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The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)- containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich's ataxia) has established that communication exists between iron metabolism in the mit0chondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism. iron sulfur cluster | heine | Friedreich's ataxia | frataxin | sideroblastic anemia doi/ 10.1073/pnas.0912925107

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane--cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the post-fusion granule membrane--plasma membrane complex. Experiments on dil-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin. doi/10.1083/jcb.200908010

Starch defines an insoluble semicrystalline form of storage polysaccharides restricted to Archaeplastida (red and green algae, land plants, and glaucophytes) and some secondary endosymbiosis derivatives of the latter. While green algae and land-plants store starch in plastids by using an ADP-glucose-based pathway related to that of cyanobacteria, red algae, glaucophytes, cryptophytes, dinoflagellates, and apicomplexa parasites store a similar type of polysaccharide named floridean starch in their cytosol or periplast. These organisms are suspected to store their floridean starch from UDP-glucose in a fashion similar to heterotrophic eukaryotes. However, experimental proof of this suspicion has never been produced. Dinoflagellates define an important group of both photoautotrophic and heterotrophic protists. We now report the selection and characterization of a low starch mutant of the heterotrophic dinoflagellate Crypthecodinium cohnii. We show that the sta 1-1 mutation of C. cohnii leads to a modification of the UDP-glucose-specific soluble starch synthase activity that correlates with a decrease in starch content and an alteration of amylopectin structure. These experimental results validate the UDP-glucose-based pathway proposed for floridean starch synthesis. alveolate | amylopectin | amylose | starch synthase doi/10.1073/pnas.0907424106

In source leaves of resistant tobacco, oxidative burst and subsequent formation of hypersensitive lesions after infection with Phytophthora nicotianae was prevented by inhibition of glucose-6-phosphate dehydrogenase (G6PDH) or NADPH oxidases. This observation indicated that plant defense could benefit from improved NADPH availability due to increased G6PDH activity in the cytosol. A plastidic isoform of the G6PDH-encoding gene, G6PD, displaying high NADPH tolerance was engineered for cytosolic expression (cP2), and introduced into a susceptible cultivar. After infection, transgenic (previously susceptible) lines overexpressing cP2 showed early oxidative bursts, callose deposition, and changes in metabolic parameters. These responses resulted in timely formation of hypersensitive lesions similar to resistant plants, although their extent varied considerably between different transgenic lines. Additional RNAi suppression of endogenous cytosolic G6PD isoforms resulted in highly uniform defense responses and also enhanced drought tolerance and flowering. Cytosolic G6PDH seems to be a crucial factor for the outcome of plant defense responses; thus, representing an important target for modulation of stress resistance. Because isoenzyme replacement of G6PDH in the cytosol was beneficial under various kinds of cues, we propose this strategy as a tool to enhance stress tolerance in general. oxidative burst | source-to-sink transition | NADPH availability | pathogen resistance | tobacco

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on [beta]-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular [Ca.sup.2+], and in particular the role of the endoplasmic reticulum (ER). In human [beta]-cells and MIN6 cells, palmitate rapidly increased cytosolic [Ca.sup.2+] through a combination of [Ca.sup.2+] store release and extracellular [Ca.sup.2+] influx. Palmitate caused a reversible lowering of ER [Ca.sup.2+], measured directly with the fluorescent protein-based ER [Ca.sup.2+] sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic [Ca.sup.2+] in palmitate-treated cells. In keeping with this observed ER [Ca.sup.2+] depletion, palmitate induced rapid phosphorylation of the ER [Ca.sup.2+] sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and [beta]-cell death. We detected little palmitate-induced insulin secretion, suggesting that these [Ca.sup.2+] signals are poorly coupled to exocytosis. In summary, we have characterized [Ca.sup.2+]-dependent mechanisms involved in altered [beta]-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER [Ca.sup.2+] and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes. diabetes; free fatty acids; fluorescence resonance energy transfer; calcium homeostasis; endoplasmic reticulum

Cytosolic [NADP.sup.+]-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/ (GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-[PK.sub.1] cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R. reduced nicotinamide adenine dinucleotide phosphate; glutathione; proximal tubular cell; acute kidney injury; oxidative stress; ROS

Autophagy is responsible for nonspecific, bulk degradation of cytoplasmic components. Recent work has revealed also that there is specific, autophagic degradation of polyubiquitinated protein aggregates, whose buildup occurs during neurodegenerative disease. Here, we report that simple mono-ubiquitination of normally long-lived cytoplasmic substrates is sufficient to target these substrates for autophagic degradation in mammalian cells. That is, upon their ubiquitination, both small [i.e., red fluorescent protein (RFP)] and large (i.e., peroxisomes) substrates are efficiently targeted to autophagosomes and then degraded within lysosomes upon autophagosome-lysosome fusion. This targeting requires the ubiquitin-binding protein, p62, and is blocked by the Class III phosphatidylinositol 3-kinase (PI3K) inhibitor, 3-methyladenine (3-MA), or by depletion of the autophagy-related-12 (Atg12) protein homolog. Mammalian cells thus use a common pathway involving ubiquitin and p62 for targeting diverse types of substrates for autophagy. autophagy | p62 | pexophagy

Direct delivery of molecules into the cytosol of live cells is required in many areas of biology and clinical research. Molecules of interest include indicator dyes, biomolecules, and pharmacological agents. In this work we describe continuous delivery of molecules into single cells using a diffusional microburet, DMB. The DMB is a pulled glass micropipette with a fine tip that contains a microscopic plug made of a hydrogel such as agar or polyacrylamide. This plug prevents flow but allows diffusive delivery of the molecule of interest from the DMB body into the cytosol, driven by its concentration gradient. This leads to a scheme of sustained intracellular dosing that is highly reproducible and quantifiable yet does not require the addition of solution volume to the cell. Potential loss of biomolecules from the cytosol through the plug of the DMB can be greatly reduced by proper choice of the pore size and tortuosity of the hydrogel in the DMB tip. The intracellular concentration of fluorescent molecules during delivery can be obtained calibration free. In this work we demonstrate dosing of Lucifer Yellow CH, LY, a charged fuorescent dye, into individual a7r5 vascular smooth muscle cells with a DMB. New types of quantitative analytical experiments on single live cells that the DMB technology enables are titration of intracellular ions and ligands, binding sites, and efflux pathways such as those that are involved in drug resistance.

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states. Hsc70 | Hsp90 | ricin

Cytosolic phospholipase [A.sub.2[[alpha] (cPL[A.sub.2][alpha]) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (SIP) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of SIP-induced AA release and determined the involvement of cPL[A.sub.2][alpha] in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. SIPinduced AA release was inhibited by the cPL[A.sub.2][alpha] inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPL[A.sub.2][alpha], and by inhibition of SIP-induced calcium flux, suggesting a significant role of cPL[A.sub.2][alpha] in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited SIP-induced calcium flux and AA release. The S l P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphkl) expression and activity. Furthermore, Rho-associated kinase, downstream of S IP3, was crucial for S IP-induced cPL[A.sub.2][alpha] activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPL[A.sub.2][alpha] and releases AA in lung epithelial cells. An understanding of SiP-induced cPL[A.sub.2][alpha] activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung. eicosanoid; calcium flux; airway inflammation

Abstract: Our previous work showed that ET-1 induced a concentration-dependent increase of cytosolic [Ca.sup.2+] ([[Ca].sub.c]) and nuclear [Ca.sup.2+] ([[Ca].sub.n]) in human aortic vascular smooth muscle cells (hVSMCs). In the present [...]

Dopamine is a potentially toxic neurotransmitter that has long been speculated to contribute to the pathogenesis of Parkinson's disease (PD). Recent work has demonstrated the importance of proper storage of dopamine in vesicles to maintain dopamine homeostasis, thus protecting neurons from the detrimental effects of dopamine accumulation and breakdown in the cytosol. These studies suggest that factors which affect dopamine storage might increase the susceptibility of dopamine neurons to further environmental or genetic insults, exacerbating the neuronal degeneration that characterizes PD. This review seeks to revisit the pathogenicity of cytosolic dopamine and further address the critical role of neurotransmitter storage in dopaminemediated neurotoxicity.

Sensory neurons can detect ischemia and transmit pain from various organs. Whereas the primary stimulus in ischemia is assumed to be acidosis, little is known about how the inevitable metabolic challenge influences neuron function. In this study we have investigated the effects of anoxia, aglycemia, and acidosis upon intracellular [Mg.sup.2+] concentration [[[Mg.sup.2+]].sub.i] and intracellular pH (p[H.sub.i]) in isolated sensory neurons. Anoxia, anoxic aglycemia, and acidosis all caused a rise in [[[Mg.sup.2+]].sub.i] and a fall in p[H.sub.i]. The rise in [[[Mg.sup.2+]].sub.i] in response to acidosis appears to be due to [H.sup.+] competing for intracellular [Mg.sup.2+] binding sites. The effects of anoxia and aglycemia were mimicked by metabolic inhibition and, in a dorsal root ganglia (DRG)-derived cell line, the rise in [[[Mg.sup.2+]].sub.i] during metabolic blockade was closely correlated with fall in intracellular ATP concentration ([[ATP].sub.i]). Increase in [[[Mg.sup.2+]].sub.i] during anoxia and aglycemia were therefore assumed to be due to MgATP hydrolysis. Even brief periods of anoxia ( magnesium; ischemia