Mitochondria are an essential source of ATP for cellular function, but when damaged, mitochondria generate a plethora of stress signals, which lead to cellular dysfunction and eventually programmed ...cell death. Thus, a major component of maintaining cellular homeostasis is the recognition and removal of dysfunctional mitochondria through autophagy-mediated degradation, i.e., mitophagy. Mitophagy further constitutes a developmental program, and undergoes a high degree of crosstalk with apoptosis. Reduced mitochondrial quality control is linked to disease pathogenesis, suggesting the importance of process elucidation as a clinical target. Recent work has revealed multiple mitophagy programs that operate independently or undergo crosstalk, and require modulated autophagy receptor activities at outer membranes of mitochondria. Here, we review these mitophagy programs, focusing on pathway mechanisms which recognize and target mitochondria for sequestration by autophagosomes, as well as mechanisms controlling pathway activities. Furthermore, we provide an introduction to the currently available methods for detecting mitophagy.
Mitochondrial outer membrane permeabilization (MOMP) is a core event in apoptosis signaling. However, the underlying mechanism of BAX and BAK pore formation remains incompletely understood. We ...demonstrate that mitochondria are globally and dynamically targeted by endolysosomes (ELs) during MOMP. In response to pro-apoptotic BH3-only protein signaling and pharmacological MOMP induction, ELs increasingly form transient contacts with mitochondria. Subsequently, ELs rapidly accumulate within the entire mitochondrial compartment. This switch-like accumulation period temporally coincides with mitochondrial BAX clustering and cytochrome c release. Remarkably, interactions of ELs with mitochondria control BAX recruitment and pore formation. Knockdown of Rab5A, Rab5C, or USP15 interferes with EL targeting of mitochondria and functionally uncouples BAX clustering from cytochrome c release, while knockdown of the Rab5 exchange factor Rabex-5 impairs both BAX clustering and cytochrome c release. Together, these data reveal that EL-mitochondrial inter-organelle communication is an integral regulatory component of functional MOMP execution during cellular apoptosis signaling.
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•EL targeting of mitochondria is a common feature during apoptosis signaling•Knockdown of Rab5 or USP15 uncouples BAX clustering from MOMP•Rab5 GEF Rabex-5 regulates MOMP upstream of BAX recruitment•EL transformation of mitochondria positively regulates BAX-mediated MOMP
Wang et al. show that upon activation of regulated cell death, endolysosomal vesicles (ELs) are targeted to mitochondria prior to and during their permeabilization by BAX-mediated pores. These inter-organelle interactions result in a biochemical transformation of mitochondrial membranes and are required for functional in situ apoptotic pore formation.
A genetic mutation in the C9orf72 gene causes the most common forms of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein, predicted ...to be a DENN-family protein, is reduced in ALS and FTD, but its functions remain poorly understood. Using a 3110043O21Rik/C9orf72 knockout mouse model, as well as cellular analysis, we have found that loss of C9orf72 causes alterations in the signaling states of central autophagy regulators. In particular, C9orf72 depletion leads to reduced activity of MTOR, a negative regulator of macroautophagy/autophagy, and concomitantly increased TFEB levels and nuclear translocation. Consistent with these alterations, cells exhibit enlarged lysosomal compartments and enhanced autophagic flux. Loss of the C9orf72 interaction partner SMCR8 results in similar phenotypes. Our findings suggest that C9orf72 functions as a potent negative regulator of autophagy, with a central role in coupling the cellular metabolic state with autophagy regulation. We thus propose C9orf72 as a fundamental component of autophagy signaling with implications in basic cell physiology and pathophysiology, including neurodegeneration.
Mitochondria are semi-autonomous organelles that supply energy for cellular biochemistry through oxidative phosphorylation. Within a cell, hundreds of mobile mitochondria undergo fusion and fission ...events to form a dynamic network. These morphological and mobility dynamics are essential for maintaining mitochondrial functional homeostasis, and alterations both impact and reflect cellular stress states. Mitochondrial homeostasis is further dependent on production (biogenesis) and the removal of damaged mitochondria by selective autophagy (mitophagy). While mitochondrial function, dynamics, biogenesis and mitophagy are highly-integrated processes, it is not fully understood how systemic control in the cell is established to maintain homeostasis, or respond to bioenergetic demands. Here we used agent-based modeling (ABM) to integrate molecular and imaging knowledge sets, and simulate population dynamics of mitochondria and their response to environmental energy demand. Using high-dimensional parameter searches we integrated experimentally-measured rates of mitochondrial biogenesis and mitophagy, and using sensitivity analysis we identified parameter influences on population homeostasis. By studying the dynamics of cellular subpopulations with distinct mitochondrial masses, our approach uncovered system properties of mitochondrial populations: (1) mitochondrial fusion and fission activities rapidly establish mitochondrial sub-population homeostasis, and total cellular levels of mitochondria alter fusion and fission activities and subpopulation distributions; (2) restricting the directionality of mitochondrial mobility does not alter morphology subpopulation distributions, but increases network transmission dynamics; and (3) maintaining mitochondrial mass homeostasis and responding to bioenergetic stress requires the integration of mitochondrial dynamics with the cellular bioenergetic state. Finally, (4) our model suggests sources of, and stress conditions amplifying, cell-to-cell variability of mitochondrial morphology and energetic stress states. Overall, our modeling approach integrates biochemical and imaging knowledge, and presents a novel open-modeling approach to investigate how spatial and temporal mitochondrial dynamics contribute to functional homeostasis, and how subcellular organelle heterogeneity contributes to the emergence of cell heterogeneity.
Programmed cell death of cardiac myocytes occurs following a bout of ischemia/reperfusion (I/R), which results in reduced function of the heart. Numerous studies, including in vivo, have shown that ...cell death occurs via necrosis and apoptosis following I/R. Recently, autophagy has emerged as a powerful mediator of programmed cell death, either opposing or enhancing apoptosis, or acting as an alternative form of programmed cell death distinct from apoptosis.
Here we review the apoptotic and autophagic signaling pathways, their influences on each other, and we discuss the relevance of autophagy in the heart.
TFEB (transcription factor EB) regulates metabolic homeostasis through its activation of lysosomal biogenesis following its nuclear translocation. TFEB activity is inhibited by mTOR phosphorylation, ...which signals its cytoplasmic retention. To date, the temporal relationship between alterations to mTOR activity states and changes in TFEB subcellular localization and concentration has not been sufficiently addressed.
mTOR was activated by renewed addition of fully-supplemented medium, or inhibited by Torin1 or nutrient deprivation. Single-cell TFEB protein levels and subcellular localization in HeLa and MCF7 cells were measured over a time course of 15 hours by multispectral imaging cytometry. To extract single-cell level information on heterogeneous TFEB activity phenotypes, we developed a framework for identification of TFEB activity subpopulations. Through unsupervised clustering, cells were classified according to their TFEB nuclear concentration, which corresponded with downstream lysosomal responses.
Bulk population results revealed that mTOR negatively regulates TFEB protein levels, concomitantly to the regulation of TFEB localization. Subpopulation analysis revealed maximal sensitivity of HeLa cells to mTOR activity stimulation, leading to inactivation of 100 % of the cell population within 0.5 hours, which contrasted with a lower sensitivity in MCF7 cells. Conversely, mTOR inhibition increased the fully active subpopulation only fractionally, and full activation of 100 % of the population required co-inhibition of mTOR and the proteasome. Importantly, mTOR inhibition activated TFEB for a limited duration of 1.5 hours, and thereafter the cell population was progressively re-inactivated, with distinct kinetics for Torin1 and nutrient deprivation treatments.
TFEB protein levels and subcellular localization are under control of a short-term rheostat, which is highly responsive to negative regulation by mTOR, but under conditions of mTOR inhibition, restricts TFEB activation in a manner dependent on the proteasome. We further identify a long-term, mTOR-independent homeostatic control negatively regulating TFEB upon prolonged mTOR inhibition. These findings are of relevance for developing strategies to target TFEB activity in disease treatment. Moreover, our quantitative approach to decipher phenotype heterogeneity in imaging datasets is of general interest, as shifts between subpopulations provide a quantitative description of single cell behaviour, indicating novel regulatory behaviors and revealing differences between cell types.
Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate ...apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.
Background: XIAP E3 ligase induces mitochondrial membrane permeabilization, resulting in intramitochondrial degradation of its inhibitor Smac.
Results: Mitochondrial XIAP action is triggered by drug inducers of apoptosis and occurs before Drp1-mediated cytochrome c release.
Conclusion: Pre-activation of mitochondrial XIAP action can protect against caspase activation.
Significance: Mitochondrial processing by XIAP constitutes a new antagonistic component of intrinsic apoptosis signaling.
Mitochondria are key regulators of apoptosis. In response to stress, BH3-only proteins activate pro-apoptotic Bcl2 family proteins Bax and Bak, which induce mitochondrial outer membrane ...permeabilization (MOMP). While the large-scale mitochondrial release of pro-apoptotic proteins activates caspase-dependent cell death, a limited release results in sub-lethal caspase activation which promotes tumorigenesis. Mitochondrial autophagy (mitophagy) targets dysfunctional mitochondria for degradation by lysosomes, and undergoes extensive crosstalk with apoptosis signaling, but its influence on apoptosis remains undetermined. The BH3-only protein Bnip3 integrates apoptosis and mitophagy signaling at different signaling domains. Bnip3 inhibits pro-survival Bcl2 members via its BH3 domain and activates mitophagy through its LC3 Interacting Region (LIR), which is responsible for binding to autophagosomes. Previously, we have shown that Bnip3-activated mitophagy prior to apoptosis induction can reduce mitochondrial activation of caspases, suggesting that a reduction to mitochondrial levels may be pro-survival. An outstanding question is whether organelle dynamics and/or recently discovered subcellular variations of protein levels responsible for both MOMP sensitivity and crosstalk between apoptosis and mitophagy can influence the cellular apoptosis decision event. To that end, here we undertook a systems biology analysis of mitophagy-apoptosis crosstalk at the level of cellular mitochondrial populations.
Based on experimental findings, we developed a multi-scale, hybrid model with an individually adaptive mitochondrial population, whose actions are determined by protein levels, embedded in an agent-based model (ABM) for simulating subcellular dynamics and local feedback via reactive oxygen species signaling. Our model, supported by experimental evidence, identified an emergent regulatory structure within canonical apoptosis signaling. We show that the extent of mitophagy is determined by levels and spatial localization of autophagy capacity, and subcellular mitochondrial protein heterogeneities. Our model identifies mechanisms and conditions that alter the mitophagy decision within mitochondrial subpopulations to an extent sufficient to shape cellular outcome to apoptotic stimuli.
Overall, our modeling approach provides means to suggest new experiments and implement findings at multiple scales in order to understand how network topologies and subcellular heterogeneities can influence signaling events at individual organelle level, and hence, determine the emergence of heterogeneity in cellular decisions due the actions of the collective intra-cellular population.
The prosurvival BCL-2 proteins are attractive yet challenging targets for medicinal chemists. Their involvement in the initiation and progression of many, if not all, tumors makes them prime targets ...for developing new anticancer therapies. We present our approach based on de novo structure-based drug design. Using known structural information from complexes engaging opposing members of the BCL-2 family of proteins, we designed peptidomimetic compounds using a benzoylurea scaffold to reproduce key interactions between these proteins. A library stemming from the initial de novo designed scaffold led to the discovery of ligands with low micromolar potency (K D = 4 μM) and selectivity for BCL-XL. These compounds bind in the canonical BH3 binding groove in a binding mode distinct from previously known BCL-2 inhibitors. The results of our study provide insight into the design of a new class of antagonists targeting a challenging class of protein–protein interactions.
In our program to develop non-invasive magnetic resonance imaging (MRI) methods for the diagnosis of Alzheimer's disease (AD), we have synthesized antibody-conjugated, superparamagnetic iron oxide ...nanoparticles (SPIONs) for use as an in vivo agent for MRI detection of amyloid-β plaques in AD. Here we report studies in AβPP/PS1 transgenic mice, which demonstrate the ability of novel anti-AβPP conjugated SPIONs to penetrate the blood-brain barrier to act as a contrast agent for MR imaging of plaques. The conspicuity of the plaques increased from an average Z-score of 5.1 ± 0.5 to 8.3 ± 0.2 when the plaque contrast to noise ratio was compared in control AD mice with AD mice treated with SPIONs. The number of MRI-visible plaques per brain increased from 347 ± 45 in the control AD mice, to 668 ± 86 in the SPION treated mice. These results indicated that our SPION enhanced amyloid-β detection method delivers an efficacious, non-invasive MRI detection method in transgenic mice.
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