Tardigrades: Trained to be hardy in the face of DNA damage.

Tardigrades withstand ionizing irradiation levels ∼500 times higher than humans can tolerate. Two recent papers shed light on how this might be achieved - via the transcriptional induction of DNA repair genes, the induction of a radioprotective DNA-binding protein, and possibly also the heightened capacity of repair proteins.

The histone chaperone SPT2 regulates chromatin structure and function in Metazoa.

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

Genetic screen identifies non-mitochondrial proteins involved in the maintenance of mitochondrial homeostasis.

The mitochondrial unfolded protein response (UPR mt ) is an important stress response that ensures the maintenance of mitochondrial homeostasis in response to various types of cellular stress. We previously described a genetic screen for Caenorhabditis elegans genes, which when inactivated cause UPR mt activation, and reported genes identified that encode mitochondrial proteins. We now report additional genes identified in the screen. Importantly, these include genes that encode non-mitochondrial proteins involved in processes such as the control of gene expression, post-translational modifications, cell signaling and cellular trafficking. Interestingly, we identified several genes that have been proposed to participate in the transfer of lipids between peroxisomes, ER and mitochondria, suggesting that lipid transfer between these organelles is essential for mitochondrial homeostasis. In conclusion, this study shows that the maintenance of mitochondrial homeostasis is not only dependent on mitochondrial processes but also relies on non-mitochondrial processes and pathways. Our results reinforce the notion that mitochondrial function and cellular function are intimately connected.

Genome-wide RNAi screen for regulators of UPRmt in Caenorhabditis elegans mutants with defects in mitochondrial fusion.

Mitochondrial dynamics plays an important role in mitochondrial quality control and the adaptation of metabolic activity in response to environmental changes. The disruption of mitochondrial dynamics has detrimental consequences for mitochondrial and cellular homeostasis and leads to the activation of the mitochondrial unfolded protein response (UPRmt), a quality control mechanism that adjusts cellular metabolism and restores homeostasis. To identify genes involved in the induction of UPRmt in response to a block in mitochondrial fusion, we performed a genome-wide RNAi screen in Caenorhabditis elegans mutants lacking the gene fzo-1, which encodes the ortholog of mammalian Mitofusin, and identified 299 suppressors and 86 enhancers. Approximately 90% of these 385 genes are conserved in humans, and one-third of the conserved genes have been implicated in human disease. Furthermore, many have roles in developmental processes, which suggests that mitochondrial function and their response to stress are defined during development and maintained throughout life. Our dataset primarily contains mitochondrial enhancers and non-mitochondrial suppressors of UPRmt, indicating that the maintenance of mitochondrial homeostasis has evolved as a critical cellular function, which, when disrupted, can be compensated for by many different cellular processes. Analysis of the subsets "non-mitochondrial enhancers" and "mitochondrial suppressors" suggests that organellar contact sites, especially between the ER and mitochondria, are of importance for mitochondrial homeostasis. In addition, we identified several genes involved in IP3 signaling that modulate UPRmt in fzo-1 mutants and found a potential link between pre-mRNA splicing and UPRmt activation.

Reduced peroxisomal import triggers peroxisomal retrograde signaling.

Maintaining organelle function in the face of stress is known to involve organelle-specific retrograde signaling. Using Caenorhabditis elegans, we present evidence of the existence of such retrograde signaling for peroxisomes, which we define as the peroxisomal retrograde signaling (PRS). Specifically, we show that peroxisomal import stress caused by knockdown of the peroxisomal matrix import receptor prx-5/PEX5 triggers NHR-49/peroxisome proliferator activated receptor alpha (PPARα)- and MDT-15/MED15-dependent upregulation of the peroxisomal Lon protease lonp-2/LONP2 and the peroxisomal catalase ctl-2/CAT. Using proteomic and transcriptomic analyses, we show that proteins involved in peroxisomal lipid metabolism and immunity are also upregulated upon prx-5(RNAi). While the PRS can be triggered by perturbation of peroxisomal ß-oxidation, we also observed hallmarks of PRS activation upon infection with Pseudomonas aeruginosa. We propose that the PRS, in addition to a role in lipid metabolism homeostasis, may act as a surveillance mechanism to protect against pathogens.

MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology.

While the analysis of mitochondrial morphology has emerged as a key tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a challenging task and bottleneck for statistically robust conclusions. Here, we present Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6 ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet in a toolbox for Windows and Linux operating systems that combines segmentation with morphological analysis.

Autophagy compensates for defects in mitochondrial dynamics.

Compromising mitochondrial fusion or fission disrupts cellular homeostasis; however, the underlying mechanism(s) are not fully understood. The loss of C. elegans fzo-1MFN results in mitochondrial fragmentation, decreased mitochondrial membrane potential and the induction of the mitochondrial unfolded protein response (UPRmt). We performed a genome-wide RNAi screen for genes that when knocked-down suppress fzo-1MFN(lf)-induced UPRmt. Of the 299 genes identified, 143 encode negative regulators of autophagy, many of which have previously not been implicated in this cellular quality control mechanism. We present evidence that increased autophagic flux suppresses fzo-1MFN(lf)-induced UPRmt by increasing mitochondrial membrane potential rather than restoring mitochondrial morphology. Furthermore, we demonstrate that increased autophagic flux also suppresses UPRmt induction in response to a block in mitochondrial fission, but not in response to the loss of spg-7AFG3L2, which encodes a mitochondrial metalloprotease. Finally, we found that blocking mitochondrial fusion or fission leads to increased levels of certain types of triacylglycerols and that this is at least partially reverted by the induction of autophagy. We propose that the breakdown of these triacylglycerols through autophagy leads to elevated metabolic activity, thereby increasing mitochondrial membrane potential and restoring mitochondrial and cellular homeostasis.

Msp1 cooperates with the proteasome for extraction of arrested mitochondrial import intermediates.

The mitochondrial AAA ATPase Msp1 is well known for extraction of mislocalized tail-anchored ER proteins from the mitochondrial outer membrane. Here, we analyzed the extraction of precursors blocking the import pore in the outer membrane. We demonstrate strong genetic interactions of Msp1 and the proteasome with components of the TOM complex, the main translocase in the outer membrane. Msp1 and the proteasome both contribute to the removal of arrested precursor proteins that specifically accumulate in these mutants. The proteasome activity is essential for the removal as proteasome inhibitors block extraction. Furthermore, the proteasomal subunit Rpn10 copurified with Msp1. The human Msp1 homologue has been implicated in neurodegenerative diseases, and we show that the lack of the Caenorhabditis elegans Msp1 homologue triggers an import stress response in the worm, which indicates a conserved role in metazoa. In summary, our results suggest a role of Msp1 as an adaptor for the proteasome that drives the extraction of arrested and mislocalized proteins at the mitochondrial outer membrane.

Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans.

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m3C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f5C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.

Compromised Mitochondrial Protein Import Acts as a Signal for UPRmt.

The induction of the mitochondrial unfolded protein response (UPRmt) results in increased transcription of the gene encoding the mitochondrial chaperone HSP70. We systematically screened the C. elegans genome and identified 171 genes that, when knocked down, induce the expression of an hsp-6 HSP70 reporter and encode mitochondrial proteins. These genes represent many, but not all, mitochondrial processes (e.g., mitochondrial calcium homeostasis and mitophagy are not represented). Knockdown of these genes leads to reduced mitochondrial membrane potential and, hence, decreased protein import into mitochondria. In addition, it induces UPRmt in a manner that is dependent on ATFS-1 but that is not antagonized by the kinase GCN-2. We propose that compromised mitochondrial protein import signals the induction of UPRmt and that the mitochondrial targeting sequence of ATFS-1 functions as a sensor for this signal.

New Imaging Tools to Analyze Mitochondrial Morphology in Caenorhabditis elegans.

Mitochondria are highly dynamic organelles that constantly fuse and divide. This process is essential as several neurodegenerative diseases have been associated with defects in mitochondrial fusion or fission. Several tools have been developed over the years to visualize mitochondria in organisms such as Caenorhabditis elegans. Combining these tools with the powerful genetics of C. elegans has led to the discovery of new regulators of mitochondrial morphology. In this chapter, we present additional tools to further characterize mitochondrial morphology as well as regulators of mitochondrial morphology. Specifically, we introduce a photoactivatable mitoGFP (PAmitoGFP) that allows to investigate the connectivity of complex mitochondrial networks. In addition, we describe an immunostaining protocol that enables localization studies of these newly identified regulators of mitochondrial morphology.

The loss of LRPPRC function induces the mitochondrial unfolded protein response.

The inactivation of the LRPPRC gene, which has previously been associated with the neurodegenerative French Canadian Leigh Syndrome, results in a decrease in the production of mitochondria-encoded subunits of complex IV, thereby causing a reduction in complex IV activity. Previously we have shown that reducing complex IV activity triggers a compensatory and conserved mitochondrial hyperfusion response. We now demonstrate that LRPPRC knock-down in mammalian cells leads to an imbalance between mitochondria-encoded and nuclear-encoded subunits of complex IV and that this imbalance triggers the mitochondrial unfolded protein response (UPR(mt)). The inactivation of the LRPPRC-like gene mma-1 in C. elegans also induces UPR(mt), which demonstrates that this response is conserved. Furthermore, we provide evidence that mitochondrial hyperfusion and UPR(mt) are coordinated but mediated by genetically distinct pathways. We propose that in the context of LRPPRC mma-1 knock-down, mitochondrial hyperfusion helps to transiently maintain mitochondrial ATP production while UPR(mt) participates in the restoration of mitochondrial proteostasis. Mitochondrial proteostasis is not only critical in pathophysiology but also during aging, as proteotoxic stress has been shown to increase with age. Therefore, we speculate that the coordination of these two mitochondrial stress responses plays a more global role in mitochondrial proteostasis.

How to analyze mitochondrial morphology in healthy cells and apoptotic cells in Caenorhabditis elegans.

Mitochondria constantly undergo fusion and fission events. A proper balance of fusion and fission is essential in healthy cells, as disrupting this balance is associated with several neurodegenerative diseases. Mitochondrial fission has also been shown to play an important role during apoptosis. Hence, the machineries that control mitochondrial morphology have both nonapoptotic and apoptotic functions. Seminal work in yeast has identified some of the key components of these machineries. However, the list is certainly not complete and new factors that are specific to metazoans are being identified every year. In this review, we describe methodologies to test whether a particular candidate gene plays a role in the control of mitochondrial morphology in healthy cells and apoptotic cells using Caenorhabditis elegans.

Age-dependent changes in mitochondrial morphology and volume are not predictors of lifespan.

Mitochondrial dysfunction is a hallmark of skeletal muscle degeneration during aging. One mechanism through which mitochondrial dysfunction can be caused is through changes in mitochondrial morphology. To determine the role of mitochondrial morphology changes in age-dependent mitochondrial dysfunction, we studied mitochondrial morphology in body wall muscles of the nematodeC. elegans. We found that in this tissue, animals display a tubular mitochondrial network, which fragments with increasing age. This fragmentation is accompanied by a decrease in mitochondrial volume. Mitochondrial fragmentation and volume loss occur faster under conditions that shorten lifespan and occur slower under conditions that increase lifespan. However, neither mitochondrial morphology nor mitochondrial volume of five- and seven-day old wild-type animals can be used to predict individual lifespan. Our results indicate that while mitochondria in body wall muscles undergo age-dependent fragmentation and a loss in volume, these changes are not the cause of aging but rather a consequence of the aging process.

Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion.

Mitochondrial morphology changes in response to various stimuli but the significance of this is unclear. In a screen for mutants with abnormal mitochondrial morphology, we identified MMA-1, the Caenorhabditis elegans homolog of the French Canadian Leigh Syndrome protein LRPPRC (leucine-rich pentatricopeptide repeat containing). We demonstrate that reducing mma-1 or LRPPRC function causes mitochondrial hyperfusion. Reducing mma-1/LRPPRC function also decreases the activity of complex IV of the electron transport chain, however without affecting cellular ATP levels. Preventing mitochondrial hyperfusion in mma-1 animals causes larval arrest and embryonic lethality. Furthermore, prolonged LRPPRC knock-down in mammalian cells leads to mitochondrial fragmentation and decreased levels of ATP. These findings indicate that in a mma-1/LRPPRC-deficient background, hyperfusion allows mitochondria to maintain their functions despite a reduction in complex IV activity. Our data reveal an evolutionary conserved mechanism that is triggered by reduced complex IV function and that induces mitochondrial hyperfusion to transiently compensate for a drop in the activity of the electron transport chain.

Recombinant expression, biophysical characterization, and cardiolipin-induced changes of two Caenorhabditis elegans cytochrome c proteins.

Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of Caenorhabditis elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein's denatured state. Both C. elegans proteins bind to CL-containing liposomes, and these interactions promote the proteins' peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved fluorescence resonance energy transfer in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c-CL interactions in live organisms.

A molecular switch that governs mitochondrial fusion and fission mediated by the BCL2-like protein CED-9 of Caenorhabditis elegans.

Depending on the cellular context, BCL2-like proteins promote mitochondrial fusion or fission. What determines which of these two opposing processes they promote has so far been unknown. Furthermore, the mechanisms through which BCL2-like proteins affect mitochondrial dynamics remain to be fully understood. The BCL2-like protein CED-9 of Caenorhabditis elegans has previously been shown to promote mitochondrial fusion by physically interacting with the mitochondrial fusion protein FZO-1. Here, we report that CED-9 also physically interacts with the mitochondrial fission protein DRP-1 and that this interaction can be enhanced when CED-9 is associated with the BH3-only protein EGL-1. In addition, we show that the EGL-1-CED-9 complex promotes mitochondrial fission by recruiting DRP-1 to mitochondria and that the egl-1 gene is required for CED-9-dependent mitochondrial fission in vivo. Based on these results, we propose that EGL-1 converts CED-9 into a mitochondrial receptor for DRP-1, thereby shifting its activity from profusion to profission. We hypothesize that BCL2-like proteins act as mitochondrial receptors for DRP-1-like proteins in higher organisms as well and that BH3-only proteins play a general role as modifiers of the function in mitochondrial dynamics of BCL2-like proteins. We speculate that this function of BCL2-like proteins may be as couplers of mitochondrial fusion and fission.

Mitochondrial involvement in cell death of non-mammalian eukaryotes.

Although mitochondria are essential organelles for long-term survival of eukaryotic cells, recent discoveries in biochemistry and genetics have advanced our understanding of the requirements for mitochondria in cell death. Much of what we understand about cell death is based on the identification of conserved cell death genes in Drosophila melanogaster and Caenorhabditis elegans. However, the role of mitochondria in cell death in these models has been much less clear. Considering the active role that mitochondria play in apoptosis in mammalian cells, the mitochondrial contribution to cell death in non-mammalian systems has been an area of active investigation. In this article, we review the current research on this topic in three non-mammalian models, C. elegans, Drosophila, and Saccharomyces cerevisiae. In addition, we discuss how non-mammalian models have provided important insight into the mechanisms of human disease as they relate to the mitochondrial pathway of cell death. The unique perspective derived from each of these model systems provides a more complete understanding of mitochondria in programmed cell death. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

New role of the BCL2 family of proteins in the regulation of mitochondrial dynamics.

Mitochondria are highly dynamic organelles that constantly fuse and divide. Dynamin-related GTPases are the core components of the machineries that mediate mitochondrial fusion and fission. The role and regulation of these machineries are currently under intense investigation. Recently, members of the BCL2 family of proteins, conserved regulators of apoptosis, have been implicated in the regulation of mitochondrial dynamics. Here, we review the functions of mitochondrial fusion and fission in apoptotic and nonapoptotic cells and how members of the BCL2 family of proteins regulate these functions.

The BCL-2-like protein CED-9 of C. elegans promotes FZO-1/Mfn1,2- and EAT-3/Opa1-dependent mitochondrial fusion.

The mammalian dynamin-related guanosine triphosphatases Mfn1,2 and Opa1 are required for mitochondrial fusion. However, how their activities are controlled and coordinated is largely unknown. We present data that implicate the BCL-2-like protein CED-9 in the control of mitochondrial fusion in Caenorhabditis elegans. We demonstrate that CED-9 can promote complete mitochondrial fusion of both the outer and inner mitochondrial membrane. We also show that this fusion is dependent on the C. elegans Mfn1,2 homologue FZO-1 and the C. elegans Opa1 homologue EAT-3. Furthermore, we show that CED-9 physically interacts with FZO-1 in vivo and that the ability of CED-9 to interact with FZO-1 is important for its ability to cause mitochondrial fusion. CED-9-induced mitochondrial fusion is not required for the maintenance of mitochondrial morphology during embryogenesis or in muscle cells, at least under normal conditions and in the absence of stress. Therefore, we propose that the BCL-2-like CED-9 acts through FZO-1/Mfn1,2 and EAT-3/Opa1 to promote mitochondrial fusion in response to specific cellular signals.