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Significance: In addition to their classical role in cellular ATP production, mitochondria are of key relevance in various (patho)physiological mechanisms including second messenger signaling, neuro-transduction, immune responses and death induction. Recent Advances: Within cells, mitochondria are motile and display temporal changes in internal and external structure ("mitochondrial dynamics"). During the last decade, substantial empirical and in silico evidence was presented demonstrating that mitochondrial dynamics impacts on mitochondrial function and vice versa. Critical Issues: However, a comprehensive and quantitative understanding of the bidirectional links between mitochondrial external shape, internal structure and function ("morphofunction") is still lacking. The latter particularly hampers our understanding of the functional properties and behavior of individual mitochondrial within single living cells. Future Directions: In this review we discuss the concept of mitochondrial morphofunction in mammalian cells, primarily using experimental evidence obtained within the last decade. The topic is introduced by briefly presenting the central role of mitochondria in cell physiology and the importance of the mitochondrial electron transport chain (ETC) therein. Next, we summarize in detail how mitochondrial (ultra)structure is controlled and discuss empirical evidence regarding the equivalence of mitochondrial (ultra)structure and function. Finally, we provide a brief summary of how mitochondrial morphofunction can be quantified at the level of single cells and mitochondria, how mitochondrial ultrastructure/volume impacts on mitochondrial bioreactions and intramitochondrial protein diffusion, and how mitochondrial morphofunction can be targeted by small molecules.

Biodegradable, semipermeable nanoreactors that are capable of undergoing cellular integration and, subsequently, function as synthetic organelles represent an exciting therapeutic technology. Polymersomal nanoreactors have been investigated as a suitable candidate for the engineering of such a system, with the chemical versatility and structural robustness required for such a demanding application. Although steps have been taken to demonstrate this capacity, there has yet to be a system presented with biochemically robust data showing therapeutic efficacy in primary human cells. The reason for this shortfall is the absence of essential criteria of the polymersomes tested so far; biodegradability, intrinsic semipermeability, and a biomedically relevant setting. Herein, we present enzyme-loaded, biodegradable poly(ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-PCLgTMC) polymersomal nanoreactors, readily fabricated using the biocompatible direct hydration methodology. Physical characterization of PEG-PCLgTMC polymersomes highlights their semipermeable membrane and ability to shield enzymatic cargo. Surface modification with cell-penetrating peptides (CPPs) directs cellular integration of enzyme-loaded PEG-PCLgTMC nanoreactors in a controlled fashion. Using HEK293T cells and human skin fibroblasts we demonstrate that biocompatible catalase nanoreactors successfully perform as a synthetic organelle, imparting activity-dependent antioxidant (reactive-oxygen-species-shielding, ROS-shielding) capacity to cells. Notably, for the first time, patient-derived human-complex-I-deficient primary fibroblasts are effectively protected against the toxicity of exogenous H2O2 by the action of internalized enzyme-loaded nanoreactors, showcasing this system in a therapeutically relevant context.

Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 (‘BAY’) triggers death of BRAFV600E melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of eve

This review presents our current understanding of the pathophysiology and potential treatment strategies with respect to mitochondrial disease in children. We focus on pathologies due to mutations in nuclear DNA-encoded structural and assembly factors of the mitochondrial oxidative phosphorylation (OXPHOS) system, with a particular emphasis on isolated mitochondrial complex I deficiency. Following a brief introduction into mitochondrial disease and OXPHOS function, an overview is provided of the diagnostic process in children with mitochondrial disorders. This includes the impact of whole-exome sequencing and relevance of cellular complementation studies. Next, we briefly present how OXPHOS mutations can affect cellular parameters, primarily based on studies in patient-derived fibroblasts, and how this information can be used for the rational design of small-molecule treatment strategies. Finally, we discuss clinical trial design and provide an overview of small molecules that are currently being developed for treatment of mitochondrial disease.

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrialmorphofunction.Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.