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Mitochondrial permeability transition (mPT) is a phenomenon that abruptly causes the flux of low molecular weight solutes (molecular weight up to 1,500) across the generally impermeable inner mitochondrial membrane. The mPT is mediated by the so-called mitochondrial permeability transition pore (mPTP), a supramolecular entity assembled at the interface of the inner and outer mitochondrial membranes. In contrast to mitochondrial outer membrane permeabilization, which mostly activates apoptosis, mPT can trigger different cellular responses, from the physiological regulation of mitophagy to the activation of apoptosis or necrosis. Although there are several molecular candidates for the mPTP, its molecular nature remains contentious. This lack of molecular data was a significant setback that prevented mechanistic insight into the mPTP, pharmacological targeting and the generation of informative animal models. In recent years, experimental evidence has highlighted mitochondrial F1Fo ATP synthase as a participant in mPTP formation, although a molecular model for its transition to the mPTP is still lacking. Recently, the resolution of the F1Fo ATP synthase structure by cryogenic electron microscopy led to a model for mPTP gating. The elusive molecular nature of the mPTP is now being clarified, marking a turning point for understanding mitochondrial biology and its pathophysiological ramifications. This Review provides an up-to-date reference for the understanding of the mammalian mPTP and its cellular functions. We review current insights into the molecular mechanisms of mPT and validated observations — from studies in vivo or in artificial membranes — on mPTP activity and functions. We end with a discussion of the contribution of the mPTP to human disease. Throughout the Review, we highlight the multiple unanswered questions and, when applicable, we also provide alternative interpretations of the recent discoveries.Mitochondrial permeability transition — mediated by the opening of the so-called mitochondrial permeability transition pore — causes abrupt flux of low molecular weight solutes across the generally impermeable inner mitochondrial membrane. Recent studies provide new insights into the molecular nature and mechanisms of the mitochondrial permeability transition pore and the physiological consequences of its opening.

A large body of evidence indicates that mitochondrial dysfunction has a major role in the pathogenesis of multiple cardiovascular disorders. Over the past 2 decades, extraordinary efforts have been focused on the development of agents that specifically target mitochondria for the treatment of cardiovascular disease. Despite such an intensive wave of investigation, no drugs specifically conceived to modulate mitochondrial functions are currently available for the clinical management of cardiovascular disease. In this Review, we discuss the therapeutic potential of targeting mitochondria in patients with cardiovascular disease, examine the obstacles that have restrained the development of mitochondria-targeting agents thus far, and identify strategies that might empower the full clinical potential of this approach.

Membrane contact sites between virtually any known organelle have been documented and, in the last decades, their study received momentum due to their importance for fundamental activities of the cell and for the subtle comprehension of many human diseases. The lack of tools to finely image inter-organelle proximity hindered our understanding on how these subcellular communication hubs mediate and regulate cell homeostasis. We develop an improved and expanded palette of split-GFP-based contact site sensors (SPLICS) for the detection of single and multiple organelle contact sites within a scalable distance range. We demonstrate their flexibility under physiological conditions and in living organisms. The authors have previously reported split-GFP-based contact site sensors (SPLICS) to document endoplasmic reticulum/mitochondria contact sites. Here they extend this work and develop a range of improved SPLICS sensors to detect single and multiple organelle contact sites at different distances.

HSCs have a fate choice when they divide; they can self-renew, producing new HSCs, or produce daughter cells that will mature to become committed cells. Technical challenges, however, have long obscured the mechanics of these choices. Advances in flow-sorting have made possible the purification of HSC populations, but available HSC-enriched fractions still include substantial heterogeneity, and single HSCs have proven extremely difficult to track and observe. Advances in single-cell approaches, however, have led to the identification of a highly purified population of hematopoietic stem cells (HSCs) that make a critical contribution to hematopoietic homeostasis through a preference for self-renewing division. Metabolic cues are key regulators of this cell fate choice, and the importance of controlling the population and quality of mitochondria has recently been highlighted to maintain the equilibrium of HSC populations. Leukemic cells also demand tightly regulated metabolism, and shifting the division balance of leukemic cells toward commitment has been considered as a promising therapeutic strategy. A deeper understanding of precisely how specific modes of metabolism control HSC fate is, therefore, of great biological interest, and more importantly will be critical to the development of new therapeutic strategies that target HSC division balance for the treatment of hematological disease.

This protocol from Bonora et al . describes three imaging techniques for examining mitochondrial permeability transition (mPT) in living cells. Mitochondrial permeability transition (mPT) refers to a sudden increase in the permeability of the inner mitochondrial membrane. Long-term studies of mPT revealed that this phenomenon has a critical role in multiple pathophysiological processes. mPT is mediated by the opening of a complex termed the mPT pore (mPTP), which is responsible for the osmotic influx of water into the mitochondrial matrix, resulting in swelling of mitochondria and dissipation of the mitochondrial membrane potential. Here we provide three independent optimized protocols for monitoring mPT in living cells: (i) measurement using a calcein–cobalt technique, (ii) measurement of the mPTP-dependent alteration of the mitochondrial membrane potential, and (iii) measurement of mitochondrial swelling. These procedures can easily be modified and adapted to different cell types. Cell culture and preparation of the samples are estimated to take ∼1 d for methods (i) and (ii), and ∼3 d for method (iii). The entire experiment, including analyses, takes ∼2 h.

Sites of close contact between mitochondria and the endoplasmic reticulum (ER) are known as mitochondria-associated membranes (MAM) or mitochondria-ER contacts (MERCs), and play an important role in both cell physiology and pathology. A growing body of evidence indicates that changes observed in the molecular composition of MAM and in the number of MERCs predisposes MAM to be considered a dynamic structure. Its involvement in processes such as lipid biosynthesis and trafficking, calcium homeostasis, reactive oxygen species production, and autophagy has been experimentally confirmed. Recently, MAM have also been studied in the context of different pathologies, including Alzheimer's disease, Parkinson’s disease, amyotrophic lateral sclerosis, type 2 diabetes mellitus and GM1-gangliosidosis. An underappreciated amount of data links MAM with aging or senescence processes. In the present review, we summarize the current knowledge of basic MAM biology, composition and action, and discuss the potential connections supporting the idea that MAM are significant players in longevity.

Significance Accumulating evidence has underscored the role of cytosolic p53 in promoting cell death. Different reports have revealed that p53 participates in apoptosis induction by acting directly at mitochondria. However, because p53 can mediate apoptosis without its DNA-binding domain (the domain proposed to be fundamental for the targeting of p53 to mitochondria), the mitochondrial localization of p53 is likely not the only transcription-independent mechanism by which p53 promotes apoptosis. Here we demonstrate that p53 at the endoplasmic reticulum (ER) and at mitochondria-associated membranes, interacting with sarco/ER Ca ²⁺-ATPase pumps, modulates ER–mitochondria cross-talk and, in turn, Ca ²⁺-dependent apoptosis. The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca ²⁺) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca ²⁺ homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca ²⁺-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca ²⁺ load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca ²⁺ overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca ²⁺ signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca ²⁺ signal-dependent apoptosis.

Calcium (Ca 2+ ) homeostasis is fundamental for cell metabolism, proliferation, differentiation, and cell death. Elevation in intracellular Ca 2+ concentration is dependent either on Ca 2+ influx from the extracellular space through the plasma membrane, or on Ca 2+ release from intracellular Ca 2+ stores, such as the endoplasmic/sarcoplasmic reticulum (ER/SR). Mitochondria are also major components of calcium signalling, capable of modulating both the amplitude and the spatio-temporal patterns of Ca 2+ signals. Recent studies revealed zones of close contact between the ER and mitochondria called MAMs (Mitochondria Associated Membranes) crucial for a correct communication between the two organelles, including the selective transmission of physiological and pathological Ca 2+ signals from the ER to mitochondria. In this review, we summarize the most up-to-date findings on the modulation of intracellular Ca 2+ release and Ca 2+ uptake mechanisms. We also explore the tight interplay between ER- and mitochondria-mediated Ca 2+ signalling, covering the structural and molecular properties of the zones of close contact between these two networks.

The integration of endocytic routes is critical to regulate receptor signaling. A nonclathrin endocytic (NCE) pathway of the epidermal growth factor receptor (EGFR) is activated at high ligand concentrations and targets receptors to degradation, attenuating signaling. Here we performed an unbiased molecular characterization of EGFR-NCE.We identified NCE-specific regulators, including the endoplasmic reticulum (ER)–resident protein reticulon 3 (RTN3) and a specific cargo, CD147. RTN3 was critical for EGFR/CD147-NCE, promoting the creation of plasma membrane (PM)–ER contact sites that were required for the formation and/or maturation of NCE invaginations. Ca2+ release at these sites, triggered by inositol 1,4,5-trisphosphate (IP₃)–dependent activation of ER Ca2+ channels, was needed for the completion of EGFR internalization. Thus, we identified a mechanism of EGFR endocytosis that relies on ER-PM contact sites and local Ca2+ signaling.