylation and sumoylation of the core machinery are thought to regulate both fusion and division of the network. These proteins can undergo several different modifications depending on cellular signals, environment and energetic demands of the cell. Proteins involved in mitochondrial morphology may also have dual roles in both dynamics and apoptosis, with regulation of these proteins under tight control of the cell to ensure correct function. The absolute reliance of the cell on a functional mitochondrial network is highlighted in neurons, which are particularly vulnerable to any changes in organelle dynamics due to their unique biochemical requirements. Recent evidence suggests that defects in the shape or distribution of mitochondria correlate with the progression of neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's disease. This review focuses on our current understanding of the mitochondrial morphology machinery in cell homeostasis, apoptosis and neurodegeneration, and the post translational modifications that regulate these processes.     

Mitochondrial dynamics: Shaping and remodeling an organelle network

Mitochondria form networks that continually remodel and adapt to carry out their cellular function. The mitochondrial network is remodeled through changes in mitochondrial morphology, number, and distribution within the cell. Mitochondrial dynamics depend directly on fission, fusion, shape transition, and transport or tethering along the cytoskeleton. Over the past several years, many of the mechanisms underlying these processes have been uncovered. It has become clear that each process is precisely and contextually regulated within the cell. Here, we discuss the mechanisms regulating each aspect of mitochondrial dynamics, which together shape the network as a whole.     

Insights into amyloid-beta-induced mitochondrial dysfunction in Alzheimer disease

Amyloid-β has long been implicated in the pathogenesis of Alzheimer disease. The focus was initially on the extracellular fibrillar deposits of amyloid-β but more recently has shifted to intracellular oligomeric forms of amyloid-β. Unfortunately, the mechanism(s) by which either extracellular or intracellular amyloid-β induces neuronal toxicity remains unclear. That said, a number of recent studies indicate that mitochondria might be an important target of amyloid-β. Neurons rely heavily on mitochondria for energy and it is well established that mitochondrial dysfunction might be an important target of amyloid-β. Mechanistically, amyloid-β aggregates in mitochondria to impair function, leading to energy hypometabolism and elevated reactive oxygen species production. Additionally, amyloid-β affects the balance of mitochondrial fission/fusion and mitochondrial transport, negatively impacting a host of cellular functions of neurons. Here, we review the role that amyloid-β plays in mitochondrial structure and function of neurons and the importance of this in the pathogenesis of Alzheimer disease.     

线粒体分裂、融合与细胞凋亡

线粒体是高度动态变化的细胞器,其在细胞内不断分裂、融合并形成网状结构。线粒体的分裂和融合是由多种蛋白质精确调控完成的。Drp1／Dnm1p,Fis1／Fis1p,Caf4p和Mdv1p参与线粒体分裂的调控;Mfn1／2／Fzo1p控制线粒体外膜的融合,而Mgm1p／OPA1则参与线粒体内膜的融合。在细胞凋亡过程中线粒体片段化,网状结构被破坏,线粒体嵴发生重构,抑制这一过程可以部分抑制细胞色素c的释放和细胞凋亡。线粒体形态对于细胞维持正常生理代谢和机体发育起着重要的作用,一旦出现障碍会导致严重的疾病。     

The role of PTEN-induced kinase 1 in mitochondrial dysfunction and dynamics

Mutations in parkin, PTEN-induced kinase 1 (PINK1) and DJ-1 can all cause autosomal recessive forms of Parkinson's disease. Recent data suggest that these recessive parkinsonism-associated genes converge within a single pathogenic pathway whose dysfunction leads to the loss of substantia nigra pars compacta neurons. The major common functional effects of all three genes relate to mitochondrial and oxidative damage, with a possible additional involvement of the ubiquitin proteasome system. This review highlights the role of the mitochondrial kinase, PINK1, in protection against mitochondrial dysfunction and how this might relate to loss of substantia nigra neurons in recessive parkinsonism.     

Mitochondrial clustering induced by overexpression of the mitochondrial fusion protein Mfn2 causes mitochondrial dysfunction and cell death

Mitochondria change their shapes dynamically mainly through fission and fusion. Dynamin-related GTPases have been shown to mediate remodeling of mitochondrial membranes during these processes. One of these GTPases, mitofusin, is anchored at the outer mitochondrial membrane and mediates fusion of the outer membrane. We found that overexpression of a mitofusin isoform, Mfn2, drastically changes mitochondrial morphology, forming mitochondrial clusters. High-resolution microscopic examination indicated that the mitochondrial clusters consisted of small fragmented mitochondria. Inhibiting mitochondrial fission prevented the cluster formation, supporting the notion that mitochondrial clusters are formed by fission-mediated mitochondrial fragmentation and aggregation. Mitochondrial clusters displayed a decreased inner membrane potential and mitochondrial function, suggesting a functional compromise of small fragmented mitochondria produced by Mfn2 overexpression; however, mitochondrial clusters still retained mitochondrial DNA. We found that cells containing clustered mitochondria lost cytochrome c from mitochondria and underwent caspase-mediated apoptosis. These results demonstrate that mitochondrial deformation impairs mitochondrial function, leading to apoptotic cell death and suggest the presence of an intricate form-function relationship in mitochondria.     

Laminar shear stress regulates mitochondrial dynamics,bioenergetics responses and PRX3 activation in endothelial cells

Endothelial cells in the vascular system are constantly subjected to the frictional force of shear stress due to the pulsatile nature of blood flow. Although several proteins form part of the shear stress mechano-sensing pathway, the identification of mechano-transducing pathways is largely unknown. Given the increasing evidence for a signaling function of mitochondria in endothelial cells, the aim of this study was to investigate their role as mechano-sensor organelles during laminar shear stress (LSS). We demonstrated that LSS activates intracellular signaling pathways that modulate not only mitochondrial dynamics but also mitochondrial function. At early time points of LSS, the fission-related protein Drp1 was recruited from the cytosol to mitochondria and activated mitochondrial fission. LSS-dependent increase in intracellular Ca^2 + concentration was indispensable for mitochondrial fission. As alterations in mitochondrial dynamics have been related to changes in bioenergetics profiles, we studied mitochondrial function after LSS. We found that LSS decreased respiration rate, increased mitochondrial membrane potential and promoted the mitochondrial generation of ROS with the subsequent oxidation and activation of the antioxidant enzyme PRX3. Our data support a novel and active role for mitochondria in endothelial cells as active players, able to transduce the mechanical force of shear stress in the vascular endothelium into a biological response.     

Mitochondrial fusion, fission and autophagy as a quality control axis: The bioenergetic view

The mitochondrial life cycle consists of frequent fusion and fission events. Ample experimental and clinical data demonstrate that inhibition of either fusion or fission results in deterioration of mitochondrial bioenergetics. While fusion may benefit mitochondrial function by allowing the spreading of metabolites, protein and DNA throughout the network, the functional benefit of fission is not as intuitive. Remarkably, studies that track individual mitochondria through fusion and fission found that the two events are paired and that fusion triggers fission. On average each mitochondrion would go though ~ 5 fusion:fission cycles every hour. Measurement of Δψ_m during single fusion and fission events demonstrates that fission may yield uneven daughter mitochondria where the depolarized daughter is less likely to become involved in a subsequent fusion and is more likely to be targeted by autophagy. Based on these observations we propose a mechanism by which the integration of mitochondrial fusion, fission and autophagy forms a quality maintenance mechanism. According to this hypothesis pairs of fusion and fission allow for the reorganization and sequestration of damaged mitochondrial components into daughter mitochondria that are segregated from the networking pool and then becoming eliminated by autophagy.     

Nuclear-encoded NCX3 and AKAP121: Two novel modulators of mitochondrial calcium efflux in normoxic and hypoxic neurons

Mitochondria are highly dynamic organelles extremely important for cell survival. Their structure resembles that of prokaryotic cells since they are composed with two membranes, the inner (IMM) and the outer mitochondrial membrane (OMM) delimitating the intermembrane space (IMS) and the matrix which contains mitochondrial DNA (mtDNA). This structure is strictly related to mitochondrial function since they produce the most of the cellular ATP through the oxidative phosphorylation which generate the electrochemical gradient at the two sides of the inner mitochondrial membrane an essential requirement for mitochondrial function. Cells of highly metabolic demand like those composing muscle, liver and brain, are particularly dependent on mitochondria for their activities. Mitochondria undergo to continual changes in morphology since, they fuse and divide, branch and fragment, swell and extend. Importantly, they move throughout the cell to deliver ATP and other metabolites where they are mostly required. Along with the capability to control energy metabolism, mitochondria play a critical role in the regulation of many physiological processes such as programmed cell death, autophagy, redox signalling, and stem cells reprogramming. All these phenomena are regulated by Ca²⁺ ions within this organelle. This review will discuss the molecular mechanisms regulating mitochondrial calcium cycling in physiological and pathological conditions with particular regard to their impact on mitochondrial dynamics and function during ischemia. Particular emphasis will be devoted to the role played by NCX3 and AKAP121 as new molecular targets for mitochondrial function and dysfunction.     

IP3 receptor-mediated Ca2+ release from acidocalcisomes regulates mitochondrial bioenergetics and prevents autophagy in Trypanosoma cruzi

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIP₃R) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIP₃R is a Ca²⁺ release channel gated by IP₃ when expressed in DT40 cells knockout for all vertebrate IP₃ receptors, and is required for Ca²⁺ uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O₂ consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIP₃R by CRISPR/Cas9 genome editing caused: a) a reduction in O₂ consumption rate and citrate synthase activity; b) decreased mitochondrial Ca²⁺ transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIP₃R overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIP₃R-mediated acidocalcisome Ca²⁺ release on cell bioenergetics in T. cruzi.     

MKK3 deletion improves mitochondrial quality

Sepsis, a severe response to infection, leads to excessive inflammation and is the major cause of mortality in intensive care units. Mitochondria have been shown to influence the outcome of septic injury. We have previously shown that MAP kinase kinase 3 (MKK3)^−/− mice are resistant to septic injury and MKK3^−/− macrophages have improved mitochondrial function. In this study we examined processes that lead to improved mitochondrial quality in MKK3^−/− mouse embryonic fibroblasts (MEFs) and specifically the role of mitophagy in mitochondrial health. MKK3^−/− MEFs had lower inflammatory cytokine release and oxidant production after lipopolysaccharide (LPS) stimulation, confirming our earlier observations. MKK3^−/− MEFs had better mitochondrial function as measured by mitochondrial membrane potential (MMP) and ATP, even after LPS treatment. We observed higher mitophagy in MKK3^−/− MEFs compared to wild type (WT). Transmission electron microscopy studies showed longer and larger mitochondria in MKK3^−/− MEFs, indicative of healthier mitochondria. We performed a SILAC (stable isotope labeling by/with amino acids in cell culture) study to assess differences in mitochondrial proteome between WT and MKK3^−/− MEFs and observed increased expression of tricarboxylic acid (TCA) cycle enzymes and respiratory complex subunits. Further, inhibition of mitophagy by Mdivi1 led to loss in MMP and increased cytokine secretion after LPS treatment in MKK3^−/− MEFs. In conclusion, this study demonstrates that MKK3 influences mitochondrial quality by affecting the expression of mitochondrial proteins, including TCA cycle enzymes, and mitophagy, which consequently regulates the inflammatory response. Based on our results, MKK3 could be a potential therapeutic target for inflammatory diseases like sepsis.     

Mitochondrial dynamics in heart cells: Very low amplitude high frequency fluctuations in adult cardiomyocytes and flow motion in non beating Hl-1 cells

The arrangement and movement of mitochondria were quantitatively studied in adult rat cardiomyocytes and in cultured continuously dividing non beating (NB) HL-1 cells with differentiated cardiac phenotype. Mitochondria were stained with MitoTracker® Green and studied by fluorescent confocal microscopy. High speed scanning (one image every 400 ms) revealed very rapid fluctuation of positions of fluorescence centers of mitochondria in adult cardiomyocytes. These fluctuations followed the pattern of random walk movement within the limits of the internal space of mitochondria, probably due to transitions between condensed and orthodox configurational states of matrix and inner membrane. Mitochondrial fusion or fission was seen only in NB HL-1 cells but not in adult cardiomyocytes. In NB HL-1 cells, mitochondria were arranged as a dense tubular network, in permanent fusion, fission and high velocity displacements of ~90 nm/s. The differences observed in mitochondrial dynamics are related to specific structural organization and mitochondria-cytoskeleton interactions in these cells.     

Redox regulation of mitophagy in the lung during murine Staphylococcus aureus sepsis

Oxidative mitochondrial damage is closely linked to inflammation and cell death, but low levels of reactive oxygen and nitrogen species serve as signals that involve mitochondrial repair and resolution of inflammation. More specifically, cytoprotection relies on the elimination of damaged mitochondria by selective autophagy (mitophagy) during mitochondrial quality control. This aim of this study was to identify and localize mitophagy in the mouse lung as a potentially upregulatable redox response to Staphylococcus aureus sepsis. Fibrin clots loaded with S. aureus (1×10⁷ CFU) were implanted abdominally into anesthetized C57BL/6 and B6.129X1-Nfe2l2tm1Ywk/J (Nrf2^−/−) mice. At the time of implantation, mice were given vancomycin (6 mg/kg) and fluid resuscitation. Mouse lungs were harvested at 0, 6, 24, and 48 h for bronchoalveolar lavage (BAL), Western blot analysis, and qRT-PCR. To localize mitochondria with autophagy protein LC3, we used lung immunofluorescence staining in LC3–GFP transgenic mice. In C57BL/6 mice, sepsis-induced pulmonary inflammation was detected by significant increases in mRNA for the inflammatory markers IL-1β and TNF-α at 6 and 24 h, respectively. BAL cell count and protein also increased. Sepsis suppressed lung Beclin-1 protein, but not mRNA, suggesting activation of canonical autophagy. Notably sepsis also increased the LC3-II autophagosome marker, as well as the lung׳s noncanonical autophagy pathway as evidenced by loss of p62, a redox-regulated scaffolding protein of the autophagosome. In LC3–GFP mouse lungs, immunofluorescence staining showed colocalization of LC3-II to mitochondria, mainly in type 2 epithelium and alveolar macrophages. In contrast, marked accumulation of p62, as well as attenuation of LC3-II in Nrf2-knockout mice supported an overall decrease in autophagic turnover. The downregulation of canonical autophagy during sepsis may contribute to lung inflammation, whereas the switch to noncanonical autophagy selectively removes damaged mitochondria and accompanies tissue repair and cell survival. Furthermore, mitophagy in the alveolar region appears to depend on activation of Nrf2. Thus, efforts to promote mitophagy may be a useful therapeutic adjunct for acute lung injury in sepsis.     

Impaired mitochondrial bioenergetics determines glutamate-induced delayed calcium deregulation in neurons

Background

Accumulation of glutamate in ischaemic CNS is thought to amplify neuronal death during a stroke. Exposure of neurons to toxic glutamate concentrations causes an initial transient increase in [Ca²⁺]_c followed by a delayed increase commonly termed delayed [Ca²⁺]_c deregulation (DCD).

Methods

We have used fluorescence imaging techniques to explore differences in glutamate-induced DCD in rat hippocampal neurons after different periods of time in culture (days in vitro; DIV).

Results

The amplitude of both the initial [Ca²⁺]_c signal and the number of cells showing DCD in response to glutamate increased with the duration of culture. The capacity of mitochondria to accumulate calcium in permeabilised neurons decreased with time in culture, although mitochondrial membrane potential at rest did not change. The rate of ATP consumption, measured as an increase in [Mg²⁺]_c following inhibition of ATP synthesis, was lower in ‘young’ neurons. The sensitivity of ‘young’ neurons to glutamate-induced DCD approximated to that of ‘old’ neurons when mitochondrial function was impaired using either FCCP or oligomycin. Further, following such treatment, cells showed a DCD-like response to increased [Ca²⁺]_c induced by KCl induced depolarisation which was never otherwise seen.

General significance

Thus, changes in cellular bioenergetics dictate the onset of DCD in response to glutamate.     

Intersection between Mitochondrial Permeability Pores and Mitochondrial Fusion/Fission

The goal of this review is to highlight recent developments in the field of mitochondrial membrane processes, which provide new insights into the relation between mitochondrial fission/fusion events and the mitochondrial permeability transition (MPT). First, we distinguish between pore opening events at the inner and outer mitochondrial membranes. Inner membrane pore opening, or iMPT, leads to membrane depolarization, release of low molecular weight compounds, cristae reorganization and matrix swelling. Outer membrane pore opening, or oMPT, allows partial release of apoptotic proteins, while complete release requires additional remodeling of inner membrane cristae. Second, we summarize recent data that supports a similar temporal and physical separation between inner and outer mitochondrial membrane fusion events. Finally, we focus on cristae remodeling, which may be the intersection between oMPT and iMPT events. Interestingly, components of fusion machinery, such as mitofusin 2 and OPA1, appear to play a role in cristae remodeling as well. Special issue dedicated to John P. Blass.     

Regulation of mitochondrial dehydrogenases by calcium ions

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.