Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

AbstractRecent studies show that mitochondria and actin filaments work together in two contexts: 1) increased cytoplasmic calcium induces cytoplasmic actin polymerization that stimulates mitochondrial fission, and 2) mitochondrial depolarization causes actin assembly around mitochondria, with roles in mitophagy. It is unclear whether these two processes utilize similar actin assembly mechanisms. Here, we show that these are distinct actin assembly mechanisms in the acute phase after treatment (<10 min). Calcium-induced actin assembly is INF2-dependent and Arp2/3 complex-independent, whereas depolarization-induced actin assembly is Arp2/3 complex-dependent and INF2-independent. The two types of actin polymerization are morphologically distinct, with calcium-induced filaments throughout the cytosol and depolarization-induced filaments as “clouds” around depolarized mitochondria. We have previously shown that calcium-induced actin stimulates increases in both mitochondrial calcium and recruitment of the dynamin GTPase Drp1. In contrast, depolarization-induced actin is temporally-associated with extensive mitochondrial dynamics that do not result in mitochondrial fission, but in circularization of the inner mitochondrial membrane (IMM). These dynamics are dependent upon the protease Oma1 and independent of Drp1. Actin cloud inhibition causes increased IMM circularization, suggesting that actin clouds limit these dynamics.Summary statementMitochondrial depolarization induces Arp2/3 complex-dependent actin clouds that restrain mitochondrial shape changes induced by Oma1 on the inner mitochondrial membrane. A distinct actin network stimulates mitochondrial fission in response to calcium.

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Mitochondrial dynamics: overview of molecular mechanisms

Essays in Biochemistry ◽

10.1042/ebc20170104 ◽

2018 ◽

Vol 62 (3) ◽

pp. 341-360 ◽

Cited By ~ 197

Author(s):

Lisa Tilokani ◽

Shun Nagashima ◽

Vincent Paupe ◽

Julien Prudent

Keyword(s):

Membrane Fusion ◽

Mitochondrial Membrane ◽

Molecular Mechanisms ◽

Calcium Influx ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Membrane Lipid ◽

Inner Mitochondrial Membrane ◽

Step Process ◽

Shape Distribution

Mitochondria are highly dynamic organelles undergoing coordinated cycles of fission and fusion, referred as ‘mitochondrial dynamics’, in order to maintain their shape, distribution and size. Their transient and rapid morphological adaptations are crucial for many cellular processes such as cell cycle, immunity, apoptosis and mitochondrial quality control. Mutations in the core machinery components and defects in mitochondrial dynamics have been associated with numerous human diseases. These dynamic transitions are mainly ensured by large GTPases belonging to the Dynamin family. Mitochondrial fission is a multi-step process allowing the division of one mitochondrion in two daughter mitochondria. It is regulated by the recruitment of the GTPase Dynamin-related protein 1 (Drp1) by adaptors at actin- and endoplasmic reticulum-mediated mitochondrial constriction sites. Drp1 oligomerization followed by mitochondrial constriction leads to the recruitment of Dynamin 2 to terminate membrane scission. Inner mitochondrial membrane constriction has been proposed to be an independent process regulated by calcium influx. Mitochondrial fusion is driven by a two-step process with the outer mitochondrial membrane fusion mediated by mitofusins 1 and 2 followed by inner membrane fusion, mediated by optic atrophy 1. In addition to the role of membrane lipid composition, several members of the machinery can undergo post-translational modifications modulating these processes. Understanding the molecular mechanisms controlling mitochondrial dynamics is crucial to decipher how mitochondrial shape meets the function and to increase the knowledge on the molecular basis of diseases associated with morphology defects. This article will describe an overview of the molecular mechanisms that govern mitochondrial fission and fusion in mammals.

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Tracking Retrograde Flow in Keratocytes: News from the Front

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0615 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1223-1231 ◽

Cited By ~ 104

Author(s):

Pascal Vallotton ◽

Gaudenz Danuser ◽

Sophie Bohnet ◽

Jean-Jacques Meister ◽

Alexander B. Verkhovsky

Keyword(s):

Actin Polymerization ◽

Leading Edge ◽

Membrane Resistance ◽

Retrograde Flow ◽

Actin Assembly ◽

Actin Network ◽

Freely Moving ◽

Contractile Forces ◽

Cell Motion ◽

Shape Changes

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1–3 μm/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization.

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Mitochondrial depolarization and electrophysiological changes during ischemia in the rabbit and human heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00437.2014 ◽

2014 ◽

Vol 307 (8) ◽

pp. H1178-H1186 ◽

Cited By ~ 9

Author(s):

Matthew S. Sulkin ◽

Bas J. Boukens ◽

Megan Tetlow ◽

Sarah R. Gutbrod ◽

Fu Siong Ng ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Human Heart ◽

Transmembrane Potential ◽

Optical Mapping ◽

Imaging Modality ◽

Ischemia Reperfusion ◽

Mitochondrial Depolarization ◽

Inner Mitochondrial Membrane ◽

Ventricular Tissue ◽

Direct Association

Instability of the inner mitochondrial membrane potential (ΔΨm) has been implicated in electrical dysfunction, including arrhythmogenesis during ischemia-reperfusion. Monitoring ΔΨm has led to conflicting results, where depolarization has been reported as sporadic and as a propagating wave. The present study was designed to resolve the aforementioned difference and determine the unknown relationship between ΔΨm and electrophysiology. We developed a novel imaging modality for simultaneous optical mapping of ΔΨm and transmembrane potential ( Vm). Optical mapping was performed using potentiometric dyes on preparations from 4 mouse hearts, 14 rabbit hearts, and 7 human hearts. Our data showed that during ischemia, ΔΨm depolarization is sporadic and changes asynchronously with electrophysiological changes. Spatially, ΔΨm depolarization was associated with action potential duration shortening but not conduction slowing. Analysis of focal activity indicated that ΔΨm is not different within the myocardium where the focus originates compared with normal ventricular tissue. Overall, our data suggest that during ischemia, mitochondria maintain their function at the expense of sarcolemmal electrophysiology, but ΔΨm depolarization does not have a direct association to ischemia-induced arrhythmias.

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Decavanadate Toxicology and Pharmacological Activities: V10or V1, Both or None?

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/6103457 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 38

Author(s):

M. Aureliano

Keyword(s):

Atpase Activity ◽

Mitochondrial Membrane ◽

Actin Polymerization ◽

Calcium Pump ◽

Mitochondrial Depolarization ◽

Pharmacological Activities ◽

Oxidative Stress Parameters ◽

Mitochondrial Membrane Depolarization

This review covers recent advances in the understanding of decavanadate toxicology and pharmacological applications. Toxicologicalin vivostudies point out that V10induces several changes in several oxidative stress parameters, different from the ones observed for vanadate (V1). Inin vitrostudies with mitochondria, a particularly potent V10effect, in comparison with V1, was observed in the mitochondrial depolarization (IC50= 40 nM) and oxygen consumption (99 nM). It is suggested that mitochondrial membrane depolarization is a key event in decavanadate induction of necrotic cardiomyocytes death. Furthermore, only decavanadate species and not V1potently inhibited myosin ATPase activity stimulated by actin (IC50= 0.75μM) whereas exhibiting lower inhibition activities for Ca2+-ATPase activity (15 μM) and actin polymerization (17 μM). Because both calcium pump and actin decavanadate interactions lead to its stabilization, it is likely that V10interacts at specific locations with these proteins that protect against hydrolysis but, on the other hand, it may induce V10reduction to oxidovanadium(IV). Putting it all together, it is suggested that the pharmacological applications of V10species and compounds whose mechanism of action is still to be clarified might involve besides V10and V1also vanadium(IV) species.

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ROMO1 is essential for glucose coupling in the pancreatic β cell of male mice

10.1101/2021.04.28.441811 ◽

2021 ◽

Author(s):

Lisa Wells ◽

Caterina Iorio ◽

Andy Cheuk-Him Ng ◽

Courtney Reeks ◽

Siu-Pok Yee ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Mitochondrial Dynamics ◽

Critical Role ◽

Physiological Role ◽

Glucose Sensing ◽

Mitochondrial Fusion ◽

Male Mice ◽

Inner Mitochondrial Membrane ◽

Β Cell ◽

Respiratory Capacity

AbstractReactive oxygen species modulator 1 (ROMO1) is a highly conserved inner mitochondrial membrane protein that senses ROS and regulates mitochondrial dynamics 1. ROMO1 is required for mitochondrial fusion in vitro, and silencing ROMO1 increases sensitivity to cell death stimuli. How ROMO1 promotes mitochondrial fusion and its physiological role remain unclear. Here we show that ROMO1 is essential for embryonic development, as ROMO1-null mice die before embryonic day 8.5, earlier than GTPases OPA1 or MFN1/2 that catalyze mitochondrial membrane fusion. Knockout of ROMO1 in adult pancreatic β cells results in impaired glucose homeostasis in male mice due to an insulin secretion defect resulting from impaired glucose sensing. Mitochondria in ROMO1 β cell KO cells were swollen and fragmented, consistent with a role for ROMO1 in mitochondrial fusion in vivo. While basal respiration was normal in ROMO1β cell KO islets, spare respiratory capacity was lost. Taken together, our data indicate that ROMO1 is required for nutrient coupling in the β cell and point to a critical role for spare respiratory capacity in the maintenance of euglycemia in males.

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Receptor-mediated Drp1 oligomerization on endoplasmic reticulum

10.1101/190538 ◽

2017 ◽

Author(s):

Wei-Ke Ji ◽

Rajarshi Chakrabarti ◽

Xintao Fan ◽

Lori Schoenfeld ◽

Stefan Strack ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Actin Polymerization ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Outer Mitochondrial Membrane ◽

Mitochondrial Division ◽

Knock Out ◽

Multiple Factors ◽

Latrunculin A

AbstractDrpl is a dynamin GTPase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51 and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is ER-associated in mammalian cells, and is distinct from mitochondrial or peroxisomal Drp1 populations. Sub-populations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, ER-bound) and mitochondrial division, while Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division.SummaryAssembly of the dynamin GTPase Drp1 into constriction-competent oligomers is a key event in mitochondrial division. Here, Ji et al show that Drp1 oligomerization can occur on endoplasmic reticulum through an ER-bound population of the tail-anchored protein Mff.Abbreviations used in this paper: Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein; INF2, inverted formin 2; KD, siRNA-mediated knock down; KI, CRISPR-mediated knock in; KO, CRISPR-mediated knock out; LatA, Latrunculin A; MDV, mitochondrially-derived vesicle; Mff, mitochondrial fission factor; MiD49 and MiD51, mitochondrial dynamics protein of 49 and 51 kDa; OMM, outer mitochondrial membrane; TA, tail-anchored.

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Role of Mitochondrial Dynamics in Microglial Activation and Metabolic Switch

10.21203/rs.3.rs-134161/v1 ◽

2021 ◽

Author(s):

Alejandro Montilla ◽

Asier Ruiz ◽

Mar Marquez ◽

Amanda Sierra ◽

Carlos Matute ◽

...

Keyword(s):

Membrane Potential ◽

Oxidative Phosphorylation ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Microglial Activation ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Metabolic Reprogramming ◽

Metabolic Switch

Abstract Microglia act as sensors of injury in the brain, favouring its homeostasis. Their activation and polarization towards a pro-inflammatory phenotype are associated to injury and disease. These processes are linked to a metabolic reprogramming of the cells, characterized by high rates of glycolysis and suppressed oxidative phosphorylation. This metabolic switch can be reproduced in vitro by microglial stimulation with lipopolysaccharide (LPS) plus interferon-γ (IFNγ). In order to understand the mechanisms regulating mitochondrial respiration abolishment, we examined potential alterations in mitochondrial features during this switch. Cells did not show any change in mitochondrial membrane potential, suggesting a limited impact in the mitochondrial viability. We provide evidence that reverse operation of F0F1-ATP synthase contributes to mitochondrial membrane potential. In addition, we studied the possible implication of mitochondrial dynamics in the metabolic switch using the mitochondrial division inhibitor-1 (Mdivi-1), which blocks Drp1-dependent mitochondrial fission. Mdivi-1 significantly reduced the expression of pro-inflammatory markers in LPS+IFNγ-treated microglia. However, this inhibition did not lead to a recovery of the oxidative phosphorylation ablation by LPS+IFNγ or to a microglia repolarization. Altogether, these results suggest that Drp1-dependent mitochondrial fission, although potentially involved in microglial activation, does not play an essential role in metabolic reprogramming and repolarization of microglia.

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Amyloid-Beta Interaction with Mitochondria

International Journal of Alzheimer s Disease ◽

10.4061/2011/925050 ◽

2011 ◽

Vol 2011 ◽

pp. 1-12 ◽

Cited By ~ 57

Author(s):

Lucia Pagani ◽

Anne Eckert

Keyword(s):

Mitochondrial Dysfunction ◽

Amyloid Beta ◽

Mitochondrial Membrane ◽

Current Knowledge ◽

Mitochondrial Dynamics ◽

Intracellular Localization ◽

Protein A ◽

Postmortem Brain ◽

Intermembrane Space ◽

Inner Mitochondrial Membrane

Mitochondrial dysfunction is a hallmark of amyloid-beta(Aβ)-induced neuronal toxicity in Alzheimer's disease (AD). The recent emphasis on the intracellular biology of Aβand its precursor protein (AβPP) has led researchers to consider the possibility that mitochondria-associated and/or intramitochondrial Aβmay directly cause neurotoxicity. In this paper, we will outline current knowledge of the intracellular localization of both Aβand AβPP addressing the question of how Aβcan access mitochondria. Moreover, we summarize evidence from AD postmortem brain as well as cellular and animal AD models showing that Aβtriggers mitochondrial dysfunction through a number of pathways such as impairment of oxidative phosphorylation, elevation of reactive oxygen species (ROS) production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. In particular, we focus on Aβinteraction with different mitochondrial targets including the outer mitochondrial membrane, intermembrane space, inner mitochondrial membrane, and the matrix. Thus, this paper establishes a modified model of the Alzheimer cascade mitochondrial hypothesis.

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Parkin Mediates Proteasome-dependent Protein Degradation and Rupture of the Outer Mitochondrial Membrane

Journal of Biological Chemistry ◽

10.1074/jbc.m110.209338 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19630-19640 ◽

Cited By ~ 377

Author(s):

Saori R. Yoshii ◽

Chieko Kishi ◽

Naotada Ishihara ◽

Noboru Mizushima

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Ubiquitin Ligase ◽

Mitochondrial Membrane ◽

Outer Membrane Proteins ◽

Matrix Proteins ◽

Mitochondrial Depolarization ◽

Inner Mitochondrial Membrane ◽

Membrane Rupture ◽

Dependent Protein

Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.

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Role of mitochondrial dynamics in microglial activation and metabolic switch

10.21203/rs.3.rs-26982/v1 ◽

2020 ◽

Author(s):

Alejandro Montilla ◽

Asier Ruiz ◽

Carlos Matute ◽

Maria Domercq

Keyword(s):

Membrane Potential ◽

Oxidative Phosphorylation ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Respiration ◽

Mitochondrial Membrane ◽

Microglial Activation ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Metabolic Reprogramming ◽

Metabolic Switch

Abstract Background Microglia are the endogenous immune cells of the central nervous system (CNS) and act as sensors of injury in the brain, favouring its homeostasis. Their activation and polarization towards a pro-inflammatory phenotype are associated to injury and disease. These processes are linked to a metabolic reprogramming of the cells, characterized by high rates of glycolytic function and suppressed levels of oxidative phosphorylation. This metabolic switch can be reproduced in vitro by stimulation with lipopolysaccharide (LPS) plus Interferon-γ (IFNγ). In an attempt to understand the mechanisms regulating mitochondrial respiration abolishment, we examined potential alterations in mitochondrial features during the metabolic switch. In addition, we studied the possible implication of mitochondrial dynamics in the metabolic switch using the mitochondrial division inhibitor-1 (Mdivi-1), which blocks Drp1-dependent mitochondrial fission. Methods Cultured microglia was treated with LPS + IFNγ to reproduce the metabolic switch under pro-inflammatory stimuli in the absence or in the presence of Mdivi-1 to block mitochondrial fission. Mitochondrial membrane potential and mitochondrial calcium were measured with living cell imaging, and microglial polarization was assessed by immunofluorescence and qRT-PCR. The metabolic profile of the cells was measured using the Seahorse XFe96 Extracellular Flux Analyzer. Results Under conditions of mitochondrial respiration abolishment, microglia did not show any change in mitochondria morphology, nor in mitochondrial membrane potential, indicative of a limited impact in its viability. We provided evidence that reverse operation of F0F1-ATP synthase contributes to mitochondrial membrane potential. On the other hand. mitochondrial fission blockage significantly reduced the expression of pro-inflammatory markers in LPS + IFNγ-treated microglia, such as the inducible nitric oxide synthase (iNOS). However, this inhibition did not lead to a recovery of the oxidative phosphorylation ablation by LPS + IFNγ or to a microglia repolarization. Conclusions Altogether, these results suggest that Drp1-dependent mitochondrial fission, although potentially involved in microglial activation, does not play an essential role in metabolic reprogramming and repolarization of microglia.

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