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The mitochondrial contact site and cristae organizing system (MICOS) is a multisubunit protein complex that is essential for the proper architecture of the mitochondrial inner membrane. MICOS plays a key role in establishing and maintaining crista junctions, tubular or slit-like structures that connect the cristaemembrane with the inner boundary membrane, thereby ensuring a contiguous inner membrane. MICOS is enriched at crista junctions, but the detailed distribution of its subunits around crista junctions is unclear because such small length scales are inaccessible with established fluorescence microscopy. By targeting individually activated fluorophores with an excitation beam featuring a central zero-intensity point, the nanoscopy method called MINFLUX delivers single-digit nanometer-scale three-dimensional (3D) resolution and localization precision. We employed MINFLUX nanoscopy to investigate the submitochondrial localization of the core MICOS subunit Mic60 in relation to two other MICOS proteins, Mic10 and Mic19. We demonstrate that dual-color 3D MINFLUX nanoscopy is applicable to the imaging of organellar substructures, yielding a 3D localization precision of ∼5 nm in human mitochondria. This isotropic precision facilitated the development of an analysis framework that assigns localization clouds to individual molecules, thus eliminating a source of bias when drawing quantitative conclusions from single-molecule localization microscopy data. MINFLUX recordings of Mic60 indicate ringlike arrangements of multiple molecules with a diameter of 40 to 50 nm, suggesting that Mic60 surrounds individual crista junctions. Statistical analysis of dual-color MINFLUX images demonstrates that Mic19 is generally in close proximity to Mic60, whereas the spatial coordination of Mic10 with Mic60 is less regular, suggesting structural heterogeneity of MICOS.

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block‐face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase‐quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age‐related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age‐related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue‐dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals. These studies have provided a more comprehensive understanding of the structural and functional changes associated with aging and loss of the MICOS complex. Together, this verifies there is an age‐related loss of both mitochondria structure and the MICOS complex, and shows that the MICOS complex affects the mitochondrial structure, thus suggesting that the MICOS complex plays an evolutionarily conserved role in age‐dependent mitochondrial structural and functional decline.

Mitochondria are organelles of endosymbiotic origin, surrounded by two membranes. The inner membrane forms invaginations called cristae that enhance its surface and are important for mitochondrial function. A recently described mitochondrial contact site and cristae organizing system (MICOS) in the inner mitochondrial membrane is crucial for the formation and maintenance of cristae structure. The MICOS complex in human mitochondria exhibits specificities and greater complexity in comparison to the yeast system. Many subunits of this complex have been previously described, but several others and their function remain to be explored. This review will summarize our present knowledge about the human MICOS complex and its constituents, while discussing the future research perspectives in this exciting and important field.

Mitochondria are the respiratory organelles of eukaryotes and their evolutionary history is deeply intertwined with that of eukaryotes. The compartmentalization of respiration in mitochondria occurs within cristae, whose evolutionary origin has remained unclear. Recent discoveries, however, have revived the old notion that mitochondrial cristae could have had a pre-endosymbiotic origin. Mitochondrial cristae are likely homologous to the intracytoplasmic membranes (ICMs) used by diverse alphaproteobacteria for harnessing energy. Because the Mitochondrial Contact site and Cristae Organizing System (MICOS) that controls the development of cristae evolved from a simplified version that is phylogenetically restricted to Alphaproteobacteria (alphaMICOS), ICMs most probably transformed into cristae during the endosymbiotic origin of mitochondria. This inference is supported by the sequence and structural similarities between MICOS and alphaMICOS, and the expression pattern and cellular localization of alphaMICOS. Given that cristae and ICMs develop similarly, alphaMICOS likely functions analogously to mitochondrial MICOS by culminating ICM development with the creation of tubular connections and membrane contact sites at the alphaproteobacterial envelope. Mitochondria thus inherited a pre-existing ultrastructure adapted to efficient energy transduction from their alphaproteobacterial ancestors. The widespread nature of purple bacteria among alphaproteobacteria raises the possibility that cristae evolved from photosynthetic ICMs.

Mitochondria are tubular double-membrane organelles essential for eukaryotic life. They form extended networks and exhibit an intricate inner membrane architecture. The MICOS (mitochondrial contact site and cristae organizing system) complex, crucial for proper architecture of the mitochondrial inner membrane, is localized primarily at crista junctions. Harnessing superresolution fluorescence microscopy, we demonstrate that Mic60, a subunit of the MICOS complex, as well as several of its interaction partners are arranged into intricate patterns in human and yeast mitochondria, suggesting an ordered distribution of the crista junctions. We show that Mic60 forms clusters that are preferentially localized in the inner membrane at two opposing sides of the mitochondrial tubules so that they form extended opposing distribution bands. These Mic60 distribution bands can be twisted, resulting in a helical arrangement. Focused ion beam milling-scanning electron microscopy showed that in yeast the twisting of the opposing distribution bands is echoed by the folding of the inner membrane. We show that establishment of the Mic60 distribution bands is largely independent of the cristae morphology. We suggest that Mic60 is part of an extended multiprotein interaction network that scaffolds mitochondria.

Mitochondria are organelles with a complex architecture. They are bounded by an envelope consisting of the outer membrane and the inner boundary membrane (IBM). Narrow crista junctions (CJs) link the IBM to the cristae. OMs and IBMs are firmly connected by contact sites (CS). The molecular nature of the CS remained unknown. Using quantitative high‐resolution mass spectrometry we identified a novel complex, the mi tochondrial co ntact s ite (MICOS) complex, formed by a set of mitochondrial membrane proteins that is essential for the formation of CS. MICOS is preferentially located at the CJs. Upon loss of one of the MICOS subunits, CJs disappear completely or are impaired, showing that CJs require the presence of CS to form a superstructure that links the IBM to the cristae. Loss of MICOS subunits results in loss of respiratory competence and altered inheritance of mitochondrial DNA. The outer and inner mitochondrial membranes are physically linked. Quantitative high resolution mass spectrometry now identifies the molecular nature of the Mitochondrial Contact Site complex (MICOS). MICOS is required for crista junctions formation, respiration and mitochondrial DNA inheritance.

Background Binding of the programmed death-1 (PD-1) receptor to its ligands (PD-L1/2) transduces inhibitory signals that promote exhaustion of activated T cells. Blockade of the PD-1 pathway is widely used for cancer treatment, yet the inhibitory signals transduced by PD-1 in T cells remain elusive. Methods Expression profiles of human CD8 + T cells in resting, activated (CD3 + CD28) and PD-1-stimulated cells (CD3 + CD28 + PD-L1-Fc) conditions were evaluated by RNA-seq. Bioinformatic analyses were used to identify signaling pathways differentially regulated in PD-1-stimulated cells. Metabolic analyses were performed with SeaHorse technology, and mitochondrial ultrastructure was determined by transmission electron microscopy. PD-1-regulated mitochondrial genes were silenced using short-hairpin RNA in primary cells. Blue native gel electrophoresis was used to determine respiratory supercomplex assembly. Results PD-1 engagement in human CD8 + T cells triggers a specific, progressive genetic program different from that found in resting cells. Gene ontology identified metabolic processes, including glycolysis and oxidative phosphorylation (OXPHOS), as the main pathways targeted by PD-1. We observed severe functional and structural alterations in the mitochondria of PD-1-stimulated cells, including a reduction in the number and length of mitochondrial cristae. These cristae alterations were associated with reduced expression of CHCHD3 and CHCHD10, two proteins that form part of the mitochondrial contact site and cristae organizing system (MICOS). Although PD-1-stimulated cells showed severe cristae alterations, assembly of respiratory supercomplexes was unexpectedly greater in these cells than in activated T cells. CHCHD3 silencing in primary CD8 + T cells recapitulated some effects induced by PD-1 stimulation, including reduced mitochondrial polarization and interferon-γ production following T cell activation with anti-CD3 and -CD28 activating antibodies. Conclusions Our results suggest that mitochondria are the main targets of PD-1 inhibitory activity. PD-1 reprograms CD8 + T cell metabolism for efficient use of fatty acid oxidation; this mitochondrial phenotype might explain the long-lived phenotype of PD-1-engaged T cells.

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.

Abstract Cristae enclose respiratory chain complexes, making them the bioenergetic subcompartments of mitochondria. The Mitochondrial contact site and Cristae Organizing System (MICOS) complex is among the inducers of membrane curvature needed for crista formation. Resembling the respiratory chain complexes, MICOS is organized around a core protein, the mitofilin-domain bearing Mic60, that was inherited from the alphaproteobacterial progenitor of mitochondria. Extant alphaproteobacteria express Mic60 to form their own bioenergetic subcompartments, demonstrating the permeance of Mic60's form and function during prokaryotic and eukaryotic evolution. Yet, unlike virtually all aerobic eukaryotes, Mic60 is not encoded within the genomes of the multifarious protists that comprise the phylum Euglenozoa, including trypanosomes. Here, we show that Mic60 has been replaced in euglenozoans by two cryptic mitofilin domain-containing MICOS subunits, Mic34 and Mic40. Contrasting alphaproteobacterial and mitochondrial Mic60, these are not integral membrane proteins. Mic34 and Mic40 are as diverged from each other as both are to canonical Mic60. Reverse genetics revealed they are intertwined with the oxidative protein folding pathway required for mitochondrial–and crista–biogenesis, veiling a potential membrane remodeling role. Nevertheless, Mic34 binds phospholipid bilayers in vitro. Mic34 and Mic40 heterologous expression remodels gammaproteobacterial cytoplasmic membranes, like Mic60. Unexpectedly, Mic34 overexpression elaborates the simplified tubular mitochondrion of a Trypanosoma brucei life cycle stage with repressed oxidative phosphorylation. Furthermore, this activity was ablated by mutations to Mic34's mitofilin domain that correspond to essential motifs found in yeast Mic60's mitofilin domain. Thus, the mitofilin protein family is more diverse than originally supposed, with two of its structurally most divergent members altering the core of euglenozoan MICOS.

Pathogenic bacteria can target host cell organelles to take control of key cellular processes and promote their intracellular survival, growth, and persistence. Mitochondria are essential, highly dynamic organelles with pivotal roles in a wide variety of cell functions. Mitochondrial dynamics and function are intimately linked. Our previous research showed that Listeria monocytogenes infection impairs mitochondrial function and triggers fission of the mitochondrial network at an early infection stage, in a process that is independent of the presence of the main mitochondrial fission protein Drp1. Here, we analyzed how mitochondrial proteins change in response to L. monocytogenes infection and found that infection raises the levels of Mic10, a mitochondrial inner membrane protein involved in formation of cristae. We show that Mic10 is important for L. monocytogenes -dependent mitochondrial fission and infection of host cells. Our findings thus offer new insight into the mechanisms used by L. monocytogenes to hijack mitochondria to optimize host infection. Mitochondrial function adapts to cellular demands and is affected by the ability of the organelle to undergo fusion and fission in response to physiological and nonphysiological cues. We previously showed that infection with the human bacterial pathogen Listeria monocytogenes elicits transient mitochondrial fission and a drop in mitochondrion-dependent energy production through a mechanism requiring the bacterial pore-forming toxin listeriolysin O (LLO). Here, we performed quantitative mitochondrial proteomics to search for host factors involved in L. monocytogenes -induced mitochondrial fission. We found that Mic10, a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex, is significantly enriched in mitochondria isolated from cells infected with wild-type but not with LLO-deficient L. monocytogenes . Increased mitochondrial Mic10 levels did not correlate with upregulated transcription, suggesting a posttranscriptional mechanism. We then showed that Mic10 is necessary for L. monocytogenes -induced mitochondrial network fragmentation and that it contributes to L. monocytogenes cellular infection independently of MICOS proteins Mic13, Mic26, and Mic27. In conclusion, investigation of L. monocytogenes infection allowed us to uncover a role for Mic10 in mitochondrial fission. IMPORTANCE Pathogenic bacteria can target host cell organelles to take control of key cellular processes and promote their intracellular survival, growth, and persistence. Mitochondria are essential, highly dynamic organelles with pivotal roles in a wide variety of cell functions. Mitochondrial dynamics and function are intimately linked. Our previous research showed that Listeria monocytogenes infection impairs mitochondrial function and triggers fission of the mitochondrial network at an early infection stage, in a process that is independent of the presence of the main mitochondrial fission protein Drp1. Here, we analyzed how mitochondrial proteins change in response to L. monocytogenes infection and found that infection raises the levels of Mic10, a mitochondrial inner membrane protein involved in formation of cristae. We show that Mic10 is important for L. monocytogenes -dependent mitochondrial fission and infection of host cells. Our findings thus offer new insight into the mechanisms used by L. monocytogenes to hijack mitochondria to optimize host infection.