Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

A genomically/chemically complete module for synthesis of lipid membrane in a minimal cell

A minimal cell is a hypothetical cell defined by the essential functions required for life. We have developed a module for the synthesis of membrane precursors for a mathematical minimal cell model. This module describes, with chemical and genomic detail the production of the constituents required to build a cell membrane and identifies the corresponding essential genes. Membranes allow selective nutrient passage, harmful substance exclusion, and energy generation. Bacterial membrane components range from lipids to fatty acids with embedded proteins and are structurally similar to eukaryotic cell membranes. Membranes are dynamic structures and experimental analyses show great variations in bacterial membrane composition. The flexibility of the model is such that different membrane compositions could be obtained in response to simulated changes in culture conditions. The model's predictions are in close agreement with the observed biological trends. The model's predictions correspond well with the experimental values of total lipid content in cells grown in chemostat culture, but less well with data from batch growth. Cell shape and size results agree especially well for data for growth rate relative to maximum growth rate larger than 0.5; and DNA, RNA, and protein predictions are consistent with experimental observations. A better understanding of the simplest bacterial membrane should lead to insights on the more complex behavior of membranes of higher species as well as identification of potential targets for antimicrobials.     

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.     

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Mitochondrial membrane permeabilization (MMP) is considered as the “point-of-no-return” in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential (ΔΨ_m). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several techniques to measure MMP by cytofluorometry and fluorescence microscopy have been developed. Here, we summarize the currently available methods for the detection of MMP, and provide a comparative analysis of these techniques.     

Mechanisms and functions of membrane lipid remodeling in plants

Lipid remodeling, defined herein as post-synthetic structural modifications of membrane lipids, play crucial roles in regulating the physicochemical properties of cellular membranes and hence their many functions. Processes affected by lipid remodeling include lipid metabolism, membrane repair, cellular homeostasis, fatty acid trafficking, cellular signaling and stress tolerance. Glycerolipids are the major structural components of cellular membranes and their composition can be adjusted by modifying their head groups, their acyl chain lengths and the number and position of double bonds. This review summarizes recent advances in our understanding of mechanisms of membrane lipid remodeling with emphasis on the lipases and acyltransferases involved in the modification of phosphatidylcholine and monogalactosyldiacylglycerol, the major membrane lipids of extraplastidic and photosynthetic membranes, respectively. We also discuss the role of triacylglycerol metabolism in membrane acyl chain remodeling. Finally, we discuss emerging data concerning the functional roles of glycerolipid remodeling in plant stress responses. Illustrating the molecular basis of lipid remodeling may lead to novel strategies for crop improvement and other biotechnological applications such as bioenergy production.     

Mitochondria isolated in nearly isotonic KCl buffer: Focus on cardiolipin and organelle morphology

Rat liver mitochondria were isolated in parallel in two different isolation buffers: a standard buffer containing mannitol/sucrose and a nearly physiological KCl based solution. The two different organelle preparations were comparatively characterized by respiratory activity, heme content, microsomal and Golgi contamination, electron microscopy and lipid analyses. The substitution of saccharides with KCl in the isolation buffer does not induce the formation of mitoplasts or disruption of mitochondria. Mitochondria isolated in KCl buffer are coupled and able to maintain a stable transmembrane charge separation. A number of biochemical and functional differences between the two organelle preparations are described; in particular KCl mitochondria exhibit lower cardiolipin content and smaller intracristal compartments in comparison with the standard mitochondrial preparation.     

鼠毛及脑线粒体DNA片段缺失与增龄的关系

以聚合酶链反应（ＰＣＲ）技术检测不同年龄Ｂａｌｂ／ｃ小鼠脑细胞线粒体ＤＮＡ片段缺失与增龄的关系．发现老年鼠脑细胞线粒体３８６７ｂｐ片段缺失率为５０％;而断奶鼠与青年鼠均无此缺失片段出现;用鼠毛为材料进行无损伤检测亦获类似的结果．有人认为线粒体ＤＮＡ片段缺失率可作为生物衰老的一种生物学标志     

Mitochondrial SIRT3: a new potential therapeutic target for metabolic syndrome

In this issue of Molecular Cell, Hirschey et?al. demonstrate that loss of the NAD(+)-dependent deacetylase SIRT3 and resultant mitochondrial protein hyperacetylation play a critical role in the pathogenesis of metabolic syndrome, providing new insights into the therapeutic potential of SIRT3.     

Imipramine and lipid phase transition in inner mitochondrial membrane

As ascertained by freeze-fracture electron microscopy, imipramine prevents lateral phase separation from taking place in inner mitochondrial membranes at sub-zero temperatures. Electron spin resonance (ESR) measurements performed on mitochondrial membranes labeled with the N-oxyl-4′,4′-dimethyloxazolidine derivative of 16-ketostearic acid, show that the spin probe motion is markedly inhibited below 0°C and that 5 mM imipramine attenuates the temperature effect. These results are explained by supposing that imipramine is able to decrease the transition temperature of the inner mitochondrial membrane lipids as it does for simple lipid systems.     

Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: Implications for aging

The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.     

Effects of membrane lipid and fluidity modifications on HIV-1 infectibility of primate lymphocytesin vitro

Although most non-human primates, except the chimpanzee and the gibbonin vivo are not infectible by HIV-1, lymphocytes of several of these species can be infected by HIV-1in vitro.In order to investigate whether thein vitro infectibility of primate lymphocytes might be attributed to plasma membrane adaptation processes or to serum factors, we compared HIV-1 infectibility of cultivated peripheral blood lymphocytes of macaques and of baboons on day one and on day ten of cultivation. These data were correlated to plasma membrane lipid composition and membrane fluidity.We found a correlation between increased HIV-1in vitro infectibility and changes in plasma membrane lipid composition resulting in decreased membrane fluidity of cultured primate lymphocytes.     

Mitochondrial remodeling in hepatic differentiation and dedifferentiation

Mitochondrial biogenesis and metabolism have recently emerged as important actors of stemness and differentiation. On the one hand, the differentiation of stem cells is associated with an induction of mitochondrial biogenesis and a shift from glycolysis toward oxidative phosphorylations (OXPHOS). In addition, interfering with mitochondrial biogenesis or function impacts stem cell differentiation. On the other hand, some inverse changes in mitochondrial abundance and function are observed during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Yet although great promises in cell therapy might generate better knowledge of the mechanisms regulating the stemness and differentiation of somatic stem cells (SSCs)—which are preferred over embryonic stem cells (ESCs) and iPSCs because of ethical and safety considerations—little interest was given to the study of their mitochondria. This study provides a detailed characterization of the mitochondrial biogenesis occurring during the hepatogenic differentiation of bone marrow-mesenchymal stem cells (BM-MSCs). During the hepatogenic differentiation of BM-MSCs, an increased abundance of mitochondrial DNA (mtDNA) is observed, as well as an increased expression of several mitochondrial proteins and biogenesis regulators, concomitant with increased OXPHOS activity, capacity, and efficiency. In addition, opposite changes in mitochondrial morphology and in the abundance of several OXPHOS subunits were found during the spontaneous dedifferentiation of primary hepatocytes. These data support reverse mitochondrial changes in a different context from genetically-engineered reprogramming. They argue in favor of a mitochondrial involvement in hepatic differentiation and dedifferentiation.     

NAD+ metabolism: A therapeutic target for age-related metabolic disease

Abstract

Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein silent regulator 2 (Sir2), the founding member of the sirtuin protein family, as being NAD⁺-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD⁺ to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD⁺ levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by poly(ADP-ribose) polymerase (PARP) enzymes or CD38/CD157, boosts NAD⁺ levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD⁺ metabolism, primarily in relation to sirtuin function. We highlight how NAD⁺ levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy.     

Changes in tissue and mitochondrial membrane composition during rapid growth, maturation and aging in rainbow trout, Oncorhynchus mykiss

Membrane compositions, particularly of mitochondria, could be critical factors in the mechanisms of growth and aging processes, especially during phases of high oxidative stress that result in molecular damage. In the present study, liver and mitochondrial membrane phospholipid (PL) compositions were analyzed in rainbow trout during its four first years of life, a period characterized by rapid growth and high oxidative stress. Specifically, farmed fish of three ages (1-, 2- and 4-years) were studied, and PL compositions of whole liver and liver mitochondria, and fatty acid compositions of individual PL classes were determined. Liver mitochondrial membranes showed a PL composition different to that of the whole tissue suggesting adaptation of cell and subcellular membranes to specific functions. Individual PL had characteristic fatty acid compositions that were similar in whole liver and mitochondrial membranes. Whole liver and mitochondria showed increased lipid peroxidation with age along with changes in membrane PL fatty acid compositions. Most PL classes showed similar changes in fatty acid composition among the age groups, with reduced proportions of docosahexaenoic acid (DHA) and, generally, concomitantly increased levels of monounsaturated fatty acids, which together resulted in reduced peroxidation index (PIn). However, total polyunsaturated fatty acid (PUFA) content did not change significantly with age due to increased eicosapentaenoic acid, docosapentaenoic acid and, in most PL, increased n−6 PUFA. These results suggest there may be oxidation of PL DHA with compensatory mechanisms to maintain membrane fluidity and function. However, modification of fatty acid composition of specific PLs, such as cardiolipin, could affect the electron transport chain efficiency and propagate the oxidative reaction throughout the cell. In addition, both the content and fatty acid composition of sphingomyelin, which has been suggested as a possible mediator of cell dysfunction and apoptosis, changed with age differently to the other PL classes. Moreover, these changes showed different trends between mitochondria and whole liver. These data suggest there is marked oxidative stress associated with rapid growth and maturation in rainbow trout. Changes observed in membrane lipids point to their possible participation in the processes involved in this species response to oxidative stress and damage accumulation rate.     

A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids

Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused a reduction in MIM potential, oxygen consumption, and ATP synthetic capacity and, ultimately, death. Additionally, we showed that FAs induce a transient increase in intramitochondrial reactive oxygen species (ROS) and lipid peroxide production, lasting roughly 30 and 120 min for the ROS and lipid peroxides, respectively. MIM permeabilization and its deleterious consequences including mitochondrial crisis and cell death were prevented by treating the cells with the mitochondrial FA uptake inhibitor etomoxir, the mitochondrion-selective superoxide and lipid peroxide antioxidants MitoTempo and MitoQ, or the lipid peroxide and reactive carbonyl scavenger l-carnosine. FAs also promoted a delayed oxidative stress phase. However, the beneficial effects of etomoxir, MitoTempo, and l-carnosine were lost by delaying the treatment by 2 h, suggesting that the initial phase was sufficient to prime the cells for the delayed MIM permeabilization and mitochondrial crisis. It also suggested that the second ROS production phase is a consequence of this loss in mitochondrial health. Altogether, our data suggest that approaches designed to diminish intramitochondrial ROS or lipid peroxide accumulation, as well as MIM permeabilization, are valid mechanism-based therapeutic avenues to prevent the loss in preadipocyte metabolic fitness associated with prolonged exposure to elevated FA levels.     

Mitochondrial outer-membrane permeabilization and remodelling in apoptosis

Many human pathologies are associated with defects in mitochondria such as diabetes, neurodegenerative diseases or cancer. This tiny organelle is involved in a plethora of processes in mammalian cells, including energy production, lipid metabolism and cell death. In the so-called intrinsic apoptotic pathway, the outer mitochondrial membrane (MOM) is premeabilized by the pro-apoptotic Bcl-2 members Bax and Bak, allowing the release of apoptogenic factors such as cytochrome c from the inter-membrane space into the cytosol. At the same time, mitochondria fragment in response to Drp-1 activation suggesting that mitochondrial fission could play a role in mitochondrial outer-membrane permeabilization (MOMP). In this review, we will discuss the link that could exist between mitochondrial fission and fusion machinery, Bcl-2 family members and MOMP.     

Mitochondrial membrane cholesterol,the voltage dependent anion channel (VDAC), and the Warburg effect

Normal cells of aerobic organisms synthesize the energy they require in the form of ATP via the process of oxidative phosphorylation. This complex system resides in the mitochondria of cells and utilizes oxygen to produce a majority of cellular ATP. However, in most tumors, especially those with elevated cholesterogenesis, there is an increased reliance on glycolysis for energy, even in conditions where oxygen is available. This aerobic glycolysis (the Warburg effect) has far reaching ramifications on the tumor itself and the cells that surround it. In this brief review, we will discuss how abnormally high membrane cholesterol levels can result in a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH-7777). We have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution and transport. Work in our laboratory demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.     

Mitochondrial Na/Ca exchanger, a new target for neuroprotection in rat hippocampal slices

We tested here the hypothesis that the pharmacological modulation of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCX) could be a new neuroprotective strategy to rescue stressed vulnerable neurons from death. We used rat hippocampal slices incubated with veratridine to cause neuronal death through a mechanism involving Na⁺ and Ca²⁺ overload. CGP37157 (CGP), an inhibitor of the mNCX, rescued veratridine vulnerable neurons from death, showing an EC₅₀ of 5 μM. This neuroprotection was associated to mitigation of veratridine-elicited overproduction of free radicals and to inhibition of the p38 MAPK-linked apoptotic pathway. These results suggest that the mNCX could become a new target to develop compounds with potential therapeutic neuroprotective actions in neurodegenerative diseases.     

Aclarubicin-induced ROS generation and collapse of mitochondrial membrane potential in human cancer cell lines

The cytotoxicity of aclarubicin (ACL) in A549 (human non-small lung), HepG2 (human hepatoma) and MCF-7 (human breast adenocarcinoma) cancer cell lines was evaluated and compared with that of doxorubicin (DOX). Changes in mitochondrial transmembrane potential (ΔΨ_m), and production of reactive oxygen species (ROS) of drug-treated cells were monitored. Moreover, morphological changes associated with apoptosis were examined using double staining with Hoechst 33258-propidium iodide (PI). The results showed that ACL was much more cytotoxic than DOX in all investigated cell lines. Furthermore, ACL induced a concentration- and time-dependent increase in ROS production and decrease in mitochondrial membrane potential. The drugs, especially ACL, also induced ROS mediated apoptosis and necrosis pathways in all cell lines depending on the length of the post-treatment time. All these processes were partially inhibited by the antioxidants: N-acetylcysteine (NAC) and α-tocopherol. Of both drugs, DOX caused considerably weaker depolarization of the mitochondrial membrane. Its 10-fold higher concentration, as compared to ACL, was required to induce a similar effect, in accordance with the highly distinct cytotoxicity of these drugs towards investigated cells. In conclusion, ROS production preceded a decrease in mitochondrial membrane potential, but only changes in ΔΨ_m were correlated with drug cytotoxicity in particular cell line. These results suggest that the impairment of ΔΨ_m and an increase in ROS level might be important mechanisms of ACL cytotoxicity in cancer cells in solid tumors.     

Population growth kinetics and bulk membrane lipid alterations in Tetrahymena pyriformis: exposure to pentachlorophenol

This study describes effects of exposure of the freshwater ciliate Tetrahymena pyriformis to the "classic" weak acid respiratory uncoupler pentachlorophenol (PCP) on the population growth kinetics and membrane lipid profiles. The assessment of growth kinetics of naive populations exposed to PCP, at concentrations eliciting <50% growth inhibition, showed generation times of exposed cultures similar to generation times of controls but preceded by a short lag phase (<2 h). Assessment of exposed cultures exhibiting >50% growth inhibition revealed generation times that increased with increasing concentrations of toxicant. In addition, the relative percentages of selected fatty acid methyl esters (FAMEs) in both pellicle and mitochondrial membranes were examined. Upon exposure to PCP the relative percentages of FAMEs 12:0, 14:0, 16:0, 16:1, and 18:0 did not change. However, with exposure to PCP a decrease was observed for FAMEs 15:0 and 17:0. Conversely, with PCP exposure there was an increase in FAME 18:1. A comparison of these results with those elicited upon exposure to the model narcotic 1-octanol reveals marked differences in both growth kinetics and fatty acid shifts. This revised version was published online in July 2006 with corrections to the Cover Date.