Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 273-295).

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 105-113). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 124-135).

In heart, Ca2+ plays an important role in maintenance of normal cardiac functions. Regulation of Ca2+ is mainly through L-type Ca2+ channel (LTCC), Ryanodine receptor (RyR) and sarcoplasmic reticulum calcium ATPase pump (SERCA) on sarcoplasmic reticulum (SR), Na+-Ca2+ exchanger (NCX), plasma membrane Ca2+ ATPase (PMCA). It has been well-accepted that PMCA plays a minor contribution to elevation of Ca2+ compared to SERCA and NCX and in regulation of cytosolic Ca2+ homeostasis. There are four isoforms of PMCA, PMCA1-4, and PMCA1 is a house-keeping gene, and abundantly distributed in heart. However, the role of PMCA1 in the murine heart has not been fully explored. With a cardiac specific knockout mouse model, the electrophysiological characteristics of PMCA1 in murine hearts, particularly in atria under normal physiological and stress conditions ([Ca2+]o overload and pacing conditions) are investigated. Firstly the complete deletion of PMCA1 in the atria in PMCA1cko mice was confirmed by Western blotting and immunostaining, also the membrane localisation of PMCA1 in the atria in PMCA1loxP/loxP mice was demonstrated. Then the phenotypes of ex vivo whole hearts between PMCA1loxP/loxP and PMCA1cko mice under physiological conditions and [Ca2+]o overload condition and with different frequencies by programmed electrical stimulation (PES) were explored. Further more, the Ca2+ handling process in single atrial myocytes between the PMCA1 deletion mice and control mice under normal physiological conditions and [Ca2+]o overload condition and stimulation with different frequencies was investigated. Finally the Ca2+ handling process in single ventricular myocytes between the PMCA1 deletion mice and control mice under normal physiological condition was investigated. At the whole heart level, the PMCA1cko hearts became more susceptible to arrhythmias with PES under physiological conditions compared with the PMCA1loxP/loxP hearts, and such arrhythmic events occurred more often and had longer pacing durations under Ca2+ overload conditions and higher frequency of pacing. At the single cellular level, the NCX current decay was significantly prolonged in PMCA1cko atrial myocytes under physiological conditions. This was further increased under Ca2+ overload conditions. With frequency-dependent stimulation, the PMCA1cko atrial myocytes showed few EAD- or DAD-type APs under physiological conditions in contrast to PMCA1loxP/loxP atrial myocytes that showed no arrhythmic events. The occurrence increased significantly under Ca2+ overload condition and/or at higher frequency of stimulation. Similar findings were observed in isolated ventricular myocytes. To conclude, the role of PMCA1 in maintaining Ca2+ homeostasis and electrical function in atrial myocytes under physiological conditions is minor. ii) PMCA1 has a critical role in maintaining Ca2+ homeostasis and electrical function in the atrium under stress conditions. This is particularly important during fast efflux of Ca2+ which is required under stress conditions.

L’objectif de cette thèse est de définir l’effet de l’environnement maternel sur la qualité des graines de A.thaliana, H.annuus, B.oleracea et H.vulgare, caractériser deux mutants d’A.thaliana affectés au niveau d’enzymes de la membrane plasmique et caractériser l’activité de ces deux protéines. Des dosages de O₂•⁻ et de radicaux ont été faits sur des graines issues de plantes ayant subi des stress thermique et/ou hydrique et n’ont pas montré d’effet des stress. Des traitements de vieillissement accéléré ont eu des effets négatifs sur la vigueur et la viabilité des graines. Des radicaux ont été identifiés par RPE haut champ au niveau de l’enveloppe et du péricarpe. Etant donné la localisation de ces radicaux, ils ne sont pas de bons marqueurs de la qualité des graines. Des mutants KO d’A.thaliana de quinones réductases (QR) et de AIR12 ont été caractérisés. Les QR sont des enzymes cytosoliques ayant une affinité pour la membrane. Elles catalysent la réduction de quinones en dihydroquinones et jouent un rôle protecteur contre le stress oxydatif. A pH alcalin par contre, les dihydroquinones se déprotonent et s’autooxydent entrainant la formation de semiquinones et de O₂•⁻. AIR12 est un cytochrome b 561 ancré à la membrane du côté de l’apoplaste réductible par l’ascorbate et le O₂•⁻. Ces protéines pourraient être impliquées dans un transfert d’électrons à travers la membrane via la vitamine K1. Les protéines recombinantes NQR et AIR12 ont été produites. A pH alcalin, AIR12 est réduit par les semiquinones et pourrait donc récupérer les électrons du produit de NQR. En présence de membrane plasmique, la production de O₂•⁻ est augmentée avec NQR et diminuée avec NQR et AIR12. Les QR semblent avoir un rôle anti- et pro-oxydant selon les cas et AIR12 un rôle anti-oxydant. Le transfert d’électrons entre les deux protéines pourrait se faire via les semiquinones à pH alcalin et via O₂•⁻. / The aim of this work is to define the effects of adverse environmental conditions on seed quality of A.thaliana, H.annuus, B.oleracea et H.vulgare), characterize two A.thaliana mutants affected in plasma membrane proteins and characterize the activity of the two proteins. Seeds were harvested from plants subjected to drought and/or thermal stress. Then O₂•⁻ and organic radicals in seeds were measured showing no impact of the stress. Accelerated ageing had a negative effect on seed vigor and viability. Radicals have been identified by high field EPR: epi-catechin and Mn(II) in the testa and melanin in the pericarp. There was no correlation between these radicals and the seed quality, therefore these radicals were not found to be good markers of seed quality. A.thaliana KO mutants of quinone reductase (QR) and AIR12 have been characterized. QR are cytosolic enzymes that have an affinity for the membrane. QR catalyze the reduction of quinone to dihydroquinone. Thus they are known to be protective enzymes against oxidative stress. However, at alkaline pH, dihydroquinone deprotonize and form semiquinones and O₂•⁻. AIR12 is a b561 cytochrome anchored to the apoplastic side of the membrane. These proteins could be implied in a transmembrane electron transport via vitamin K1. Recombinant proteins and NQR and AIR12 were produced. At alkaline pH, AIR 12 was reduced by the semiquinone, AIR12 could form a redox couple with vitamin K1 as electron shuttle across the membrane. In the presence of plasma membrane, the production O₂•⁻ was increased with NQR and reduced with NQR and AIR12. QR appear to have an anti- and pro-oxidant according to the conditions and AIR12 an anti-oxidant role. Electron transfer between the two proteins could be done via the semiquinone at alkaline pH and via O₂•⁻.

Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...

The plasma membrane Ca2+-pump (PMCA) is a Ca2+-Mg2+- ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+. Four genes (PMCAl to PMCA4) encode various isoforms ofthis pump. Caloxin is a novel peptide that was selected for binding to the second putative extracellular domain of PMCAl. Caloxin inhibits the Ca2+- Mg2+-ATPase in human erythrocyte leaky ghosts which primarily contain PMCA4. The objectives of this thesis are to delineate the mechanism of this inhibition and to determine its PMCA isoform selectivity. Caloxin inhibition of the PMCA pump in erythrocyte ghosts is non-competitive with respect to the substrates Ca2+ and ATP and the activator calmodulin. This was expected because the high affinity binding site for Ca2+ and sites for ATP and calmodulin are intracellular whereas caloxin is a peptide selected for binding to the second extracellular domain of the pump. In the reaction cycle, PMCA forms a 140 kDa acylphosphate enzyme intermediate from ATP (forward reaction) or orthophosphate (reverse reaction). Caloxin inhibits the acylphosphate formation in the forward but not in the reverse reaction. These results suggest that caloxin inhibits conformational changes required during the reaction cycle of the pump. In COS-M6 cells overexpressing PMCA4, caloxin inhibited the Ca2+ -Mg2+ATPase but with a marginally lower affinity than in the erythrocyte ghosts. Caloxin inhibition was also observed in insect cells overexpressing PMCA2. Several unsuccessful attempts were made to overexpress functional PMCAl. The work on isoform selectivity would require high level of expression of various PMCA isoforms and problems related to this requirement are discussed. Caloxin appears to inhibit the PMCA pumps by binding to the second putative extracellular domain and thus affecting the conformation of the protein. This is the first identified role of an extracellular domain of a PMCA pump. / Thesis / Master of Science (MSc)

Proteins of DREPP family (20-25 kDa, syn. PCaP1 in Arabidopsis thaliana) first appeared in ferns and we have shown that several independent duplications of DREPP protein occurred during evolution of large families (Poaceae, Brassicaceae, Solanaceae and Asteraceae) and in group Coniferophyta. Secondary losses of one paralogue occurred in subfamilies Pooideae and Solanoideae.We have also detected two large-scale modification of DREEP protein in Asparagales and Brassicaceae (this divergent paralogue was previously described as MAP18 protein). We have examined colinearity of chromosome fragments in vicinity both PCaP1 and MAP18 paralogues in Arabidopsis thaliana and we hypothesize that MAP18 gene arose during genome duplication on the origin of Brassicaceae family. DREPP protein was previously identified in detergent-resistant membrane microdomain fraction and a myristyl anchor was shown to be necessary for their membrane localization. Membrane association was shown to be modified by the interaction of unique N-terminal domain with PtdInsPs, which was inhibited by binding of Ca-calmodulin (Nagasaki et al., 2008). The mutation of Gly2 by Ala in the myristilation site, or C-terminal GFP-fusion (GFP-DREPP), affect membrane association in Arabidopsis thaliana (Nagasaki et al., 2008). Several DREPP paralogues in...

Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration. Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo. The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA. The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface. The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed. In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.