ABSTRACT
MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, providing photon-efficient fluorophore localizations, has brought about three-dimensional resolution at nanometer scales. However, by using an intrinsic on-off switching process for single fluorophore separation, initial MINFLUX implementations have been limited to two color channels. Here we show that MINFLUX can be effectively combined with sequentially multiplexed DNA-based labeling (DNA-PAINT), expanding MINFLUX nanoscopy to multiple molecular targets. Our method is exemplified with three-color recordings of mitochondria in human cells.
Subject(s)
DNA , Fluorescent Dyes , Humans , Microscopy, Fluorescence/methods , Mitochondria , PhotonsABSTRACT
The ion pump Na+,K+-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na+,K+-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg2+. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na+,K+-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na+,K+-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.
Subject(s)
Epilepsy/genetics , Mutation, Missense , Sodium-Potassium-Exchanging ATPase/genetics , Animals , Anticonvulsants/pharmacology , Brain/drug effects , Brain/metabolism , Brain/pathology , Cells, Cultured , Drug Resistance , Epilepsy/drug therapy , Epilepsy/pathology , Humans , Infant , Molecular Dynamics Simulation , Mutation, Missense/drug effects , Protein Subunits/analysis , Protein Subunits/genetics , Sodium-Potassium-Exchanging ATPase/analysis , XenopusABSTRACT
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.
Subject(s)
Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Humans , Saccharomycetales/metabolismABSTRACT
The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells.
Subject(s)
Apoptosis , Mitochondria/enzymology , Mitochondrial Membranes/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Protein Multimerization , bcl-2-Associated X Protein/metabolism , Cell Line , Cytochromes c/metabolism , Humans , Microscopy, Fluorescence , Mitochondria/physiology , Mitochondrial Membranes/physiology , PermeabilityABSTRACT
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.
Subject(s)
Microscopy, Fluorescence/methods , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Multiprotein Complexes/metabolism , Muscle Proteins/metabolism , Animals , Chlorocebus aethiops , Fibroblasts , HeLa Cells , Humans , Microscopy, Electron , Microscopy, Immunoelectron , Mitochondrial Membranes/ultrastructure , Nanotechnology , Saccharomyces cerevisiae , Vero CellsABSTRACT
One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same spatiotemporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wild type cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK- single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings in both wild type as well as single-knockout cells corroborates the toroidal apoptotic pore model.
Subject(s)
Apoptosis , Mitochondria , bcl-2 Homologous Antagonist-Killer Protein , bcl-2-Associated X Protein , Animals , Humans , Mice , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/genetics , Mitochondria/metabolism , Mitochondrial Membranes/metabolismABSTRACT
Mitochondria are highly dynamic organelles that interchange their contents mediated by fission and fusion. However, it has previously been shown that the mitochondria of cultured human epithelial cells exhibit a gradient in the relative abundance of several proteins, with the perinuclear mitochondria generally exhibiting a higher protein abundance than the peripheral mitochondria. The molecular mechanisms that are required for the establishment and the maintenance of such inner-cellular mitochondrial protein abundance gradients are unknown. We verified the existence of inner-cellular gradients in the abundance of clusters of the mitochondrial outer membrane protein Tom20 in the mitochondria of kidney epithelial cells from an African green monkey (Vero cells) using STED nanoscopy and confocal microscopy. We found that the Tom20 gradients are established immediately after cell division and require the presence of microtubules. Furthermore, the gradients are abrogated in hyperfused mitochondrial networks. Our results suggest that inner-cellular protein abundance gradients from the perinuclear to the peripheral mitochondria are established by the trafficking of individual mitochondria to their respective cellular destination.
ABSTRACT
The mitochondrial presequence translocation machinery (TIM23 complex) is conserved between the yeast Saccharomyces cerevisiae and humans; however, functional characterization has been mainly performed in yeast. Here, we define the constituents of the human TIM23 complex using mass spectrometry and identified ROMO1 as a new translocase constituent with an exceptionally short half-life. Analyses of a ROMO1 knockout cell line revealed aberrant inner membrane structure and altered processing of the GTPase OPA1. We show that in the absence of ROMO1, mitochondria lose the inner membrane YME1L protease, which participates in OPA1 processing and ROMO1 turnover. While ROMO1 is dispensable for general protein import along the presequence pathway, we show that it participates in the dynamics of TIM21 during respiratory chain biogenesis and is specifically required for import of YME1L. This selective import defect can be linked to charge distribution in the unusually long targeting sequence of YME1L. Our analyses establish an unexpected link between mitochondrial protein import and inner membrane protein quality control.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Gene Knockdown Techniques , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Metalloendopeptidases/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Protein Transport/physiology , Saccharomyces cerevisiaeABSTRACT
Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space toward the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the mitochondrial contact site and cristae organizing system (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria, the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane.
Subject(s)
Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Protein Interaction Maps , Carrier Proteins/metabolism , HEK293 Cells , Humans , Mitochondrial Precursor Protein Import Complex Proteins , Proteomics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.
Subject(s)
Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Protein Interaction Maps , Proteome/metabolism , Breast/pathology , Breast Neoplasms/genetics , Breast Neoplasms/immunology , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/immunology , DNA Copy Number Variations , Datasets as Topic , Female , Gene Expression Profiling , Humans , Oligonucleotide Array Sequence Analysis , Proteogenomics/methods , Proteome/genetics , Proteome/immunology , RNA, Messenger/metabolismABSTRACT
The amyloid ß-peptide (Aß) is a physiological ubiquitously expressed peptide suggested to be involved in synaptic function, long-term potentiation, and memory function. The 42 amino acid-long variant (Aß42) forms neurotoxic oligomers and amyloid plaques and plays a key role in the loss of synapses and other pathogenic events of Alzheimer disease. Still, the exact localization of Aß42 in neurons and at synapses has not been reported. Here, we used super-resolution microscopy and show that Aß42 was present in small vesicles in presynaptic compartments, but not in postsynaptic compartments, in the neurites of hippocampal neurons. Some of these vesicles appeared to lack synaptophysin, indicating that they differ from the synaptic vesicles responsible for neurotransmitter release. The Aß42-containing vesicles existed in presynapses connected to stubby spines and mushroom spines, and were also present in immature presynapses. These vesicles were scarce in other parts of the neurites, where Aß42 was instead present in large, around 200-600 nm, vesicular structures. Three-dimensional super-resolution microscopy confirmed that Aß42 was present in the presynapse and absent in the postsynapse.
ABSTRACT
The inner membrane (IM) of mitochondria displays an intricate, highly folded architecture and can be divided into two domains: the inner boundary membrane adjacent to the outer membrane and invaginations toward the matrix, called cristae. Both domains are connected by narrow, tubular membrane segments called cristae junctions (CJs). The formation and maintenance of CJs is of vital importance for the organization of the mitochondrial IM and for mitochondrial and cellular physiology. The multisubunit mitochondrial contact site and cristae organizing system (MICOS) was found to be a major factor in CJ formation. In this study, we show that the MICOS core component Mic60 actively bends membranes and, when inserted into prokaryotic membranes, induces the formation of cristae-like plasma membrane invaginations. The intermembrane space domain of Mic60 has a lipid-binding capacity and induces membrane curvature even in the absence of the transmembrane helix. Mic60 homologues from α-proteobacteria display the same membrane deforming activity and are able to partially overcome the deletion of Mic60 in eukaryotic cells. Our results show that membrane bending by Mic60 is an ancient mechanism, important for cristae formation, and had already evolved before α-proteobacteria developed into mitochondria.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Biological Transport/physiology , Eukaryotic Cells/metabolism , Protein Binding/physiology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
We here report for the first time the synergistic implementation of structured illumination microscopy (SIM) and multifocus microscopy (MFM). This imaging modality is designed to alleviate the problem of insufficient volumetric acquisition speed in super-resolution biological imaging. SIM is a wide-field super-resolution technique that allows imaging with visible light beyond the classical diffraction limit. Employing multifocus diffractive optics we obtain simultaneous wide-field 3D imaging capability in the SIM acquisition sequence, improving volumetric acquisition speed by an order of magnitude. Imaging performance is demonstrated on biological specimens.
ABSTRACT
Hydrophobic inner mitochondrial membrane proteins with internal targeting signals, such as the metabolite carriers, use the carrier translocase (TIM22 complex) for transport into the inner membrane. Defects in this transport pathway have been associated with neurodegenerative disorders. While the TIM22 complex is well studied in baker's yeast, very little is known about the mammalian TIM22 complex. Using immunoprecipitation, we purified the human carrier translocase and identified a mitochondrial inner membrane protein TIM29 as a novel component, specific to metazoa. We show that TIM29 is a constituent of the 440 kDa TIM22 complex and interacts with oxidized TIM22. Our analyses demonstrate that TIM29 is required for the structural integrity of the TIM22 complex and for import of substrate proteins by the carrier translocase.
Subject(s)
Mitochondrial Membrane Transport Proteins/metabolism , Amino Acid Sequence , HEK293 Cells , HeLa Cells , Humans , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Precursor Protein Import Complex Proteins , Oxidation-Reduction , Protein TransportABSTRACT
RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.
Subject(s)
Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Microscopy, Fluorescence/methods , Nanotechnology/methods , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Animals , Cell Line , Protein Structure, TertiaryABSTRACT
The function of mitochondria depends on the proper organization of mitochondrial membranes. The morphology of the inner membrane is regulated by the recently identified mitochondrial contact site and crista organizing system (MICOS) complex. MICOS mutants exhibit alterations in crista formation, leading to mitochondrial dysfunction. However, the mechanisms that underlie MICOS regulation remain poorly understood. MIC19, a peripheral protein of the inner membrane and component of the MICOS complex, was previously reported to be required for the proper function of MICOS in maintaining the architecture of the inner membrane. Here, we show that human and Saccharomyces cerevisiae MIC19 proteins undergo oxidation in mitochondria and require the mitochondrial intermembrane space assembly (MIA) pathway, which couples the oxidation and import of mitochondrial intermembrane space proteins for mitochondrial localization. Detailed analyses identified yeast Mic19 in two different redox forms. The form that contains an intramolecular disulfide bond is bound to Mic60 of the MICOS complex. Mic19 oxidation is not essential for its integration into the MICOS complex but plays a role in MICOS assembly and the maintenance of the proper inner membrane morphology. These findings suggest that Mic19 is a redox-dependent regulator of MICOS function.
Subject(s)
Membrane Proteins/metabolism , Mitochondrial Membranes/metabolism , Multiprotein Complexes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Line , HEK293 Cells , Humans , Membrane Proteins/genetics , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Oxidation-Reduction , Protein Binding , RNA Interference , RNA, Small Interfering , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The mitochondrial inner membrane is highly folded and displays a complex molecular architecture. Cristae junctions are highly curved tubular openings that separate cristae membrane invaginations from the surrounding boundary membrane. Despite their central role in many vital cellular processes like apoptosis, the details of cristae junction formation remain elusive. Here we identify Mic10, a core subunit of the recently discovered MICOS complex, as an inner mitochondrial membrane protein with the ability to change membrane morphology in vitro and in vivo. We show that Mic10 spans the inner membrane in a hairpin topology and that its ability to sculpt membranes depends on oligomerization through a glycine-rich motif. Oligomerization mutants fail to induce curvature in model membranes, and when expressed in yeast, mitochondria display an altered inner membrane architecture characterized by drastically decreased numbers of cristae junctions. Thus, we demonstrate that membrane sculpting by Mic10 is essential for cristae junction formation.
Subject(s)
Membrane Proteins/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Membrane Proteins/analysis , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/ultrastructure , Mitochondrial Proteins , Molecular Sequence Data , Protein Multimerization , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/analysis , Sequence AlignmentABSTRACT
The inner membrane of mitochondria is especially protein rich and displays a unique morphology characterized by large invaginations, the mitochondrial cristae, and the inner boundary membrane, which is in proximity to the outer membrane. Mitochondrial inner membrane proteins appear to be not evenly distributed in the inner membrane, but instead organize into functionally distinct subcompartments. It is unknown how the organization of the inner membrane is achieved. We identified MINOS1/MIO10 (C1orf151/YCL057C-A), a conserved mitochondrial inner membrane protein. mio10-mutant yeast cells are affected in growth on nonfermentable carbon sources and exhibit altered mitochondrial morphology. At the ultrastructural level, mutant mitochondria display loss of inner membrane organization. Proteomic analyses reveal MINOS1/Mio10 as a novel constituent of Mitofilin/Fcj1 complexes in human and yeast mitochondria. Thus our analyses reveal new insight into the composition of the mitochondrial inner membrane organizing machinery.
Subject(s)
Membrane Proteins/metabolism , Mitochondria/enzymology , Mitochondria/ultrastructure , Mitochondrial Proteins/metabolism , Muscle Proteins/metabolism , Proton-Translocating ATPases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , HEK293 Cells , Humans , Membrane Proteins/genetics , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/ultrastructure , Mitochondrial Proteins/genetics , Molecular Sequence Data , Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.