Background: Cellular stress leading to cell death induces the formation of lipid droplets. Results: Nutrient deprivation induces LD biogenesis and mobilization, fueling fatty acid oxidation to sustain cell viability. Conclusion: ␤-Oxidation requires biogenesis and mobilization of LD. Significance: The role of LD in cell survival and ␤-oxidation might provide new potential targets for antitumor therapy.

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A 2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A 2 (cPLA 2 ␣). Knocking down cPLA 2 ␣ expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA 2 ␣ fusion protein (EGFP-cPLA 2 ) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA 2 ␣ at Ser 505 . Transfection of a S505A mutant cPLA 2 ␣ showed that phosphorylation at Ser 505 is key for enzyme activity and LD formation. cPLA 2 ␣ contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA 2 ␣ inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA 2 ␣ in the formation of nascent LD from the endoplasmic reticulum.

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation. INTRODUCTIONRecent observations from several laboratories indicate that membrane lipids regulate intracellular membrane transport, particularly in distal stages of the secretory pathway. Diacylglycerol (DAG) is a simple and small sized signal-transducing lipid which among other functions is necessary for protein transport from the Golgi complex to the cell surface both in yeast and in mammals. Thus, in budding yeast phosphatidylinositol (PI)-transfer Sec14p protein directly regulates DAG homeostasis in the Golgi complex and protein secretion (Bankaitis et al., 1990;Kearns et al., 1997;Huijbregts et al., 2000). In mammals, the reduction of DAG levels at the Golgi caused by the depletion of Nir2 (a peripheral Golgi protein containing a PI-transfer domain) inhibits post-Golgi protein transport (Litvak et al., 2005). DAG acts in the trans-Golgi network (TGN) as a membrane acceptor for specific proteins such as the protein kinase C (PKC) family member PKD/ PKC (Prestle et al., 1996;Liljedahl et al., 2001;Baron andMalhotra, 2002), and Hmun13 (Speight andSilverman, 2005).PKD together with PKC, the trimeric G-protein subunits ␤/␥ (Díaz Anel and Malhotra, 2005), and phosphatidylinositol-4 kinase III␤ (Hausser et al., 2005) directly participate in the post-Golgi transport of plasma membrane proteins containing basolateral sorting information (Yeaman et al., 2004). On the other hand, Hmun13, through the recruitment of Rab34, participates in the Golgi-lysosome protein trafficking (Speight and Silverman, 2005). DAG also promotes the Golgi membrane targeting and activation of other C1 domain-containing signaling molecules such as other PKC isoforms (PKC, PKC, and PKC␦;Maissel et al., 2006;Lehel et al., 1995; Wang et al., 1999, respectively) and Ras guanosine nucleotide-releasing proteins (RasGRPs;Caloca et al., 2003), whose potential involvement in Golgi-associated transport functions remains unexplored.The aforementioned Go...

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A 2 (iPLA 2 -VIA). Induction of stress with acidic pH, C 2 -ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA 2 -VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA 2 (cPLA 2 ␣). The results demonstrate that iPLA 2 -VIA provides fatty acids for TAG synthesis while cPLA 2 ␣ allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates.

Light-triggered reversible modulation of physiological functions offers the promise of enabling on-demand spatiotemporally controlled therapeutic interventions. Optogenetics has been successfully implemented in the heart, but significant barriers to its use in the clinic remain, such as the need for genetic transfection. Herein, we present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. The molecule, named PAI, was designed by introduction of a photoswitch into the molecular structure of an M2 mAChR agonist. In vitro assays revealed that PAI enables light-dependent activation of M2 mAChRs. To validate the method, we show that PAI photoisomers display different cardiac effects in a mammalian animal model, and demonstrate reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wavelength illumination, and offers new opportunities for intravital imaging and control of cardiac function.

Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCTα-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCTα is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCTα-deficient cells. Pro–tumor necrosis factor α and interleukin-6 remain lodged in the secretory compartment of CCTα-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1β secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCTα-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro–tumor necrosis factor α or interleukin-6. These results identify CCTα as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A 2 (cPLA 2 ␣). This work dissects the pathway leading to cPLA 2 ␣ activation and LD biogenesis. Both processes were Ca 2؉ -independent, as they took place after pharmacological blockade of Ca 2؉ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA 2 ␣, which abrogates its Ca 2؉ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca 2؉ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA 2 ␣ at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA 2 ␣ at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA 2 ␣ and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.

Red-shifted azobenzene scaffolds have emerged as useful molecular photoswitches to expand potential applications of photopharmacological tool compounds. As one of them, tetra-ortho-fluoro azobenzene is well compatible for the design of visible-light-responsive systems, providing stable and bidirectional photoconversions and tissue-compatible characteristics. Using the unsubstituted azobenzene core and its tetra-ortho-fluorinated analogue, we have developed a set of uni- and bivalent photoswitchable toolbox derivatives of the highly potent muscarinic acetylcholine receptor agonist iperoxo. We investigated the impact of the substitution pattern on receptor activity and evaluated the different binding modes. Compounds 9b and 15b show excellent photochemical properties and biological activity as fluorination of the azobenzene core alters not only the photochromic behavior but also the pharmacological profile at the muscarinic M1 receptor. These findings demonstrate that incorporation of fluorinated azobenzenes not just may alter photophysical properties but can exhibit a considerably different biological profile that has to be carefully investigated.