Cell migration is required for a number of physiological processes and is implicated in pathologies such as tumor metastasis. Cell motility is dependent upon dynamic actin reorganization, and is regulated by the Rho family of small GTPases. Rho GTPases are molecular switches that cycle between their active and inactive conformations. The best-studied members of this family are Rac1, RhoA, and Cdc42. Each is responsible for the formation of specific actin structures. Diacylglycerol kinases (DGKs) act at the membrane to convert diacylglycerol (DAG) and phosphatidic acid (PA), maintaining the balance of these two lipid second messengers. Previous studies from our lab have demonstrated that the ζ isoform of DGK facilitates the release of Rac1 and RhoA from their inhibitor, RhoGDI. Here we studied a closely related isoform, DGKι, using mouse embryonic fibroblasts (MEFS) in which the gene for DGKι had been deleted. Aberrations in cell morphology, spreading, and migration were identified in DGKι-null MEFs. We showed that the activity of Rac1 and RhoC, but not RhoA, was impaired in the absence of DGKι, yet only RhoC protein levels were affected. Reduced activation of these Rho GTPases was accompanied by defects in Rac1- and RhoC- related actin structures. These data demonstrate that DGKι, in addition to DGKζ, contributes to the regulation of GTPase activation and remodeling of the actin cytoskeleton.

The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. This neutral lipid, TAG is utilized by plants for the carbon and energy source when stores in seed tissue. There is significant human nutritional demand for vegetable oil, but its use in production of renewable biomaterials and fuels has intensified the need to increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). Both DGAT and PDAT contribute to seed TAG biosynthesis in an independent or overlapping manner, depending on the species. However, the regulation of TAG biosynthesis is not wellstudied in nonseed tissues such as mesocarp of avocado. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 was identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGAT1s. These data reveal that the putative DGATs of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For further functional analysis, the avocado DGAT1 was expressed in H1246, a TAG-deficient yeast strain and lipotoxicity rescue assays, TLC analysis, Nile Red staining were conducted. The complementation of this yeast strain confirmed enzyme activity and supported the possible role of avocado DGAT1 in TAG biosynthesis. Finally, substrate specificity of DGAT was determined by incubating microsomes with different radiolabeled substances and found that avocado DGAT1 has a preference toward oleic acid (18:1) compare to palmitic acid (16:0) while it is converting diacylglycerol (DAG) to triacylglycerol. All these data suggested that avocado DGAT1 is functional and making TAG with high preference of oleic acid over palmitic acid.

The molecular species of diacylglycerol and alkylacylglycerol of Madin-Darby canine Kidney (MDCK) cells were analyzed to determine the sources of diradylglycerols generated during cell growth and phorbol ester stimulation. MDCK cells in log phase growth contained higher levels of diacylglycerol and alkylacylglycerol than confluent cells. Both subclasses of diradylglycerol showed higher levels of saturated and monoenoic species during log phase. Glycerol incorporation into diradylglycerols was increased during growth, consistent with an increase in their synthesis de novo. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, caused an increase in the level of diacylglycerol but not alkylacylglycerol. Log phase MDCK cells showed a greater response to TPA treatment than confluent cells. The molecular species of diacylglycerol generated during stimulation with either TPA or dioctanoylglycerol closely resembled the species of phosphatidylcholine. These results indicate that TPA and synthetic diacylglycerol stimulate endogenous diacylglycerol production through the hydrolysis of phosphatidylcholine. In contrast, the higher content of diacylglycerol and alkylacylglycerol in replicating MDCK cells is the result of an increase in their synthesis de novo.

<p>The two lipid kinases, diacylglycerol kinase (DGK) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K), are vital players of the phosphatidylinositol cycle. DGK regulates the intracellular balance between two important lipid signaling molecules, diacylglycerol and phosphatidic acid. PIP5K produces another key signal messenger, phosphatidylinositol 4,5-bisphosphate. We studied several fundamental aspects of DGK and PIP5K properties. We investigated the topology of the hydrophobic segment of FLAG-tagged DGK epsilon, and showed that a single amino acid mutation P32A caused the hydrophobic segment to favor a transmembrane orientation. We demonstrated that DGKε is localized in both the plasma membrane and endoplasmic reticulum. Our work helped to better elucidate the substrate specificity of DGKε and PIP5K isoforms, and it lead us to discover the motif that is common for several enzymes that exhibit specificity for substrates containing polyunsaturated fatty acids. We studied the organ distribution of murine DGK isoforms, and also expanded our knowledge of DGK expression in diabetic animals, showing that the expression profiles of several DGK isoforms are altered in adipocytes isolated from diabetic mice. Moreover, DGK expression profiles change dramatically during adipocyte differentiation. Taken together, our findings contribute to the growing knowledge about two enzymes, DGK and PIP5K, by providing the fundamental information about the structural and functional properties of these lipid kinases. Both PIP5K and DGK enzymes have a strong potential for use as drug targets. Although at present their clinical importance has not been completely assessed, we believe that their significance as drug targets will be recognized in the nearest future. <strong></strong></p> / Doctor of Philosophy (PhD)

<p> An area of current research interest involves the diacylglycerol kinase (DGK) family. Diacylglycerol kinases (DGKs) are a group of enzymes that phosphorylate diacylglycerol (DAG), a second messenger involved in cell signaling. The product of this reaction, phosphatidic acid (PA), also has signaling roles. An interesting isoform is DGKε, that although it has no identifiable regulatory domains other than the C1 domains. In addition, the catalytic domain is homologous to that of other DGK isoforms; however, DGKε exhibits an unusual specificity toward acyl chains of DAG, selectively phosphorylating an arachidonoyl-DAG substituted at the sn-2 position. Recently, researchers have identified an N-terminal hydrophobic domain of about 19 amino-acids in human DGKε. The present study attempted to identify the function of the N-terminal putative transmembrane domain of human DGKε and its relationship to the activity and substrate specificity of this enzyme by designing a truncated form of DGKε lacking the putative transmembrane domain.</p> <p> We have shown that the putative transmembrane domain of DGKε is not required for enzyme activity or for substrate specificity. In a mixed micellar assay the enzyme-catalyzed reaction followed surface dilution kinetics with respect to diacylglycerol and followed Michaelis-Menten kinetics with respect to ATP. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increased the catalytic rate constant for all three substrates used in this study. It appears unlikely that the putative transmembrane domain, a segment unique to DGKε, has no functional role. It is possible that the hydrophobic segment may have a role in enzyme regulation by associating the enzyme in oligomers that are inactive in quiescent cells and get activated upon dissociation into monomers by increased levels of DAG in the membrane. We have shown that the presence of higher molecular species in the gel is not dependent on the presence or absence of the putative transmembrane domain. The only difference between the full-length and truncated enzyme is the monomer to dimer ratio. It appears likely that another segment of DGKε besides the putative transmembrane domain may be involved in oligomerization and that oligomerization is either transient or very weak. The absence of the hydrophobic domain of DGKε seems to cause no drastic changes either in the activity, the substrate specificity, or the state of oligomerization of the enzyme.</p> <p> Therefore, the next question is whether the hydrophobic domain of DGKε inserts itself in the membrane as a transmembrane helix or it only helps associate the enzyme to the surface of the membrane. We studied the topology of theN-terminal domain of DGKε in intact and permeabilized cells by indirect immunofluorescent microscopy. The results show that the N-terminal domain of the protein is present in the cytosol. The data supports a model in which the hydrophobic domain of DGKε forms a hydrophobic loop that attaches to the inner layer of the plasma membrane or that the hydrophobic domain attaches to the inner leaflet through its nonpolar surface of a horizontal helix. The first hypothesis is supported by the presence of a Pro residue in the middle of the hydrophobic domain. This Pro would introduce a kink in the helix creating a loop, but the absence of one or more glycine residues proximal to proline may hinder the formation of the loop. The second hypothesis is sustained by the presence of a polar surface on one side of the helical wheel. This orientation indicates the presence of a slightly horizontal helix attached to the surface of the inner layer of the plasma membrane.</p> <p> Regardless of the orientation of the helix, the weak association of the enzyme with the membrane is supported by previous data on the ease of extractability of the enzyme with high salts and on the Triton X-114 phase partitioning.</p> / Thesis / Master of Science (MSc)

Die vorliegende Arbeit beschäftigt sich mit den physiologischen Veränderungen porciner Spermatozoen, die durch einen metabolischen Einbau von Fettsäuren in Spermatozoenlipide hervorgerufen werden. Ziel dieser Arbeit war die Untersuchung der metabolischen Aufnahme von Fettsäuren in die Spermatozoenlipide und die Bewertung des physiologischen Zustandes porciner Spermatozoen mit Hinblick auf die Niedrigtemperaturlagerung. Alle in den porcinen Spermatozoen vorkommenden Lipide wurden mittels GC und MALDI-TOF-MS analysiert. Hauptvertreter der polaren Lipidklassen sind Glycerophospholipide (GPC, GPE). Der Hauptvertreter der neutralen Lipidklassen ist Diacylglycerol (DAG). Die metabolische Aufnahme von Fettsäuren in die Lipide wurde durch die Supplementierung des Flüssigkonservierungsmediums mit [14C]-Octadecadiensäure radiochemisch untersucht. Anhand dieser Experimente wurde gezeigt, dass die Temperatur und die Inkubationsdauer wichtige Faktoren für die metabolische Aufnahme dieser Radiochemikalie in die Spermatozoenlipide sind. Die zugesetzten Fettsäuren werden sowohl in die neutralen (DAG) als auch in die polaren Lipide (diacyl-GPC) der Spermatozoen eingebaut. Nach Supplementierung mit 13C-markierter Octadecadiensäure wurden die Lipide mittels MALDI- und Q-TOF-MS als DAG (18:2/18:2), GPC (16:0/18:2) und GPC (18:2/18:2) charakterisiert. Die gleichen Ergebnisse wurden auch für die in den Spermatozoenlipiden vorkommenden Hexadecen-, Octadecen-, und Octadecatriensäure erhalten. Bei der Untersuchung des physiologischen Zustandes von Spermatozoen wurde gezeigt, dass insbesondere Supplementierungsvarianten mit endogen vorkommenden Fettsäuren zu einer besseren Spermatozoenvitalität und Motilität bei Niedrigtemperaturlagerung führten. Gleichzeitig wurde eine Verminderung des Auftretens von akrosomalen Schäden festgestellt. Damit stellt eine Supplementierung der Spermatozoen mit ausgewählten Fettsäuren eine effektive Maßnahme zur Lagerung von Spermatozoen bei 4 bis 6°C dar. / This study examines the metabolic incorporation of selected fatty acids into the lipids of porcine spermatozoa and evaluates the physiological state of spermatozoa subsequent to low temperature storage supplementation with selected free fatty acids. The aim was to understand the role of fatty acids in relation to the (cryo-)preservation of spermatozoa and successful reproduction in more detail. All lipids present in porcine spermatozoa were analysed using gas chromatography (GC) and mass spectrometry (MALDI-TOF-MS). The main representatives of the polar lipid classes are glycerophospholipids (in particular GPC and GPE). The main representatives of the neutral lipid classes are diacylglycerols (DAG). Metabolic incorporation of fatty acids into lipids was radiochemically monitored using [14C]-octadecadienoic acid in the supplied spermatozoa-preservation medium. Temperature and incubation time were shown to be particularly important determinants. The added fatty acids were incorporated into both the spermatozoas’ neutral (DAG) and polar lipids (diacyl-GPC). The affected lipids were characterised by means of MALDI- and Q-TOF-MS subsequent to the supplementation of uniformly 13C-labelled octadecadienoic acid. DAG (18:2/18:2), GPC (16:0/18:2) and GPC (18:2/18:2) could be identified and a de-novo biosynthesis of DAG (18:2/18:2) could be proven. The same results were obtained when spermatozoa were supplemented with hexadecenoic, octadecenoic and octadecatrienoic acids. Finally, it was shown that the physiological state of the spermatozoa, especially those supplemented with endogeneously present fatty acids, led to an enhanced vitality and motility in spermatozoa subsequent to low temperature storage. It was also observed that acrosomal damage was reduced and that hexadecenoic acid significantly stabilised all the vitality parameters. In conclusion, supplementing spermatozoa with selected fatty acids is an effective solution for the storage of spermatozoa at 4 to 6°C.

Techn. Hochsch., Diss., 2002--Aachen.

La thèse est consacrée à l'étude de la signalisation lipidique comme un mécanisme universel de médiation des réponses cellulaires à phytohormones et élicitors jouant ainsi un rôle clé dans la réorganisation de métabolisme cellulaire pendant l'adaptation de la plante aux changements environnementaux.Phospholipase D (PLD) et son produit acide phosphatidic (PA) ont étés impliqués au cascades de signalisation induites par l’acide salicylique (SA) dans les cellules de garde de Arabidopsis thaliana. On a trouvé une activation de PLD et la production de PA dans les feuilles des plantes après le traitement par SA. En utilisant le marquage radioactif des phospholipides, l'analyse histochimique, les inhibiteurs de la signalisation lipidique et des lignées transgénique des plantes, nous avons montré la participation de la PLD et la NADPH-oxidase RbohD à la formation du superoxyde dans les tissues d’Arabidopsis et à la fermeture des stomates induite par SA.La cooperation entre le SA et l’acide abscisic (ABA) dans la réorganisation de transcriptome induite par ces hormones a été examinée dans la culture de la suspension cellulaire. Tant SA que l'ABA ont inhibé l'activité basale in vivo de phospholipase C dépendante de phosphatidylinositol (PI-PLC), tandis que SA (mais pas ABA) a incité aussi le phosphorylation de phosphatidylinositols. Les transcriptomes de cellules après le traitement par SA ou ABA ont été comparé à ceux obtenus aprés le traitement avec U73122 ou wortmannin. Nous avons trouvé des groupes de gènes, pour qui l'effet d'ABA et des inhibiteurs était semblable; des gènes dependants du SA via l'équilibre des phosphoinositides et des gènes dependants du SA via l’activité de PLD. Basé sur l'analyse bioinformatique de toutes les groupes de gènes choisis, nous proposons le règlement du niveaux des phosphoinositides comme un facteur important dans la regulation du transcriptome basal et également dans les changements du profile transcriptomique induits par l'effet du SA ou d'ABA.L'effet du peptide bactérien flg22 sur l’équilibre des phospholipides a été détecté tant dans des cellules de suspension que dans des plantules. Flg22 a induit l'accumulation de PA par l'activation de PI-PLC couplée a la diacylglycerolkinase 5 (DGK5), et egalement la diminution de niveau de phosphatidylinositol-4,5-biphosphate, qui est un substrat de PI-PLC. L'analyse des effects des inhibiteurs a révélé la participation des DGK et PI-PLC dans la production des espèces d'oxygène réactive (ROS) induite par flg22. La production du PA a été placée dans la cascade de la signalisation en aval de la reconnaissance du flg22 par le complexe de récepteur FLS2-BAK1, mais aprés la formation du ROS par NADPH-oxydase RbohD. Le rôle de DGK5 a été caractérisé dans la regulation du transcriptome; dans l’accumulation du callose induite par flg22 dans l’apoplast et dans la résistance au pathogène biotrophique Pseudomonas syringae pv tomato DC3000. Finalement, nous avons proposé un nouveau modèle de perception du flagellin qui inclut PI-PLC et DGK5.Le rôle de phosphoinositides dans les cascades de la signalisation d’auxin et cytokinin a été révélé dans la morphogenesis racinaire dans le mutant d'Arabidopsis pi4kb1b2 (muté dans deux isoformes de PI4K) et pi4kb1b2sid2 (contient la mutation supplémentaire de l'enzyme de biosynthèse du SA, permettant de séparer les effets du mutation en PI4K qui dependent du SA). Nous avons analysé l'anatomie de la meristem des racines, l'allongement de cellules corticales, la réponse gravitropic, les réponses aux hormones exogènes et nous avons montré la connexion entre l'activité PI4K avec les effets d’auxin et de cytokinin pendant le morphogenesis racinaire et gravitropism. Nos résultats élargissent la connaissance de la nature de la signalisation phytohormonale dans les plantes et peuvent être utilisés comme une base pour augmenter la résistance de céréales agricolement importantes aux contraintes de l’environnement / Thesis is devoted to the investigation of lipid signaling processes as a universal mechanism mediating cellular responses to phytohormones and elicitors thus playing a key role in cell metabolism remodeling during plant adaptation to environmental changes. Phospholipase D (PLD) and its product phosphatidic acid (PA) were found to be involved to the SA-induced signaling cascades in Arabidopsis thaliana guard cells. Using radioactive labeling of phospholipids we found an activation of PLD and production of PA in leaves of 4-week old plants after salicylic acid (SA) treatment. Using histochemical assay, inhibitor assay and transgenic lines knock-out by different isoforms of NADPH-oxidases, we showed the involvement of PLD and NADPH-oxidase RbohD to PA-mediated superoxide formation in Arabidopsis tissues infiltrated by SA and SA-induced stomatal closure. SA- crosstalk with abscisic acid (ABA) in transcriptome remodeling induced by these hormones was investigated in suspension cell culture. Both SA and ABA inhibited basal activity of phosphatidylinositol dependent phospholipase C (PI-PLC) in vivo, while SA (but not ABA) also induced the phosphorylation of phosphatidylinositols. Total transcriptomes of suspension cells after SA or ABA treatment were compared to those obtained from suspension cells treated with U73122 (PI-PLC inhibitor) or wortmannin (inhibitor of phosphatidylinositol-4-kinases (PI4K) that provide the substrate for PI-PLC catalyzed reactions). We found a specific gene clusters, for those the effect of ABA and inhibitors was similar; SA-dependent genes, regulated via the balance of phosphoinositides, and SA-dependent genes, regulated via PLD-mediated pathway. Based on the bioinformatic analysis of the promoters of all selected gene sets, we claim a phosphoinositides level regulation to be an important factor mediating basal cell transcriptome and expression changes induced by SA and ABA.The effect of bacterial peptide flg22 on phospholipid turnover was detected in both suspension cells and seedlings. Flg22 induced accumulation of PA by the activation of PI-PLC coupled with diacylglycerolkinase (DGK) and a corresponding parallel increase of phosphatidylinositol-4,5-biphosphate content, that is a substrate of PI-PLC. Inhibitor analysis revealed the involvement of Ca2+ ions in lipid signaling enzymes reaction to flagellin treatment. We showed the role of DGK and PI-PLC in production of reactive oxygen species (ROS) induced by flg22. PA-production was placed in signaling cascade downstream of flagellin recognition by FLS2-BAK1 receptor complex receptor, but upstream or ROS formation by NADPH-oxidase RbohD. DGK5 was found to be the main source of the detected PA. The role of DGK5 was characterized in basal transcriptome regulation and its flagellin-induced remodeling; in flg22-induced callose accumulation in apoplast and resistance to biotrophic pathogen Pseudomonas syringae pv tomato DC3000. We proposed a new model of flagellin perception that includes PI-PLC and DGK5. Role of phosphoinositides in auxin and cytokinin signaling cascades was revealed studying root morphogenesis in Arabidopsis mutant pi4kb1b2 deficient for two PI4K genes, and pi4kb1b2sid2 that had additional mutation it key enzyme of SA biosynthesis, thus allowing us to separate SA-dependent and independent effects of the PI4K deficiency. pi4kb1b2 mutant plants exhibit the dwarf phenotype both in leaf and root parts, while pi4kb1b2sid2 show the normal rosette growth compared to WT, but still shorter roots. We analyzed root meristem anatomy, cortical cells elongation, gravitropic response, responses to exogenic hormones and firstly showed the connection of PI4K activity with auxin and cytokinin effects during root morphogenesis and gravitropism. Our results broaden the knowledge about the nature of plant phytohormonal signaling and can be used as a basis for increasing the resistance of agriculturally important crop plants to environmental stresses

Triacylglycerols (TAG) or storage oils in plants are utilized by humans for nutrition, production of biomaterials and fuels. Since nonseed tissues comprise the bulk biomass, it is pertinent to understand how to improve their TAG content. Typically, the final step in TAG biosynthesis is catalyzed by diacylglycerol (DAG) acyltransferases (DGAT) and/or phospholipid: diacylglycerol acyltransferases (PDAT), which also determine the content and composition of TAG. Besides enzymatic regulation of TAG synthesis, transcription factors such as WRINKLED1 (WRI1) play a critical role during fatty acid synthesis. In this study, mesocarp of Persea americana, with > 60% TAG by dry weight and oleic acid as the major constituent was used as a model system to explore TAG synthesis in nonseed tissues. Based on the transcriptome data of avocado, it was hypothesized that both DGAT and PDAT are likely to catalyze the conversion of DAG to TAG, and orthologs of WRI1 transcription factors regulate fatty acid biosynthesis. Here, with comprehensive in silico analyses, putative PamDGAT1 and 2 (Pam; Persea americana), PamPDAT1, and PamWRI1 and 2 were identified. When acyltransferases were expressed into TAG-deficient mutant yeast strain (H1246), only DGAT1 restored TAG synthesis capacity, with a preference for oleic acid. However, in planta, when transiently expressed in Nicotiana benthamiana leaves, PamDGAT1, PamPDAT1, PamWRI1, and PamWRI2 increased lipid contents, PamDGAT2 remained inactive. The data reveals that putative PamDGAT1, PamPDAT1 are functional and preferred acyltransferases in avocado and both PamWRI1 and 2 regulate fatty acid synthesis. In conclusion, while nonseed tissue of a basal angiosperm has certain distinct regulatory features, DAG to TAG conversion remains highly conserved.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Timothy P. Durrett / The membrane bound O-acyltransferase (MBOAT) family contains multi-pass membrane proteins that add fatty acids to different compounds. Despite their importance in economic activity and human health, little is known about the localization of the active site and regions important for determining substrate specificity of MBOATs. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) is the only known MBOAT enzyme that exhibits a high preference for acetyl-CoA, the shortest possible acyl-CoA. EaDAcT catalyzes the transfer of the acetate group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol. Our goal was to investigate the structural features and the amino acid residues that define substrate specificity of EaDAcT to provide insights into the mechanism by which MBOAT family controls substrate selection. By mapping the membrane topology of EaDAcT we obtained the first experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains with both the N- and C- termini oriented toward the endoplasmic reticulum lumen. The MBOAT signature region including the putative active site His-257 of the protein is embedded in the third transmembrane domain close to the interface between the membrane and the cytoplasm. In order to identify amino acid residues important for acetyltransferase activity, we isolated and characterized orthologs of EaDAcT from other acetyl-TAG producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved residues of DAcTs revealed that Ser-253, His-257 and Asp-258 are essential for enzyme activity of EaDAcT, suggesting their involvement in the enzyme catalysis. Alteration of residues unique to acetyltransferases did not alter the acyl donor specificity of EaDAcT, implying that multiple amino acids are important for substrate recognition. Together, this work identifies the structural features of EaDAcT and offers an initial view of the amino acids important for activity of the enzyme.