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Mitochondria lacking Sam50 show an altered structure of cristae. (A and B) sam50kd-2 and pLV-THM cells were induced with doxycycline for 7 days and
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Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid
composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role
of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sort...
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... then analyzed the mitochondrial ultrastructure in sam50kd-2 cells by transmission electron microscopy (TEM). Vir- tually all mitochondria in Dox-treated cells lacked the typical structure of cristae, although remnants of the membranes of cris- tae were preserved. The IMM often appeared in the form of one or more concentric circles ( Fig. 2A). In contrast, mitochondria of the untreated sam50kd-2 cells revealed conventional cristae ( Fig. 2A). In the sam50kd-3 cell line, we again observed a striking rearrange- ment of the structure of mitochondrial cristae after Sam50 deple- tion (data not shown). By using the empty vector cell line pLV- THM as a control, we could rule out ...
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... microscopy (TEM). Vir- tually all mitochondria in Dox-treated cells lacked the typical structure of cristae, although remnants of the membranes of cris- tae were preserved. The IMM often appeared in the form of one or more concentric circles ( Fig. 2A). In contrast, mitochondria of the untreated sam50kd-2 cells revealed conventional cristae ( Fig. 2A). In the sam50kd-3 cell line, we again observed a striking rearrange- ment of the structure of mitochondrial cristae after Sam50 deple- tion (data not shown). By using the empty vector cell line pLV- THM as a control, we could rule out that the Dox treatment induced the described alterations (Fig. 2B). Depletion of Sam50 results in a ...
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... sam50kd-2 cells revealed conventional cristae ( Fig. 2A). In the sam50kd-3 cell line, we again observed a striking rearrange- ment of the structure of mitochondrial cristae after Sam50 deple- tion (data not shown). By using the empty vector cell line pLV- THM as a control, we could rule out that the Dox treatment induced the described alterations (Fig. 2B). Depletion of Sam50 results in a reduction in the amounts of its substrates, the -barrel proteins Tom40 and voltage-dependent anion-selective channel (VDAC), and of other components of the SAM complex, metaxins 1 and 2 (24). We therefore investigated whether the reduction of any of these proteins had an impact on the structure of ...
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... visibly less mitochondria than the same number of untreated cells. Therefore, we determined the size of mitochon- dria. Whereas mitochondria in the noninduced cells had an aver- age size of 0.3 (0.2) m 2 , we measured an average size of 0.7 (0.7) m 2 of mitochondria in Dox-treated cells, displaying a variability ranging between 0.1 and 5.6 m 2 (Fig. 2C). In compar- ison, mitochondria from Tom40 knockdown cells revealed an av- erage size of 0.4 (0.3) m 2 in both Dox-treated and untreated cells (not shown). This suggests a fission/fusion abnormality in Sam50-depleted cells. Considering our previous observation that mitochondria appear fragmented after Sam50 knockdown, we de- termined ...
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... in Sam50-depleted cells. Considering our previous observation that mitochondria appear fragmented after Sam50 knockdown, we de- termined the amounts of proteins involved in mitochondrial fu- sion (mitofusins 1 and 2) and of a known organizer of crista junc- tion, OPA1. The levels of all three proteins were unchanged in mitochondria lacking Sam50 (Fig. 2D). Therefore, we conclude that Sam50 plays a role in maintenance of both the mitochondrial network and the structure of cristae, which is likely independent of its function in the assembly of -barrel ...
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... species was moderately changed-the amounts of cardiolipins with shorter acyl chains were reduced, whereas those cardiolipin species with prevalent longer acyl chains were enriched in knockdown samples (Fig. 11). However, none of these differences were striking enough to ex- plain the complete loss of cristae we observe in the absence of Sam50 (Fig. ...
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... even a moderate Sam50 reduction leads to the com- plete loss of cristae ( Fig. 2 and 4D). In addition, Sam50 absence leads to the depletion of respiratory complexes (Table 1). We do not believe that mild changes in cardiolipin levels and species that occur after Sam50 knockdown are responsible for this (Fig. 11). It has in fact been shown that the biogenesis of cardiolipin depends on the intact and functional respiratory ...
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... the initial stages of Sam50 depletion, levels of both CHCHD3 and mitofilin are nearly unaffected, but mitochon- drial cristae are completely lost (Fig. 4A and D). The amount of OPA1 is also unchanged at the similar stage (Fig. 2D). This ex- cludes reduction in the mitofilin/CHCHD3/OPA1 complex as the main cause for the observed defect in cristae as proposed by Darshi et al. (6) and points to the importance of the communication of the OMM and IMM components of the MIB complex in keeping cristae properly structured. Metaxins 1 and 2, found by Xie and colleagues in ...
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... and in this way influences the amount of Sam50. This could par- tially explain why the observed phenotype of cristae after mito- filin depletion, which consists of multiple layers of IMM form- ing an onion-like structure (21; data not shown), does not entirely resemble the defect in cristae detected after the knock- down of CHCHD3 (6) or Sam50 ( Fig. 2A). and CoxVakd-2 (single clone) cells were grown for 7 days in the absence (Dox) or presence (Dox) of doxycycline. Cells were then analyzed by TEM (lower panels), and mitochondria were isolated in parallel and analyzed by SDS-PAGE and Western blotting. Scale bar, 1 m. For designations, see the text and the legends for Fig. 1, 3, and 4. ...
Citations
... In addition to its role in cristae organization, MICOS is a "master regulator" of mitochondrial function, forming an extensive protein interaction network with protein complexes and machineries on the IMM and OMM (23)(24)(25)(26)(27)(28)(29). Among these interactions, MICOS forms a supercomplex, the mitochondrial intermembrane space bridging complex (MIB), with OMM protein biogenesis machinery (30,31). Thus, in addition to its role as a key determinant of cristae architecture and respiratory function, MICOS is central to overall mitochondrial organization. ...
... ; https://doi.org/10.1101/2024.06.21.600105 doi: bioRxiv preprint relative to TIMM23 at the distal tips of mitochondria (Fig. 1D). MICOS is part of the MIB supercomplex with OMM proteins, including the Sorting and Assembly Machinery (SAM) complex and DNAJC11 (30,31). Both the SAM subunit SAMM50 and DNAJC11 were also co-enriched with MIC60 at these mitochondria ( Fig. S1B-S1C). ...
Local metabolic demand within cells varies widely and the extent to which individual mitochondria can be specialized to meet these functional needs is unclear. We examined the subcellular distribution of MICOS, a spatial and functional organizer of mitochondria, and discovered that it dynamically enriches at the tip of a minor population of mitochondria in the cell periphery that we term METEORs. METEORs have a unique composition; MICOS enrichment sites are depleted of mtDNA and matrix proteins and contain high levels of the Ca2+ uniporter MCU, suggesting a functional specialization. METEORs are also enriched for the myosin MYO19, which promotes their trafficking to a small subset of filopodia. We identify a positive correlation between the length of filopodia and the presence of METEORs and show that elimination of mitochondria from filopodia impairs cellular motility. Our data reveal a novel type of mitochondrial heterogeneity and suggest compositionally specialized mitochondria support cell migration.
... As a key regulatory subunit of mitochondrial integrity, mitochondrial inner membrane protein (IMMT), also known as Mic-60 or Mitofilin 8 , is not only an important part of the mitochondrial contact sites and cristae organization system (MICOS) complex, but it also interacts with members of the SAM (The Sorting and Assembly Machinery) complex to form the mitochondrial membrane space bridging complex 9 . Recent studies have linked IMMT to the onset of various diseases, including cardiomyopathy 10 , degenerative encephalopathy 11 and kidney injury 12 . ...
Metabolic reprogramming is widely recognized as a hallmark of malignant tumors, and the targeting of metabolism has emerged as an appealing approach for cancer treatment. Mitochondria, as pivotal organelles, play a crucial role in the metabolic regulation of tumor cells, and their morphological and functional alterations are intricately linked to the biological characteristics of tumors. As a key regulatory subunit of mitochondria, mitochondrial inner membrane protein (IMMT), plays a vital role in degenerative diseases, but its role in tumor is almost unknown. The objective of this research was to investigate the roles that IMMT play in the development and progression of breast cancer (BC), as well as to elucidate the underlying biological mechanisms that drive these effects. In this study, it was confirmed that the expression of IMMT in BC tissues was significantly higher than that in normal tissues. The analysis of The Cancer Genome Atlas (TCGA) database revealed that IMMT can serve as an independent prognostic factor for BC patients. Additionally, verification in clinical specimens of BC demonstrated a positive association between high IMMT expression and larger tumor size (> 2 cm), Ki-67 expression (> 15%), and HER-2 status. Furthermore, in vitro experiments have substantiated that the suppression of IMMT expression resulted in a reduction in cell proliferation and alterations in mitochondrial cristae, concomitant with the liberation of cytochrome c, but it did not elicit mitochondrial apoptosis. Through Gene Set Enrichment Analysis (GSEA) analysis, we have predicted the associated metabolic genes and discovered that IMMT potentially modulates the advancement of BC through its interaction with 16 metabolic-related genes, and the changes in glycolysis related pathways have been validated in BC cell lines after IMMT inhibition. Consequently, this investigation furnishes compelling evidence supporting the classification of IMMT as prognostic marker in BC, and underscoring its prospective utility as a novel target for metabolic therapy.
... Table 1). We also identified GOP-3, the orthologue of the outer membrane complex protein SAMM50 [32], which is required for threading of mitochondrial β-barrel proteins into the outer and inner membrane bilayers [33], along with several small molecule transporters: VDAC-1, ANT-1.1 and MTCH-1 (Fig. 1B, Supp. Table 1). ...
Loss of proteostasis is a highly conserved feature of aging across model organisms and results in the accumulation of insoluble protein aggregates. Protein insolubility is also a unifying feature of major age-related neurodegenerative diseases, including Alzheimer's Disease (AD), in which hundreds of insoluble proteins associate with aggregated amyloid beta (Aβ) in senile plaques. Despite the connection between aging and AD risk, therapeutic approaches to date have overlooked aging-driven generalized protein insolubility as a contributing factor. However, proteins that become insoluble during aging in model organisms are capable of accelerating Aβ aggregation in vitro and lifespan in vivo. Here, using an unbiased proteomics approach, we questioned the relationship between Aβ and age-related protein insolubility. Specifically, we uncovered that Aβ expression drives proteome-wide protein insolubility in C. elegans , even in young animals, and this insoluble proteome is highly similar to the insoluble proteome driven by normal aging, this vulnerable sub-proteome we term the core insoluble proteome (CIP). We show that the CIP is enriched with proteins that modify Aβ toxicity in vivo, suggesting the possibility of a vicious feedforward cycle in the context of AD. Importantly, using human genome-wide association studies (GWAS), we show that the CIP is replete with biological processes implicated not only in neurodegenerative diseases but also across a broad array of chronic, age-related diseases (CARDs). This provides suggestive evidence that age-related loss of proteostasis could play a role in general CARD risk. Finally, we show that the geroprotective, gut-derived metabolite, Urolithin A, relieves Aβ toxicity, supporting its use in clinical trials for dementia and age-related diseases.
... Crista junctions with their high local membrane curvature require for their stability the mitochondrial contact site and cristae organizing system (MICOS) (Harner et al, 2011;Hoppins et al, 2011;Malsburg et al, 2011;Friedman & Nunnari, 2014;Kozjak-Pavlovic, 2017;Rampelt et al, 2017;Wollweber et al, 2017). The MICOS complex is highly conserved in evolution and consists of at least six different genuine subunits in yeast and seven in mammals that are organized in two distinct modules Harner et al, 2011;Hoppins et al, 2011;Malsburg et al, 2011;Alkhaja et al, 2012;Ott et al, 2012;Pfanner et al, 2014;Guarani et al, 2015;Muñoz-Gómez et al, amphipathic helix within its intermembrane space domain (Hessenberger et al, 2017;Tarasenko et al, 2017). The other subcomplex is composed of large oligomers of Mic10, a small inner membrane protein with an intrinsic membrane-bending activity, together with Mic12/QIL1, Mic26 and Mic27 (Barbot et al, 2015;Bohnert et al, 2015;Friedman et al, 2015;Guarani et al, 2015). ...
The boundary and cristae domains of the mitochondrial inner membrane are connected by crista junctions. Most cristae membrane proteins are nuclear-encoded and inserted by the mitochondrial protein import machinery into the inner boundary membrane. Thus, they must overcome the diffusion barrier imposed by crista junctions to reach their final location. Here, we show that respiratory chain complexes and assembly intermediates are physically connected to the mitochondrial contact site and cristae organizing system (MICOS) that is essential for formation and stability of crista junctions. We identify the inner membrane protein Mar26 (Fmp10) as determinant in the biogenesis of the cytochrome bc 1 complex (complex III). Mar26 couples a Rieske Fe/S protein-containing assembly intermediate to MICOS. Our data indicate that Mar26 maintains an assembly-competent Rip1 pool at crista junctions where complex III maturation likely occurs. MICOS facilitates efficient Rip1 assembly by recruitment of complex III assembly intermediates to crista junctions. We propose that MICOS, via interaction with assembly factors such as Mar26, directly contributes to the spatial and temporal coordination of respiratory chain biogenesis.
... However, these effects are also influenced by Sam50 mRNA level. To clarify the role of these two proteins we shifted to cells and used Hela doxycycline-inducible SAMM50 KD cells 24 . In these cells, SAMM50 KD induces mtDNA leakage and robust mitophagy response 25,26 . ...
Mitochondrial DNA is exposed to multiple insults produced by normal cellular function. Upon mtDNA replication stress the mitochondrial genome transfers to endosomes where it is degraded. Here, using proximity proteomics we found that mtDNA replication stress leads to the rewiring of the mitochondrial proximity proteome, increasing mitochondria association with lysosomal and vesicle-associated proteins, such as the GTPase RAB10 and the retromer. We found that upon mtDNA replication stress, RAB10 enhances mitochondrial fragmentation and relocates from the ER to lysosomes containing mtDNA. The retromer enhances and coordinates the expulsion of mitochondrial matrix components through mitochondrial-derived vesicles, and mtDNA with direct transfer to lysosomes. Using a Drosophila model carrying a long deletion on the mtDNA (deltamtDNA), we evaluated in vivo the role of the retromer in mtDNA extraction and turnover in the larval epidermis. The presence of deltamtDNA elicits the activation of a specific transcriptome profile related to counteract mitochondrial damage. Expression of the retromer component Vps35 is sufficient to restore mtDNA homoplasmy and mitochondrial defects associated with deltamtDNA. Our data reveal novel regulators involved in the specific elimination of mtDNA. We demonstrate that modulation of the retromer in vivo is a successful mechanism to restore mitochondrial function associated with mtDNA damage.
... The Sorting and Assembly Machinery (SAM) complex in the mitochondrial outer membrane consists of three core components: MTX1, MTX2 and Sam50 [13], which helps the β-barrel proteins to fold correctly and assemble into the mitochondrial outer membrane [14,15]. The SAM complex and the mitochondrial contact site and cristae organizing system (MICOS) complex located in the inner mitochondrial membrane together constitute the core components of the mitochondrial intermembrane space bridging (MIB) complex, which is associated with the maintenance and regulation of mitochondrial cristae [16,17]. In addition, MTX1 and MTX2 are involved in TNF-α induced apoptosis [18][19][20][21]. ...
Proteinuria is a common and important clinical manifestation of chronic kidney disease (CKD) and an independent risk factor for the progression of kidney disease. As a component of the glomerular filtration barrier (GFB), podocyte plays a key role in the pathogenesis of glomerular diseases and proteinuria. However, the pathophysiology of glomerular diseases associated with mitochondrial function is incompletely understood. Here, we identified three novel mutations in MTX2, encoding a membrane protein in mitochondria, associated with multisystem manifestations including nephrotic proteinuria and kidney injury in two Chinese patients. Conditional podocyte-specific Mtx2 knockout (Pod-Mtx2-KO) mice present a series of podocyte and glomerular abnormalities from 8 weeks to old age, including microalbuminuria, glomerular mesangial hyperplasia, fusion and effacement of foot process. MTX2 deficiency impaired podocyte functions in vitro, manifested by reductions of adhesion, migration and endocytosis, which were further restored by overexpression of MTX2. Moreover, MTX2 defects led to abnormal mitochondrial structure and dysfunction, evidenced with defects of complex I and III, increased production of reactive oxygen species (ROS), and decreased protein levels of Sam50-CHCHD3-Mitofilin axis in the mitochondrial intermembrane space bridging (MIB) complex which is responsible for maintaining mitochondrial cristae morphology. Collectively, these findings reveal that the normal expression of MTX2 in glomerulus plays an important role in the adhesion, migration, endocytosis, proliferation and other physiological functions of podocytes, which may be realized by maintaining the morphological structure and function of mitochondria. Abnormal expression of MTX2 can lead to mitochondrial dysfunction and structural abnormalities by Sam50-CHCHD3-Mitofilin axis in podocyte, which further induces podocyte injury, glomerular lesions and proteinuria.
... Apart from the MICOS complex, a variety of other molecules present in the OM and the IM also regulate IM remodeling. The mitochondrial intermembrane space bridging (MIB) complex was discovered when the role of sorting and assembly machinery 50 kDa (SAMM50) was studied in detail (97). SAMM50 depletion resulted in a severe loss of cristae, which also led to loss of MTX1, MIC19, and MIC60 (96,97). ...
... The mitochondrial intermembrane space bridging (MIB) complex was discovered when the role of sorting and assembly machinery 50 kDa (SAMM50) was studied in detail (97). SAMM50 depletion resulted in a severe loss of cristae, which also led to loss of MTX1, MIC19, and MIC60 (96,97). BN-PAGE analysis and complexome profiling overall showed that SAMM50, together with the MICOS complex, forms the MIB complex, which has reported apparent sizes of 1.1-1.4 ...
... BN-PAGE analysis and complexome profiling overall showed that SAMM50, together with the MICOS complex, forms the MIB complex, which has reported apparent sizes of 1.1-1.4 MDa and 2.2 MDa (61,97). Dissipation of the m or depletion of SAMM50 was reported to cause proteolytic processing of MIC19 by OMA1 protease (124). ...
Mitochondria are essential organelles performing important cellular functions ranging from bioenergetics and metabolism to apoptotic signaling and immune responses. They are highly dynamic at different structural and functional levels. Mitochondria have been shown to constantly undergo fusion and fission processes and dynamically interact with other organelles such as the endoplasmic reticulum, peroxisomes, and lipid droplets. The field of mitochondrial dynamics has evolved hand in hand with technological achievements including advanced fluorescence super-resolution nanoscopy. Dynamic remodeling of the cristae membrane within individual mitochondria, discovered very recently, opens up a further exciting layer of mitochondrial dynamics. In this review, we discuss mitochondrial dynamics at the following levels: ( a) within an individual mitochondrion, ( b) among mitochondria, and ( c) between mitochondria and other organelles. Although the three tiers of mitochondrial dynamics have in the past been classified in a hierarchical manner, they are functionally connected and must act in a coordinated manner to maintain cellular functions and thus prevent various human diseases.
Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Mic60, along with its partner Mic19, the only non-membrane integral member of the complex, are proposed to stabilize cristae junctions by forming a supportive vault 12 . Additionally, Mic60 and Mic19 interact with the OMM beta-barrel assembly SAM complex [13][14][15][16] . This trans-boundary complex between MICOS, SAM, and other OMM proteins, termed the mitochondrial intermembrane space bridging complex (MIB), presents an opportunity for communication between other organelles such as the endoplasmic reticulum and the inside of mitochondria 17 . ...
Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial inner membrane-localized MICOS complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS complexes to promote normal mitochondrial morphology and respiratory function. Bioinformatic analyses reveal that Mmc1 is a distant relative of the Dynamin-Related Protein (DRP) family of GTPases, which are well established to shape and remodel membranes. We find that, like DRPs, Mmc1 self-associates and forms high molecular weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting it does not dynamically remodel membranes. These data are consistent with a model in which Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans.
... The mitochondrial contact site and cristae-organizing system (MICOS) is crucial to maintain the typical inner membrane architecture, comprising the inner boundary membrane adjacent to the outer membrane and the folded cristae membranes [125]. MICOS is enriched at crista junctions (which link inner boundary membrane and cristae membranes) and associates with TOM and SAM complexes, constituting contact sites between both mitochondrial membranes ( Figure 4) [28,[126][127][128]. By coupling to protein translocases, MICOS supports protein transport into mitochondria. ...
... Our results show that ORF3a, ORF9c and ORF10 induce changes in the shape and density of mitochondrial cristae, suggesting that these accessory proteins may directly perturb either mitochondrial shape-determining proteins. Genes for mitochondrial proteins that determine the shape, length and function of mitochondrial cristae 37,39 , are variously downregulated by the accessory proteins studied here, particularly by ORF3a, which caused a significant downregulation of OPA1 and SAMM50. The other accessory proteins caused a downregulation of different sets of genes for proteins that also play important roles in mitochondrial and cristae morphology, The predicted upregulation of bile acid and ether lipid biosynthesis pathways triggered by ORF3a and of one-carbon, fatty acid and amino acid metabolism induced by ORF10, led us to infer potential targets for intervention in order to revert these metabolic perturbations. ...
Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.
One-Sentence Summary
Mitochondria and metabolic alterations induced by SARS- CoV-2 accessory proteins ORF3a, ORF9b, ORF9c, ORF10 in pulmonary cells unravel new targets of intervention.
