Assessing the effects of mitofusin 2 deficiency in the adult heart using 3D electron tomography

The effects of mitofusin 2 (MFN2) deficiency, on mitochondrial morphology and the mitochondria-junctional sarcoplasmic reticulum (jSR) complex in the adult heart, have been previously investigated using 2D electron microscopy, an approach which is unable to provide a 3D spatial assessment of these imaging parameters. Here, we use 3D electron tomography to show that MFN2-deficient mitochondria are larger in volume, more elongated, and less rounded; have fewer mitochondria-jSR contacts, and an increase in the distance between mitochondria and jSR, when compared to WT mitochondria. In comparison to 2D electron microscopy, 3D electron tomography can provide further insights into mitochondrial morphology and the mitochondria-jSR complex in the adult heart

Deletion of heat shock protein 60 in adult mouse cardiomyocytes perturbs mitochondrial protein homeostasis and causes heart failure.

To maintain healthy mitochondrial enzyme content and function, mitochondria possess a complex protein quality control system, which is composed of different endogenous sets of chaperones and proteases. Heat shock protein 60 (HSP60) is one of these mitochondrial molecular chaperones and has been proposed to play a pivotal role in the regulation of protein folding and the prevention of protein aggregation. However, the physiological function of HSP60 in mammalian tissues is not fully understood. Here we generated an inducible cardiac-specific HSP60 knockout mouse model, and demonstrated that HSP60 deletion in adult mouse hearts altered mitochondrial complex activity, mitochondrial membrane potential, and ROS production, and eventually led to dilated cardiomyopathy, heart failure, and lethality. Proteomic analysis was performed in purified control and mutant mitochondria before mutant hearts developed obvious cardiac abnormalities, and revealed a list of mitochondrial-localized proteins that rely on HSP60 (HSP60-dependent) for correctly folding in mitochondria. We also utilized an in vitro system to assess the effects of HSP60 deletion on mitochondrial protein import and protein stability after import, and found that both HSP60-dependent and HSP60-independent mitochondrial proteins could be normally imported in mutant mitochondria. However, the former underwent degradation in mutant mitochondria after import, suggesting that the protein exhibited low stability in mutant mitochondria. Interestingly, the degradation could be almost fully rescued by a non-specific LONP1 and proteasome inhibitor, MG132, in mutant mitochondria. Therefore, our results demonstrated that HSP60 plays an essential role in maintaining normal cardiac morphology and function by regulating mitochondrial protein homeostasis and mitochondrial function

Ltc1 is an ER-localized sterol transporter and a component of ER-mitochondria and ER-vacuole contacts.

Organelle contact sites perform fundamental functions in cells, including lipid and ion homeostasis, membrane dynamics, and signaling. Using a forward proteomics approach in yeast, we identified new ER-mitochondria and ER-vacuole contacts specified by an uncharacterized protein, Ylr072w. Ylr072w is a conserved protein with GRAM and VASt domains that selectively transports sterols and is thus termed Ltc1, for Lipid transfer at contact site 1. Ltc1 localized to ER-mitochondria and ER-vacuole contacts via the mitochondrial import receptors Tom70/71 and the vacuolar protein Vac8, respectively. At mitochondria, Ltc1 was required for cell viability in the absence of Mdm34, a subunit of the ER-mitochondria encounter structure. At vacuoles, Ltc1 was required for sterol-enriched membrane domain formation in response to stress. Increasing the proportion of Ltc1 at vacuoles was sufficient to induce sterol-enriched vacuolar domains without stress. Thus, our data support a model in which Ltc1 is a sterol-dependent regulator of organelle and cellular homeostasis via its dual localization to ER-mitochondria and ER-vacuole contact sites

Evidence for the involvement of lipid rafts localized at the ER-mitochondria associated membranes in autophagosome formation

Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic “brick” of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome. Introduction The interaction of the endoplasmic reticulum (ER) with mito- chondria occurs via certain subdomains of the ER, named mitochondria-associated membranes (MAMs), which allow membrane “scrambling” between these organelles and contrib- utes to the complex series of ER functions.1-3 Indeed, several regions of close apposition between the ER and mitochondria were detected by studies carried out several years ago.4,5 How- ever, since these studies provided only ultrastructural observa- tions, these reports remained neglected for a long time. In particular, while morphological evidence of the physical juxta- position between ER and mitochondria was described since 1959,6 it was experimentally proven only 30 y later. In fact, ana- lyzing ER fractions copurified with mitochondria in velocity sedimentation assays, mainly from rat liver cells, it was observed that mitochondria can tightly be associated with ele- ments of the ER and that the communication and intermixing between ER and mitochondria can be mediated by MAMs.7-12 These works also showed that these cosedimenting fractions were enriched in enzymes responsible for the synthesis of lipids. These findings suggested that MAMs could act as sites

A study of mitochondrial membranes in relation to elementary particles

Elementary particles that commonly have been seen by electron microscopy to be attached by stalks to mitochondrial cristae in negatively stained preparations, were not apparent in similarly stained mitochondria from exponentially growing wild-type Neurospora crassa when these were isolated in sucrose solution containing 1 x 10^-3 M EDTA. However, elementary particles were easily demonstrable in electron micrographs if the mitochondria were isolated without EDTA in the sucrose solution. A biochemical study indicated that both kinds of mitochondrial preparations, isolated in the presence or absence of EDTA, had about the same capacity for oxidative phosphorylation. Observations on rat-liver mitochondria also suggested that the stalked elementary particles were more easily demonstrated if the preparation was made in the absence of EDTA. It was difficult to demonstrate elementary particles in wild-type Neurospora mitochondria isolated with or without EDTA and subsequently prepared for electron microscopy by spreading on the surface of an aqueous solution of potassium phosphotungstate. Elementary particles could be demonstrated in poky Neurospora mitochondria isolated with EDTA if the mitochondria were spread on the surface of an aqueous solution of phosphotungstate. It was concluded that biochemical functions associated with elementary particles are independent of structural configuration as seen by electron microscopy

How proteins are transported into mitochondria

Most mitochondrial polypeptides are synthesized outside the organelle as precursors which are usually larger than the ‘mature’ polypeptides found within mitochondria. The precursors are imported into the mitochondria by a process which is independent of protein synthesis but dependent on high-energy phosphate bonds inside the mitochondria. This mechanism is basically different from that which governs the movement of secretory polypeptides across the membrane of the endoplasmic reticulum

Localization of TCA cycle dehydrogenases in the mitochondria

The site of localization of TCA cycle dehydrogenases in mitochondria has been investigated by observing the dehydrogenase activities and fine structure of the fractionated samples after freezing and thawing or sonication of beef heart and rat liver mitochondria. 1. In the sonicated mitochondria, activities of malic and isocitric dehydrogenases were highest in the supernatant fraction centrifuged at 198,000 x g for 60 minutes, while the specific activity of a-ketoglutaric dehydrogenase was higher in the fluffy or residue fraction. The distribution of the activity of pyruvic dehydrogenase was similar to that of a-ketoglutaric dehydrogenase. 2. In a sucrose density gradient fractionation of the fluffy fraction obtained by centifugation of sonicated mitochondria at 198, 000 x g for 60 minutes, the activities of malic and pyruvic dehydrogenase were observed in the top (or low density) layer in the form of fine particles, while that of a-ketoglutaric dehydrogenase was observed in the middle (or medium density) layers in the form of aggregates of fine particles and membranous fragments. 3. In the samples fractionated after freezing and thawing of mitochondria, which were considered to be a relatively mild disruption, the specific activity of a-ketoglutaric dehydrogenase was higher in the residue (submitochondria) fraction than that in the supernatant fraction (centrifuged at 144,000 x g, 30 minutes), and the activity of malic dehydrogenase still remained significantly high in the residue fraction. 4. It was deduced that the TCA cycle dehydrogenases could be localized in the matrix of the mitochondria by a loose binding to the inner membrane.</p

Humanin G (HNG) protects age-related macular degeneration (AMD) transmitochondrial ARPE-19 cybrids from mitochondrial and cellular damage.

Age-related macular degeneration (AMD) ranks third among the leading causes of visual impairment with a blindness prevalence rate of 8.7%. Despite several treatment regimens, such as anti-angiogenic drugs, laser therapy, and vitamin supplementation, being available for wet AMD, to date there are no FDA-approved therapies for dry AMD. Substantial evidence implicates mitochondrial damage and retinal pigment epithelium (RPE) cell death in the pathogenesis of AMD. However, the effects of AMD mitochondria and Humanin G (HNG), a more potent variant of the mitochondrial-derived peptide (MDP) Humanin, on retinal cell survival have not been elucidated. In this study, we characterized mitochondrial and cellular damage in transmitochondrial cybrid cell lines that contain identical nuclei but possess mitochondria from either AMD or age-matched normal (Older-normal (NL)) subjects. AMD cybrids showed (1) reduced levels of cell viability, lower mtDNA copy numbers, and downregulation of mitochondrial replication/transcription genes and antioxidant enzyme genes; and (2) elevated levels of genes related to apoptosis, autophagy and ER-stress along with increased mtDNA fragmentation and higher susceptibility to amyloid-β-induced toxicity compared to NL cybrids. In AMD cybrids, HNG protected the AMD mitochondria, reduced pro-apoptosis gene and protein levels, upregulated gp130 (a component of the HN receptor complex), and increased the protection against amyloid-β-induced damage. In summary, in cybrids, damaged AMD mitochondria mediate cell death that can be reversed by HNG treatment. Our results also provide evidence of Humanin playing a pivotal role in protecting cells with AMD mitochondria. In the future, it may be possible that AMD patient's blood samples containing damaged mitochondria may be useful as biomarkers for this condition. In conclusion, HNG may be a potential therapeutic target for treatment of dry AMD, a debilitating eye disease that currently has no available treatment. Further studies are needed to establish HNG as a viable mitochondria-targeting therapy for dry AMD

Identity and Function of a Cardiac Mitochondrial Small Conductance Ca2+-Activated K+ Channel Splice Variant

We provide evidence for location and function of a small conductance, Ca2+-activated K+ (SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCa agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCa antagonists worsened IR injury. Intravenous infusion of a SKCa channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca2+ calmodulin binding domain is required for Ca2+ sensing to induce mK+ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK+ influx was driven by adding CaCl2. Moreover, there was a greater fall in membrane potential (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCa channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCa channel opening remains unclear. SKCa channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+ overload

Depletion of mitochondria in mammalian cells through enforced mitophagy

Mitochondria are not only the 'powerhouse' of the cell; they are also involved in a multitude of processes that include calcium storage, the cell cycle and cell death. Traditional means of investigating mitochondrial importance in a given cellular process have centered upon depletion of mtDNA through chemical or genetic means. Although these methods severely disrupt the mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here we describe a straightforward protocol to generate mammalian cell populations with low to nondetectable levels of mitochondria. Ectopic expression of the ubiquitin E3 ligase Parkin, combined with short-term mitochondrial uncoupler treatment, stimulates widespread mitophagy and effectively eliminates mitochondria. In this protocol, we explain how to generate Parkin-expressing, mitochondria-depleted cells from scratch in 23 d, as well as offer a variety of methods for confirming mitochondrial clearance. Furthermore, we describe culture conditions to maintain mitochondrial-depleted cells for up to 30 d with minimal loss of viability, for longitudinal studies. This method should prove useful for investigating the importance of mitochondria in a variety of biological processes