Expression of phosphatidylinositol (4,5) bisphosphate-specific pleckstrin homology domains alters direction but not the level of axonal transport of mitochondria

Axonal transport of membranous organelles such as mitochondria is essential for neuron viability and function. How signaling mechanisms regulate or influence mitochondrial distribution and transport is still largely unknown. We observed an increase in the distal distribution of mitochondria in neurons upon the expression of pleckstrin homology (PH) domains of phospholipase Cdelta1 (PLCdelta-PH) and spectrin (spectrin-PH). Quantitative analysis of mitochondrial transport showed that specific binding of PH domains to phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) but not 3' phosphorylated phosphatidylinositol species enhanced plus-end-directed transport of mitochondria two- to threefold and at the same time decreased minus-end-directed transport of mitochondria along axonal microtubules (MTs) without altering the overall level of motility. Further, the velocity and duration of mitochondrial transport plus the association of molecular motors with mitochondria remained unchanged by the expression of PH domains. Thus, PtdIns(4,5)P2-specific PH domains caused an increase in distal mitochondria by disturbing the balance of plus- and minus-end-directed transport rather than directly affecting the molecular machinery involved. Taken together our data reveal that level and directionality of transport are separable and that PtdIns(4,5)P2 has a novel role in regulation of the directionality of axonal transport of mitochondria.     

cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells

We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.     

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.     

The role of mitochondrial transport in energy metabolism

Since mitochondria are closed spaces in the cell, metabolite traffic across the mitochondrial membrane is needed to accomplish energy metabolism. The mitochondrial carriers play this function by uniport, symport and antiport processes. We give here a survey of about 50 transport processes catalysed by more than 30 carriers with a survey of the methods used to investigate metabolite transport in isolated mammalian mitochondria. The role of mitochondria in metabolic pathways including ammoniogenesis, amino acid metabolism, mitochondrial shuttles etc. is also reported in more detail, mainly in the light of the existence of new transport processes.     

Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent

Mitochondria are distributed within cells to match local energy demands. We report that the microtubule-dependent transport of mitochondria depends on the ability of milton to act as an adaptor protein that can recruit the heavy chain of conventional kinesin-1 (kinesin heavy chain [KHC]) to mitochondria. Biochemical and genetic evidence demonstrate that kinesin recruitment and mitochondrial transport are independent of kinesin light chain (KLC); KLC antagonizes milton's association with KHC and is absent from milton-KHC complexes, and mitochondria are present in klc (-/-) photoreceptor axons. The recruitment of KHC to mitochondria is, in part, determined by the NH(2) terminus-splicing variant of milton. A direct interaction occurs between milton and miro, which is a mitochondrial Rho-like GTPase, and this interaction can influence the recruitment of milton to mitochondria. Thus, milton and miro are likely to form an essential protein complex that links KHC to mitochondria for light chain-independent, anterograde transport of mitochondria.     

Inactivation of mitochondrial electron transport by photosensitization of a pheophorbide a derivative

We examined the damage to mitochondrial electron transport caused by photosensitization of a pheophorbide a derivative, DH-I-180-3, shown recently to induce necrosis of lung carcinoma cells with low dark toxicity. Confocal microscopy showed that DH-I-180-3 co-localized with dihydrorhodamine-123 suggesting that it mainly accumulates in mitochondria. The photosensitizer alone in the dark did not affect mitochondrial electron transport. Illumination of isolated mitochondria in the presence of DH-I-180-3 resulted in inhibition of both NADH- and succinate-dependent respiration. Measurement of the activity of each component of the electron transport chain revealed that Complex I and III were very susceptible to the treatment whereas Complex IV was resistant. We conclude that the photosensitizer is localized in mitochondria and, upon illumination, produces reactive oxygen species that inactivate Complexes I and III.     

Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila

The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic demands. Consequently, it is important to carefully define the conditions facilitating live imaging of mitochondrial transport in dissected animal preparations. In this study, we examined Schneider's and the haemolymph-like solutions HL3 and HL6 for their suitability to image mitochondrial transport in motor axons of dissected Drosophila melanogaster larvae. Overall, mitochondrial transport kinetics in larval motor axons appeared similar among all three solutions. Unexpectedly, HL3 solution selectively increased the length of mitochondria in the context of the net-direction of transport. We also found that mitochondrial transport is sensitive to the extracellular Ca(2+) but not glutamate concentration. High concentrations of extracellular glutamate affected only the ratio between motile and stationary mitochondria. Our study offers a valuable overview of mitochondrial transport kinetics in larval motor axons of Drosophila under various conditions, guiding future studies genetically dissecting mechanisms of mitochondrial transport.     

Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos

In Drosophila, polar plasm contains polar granules, which deposit the factors required for the formation of pole cells, germ line progenitors. Polar granules are tightly associated with mitochondria in early embryos, suggesting that mitochondria could contribute to pole cell formation. We have previously reported that mitochondrial large and small rRNAs (mtrRNAs) are transported from mitochondria to polar granules prior to pole cell formation and the large rRNA is essential for pole cell formation. Here we show that the localization of mtrRNAs is diminished in embryos laid by tudor mutant females, although the polar granules are maintained. We also found that Tud protein was colocalized with mtrRNAs at the boundaries between mitochondria and polar granules when the transport of mtrRNAs takes place. These observations suggest that Tud mediates the transport of mtrRNAs from mitochondria to polar granules.     

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata

Recent studies suggest that intercellular transport via plasmodesmata (PD) is regulated by cellular redox state. Until now, this relationship has been unclear, as increased production of reactive oxygen species (ROS) has been associated with both increased and decreased intercellular transport via PD. Here, we show that silencing two genes that both increase transport via PD, INCREASED SIZE EXCLUSION LIMIT1 (ISE1) and ISE2, alters organelle redox state. Using redox-sensitive green fluorescent proteins targeted to the mitochondria or plastids, we show that, relative to wild-type leaves, plastids are more reduced in both ISE1- and ISE2-silenced leaves, whereas mitochondria are more oxidized in ISE1-silenced leaves. We further show that PD transport is positively regulated by ROS production in mitochondria following treatment with salicylhydroxamic acid but negatively regulated by an oxidative shift in both chloroplasts and mitochondria following treatment with paraquat. Thus, oxidative shifts in the mitochondrial redox state positively regulate intercellular transport in leaves, but oxidative shifts in the plastid redox state counteract this effect and negatively regulate intercellular transport. This proposed model reconciles previous contradictory evidence relating ROS production to PD transport and supports accumulating evidence that mitochondria and plastids are crucial regulators of PD function.     

Vps39 is required for ethanolamine-stimulated elevation in mitochondrial phosphatidylethanolamine

Mitochondrial membrane biogenesis requires the import of phospholipids; however, the molecular mechanisms underlying this process remain elusive. Recent work has implicated membrane contact sites between the mitochondria, endoplasmic reticulum (ER), and vacuole in phospholipid transport. Utilizing a genetic approach focused on these membrane contact site proteins, we have discovered a ‘moonlighting’ role of the membrane contact site and vesicular fusion protein, Vps39, in phosphatidylethanolamine (PE) transport to the mitochondria. We show that the deletion of Vps39 prevents ethanolamine-stimulated elevation of mitochondrial PE levels without affecting PE biosynthesis in the ER or its transport to other sub-cellular organelles. The loss of Vps39 did not alter the levels of other mitochondrial phospholipids that are biosynthesized ex situ, implying a PE-specific role of Vps39. The abundance of Vps39 and its recruitment to the mitochondria and the ER is dependent on PE levels in each of these organelles, directly implicating Vps39 in the PE transport process. Deletion of essential subunits of Vps39-containing complexes, vCLAMP and HOPS, did not abrogate ethanolamine-stimulated PE elevation in the mitochondria, suggesting an independent role of Vps39 in intracellular PE trafficking. Our work thus identifies Vps39 as a novel player in ethanolamine-stimulated PE transport to the mitochondria.     

Attachment of mitochondria to intermediate filaments in adrenal cells: relevance to the regulation of steroid synthesis.

The rate of steroid synthesis is regulated by the rate of transport of cholesterol from lipid droplets to mitochondria. We have previously demonstrated that lipid droplets in adrenal cells are tightly attached to intermediate filaments. Here we now show that mitochondria colocalize with intermediate filaments in modified double indirect immunofluorescence and by electron microscopy of extracted adrenal cells. Direct contact between mitochondria and intermediate filaments was established by examination of stereo pairs of electron micrographs from extracted cells. The attachment of both droplets and mitochondria to intermediate filaments suggests possible mechanisms for this form of intracellular transport of cholesterol to mitochondria and hence for the regulation of steroid synthesis.     

A Highlights from MBoC Selection: APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations.     

Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential.

Two different functions have been proposed for the phosphate carrier protein/p32 of Saccharomyces cerevisiae mitochondria: transport of phosphate and requirement for import of precursor proteins into mitochondria. We characterized a yeast mutant lacking the gene for the phosphate carrier/p32 and found both a block in the import of phosphate and a strong reduction in the import of preproteins transported to the mitochondrial inner membrane and matrix. Binding of preproteins to the surface of mutant mitochondria and import of outer membrane proteins were not inhibited, indicating that the inhibition of protein import occurred after the recognition step at the outer membrane. The membrane potential across the inner membrane of the mutant mitochondria was strongly reduced. Restoration of the membrane potential restored preprotein import but did not affect the block of phosphate transport of the mutant mitochondria. We conclude that the inhibition of protein import into mitochondria lacking the phosphate carrier/p32 is indirectly caused by a reduction of the mitochondrial membrane potential (delta(gamma)), and we propose a model that the reduction of delta(psi) is due to the defective phosphate import, suggesting that phosphate transport is the primary function of the phosphate carrier/p32.     

Characteristics of intermittent mitochondrial transport in guinea pig enteric nerve fibers

Enteric neurons controlling various gut functions are prone to oxidative insults that might damage mitochondria (e.g., intestinal inflammation). To resume local energy supply, mitochondria need to be transported. We used MitoTracker dyes and confocal microscopy to investigate basic characteristics of mitochondrial transport in guinea pig myenteric neurites. During a 10-s observation of 1 mm nerve fiber, on average, three mitochondria were transported at an average speed of 0.41 +/- 0.02 microm/s. Movement patterns were clearly erratic, and velocities were independent of mitochondrial size. The velocity oscillated periodically ( approximately 6 s) but was not consistently affected by structures such as en route boutons, bifurcations, or stationary mitochondria. Also, mitochondria transported in opposite directions did not necessarily affect each others' mobility. Transport was blocked by microtubule disruption (100 microM colchicine), and destabilization (1 microM cytochalasin-D) or stabilization (10 microM phalloidin) of actin filaments, respectively, decreased (0.22 +/- 0.02 microm/s, P < 0.05) or increased (0.53 +/- 0.02 microm/s, P < 0.05) transport speed. Transport was inhibited by TTX (1 microM), and removal of extracellular Ca(2+) (plus 2 mM EGTA) had no effect. However, depletion of intracellular stores (thapsigargin) reduced (to 33%) and slowed the transport significantly (0.18 +/- 0.02 microm/s, P < 0.05), suggesting an important role for stored Ca(2+) in mitochondrial transport. Transport was also reduced (to 21%) by the mitochondrial uncoupler FCCP (1 microM) in a time-dependent fashion and slowed by oligomycin (10 microM). We conclude that mitochondrial transport is remarkably independent of structural nerve fiber properties. We also show that mitochondrial transport is TTX sensitive and speeds up by stabilizing actin and that functional Ca(2+) stores are required for efficient transport.     

Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150(Glued) suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ("organelle jams" or "clogs") caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. We speculate that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration.     

植物核编码线粒体蛋白转动的研究进展

植物核编码蛋白向线粒体内的转运对于线粒体正常功能的发挥有着不可忽视的作用。目前在诸如前序列、前体蛋白加工、分子伴侣及线粒体与质体（或叶绿体）之间分选等几方面开展了许多研究。本综述以上方面的研究进展。     

Effect of Osmotic Stress on Ion Transport Processes and Phospholipid Composition of Wheat (Triticum aestivum L.) Mitochondria

The effect of osmotic stress on wheat (Triticum aestivum L.) mitochondrial activity and phospholipid composition was investigated. Preliminary growth measurements showed that osmotic stress (−0.25 or −0.5 megapascal external water potential) inhibited the rate of shoot dry matter accumulation while root dry matter accumulation was less sensitive. We have determined that differences in sensitivity to osmotic stress existed between tissues at the mitochondrial level. Mitochondria isolated from roots or shoots of stressed seedlings showed respiratory control and ADP/O ratios similar to control seedlings which indicates that stressed mitochondria were well coupled. However, under passive swelling conditions in a KCl reaction mixture, the rate and extent of valinomycin-induced swelling of shoot mitochondria were increased by osmotic stress while root mitochondria were largely unaffected. Active ion transport studies showed efflux transport by stressed-shoot mitochondria to be partially inhibited since mitochondrial contraction required the addition of N-ethylmaleimide or nigericin. Efflux ion transport by root mitochondria was not inhibited by osmotic stress which indicates that stress-induced changes in ion transport were largely limited to shoot mitochondria. Characterization of mitochondrial fatty acid and phospholipid composition showed an increase in the percentage of phosphatidylcholine in stressed shoot mitochondria compared to the control. Mitochondrial fatty acid composition was not markedly altered by stress. No significant changes in either the phospholipid or fatty acid composition of stressed root mitochondria were observed. Hence, these results suggest that a tissue-specific response to osmotic stress exists at the mitochondrial level.     

Ischemic preconditioning does not protect via blockade of electron transport.

Ischemic preconditioning (IPC) before sustained ischemia decreases myocardial infarct size mediated in part via protection of cardiac mitochondria. Reversible blockade of electron transport at complex I immediately before sustained ischemia also preserves mitochondrial respiration and decreases infarct size. We proposed that IPC would attenuate electron transport from complex I as a potential effector mechanism of cardioprotection. Isolated, Langendorff-perfused rat hearts underwent IPC (3 cycles of 5-min 37 degrees C global ischemia and 5-min reperfusion) or were perfused for 40 min without ischemia as controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated. IPC did not decrease ADP-stimulated respiration measured in intact mitochondria using substrates that donate reducing equivalents to complex I. Maximally expressed complex I activity measured as rotenone-sensitive NADH:ubiquinone oxidoreductase in detergent-solubilized mitochondria was also unaffected by IPC. Thus the protection of IPC does not occur as a consequence of a partial decrease in complex I activity leading to a decrease in integrated respiration through complex I. IPC and blockade of electron transport both converge on mitochondria as effectors of cardioprotection; however, each modulates mitochondrial metabolism during ischemia by different mechanisms to achieve cardioprotection.     

Blockade of electron transport during ischemia protects cardiac mitochondria

Subsarcolemmal mitochondria sustain progressive damage during myocardial ischemia. Ischemia decreases the content of the mitochondrial phospholipid cardiolipin accompanied by a decrease in cytochrome c content and a diminished rate of oxidation through cytochrome oxidase. We propose that during ischemia mitochondria produce reactive oxygen species at sites in the electron transport chain proximal to cytochrome oxidase that contribute to the ischemic damage. Isolated, perfused rabbit hearts were treated with rotenone, an irreversible inhibitor of complex I in the proximal electron transport chain, immediately before ischemia. Rotenone pretreatment preserved the contents of cardiolipin and cytochrome c measured after 45 min of ischemia. The rate of oxidation through cytochrome oxidase also was improved in rotenone-treated hearts. Inhibition of the electron transport chain during ischemia lessens damage to mitochondria. Rotenone treatment of isolated subsarcolemmal mitochondria decreased the production of reactive oxygen species during the oxidation of complex I substrates. Thus, the limitation of electron flow during ischemia preserves cardiolipin content, cytochrome c content, and the rate of oxidation through cytochrome oxidase. The mitochondrial electron transport chain contributes to ischemic mitochondrial damage that in turn augments myocyte injury during subsequent reperfusion.