mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis.

Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS, essential in near-all eukaryotes. Packed into distinct foci (nucleoids) inside mitochondria, the number of mtDNA copies differs between cell-types and is affected in several human diseases. Currently, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we present mtFociCounter, a single-cell image analysis tool for reproducible quantification of nucleoids and other foci. mtFociCounter is a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition, mtFociCounter quantifies mitochondrial content and our results show good correlation (R = 0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstrate mtFociCounter readily detects differences in foci-numbers upon sample treatment, and applies to Mitochondrial RNA Granules and superresolution microscopy. mtFociCounter provides a versatile solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial foci analysis.

A mouse model of human mitofusin-2-related lipodystrophy exhibits adipose-specific mitochondrial stress and reduced leptin secretion.

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology.

Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane-localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.

TMEM63C mutations cause mitochondrial morphology defects and underlie hereditary spastic paraplegia.

The hereditary spastic paraplegias (HSP) are among the most genetically diverse of all Mendelian disorders. They comprise a large group of neurodegenerative diseases that may be divided into 'pure HSP' in forms of the disease primarily entailing progressive lower-limb weakness and spasticity, and 'complex HSP' when these features are accompanied by other neurological (or non-neurological) clinical signs. Here, we identified biallelic variants in the transmembrane protein 63C (TMEM63C) gene, encoding a predicted osmosensitive calcium-permeable cation channel, in individuals with hereditary spastic paraplegias associated with mild intellectual disability in some, but not all cases. Biochemical and microscopy analyses revealed that TMEM63C is an endoplasmic reticulum-localized protein, which is particularly enriched at mitochondria-endoplasmic reticulum contact sites. Functional in cellula studies indicate a role for TMEM63C in regulating both endoplasmic reticulum and mitochondrial morphologies. Together, these findings identify autosomal recessive TMEM63C variants as a cause of pure and complex HSP and add to the growing evidence of a fundamental pathomolecular role of perturbed mitochondrial-endoplasmic reticulum dynamics in motor neurone degenerative diseases.

A Dictyostelium model for BPAN disease reveals a functional relationship between the WDR45/WIPI4 homolog Wdr45l and Vmp1 in the regulation of autophagy-associated PtdIns3P and ER stress.

PROPPINs are conserved PtdIns3P-binding proteins required for autophagosome biogenesis that fold into a characteristic group of seven-bladed beta-propellers. Mutations in WDR45/WIPI4, a human member of this family, lead to BPAN, a rare form of neurodegeneration. We have generated mutants for the two PROPPIN proteins present in the model system Dictyostelium discoideum (Atg18 and Wdr45l) and characterized their function. Lack of Wdr45l greatly impairs autophagy, while Atg18 only causes subtle defects in the maturation of autolysosomes. The strong phenotype of the Wdr45l mutant is strikingly similar to that observed in Dictyostelium cells lacking Vmp1, an ER protein required for omegasome formation. Common phenotypes include impaired growth in axenic medium, lack of aggregation, and local enrichment of PtdIns3P as determined by the use of lipid reporters. In addition, Vmp1 and Wdr45l mutants show a chronically active response to ER stress. For both mutants, this altered PtdIns3P localization can be prevented by the additional mutation of the upstream regulator Atg1, which also leads to recovery of axenic growth and reduction of ER stress. We propose that, in addition to an autophagy defect, local autophagy-associated PtdIns3P accumulation might contribute to the pathogenesis of BPAN by disrupting ER homeostasis. The introduction of BPAN-associated mutations in Dictyostelium Wdr45l reveals the impact of pathogenic residues on the function and localization of the protein.

The Complex Dance of Organelles during Mitochondrial Division.

Mitochondria are dynamic organelles that undergo cycles of fission and fusion events depending on cellular requirements. During mitochondrial division, the GTPase dynamin-related protein-1 is recruited to endoplasmic reticulum (ER)-induced mitochondrial constriction sites where it drives fission. However, the events required to complete scission of mitochondrial membranes are not well understood. Here, we emphasize the recently described roles for Golgi-derived phosphatidylinositol 4-phosphate (PI4P)-containing vesicles in the last steps of mitochondrial division. We then propose how trans-Golgi network vesicles at mitochondria-ER contact sites and PI4P generation could mechanistically execute mitochondrial division, by recruiting PI4P effectors and/or the actin nucleation machinery. Finally, we speculate on mechanisms to explain why such a complex dance of different organelles is required to facilitate the remodelling of mitochondrial membranes.

The last wall of defense to prevent extreme and deleterious mitochondrial fusion.

Mitochondria are dynamic organelles adapting their morphology by cycles of fission and fusion events to control cellular homeostasis. In this issue of The EMBO Journal, Murata and colleagues (2020) show that lack of mitochondrial division leads to safeguard mechanisms, induced by transient mitochondrial membrane depolarization and activation of the metalloprotease OMA1, to prevent extreme mitochondrial fusion and to maintain optimal mitochondrial bioenergetics.

Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial division.

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIß [PI(4)KIIIß], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission.

Evidence for an evolutionary relationship between Vmp1 and bacterial DedA proteins.

VMP1 and DedA proteins are conserved families of transmembrane proteins in eukaryotes and prokaryotes respectively. Despite numerous reports involving these proteins in multiple cellular processes, their molecular function is still unknown. They share the domain of unknown function PF09335, suggesting a possible functional relationship between these protein families. Here we show that VMP1 from different species contain two short motifs conserved in the bacterial DedA proteins and the yeast protein Tvp38. The hallmark of one of these motifs is a glycine residue previously shown to be strictly conserved in all the DedA proteins. Substitution of this residue to leucine, glutamate or arginine in Dictyostelium Vmp1 inactivates the protein, as shown by the inability of the mutants to rescue the phenotypes associated with the lack of Vmp1 including development and lipid homeostasis. This is the first experimental approach that supports an evolutionary relationship between Vmp1 and DedA proteins and highlights the importance of the conserved glycine residue in the PF09335 domain.

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)-domains associated with diverse ER-organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1-enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1-deficient mammalian cells and other model systems.

VMP1 Establishes ER-Microdomains that Regulate Membrane Contact Sites and Autophagy.

The endoplasmic reticulum (ER) regulates organelle dynamics through the formation of membrane contact sites (MCS). Here we describe that VMP1, a multispanning ER-resident protein involved in autophagy, is enriched in ER micro-domains that are in close proximity to diverse organelles in HeLa and Cos-7 cells. These VMP1 puncta are highly dynamic, moving in concert with lipid droplets, mitochondria and endosomes. Some of these micro-domains are associated with ER sliding events and also with fission events of mitochondria and endosomes. VMP1-depleted cells display increased ER-mitochondria MCS and altered mitochondria morphology demonstrating a role in the regulation of MCS. Additional defects in ER structure and lipid droplets size and distribution are consistent with a more general function of VMP1 in membrane remodeling and organelle function. We hypothesize that in autophagy VMP1 is required for the correct morphogenesis of the omegasome by regulating MCS at the site of autophagosome formation.

Mitochondria-targeted therapies for acute kidney injury.

Acute kidney injury (AKI) is a serious clinical condition with no effective treatment. Tubular cells are key targets in AKI. Tubular cells and, specifically, proximal tubular cells are extremely rich in mitochondria and mitochondrial changes had long been known to be a feature of AKI. However, only recent advances in understanding the molecules involved in mitochondria biogenesis and dynamics and the availability of mitochondria-targeted drugs has allowed the exploration of the specific role of mitochondria in AKI. We now review the morphological and functional mitochondrial changes during AKI, as well as changes in the expression of mitochondrial genes and proteins. Finally, we summarise the current status of novel therapeutic strategies specifically targeting mitochondria such as mitochondrial permeability transition pore (MPTP) opening inhibitors (cyclosporine A (CsA)), quinone analogues (MitoQ, SkQ1 and SkQR1), superoxide dismutase (SOD) mimetics (Mito-CP), Szeto-Schiller (SS) peptides (Bendavia) and mitochondrial division inhibitors (mdivi-1). MitoQ, SkQ1, SkQR1, Mito-CP, Bendavia and mdivi-1 have improved the course of diverse experimental models of AKI. Evidence for a beneficial effect of CsA on human cardiac ischaemia-reperfusion injury derives from a clinical trial; however, CsA is nephrotoxic. MitoQ and Bendavia have been shown to be safe for humans. Ongoing clinical trials are testing the efficacy of Bendavia in AKI prevention following renal artery percutaneous transluminal angioplasty.