eLife最新文献

The mitochondrial transcription factor A (TFAM) is essential for mitochondrial genome maintenance. It binds to mitochondrial DNA (mtDNA) and determines the abundance, packaging, and stability of the mitochondrial genome. Because its function is tightly associated with mtDNA, TFAM has a protective role in mitochondrial diseases, and supportive studies demonstrate reversal of disease phenotypes by TFAM overexpression. In addition, TFAM deficiency has been shown to cause release of mtDNA into the cytosol and activation of the cGAS/STING innate immune response pathway. As such, TFAM presents as a unique target for therapeutic intervention, but limited efforts for activators have been reported. Herein, we disclose novel TFAM small-molecule modulators with sub-micromolar activity. Our results demonstrate that these compounds result in an increase of TFAM protein levels and mtDNA copy number. This results in inhibition of a mtDNA stress-mediated inflammatory response by preventing mtDNA escape into the cytosol. Furthermore, we see beneficial effects in cellular disease models in which boosting TFAM activity has been advanced as a disease-modifying strategy including improved energetics in MELAS cybrid cells and a decrease of fibrotic markers in systemic sclerosis fibroblasts. These results highlight the therapeutic potential of using small-molecule TFAM activators in indications characterized by mitochondrial dysfunction.

Fungi exhibit remarkable morphological plasticity, which allows them to undergo reversible transitions between distinct cellular states in response to changes in their environment. This phenomenon, termed fungal morphogenesis, is critical for fungi to survive and colonize diverse ecological niches and establish infections in a variety of hosts. Despite significant advancements in the field with respect to understanding the gene regulatory networks that control these transitions, the metabolic determinants of fungal morphogenesis remain poorly characterized. In this study, we uncover a previously uncharacterized, conserved dependency between central carbon metabolism and de novo biosynthesis of sulfur-containing amino acids that is critical for fungal morphogenesis in two key fungal species. Using a multidisciplinary approach, we demonstrate that glycolytic flux is crucial to drive fungal morphogenesis in a cAMP-independent manner and perturbation of this pathway leads to a significant downregulation in the expression of genes involved in de novo biosynthesis of sulfur-containing amino acids. Remarkably, exogenous supplementation of sulfur-containing amino acids robustly rescues the morphogenesis defect induced by the perturbation of glycolysis in both Saccharomyces cerevisiae and Candida albicans, underscoring the pivotal role of de novo biosynthesis of sulfur-containing amino acids as a downstream effector of morphogenesis. Furthermore, a C. albicans mutant lacking the glycolytic enzyme, phosphofructokinase-1 (Pfk1), exhibited significantly reduced survival within murine macrophages and attenuated virulence in a murine model of systemic candidiasis. Overall, our work elucidates a previously uncharacterized coupling between glycolysis and sulfur metabolism that is critical for driving fungal morphogenesis, contributing to our understanding of this conserved phenomenon.

Evolutionary adaptation to diurnal vision in ground squirrels has led to the development of a cone-dominant retina, in stark contrast to the rod-dominant retinas of most mammals. The molecular mechanisms driving this shift remain largely unexplored. Here, we perform single-cell RNA sequencing and chromatin accessibility profiling (scATAC-Seq) across developmental retinal neurogenesis in the 13-lined ground squirrel (13LGS) to uncover the regulatory basis of this adaptation. We find that 13LGS cone photoreceptors arise not only from early-stage neurogenic progenitors, as seen in rod-dominant species like mice, but also from late-stage neurogenic progenitors. This extended period of cone generation is driven by a heterochronic shift in transcription factor expression, with cone-promoting factors such as Onecut2, Pou2f1, and Zic3 remaining active in late-stage progenitors, and factors that promote cone differentiation such as Thrb, Rxrg, and Mef2c expressed precociously in late-stage neurogenic progenitors. Functional analyses reveal that Zic3 and Mef2c are sufficient to promote cone and repress rod photoreceptor-specific gene expression and act through species-specific regulatory elements that drive their expression in late-stage progenitors. These results demonstrate that modifications to gene regulatory networks underlie the development of cone-dominant retinas and provide insight into mechanisms of sensory adaptation and potential strategies for cone photoreceptor regeneration in vision disorders.

Although several open-source, easy-to-assemble light-sheet microscope platforms already exist-such as mesoSPIM, OpenSPIM, and OpenSpin-they are optimized for imaging large specimens and lack the resolution required to visualize subcellular features, such as organelles or cytoskeletal architectures. In contrast, lattice light-sheet microscopy (LLSM) achieves the resolution necessary to resolve such fine structures but, in its open-source implementation, can be alignment- and maintenance-intensive, often requiring specialist expertise. To address this gap, we developed Altair light-sheet fluorescence microscopy (LSFM), a high-resolution, open-source, sample-scanning light-sheet microscope specifically designed for subcellular imaging. By optimizing the optical pathway in silico, we created a custom baseplate that greatly simplifies alignment and assembly. The system integrates streamlined optoelectronics and optomechanics with seamless operation through our open-source software, navigate. Altair-LSFM achieves lateral and axial resolutions of approximately 235 and 350 nm, respectively, across a 266 µm field of view after deconvolution. We validate the system's capabilities by imaging sub-diffraction fluorescent nanospheres and visualizing fine structural details in mammalian cells, including microtubules, actin filaments, nuclei, and Golgi apparatus. We further demonstrate its live-cell imaging capabilities by visualizing microtubules and vimentin intermediate filaments in actively migrating cells.

Transition metals, such as iron and zinc, are indispensable trace elements for eukaryotic life, acting as co-factors in essential processes ranging from metabolism to DNA replication. These metals can be transported into cells by an evolutionary-conserved family of metal transporters; however, how the ubiquitous mammalian parasite Toxoplasma gondii acquires essential metals has been unknown. Here, we have identified and characterised the first iron and zinc importer in T. gondii. This transporter, named ZFT, localised to the parasite plasma membrane and is essential for the parasite's life cycle. We find ZFT is regulated by iron availability and overexpression sensitises cells to excess iron and zinc. Using a conditional knockdown system, we find that knockdown of ZFT leads to reduction in mitochondrial respiration and a switch to a more quiescent lifecycle stage. To confirm transport activity, we find that knockdown of ZFT leads to a reduction in parasite-associated zinc and iron, and ZFT expression complements loss of zinc transporter activity in a yeast model. Further, expression of ZFT in Xenopus oocytes demonstrates direct uptake of iron, which is outcompeted in the presence of zinc. Overall, we have identified the first metal uptake transporter in T. gondii and demonstrated the importance of iron and zinc to the parasite. This finding advances our understanding of how this obligate intracellular parasite acquires nutrients from its host.

Axolotls (Ambystoma mexicanum) exhibit a remarkable ability to regenerate limbs. Classical experiments have suggested that contact between cells derived from distinct orientations-dorsal, ventral, anterior, and posterior-within the regenerating blastema is necessary for accurate limb pattern formation. However, the molecular basis for this requirement has remained largely unknown. Here, we demonstrate that both dorsal and ventral tissues are required for limb formation via induction of Shh expression, which plays a crucial role in limb patterning. Using the accessory limb model, we induced position-specific blastemas lacking cells derived from a single orientation (anterior, posterior, dorsal, or ventral). Limb patterning occurred only in blastemas containing both dorsal- and ventral-derived cells. We further observed that Shh expression requires dorsoventral contact within a blastema, highlighting the necessity of dorsoventral contact for inducing Shh expression. Additionally, we identified WNT10B and FGF2 as dorsal- and ventral-mediated signals, respectively, that create the inductive environment for Shh expression. Our findings clarify the role of dorsal and ventral cells in inducing Shh, a mechanism that has rarely been studied in the context of limb regeneration and pattern formation. This model provides new insights into how cells with different positional identities drive the regeneration process.

The protective correlates of Mycobacterium tuberculosis (Mtb) infection-elicited host immune responses are incompletely understood. Here, we report pro-pathogenic crosstalk involving Ly6G⁺ granulocytes (Ly6G⁺Gra), IL-17, and COX2. We show that in the lungs of Mtb-infected wild-type mice, either BCG-vaccinated or not, most intracellular bacilli are Ly6G⁺Gra-resident 4 weeks post-infection onwards. In the genetically susceptible ifng^-/- mice, excessive Ly6G⁺Gra infiltration correlates with severe bacteremia. Neutralizing IL-17 (anti-IL17mAb) and COX2 inhibition by celecoxib reverse Ly6G⁺Gra infiltration, associated pathology, and death in ifng^-/- mice. Surprisingly, Ly6G⁺Gra also serves as the major source of IL-17 in the lungs of Mtb-infected WT or ifng^-/- mice. The IL-17-COX2-Ly6G⁺Gra interplay also operates in WT mice. Inhibiting RORγt, the key transcription factor for IL-17 production or COX2, reduces the bacterial burden in Ly6G⁺Gra, leading to reduced bacterial burden and pathology in the lungs of WT mice. In the Mtb-infected WT mice, COX2 inhibition abrogates IL-17 levels in the lung homogenates and significantly enhances BCG's protective efficacy, mainly by targeting the Ly6G⁺Gra-resident Mtb pool, a phenotype also observed when IL-17 is blocked by RORγt inhibitor. Furthermore, in pulmonary TB patients, high neutrophil count and IL-17 correlated with adverse treatment outcomes. Together, our results suggest that IL-17 and PGE2 are the negative correlates of protection, and we propose targeting the pro-pathogenic IL-17-COX2-Ly6G⁺Gra axis for TB prevention and therapy.

The behaviour of a receptor protein can be influenced by the presence of certain lipids in the membrane it is embedded in.

Even seemingly homogeneous populations of cells can express phenotypic diversity in response to environmental changes. Thus, X-ray irradiation of tissues composed of diverse cell types can have complex outcomes. We have used single-cell RNA sequencing to study the effects of X-ray radiation on the Drosophila wing imaginal disc, a relatively simple tissue composed mostly of epithelial cells. Transcriptomic clustering of cells collected from the wing disc generates clusters that are mainly grouped based on proximodistal cell location. To quantify heterogeneity of gene expression among clusters, we adapted a metric used to study market concentration, the Herfindahl-Hirschman Index. Genes involved in DNA damage repair, defense against reactive oxygen species, cell cycle progression, and apoptosis are expressed relatively uniformly. In contrast, genes encoding a subset of ligands, notably cytokines that activate the JAK/STAT pathway, some transcription factors, including Ets21C, previously implicated in regeneration, and several signaling proteins are expressed more regionally. Though the radiation-responsive transcription factor p53 is expressed relatively uniformly in the wing disc, several regionally induced genes still require p53 function, indicating that regional and radiation-induced factors combine to regulate their expression. We also examined heterogeneity within regions using a clustering approach based on cell cycle gene expression. A subpopulation of cells, characterized by high levels of tribbles expression, is amplified in irradiated discs. Remarkably, this subpopulation accounts for a considerable fraction of radiation-induced gene expression, indicating that cellular responses are non-uniform even within regions. Thus, both inter-regional and intra-regional heterogeneity are important features of tissue responses to X-ray radiation.