Gilad Twig

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within β‐cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a ‘kiss and run’ pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (Δψm) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non‐fusing mitochondria that were found to have reduced Δψm and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1K38A or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre‐proteolysis stage reveal that before autophagy mitochondria lose Δψm and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.

Mitochondrial fusion, fission and autophagy as a quality control axis: The bioenergetic view

The mitochondrial life cycle consists of frequent fusion and fission events. Ample experimental and clinical data demonstrate that inhibition of either fusion or fission results in deterioration of mitochondrial bioenergetics. While fusion may benefit mitochondrial function by allowing the spreading of metabolites, protein and DNA throughout the network, the functional benefit of fission is not as intuitive. Remarkably, studies that track individual mitochondria through fusion and fission found that the two events are paired and that fusion triggers fission. On average each mitochondrion would go though approximately 5 fusion:fission cycles every hour. Measurement of Deltapsi(m) during single fusion and fission events demonstrates that fission may yield uneven daughter mitochondria where the depolarized daughter is less likely to become involved in a subsequent fusion and is more likely to be targeted by autophagy. Based on these observations we propose a mechanism by which the integration of mitochondrial fusion, fission and autophagy forms a quality maintenance mechanism. According to this hypothesis pairs of fusion and fission allow for the reorganization and sequestration of damaged mitochondrial components into daughter mitochondria that are segregated from the networking pool and then becoming eliminated by autophagy.

Mitochondrial Networking Protects β-Cells From Nutrient-Induced Apoptosis

OBJECTIVE Previous studies have reported that β-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating β-cell mitochondrial morphology and function, remains unclear. In this article, we investigate β-cell mitochondrial fusion and fission in detail and report alterations in response to various combinations of nutrients. RESEARCH DESIGN AND METHODS Using matrix-targeted photoactivatable green fluorescent protein, mitochondria were tagged and tracked in β-cells within intact islets, as isolated cells and as cell lines, revealing frequent fusion and fission events. Manipulations of key mitochondrial dynamics proteins OPA1, DRP1, and Fis1 were tested for their role in β-cell mitochondrial morphology. The combined effects of free fatty acid and glucose on β-cell survival, function, and mitochondrial morphology were explored with relation to alterations in fusion and fission capacity. RESULTS β-Cell mitochondria are constantly involved in fusion and fission activity that underlies the overall morphology of the organelle. We find that networking activity among mitochondria is capable of distributing a localized green fluorescent protein signal throughout an isolated β-cell, a β-cell within an islet, and an INS1 cell. Under noxious conditions, we find that β-cell mitochondria become fragmented and lose their ability to undergo fusion. Interestingly, manipulations that shift the dynamic balance to favor fusion are able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis. CONCLUSIONS These data suggest that alterations in mitochondrial fusion and fission play a critical role in nutrient-induced β-cell apoptosis and may be involved in the pathophysiology of type 2 diabetes.

Frequency and Selectivity of Mitochondrial Fusion Are Key to Its Quality Maintenance Function

Turnover of mitochondria by autophagy constitutes an essential quality maintenance mechanism. Recent studies have demonstrated that efficient clearance of damaged mitochondrial components depends on mitochondrial dynamics, a process characterized by frequent fusion and fission events that enable the redistribution of mitochondrial components across a population of hundreds of individual mitochondria. The presented simulation identifies kinetic parameters of fusion and fission that may influence the maintenance of mitochondrial function. The program simulated repetitive cycles of fusion and fission events in which intact and damaged mitochondrial contents were redistributed between fusion mates. Redistribution impacted mitochondrial function, thereby influencing the fate of each mitochondrion, to be either destined for a subsequent fusion or eliminated by autophagy. Our findings indicate that, when paired with fission, fusion events may serve to accelerate the removal of damaged mitochondrial components by autophagy. The model predicts the existence of an optimal frequency of fusion and fission events that can maintain respiratory function at steady-state levels amid the existence of a continuous damaging process that inactivates mitochondrial components. A further elevation of the fusion frequency can increase the clearance efficiency of damaged content. However, this requires fusion to be a selective process in which depolarized mitochondria are excluded from the fusing population. The selectivity of fusion was found to be particularly beneficial in conditions of elevated rate of damage, because it permits the increase of fusion frequency without compromising the removal of damaged content by autophagy.

What can mitochondrial heterogeneity tell us about mitochondrial dynamics and autophagy

A growing body of evidence shows that mitochondria are heterogeneous in terms of structure and function. Increased heterogeneity has been demonstrated in a number of disease models including ischemia-reperfusion and nutrient-induced beta cell dysfunction and diabetes. Subcellular location and proximity to other organelles, as well as uneven distribution of respiratory components have been considered as the main contributors to the basal level of heterogeneity. Recent studies point to mitochondrial dynamics and autophagy as major regulators of mitochondrial heterogeneity. While mitochondrial fusion mixes the content of the mitochondrial network, fission dissects the mitochondrial network and generates depolarized segments. These depolarized mitochondria are segregated from the networking population, forming a pre-autophagic pool contributing to heterogeneity. The capacity of a network to yield a depolarized daughter mitochondrion by a fission event is fundamental to the generation of heterogeneity. Several studies and data presented here provide a potential explanation, suggesting that protein and membranous structures are unevenly distributed within the individual mitochondrion and that inner membrane components do not mix during a fusion event to the same extent as the matrix components do. In conclusion, mitochondrial subcellular heterogeneity is a reflection of the mitochondrial lifecycle that involves frequent fusion events in which components may be unevenly mixed and followed by fission events generating disparate daughter mitochondria, some of which may fuse again, others will remain solitary and join a pre-autophagic pool.

Hormone‐induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure

Adrenergic stimulation of brown adipocytes (BA) induces mitochondrial uncoupling, thereby increasing energy expenditure by shifting nutrient oxidation towards thermogenesis. Here we describe that mitochondrial dynamics is a physiological regulator of adrenergically‐induced changes in energy expenditure. The sympathetic neurotransmitter Norepinephrine (NE) induced complete and rapid mitochondrial fragmentation in BA, characterized by Drp1 phosphorylation and Opa1 cleavage. Mechanistically, NE‐mediated Drp1 phosphorylation was dependent on Protein Kinase‐A (PKA) activity, whereas Opa1 cleavage required mitochondrial depolarization mediated by FFAs released as a result of lipolysis. This change in mitochondrial architecture was observed both in primary cultures and brown adipose tissue from cold‐exposed mice. Mitochondrial uncoupling induced by NE in brown adipocytes was reduced by inhibition of mitochondrial fission through transient Drp1 DN overexpression. Furthermore, forced mitochondrial fragmentation in BA through Mfn2 knock down increased the capacity of exogenous FFAs to increase energy expenditure. These results suggest that, in addition to its ability to stimulate lipolysis, NE induces energy expenditure in BA by promoting mitochondrial fragmentation. Together these data reveal that adrenergically‐induced changes to mitochondrial dynamics are required for BA thermogenic activation and for the control of energy expenditure.

Organellar vs cellular control of mitochondrial dynamics.

Mitochondrial dynamics, the fusion and fission of individual mitochondrial units, is critical to the exchange of the metabolic, genetic and proteomic contents of individual mitochondria. In this regard, fusion and fission events have been shown to modulate mitochondrial bioenergetics, as well as several cellular processes including fuel sensing, ATP production, autophagy, apoptosis, and the cell cycle. Regulation of the dynamic events of fusion and fission occur at two redundant and interactive levels. Locally, the microenvironment of the individual mitochondrion can alter its ability to fuse, divide or move through the cell. Globally, nuclear-encoded processes and cellular ionic and second messenger systems can alter or activate mitochondrial proteins, regulate mitochondrial dynamics and concomitantly change the condition of the mitochondrial population. In this review we investigate the different global and local signals that control mitochondrial biology. This discussion is carried out to clarify the different signals that impact the status of the mitochondrial population.

MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age

To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.

Autophagy in the Homeostasis of Pancreatic β-Cells

Autophagy is increasingly recognized as a key process for the homeostasis in β-cells. In vivo evidence in pancreata of diabetic patients suggests that autophagy is impaired, while selective arrest of autophagy in β-cells of mice alters insulin secretion, followed by hyperglycemia. Several cellular pathways have been implicated to be tightly coupled to autophagy. Autophagy is critical for mitochondrial homeostasis in β-cells and selectively removes mitochondria at a sustained lower energetic level. As mitochondria are critical for insulin secretion, arrest of their removal leads to accumulation of mitochondrial dysfunction and results in impaired insulin secretion. In addition, various lysosomal enzymes that are associated with various stages of autophagy are co-localized on insulin granules. Hence, autophagy also plays a role in regulating insulin granules number and total insulin content.