Peter Rippstein

Cargo-Selected Transport from the Mitochondria to Peroxisomes Is Mediated by Vesicular Carriers

Mitochondria and peroxisomes share a number of common biochemical processes, including the beta oxidation of fatty acids and the scavenging of peroxides. Here, we identify a new outer-membrane mitochondria-anchored protein ligase (MAPL) containing a really interesting new gene (RING)-finger domain. Overexpression of MAPL leads to mitochondrial fragmentation, indicating a regulatory function controlling mitochondrial morphology. In addition, confocal- and electron-microscopy studies of MAPL-YFP led to the observation that MAPL is also incorporated within unique, DRP1-independent, 70-100 nm diameter mitochondria-derived vesicles (MDVs). Importantly, vesicles containing MAPL exclude another outer-membrane marker, TOM20, and vesicles containing TOM20 exclude MAPL, indicating that MDVs selectively incorporate their cargo. We further demonstrate that MAPL-containing vesicles fuse with a subset of peroxisomes, marking the first evidence for a direct relationship between these two functionally related organelles. In contrast, a distinct vesicle population labeled with TOM20 does not fuse with peroxisomes, indicating that the incorporation of specific cargo is a primary determinant of MDV fate. These data are the first to identify MAPL, describe and characterize MDVs, and define a new intracellular transport route between mitochondria and peroxisomes.

A Vesicular Transport Pathway Shuttles Cargo from Mitochondria to Lysosomes

Mitochondrial respiration relies on electron transport, an essential yet dangerous process in that it leads to the generation of reactive oxygen species (ROS). ROS can be neutralized within the mitochondria through enzymatic activity, yet the mechanism for steady-state removal of oxidized mitochondrial protein complexes and lipids is not well understood. We have previously characterized vesicular profiles budding from the mitochondria that carry selected cargo. At least one population of these mitochondria-derived vesicles (MDVs) targets the peroxisomes; however, the fate of the majority of MDVs was unclear. Here, we demonstrate that MDVs carry selected cargo to the lysosomes. Using a combination of confocal and electron microscopy, we observe MDVs in steady state and demonstrate that they are stimulated as an early response to oxidative stress, the extent of which is determined by the respiratory status of the mitochondria. Delivery to the lysosomes does not require mitochondrial depolarization and is independent of ATG5 and LC3, suggesting that vesicle delivery complements mitophagy. Consistent with this, ultrastructural analysis of MDV formation revealed Tom20-positive structures within the vesicles of multivesicular bodies. These data characterize a novel vesicle transport route between the mitochondria and lysosomes, providing insights into the basic mechanisms of mitochondrial quality control.

The SUMO protease SENP5 is required to maintain mitochondrial morphology and function

Mitochondria are dynamic organelles that undergo regulated fission and fusion events that are essential to maintain metabolic stability. We previously demonstrated that the mitochondrial fission GTPase DRP1 is a substrate for SUMOylation. To further understand how SUMOylation impacts mitochondrial function, we searched for a SUMO protease that may affect mitochondrial dynamics. We demonstrate that the cytosolic pool of SENP5 catalyzes the cleavage of SUMO1 from a number of mitochondrial substrates. Overexpression of SENP5 rescues SUMO1-induced mitochondrial fragmentation that is partly due to the downregulation of DRP1. By contrast, silencing of SENP5 results in a fragmented and altered morphology. DRP1 was stably mono-SUMOylated in these cells, suggesting that SUMOylation leads to increased DRP1 mediated fission. In addition, the reduction of SENP5 levels resulted in a significant increase in the production of free radicals. Reformation of the mitochondrial tubules by expressing the dominant interfering DRP1 or by RNA silencing of endogenous DRP1 protein rescued both the morphological aberrations and the increased production of ROS induced by downregulation of SENP5. These data demonstrate the importance of SENP5 as a new regulator of SUMO1 proteolysis from mitochondrial targets, impacting mitochondrial morphology and metabolism.

Dissociating the dual roles of apoptosis‐inducing factor in maintaining mitochondrial structure and apoptosis

The mitochondrial protein apoptosis‐inducing factor (AIF) translocates to the nucleus and induces apoptosis. Recent studies, however, have indicated the importance of AIF for survival in mitochondria. In the absence of a means to dissociate these two functions, the precise roles of AIF remain unclear. Here, we dissociate these dual roles using mitochondrially anchored AIF that cannot be released during apoptosis. Forebrain‐specific AIF null (tel. AifΔ) mice have defective cortical development and reduced neuronal survival due to defects in mitochondrial respiration. Mitochondria in AIF deficient neurons are fragmented with aberrant cristae, indicating a novel role of AIF in controlling mitochondrial structure. While tel. AifΔ Apaf1−/− neurons remain sensitive to DNA damage, mitochondrially anchored AIF expression in these cells significantly enhanced survival. AIF mutants that cannot translocate into nucleus failed to induce cell death. These results indicate that the proapoptotic role of AIF can be uncoupled from its physiological function. Cell death induced by AIF is through its proapoptotic activity once it is translocated to the nucleus, not due to the loss of AIF from the mitochondria.

Reconstitution of Mitochondria Derived Vesicle Formation Demonstrates Selective Enrichment of Oxidized Cargo

The mechanisms that ensure the removal of damaged mitochondrial proteins and lipids are critical for the health of the cell, and errors in these pathways are implicated in numerous degenerative diseases. We recently uncovered a new pathway for the selective removal of proteins mediated by mitochondrial derived vesicular carriers (MDVs) that transit to the lysosome. However, it was not determined whether these vesicles were selectively enriched for oxidized, or damaged proteins, and the extent to which the complexes of the electron transport chain and the mtDNA-containing nucloids may have been incorporated. In this study, we have developed a cell-free mitochondrial budding reaction in vitro in order to better dissect the pathway. Our data confirm that MDVs are stimulated upon various forms of mitochondrial stress, and the vesicles incorporated quantitative amounts of cargo, whose identity depended upon the nature of the stress. Under the conditions examined, MDVs did not incorporate complexes I and V, nor were any nucleoids present, demonstrating the specificity of cargo incorporation. Stress-induced MDVs are selectively enriched for oxidized proteins, suggesting that conformational changes induced by oxidation may initiate their incorporation into the vesicles. Ultrastructural analyses of MDVs isolated on sucrose flotation gradients revealed the formation of both single and double membranes vesicles of unique densities and uniform diameter. This work provides a framework for a reductionist approach towards a detailed examination of the mechanisms of MDV formation and cargo incorporation, and supports the emerging concept that MDVs are critical contributors to mitochondrial quality control.

Immunohistochemistry and ultrastructure of myoepithelium and modified myoepithelium of the ducts of human major salivary glands: Histogenetic implications for salivary gland tumors

The organization of salivary gland ducts, especially the presence or absence of myoepithelial cells, is central to histogenetic approaches to the classification of salivary gland tumors. Striated and excretory ducts are reported to be devoid of myoepithelial cells but do contain basal cells. To investigate the nature of such basal cells, tissue sections of normal human salivary glands were examined by means of immunohistochemical, ultrastructural, and fluorescent microscopic techniques. With the use of a mouse monoclonal anticytokeratin antibody (3 12C8-1) that, in salivary glands, is specific for myoepithelial cells, these cells associated with acini and intercalated ducts were strongly stained, as were the basal cells of striated and excretory ducts in each case. Ultrastructurally, some basal cells of both striated and excretory ducts had narrow, elongated cellular processes or the main portion of the cell containing parallel arrays of microfilaments with linear densities and micropinocytotic vesicles, whereas in other basal cells tonofilament bundles predominated. A similar range of cytoplasmic features existed in myoepithelial cells associated with acinar and intercalated duct cells. In addition, some duct basal cells have a complement of actin filaments similar to classic myoepithelium of acini and intercalated ducts. Striated and excretory ducts of human salivary glands, therefore, contain fully differentiated and modified myoepithelial cells, both of which express a specific cytokeratin polypeptide that is absent from duct luminal and acinar cells. Differentiation patterns in the intralobular and interlobular ducts suggest that these regions of salivary gland parenchyma cannot be excluded as histogenetic sites for the induction of salivary gland tumors in which neoplastic myoepithelial cells have been shown to have a major role.

Dominant-negative β1 integrin mice have region-specific myelin defects accompanied by alterations in MAPK activity

Recent studies have demonstrated the importance of β1 integrin in oligodendrocyte maturation in vitro. Similar studies in vivo have been difficult due to the embryonic and perinatal lethality of null mutations in integrin subunits. Here, we have generated transgenic mouse models that overexpress full length β1 integrin or express a dominant‐negative β1 integrin ΔC (lacking the C‐terminal tail) under the control of the proteolipid protein (PLP) promoter. We demonstrate that these transgenes are expressed predominantly in CNS tissues and more specifically in oligodendrocytes. Further analysis reveals that the dominant‐negative β1 integrin ΔC transgenic mice, but not the full length β1 integrin mice, have hypomyelinated axons in spinal cords and optic nerves. In addition, there is a significant increase in the number of unmyelinated axons within the spinal cords and optic nerves of the β1 integrin ΔC mice. In contrast, the corpus callosum from these mice did not show similar myelin defects. To assess if remyelination would be affected in the corpus callosum, mice were subjected to a cuprizone‐induced demyelination. Interestingly, the dominant‐negative mice recovered from this insult in a manner similar to the wild type littermates. Axons within the corpus callosum that were remyelinated had normal g‐ratios; however, the actual percentage of myelinated axons was significantly reduced compared with wild type mice. We also show that the defects observed in the dominant‐negative β1 integrin ΔC mice are accompanied by disruption of the MAP‐kinase signaling pathway. Our work highlights the importance of β1 integrin‐mediated signaling in CNS myelination in vivo.

Glutaredoxin-2 Is Required to Control Oxidative Phosphorylation in Cardiac Muscle by Mediating Deglutathionylation Reactions

Background: Mitochondrial proteins are controlled by glutaredoxin-2 (Grx2)-mediated deglutathionylation reactions. Results: Grx2 deficiency compromises cardiac mitochondrial functions leading to hypertrophy and fibrosis in male mice. This is associated with deregulated glutathionylation reactions and mitochondrial dysfunction. Conclusion: Through deglutathionylation, Grx2 controls mitochondrial oxidative phosphorylation in cardiac muscle. Significance: Deregulated glutathionylation in heart can have pathological consequences. Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2−/−) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2−/− hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2+/−) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2+/− mice were far less hypertensive than Grx2−/− mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease.

A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators

BackgroundMitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques.ResultsIn order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction.ConclusionsThe adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions.See Commentary: http://www.biomedcentral.com/1741-7007/8/99

Nanosilver cytotoxicity in rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes

Silver nanoparticles (AgNPs) are present in a multitude of consumer and medical products; however, the toxicity of AgNPs is not fully understood. This research aimed to elucidate the relationship between AgNP cytotoxicity and oxidative stress and damage in rainbow trout (Oncorhynchus mykiss) hepatocytes and erythrocytes in comparison to silver ions (Ag(+)). Generally the cytotoxicity of AgNPs and Ag(+) was similar, such that both silver types generated reactive oxygen species, decreased glutathione levels, and decreased activities of glutathione reductase and glutathione-S-transferase. Nonetheless, the two silver types had different cellular targets; AgNPs increased lipid peroxidation without apparent uptake into the cells whereas Ag(+) increased DNA damage. Furthermore, the toxicity of both silver types was generally decreased in cells treated with cysteine while treatment with buthionine sulfoximine increased the toxicity of both silver types.