The polarization of exocytosis in yeast and animals is assisted by the exocyst - an octameric vesicle tethering complex and an effector of Rab and Rho GTPases. Recently, the exocyst was described as a functional complex involved in morphogenesis also in plants. Hála et al. (2008) described involvement of exocyst complex in pollen tube growth and hypocotyls elongation in dark grown seedlings, Fendrych et al. (2010) uncovered key role of exocyst in cell plate formation, Kulich et al. (2010) emphasized the participation of exocyst in seed coat generation and Pečenková et al. (2011) described the contribution of exocyst subunits in plant defense towards the pathogens. All these processes are intimately linked to polarized secretion. Here we show involvement of exocyst in auxin efflux carriers PINs recycling. Using direct auxin transport measurement and GFP-tagged proteins, we showed that the exocyst is involved in recycling and polarization of PIN proteins and polar auxin transport regulation. Rootward polar auxin transport is compromised in loss-of-function mutants in exocyst subunits EXO70A1. On the cellular level we have detected small portion of PIN2:GFP in the "BFA-like" FM4-64 labelled compartments distinct from VHAa1 labeled endosoms. Moreover recycling of PIN1 and PIN2 is retarded in roots of...

Le mode de coordination parmi les différentes molécules qui régulent la migration reste très peu connu. Ce travail traite de deux voies de transduction régulant la migration: la voie Rac1/WRC (Wave Regulatory Complex) qui contrôle la formation du réseau d’actine au front des cellules migrantes, et la voie RalB/exocyst, dont les mécanismes moléculaires de son implication dans la motilité cellulaire étaient inconnus au début de cette thèse. Rac1 et RalB sont des petites protéines G des familles Rho et Ras, respectivement. Les complexes WRC et exocyst sont leurs effecteurs directs.Au cours de la recherche de connexions entre l’exocyst et des régulateurs de la migration, nous avons trouvé que deux sous-unités de l’exocyst, Exo70 et Sec6, interagissent directement in vitro avec Abi et Cyfip, respectivement, deux sous unités du WRC. De plus, nous avons trouvé que les sous-unités de l’exocyst peuvent interagir in vitro avec le WRC entier. Nous avons également montré que ces deux complexes s’associent in vivo. Sur le plan fonctionnel, l’exocyst est requis pour le positionnement du complexe WRC au front des cellules migrantes. D’autre part, nous avons également trouvé que deux autres sous- unités de l’exocyst Sec8 et Exo84, interagissent avec SH3BP1 (une RhoGAP) en double hybride et en co-immunoprécipitation. SH3BP1 se localise au front des cellules migrantes, et cette localisation dépend de l’exocyst. De façon intéressante, in vivo, la voie RalB/exocyst/SH3BP1 cible spécifiquement Rac1, et non Cdc42. Grâce à plusieurs approches, nous concluons que SH3BP1 est requis pour inactiver Rac1 au front. Dans notre modèle nous proposons que RalB/exocyst règulerait la migration cellulaire en véhiculant au front de migration deux éléments majeurs de la signalisation de Rac1 : son complexe effecteur WRC, qui stimule la nucléation de filaments d’actine et son régulateur négatif SH3BP1, une GAP qui promeut l’inactivation et le cycle GDP/GTP de Rac1. En conclusion, ce travail fournit de nouvelles connexions moléculaires et fonctionnelles entre l’exocytose polarisée et la dynamique de l’actine au cours de la motilité cellulaire. / Very little is known about the coordination and the integration among the different regulators of the motility process. This work deals with two migration-regulatory pathways: the Rac1/WRC (Wave Regulatory Complex) pathway that drives the formation of the actin polymerization network at the front of motile cells; and RalB/exocyst pathway for which the molecular mechanisms underlying its implication in cell motility were still largely unknown at the beginning of this thesis. Rac1 and RalB are small GTPases of the Rho and Ras family, respectively. WRC and exocyst complexes are their direct effectors.In searching for connections between the exocyst and migration regulators, we found that two subunits of the exocyst, Exo70 and Sec6, interact directly in vitro with two subunits of the WRC, Abi and Cyfip, respectively. Moreover, we found that exocyst subunits can interact in vitro with the whole fully-assembled WRC complex. We also showed that these two complexes associate in vivo. Functionally, the exocyst was required for WRC complex positioning at the front of migrating cells.On the other hand, we also found that two other subunits of the exocyst, Sec8 and Exo84, interact with SH3BP1 (a RhoGAP protein) by two-hybrid assay and by co-immunoprecipitation. SH3BP1 localizes at the leading edge and this localization is dependent on the exocyst. Interestingly, in vivo, the RalB/exocyst/SH3BP1 pathway specifically targets Rac1, and not Cdc42. By a combination of approaches we concluded that SH3BP1 is required to inactivate Rac1 at the front.In our model we propose that RalB/exocyst regulates cell migration by driving to the leading edge two key signaling elements of the Rac1 pathway: its effector WRC, that stimulates actin filament nucleation, and its negative regulator SH3BP1, a GAP promoting Rac1 inactivation and GDP/GTP cycling. In conclusion, this work provides novel molecular and functional links between polarized exocytosis and actin dynamics during cell motility.

Stigmas of Arabidopsis thaliana carrying the exo70A1-1 mutation are defective in early pollen-pistil interactions; pollen grain adhesion to the stigma, pollen hydration, and penetration of the stigmatic surface by the pollen tube. Exo70 function in directed secretion has been linked to its ability to bind the phosphatidylinositides. To provide support that the classical, octomeric exocyst complex, which contains the Exo70 subunit, participates in compatible pollen-pistil interactions, this process was analyzed in plants deficient in Sec15, another subunit of the exocyst. Additionally, the role of phosphatidylinositol-4-phosphate (PI-4-P) in female fertility was evaluated through the use of the mutants ROOT HAIR DEFECTIVE 4-1 and PI4Kβ1β2 -/-, which have increased and decreased levels of PI-4-P respectively. Reduction of Sec15b levels or perturbation of PI-4-P in the stigma resulted in a reduced ability of of the transgenic/mutant stigmas to support pollen grain hydration; though all other stages of early pollen pistil interactions were unaffected.

Stigmas of Arabidopsis thaliana carrying the exo70A1-1 mutation are defective in early pollen-pistil interactions; pollen grain adhesion to the stigma, pollen hydration, and penetration of the stigmatic surface by the pollen tube. Exo70 function in directed secretion has been linked to its ability to bind the phosphatidylinositides. To provide support that the classical, octomeric exocyst complex, which contains the Exo70 subunit, participates in compatible pollen-pistil interactions, this process was analyzed in plants deficient in Sec15, another subunit of the exocyst. Additionally, the role of phosphatidylinositol-4-phosphate (PI-4-P) in female fertility was evaluated through the use of the mutants ROOT HAIR DEFECTIVE 4-1 and PI4Kβ1β2 -/-, which have increased and decreased levels of PI-4-P respectively. Reduction of Sec15b levels or perturbation of PI-4-P in the stigma resulted in a reduced ability of of the transgenic/mutant stigmas to support pollen grain hydration; though all other stages of early pollen pistil interactions were unaffected.

Thesis supervisor: Charles A. Yeaman. Includes bibliographic references (p. 128-139).

Complex Exocyst consists of eight proteins and it is known as a Sec6/8. Its composition is evolutionarily highly conserved amongst all the species. This complex is involved in vesicle trafficking as a part of attaching mechanism to a specific place on the plasma membrane. EXO70 subunit has been found in 23 copies in Arabidopsis thaliana genome. In this study we have been examine paraloges EXO70H7 and EXO70H8. There have been suggestions that these genes are important in development of roots according to the previous studies. We have not been able to identify any significant phenotype within the mutant plants in these genes. There has been studied other mutant appearance during the stress experiments. Most of these experiments did not identify any divergence. Only experiments with germination during stress conditions revealed significantly worse germination of exo70H7 mutant seeds on the medium containing sorbitol. This suggests that mutant seeds have a worse protection against osmotic stress. Significantly worse germination of exo70H8 seeds on the medium with excess NaCl indicates that these seeds incriminate to higher absorption of sodium ions. Analysis of the cell localization of GFP constructs brought knowledge of appearance EXO70H7 and EXO70H8 proteins. EXO70H7 locates in the cytoplasm and in...

Organization of endomembrane compartments in all eukaryotic cells is dependent on continuous transport of membrane vesicles. Major part of the core regulators of intracellular membrane transport is represented by small GTPases from the Rab family. Rab GTPases cycle between the GTP-bound "active" and GDP-bound "inactive" forms. In their active form, they are able to interact with specific effectors and perform their functions. Exocyst is an octameric complex involved in regulation of secretion. It functions as an effector of Rab GTPases in yeast and mammals and tethers secretory vesicles to the plasma membrane prior to the actual membrane fusion. Using publicly available expression data, we have identified candidates from Rab GTPase family for the interaction with exocyst subunit AtSEC15b in plants and demonstrated that AtSEC15b specifically interacts with AtRABA4a GTPase. We also showed that, like in yeast and mammals, Arabidopsis Sec15b binds Rab GTPase also probably in GTP-dependent manner, implying that this interaction is well conserved throughout the eukaryotic kingdoms. We also successfully demonstrated the complementation of yeast thermo-sensitive mutant strain, sec15-1. Based on this observation we concluded that AtSEC15b is able to substitute the function of yeast SEC15 and restore the phenotype....

Many studies in different eukaryotes have shown the importance of the vesicle-tethering exocyst complex for cellular processes dependent on intensive polarized secretion. The plant exocyst complex is crucial for regulation of cell polarity, morphogenesis, and defence. In land plants, gene encoding the EXO70 exocyst subunit multiplied into many paralogs, but only a few of them have been functionally described. In this thesis, the EXO70A2 isoform, a member of the EXO70.1 subfamily, was found to be the main EXO70 exocyst subunit involved in the canonical function of the exocyst complex in Arabidopsis pollen. EXO70A2 is important for several stages of pollen development-pollen grain maturation, germination, and pollen tube growth. Pollen-expressed EXO70A2 was the only EXO70 isoform able to substitute for the function of EXO70A1 in the sporophyte, but not vice-versa. This indicates partial functional redundancy of these two closely related isoforms and a high specificity for pollen-related processes. The finding that the exocyst is targeted to the plasma membrane via EXO70A1 subunit is further elaborated in the thesis. EXO70A1 binds plasma membrane via interactions with specific phospholipids that form a unique plasma membrane-lipid signature in plants. Other isoform, EXO70B1 from the EXO70.2 subfamily,...

Arabidopsis thaliana trichomes are large unicellular epidermal outgrowths with a specific development and intriguing shape, which makes them an excellent cell type for our research of cell polarization mecha- nisms. Cell polarity is essential for plant development and the exocyst complex is one of its key regulators. It is an octameric protein complex that mediates polarized exocytosis and growth by targeted tethering of secretory vesicles to the plasma membrane. Its EXO70 subunit functions as a landmark for exocytosis site and physically binds the target membrane through interaction with phospholipids. A remarkable multipli- cation of EXO70 subunit paralogs in land plant genomes is well documented, but the functional diversity of these paralogs remains to be described. In trichomes we revealed the specific role of the EXO70H4 paralog in secondary cell wall deposi- tion, especially in callose synthase delivery. We documented formation of a thick secondary cell wall during the maturation phase of wild type trichome development and a lack of it in the exo70H4 mutant. Moreover, we showed evidence for silica deposition dependency on callose synthesis. Further, we unveiled the formation of apical and basal plasma membrane domains, which differ in their phospholipid compo- sition and ability to bind...

Cell polarity is the asymmetric distribution of organelles that almost all cells use to separate individual processes and perform complex functions. Although the manner in which cells are polarized is very diverse, the processes necessary to assume polarized phenotypes are similar in many cell types. Epithelial cell polarization is of particular importance, as these cells serve form linings of organs and act as barriers distinguishing different compartments. Furthermore, loss of epithelial polarization occurs in some disease states and may result in cell invasion through underlying matrix. During initial polarization, vesicle trafficking is indispensible for assembly of structures, including apical junctional complex formation. Trafficking of new membrane and associated proteins to leading edges is also necessary for cell migration. RalA and RalB are members of the Ras superfamily of GTPases and have been implicated in several processes, including vesicle trafficking. Only 5 Ral effectors have been identified, two of which are members of the Exocyst complex, a hetero-octameric complex also involved with vesicle trafficking. I hypothesized that Ral GTPases were necessary for several aspects of cell polarization, and that they engage the Exocyst complex to mediate these processes. Initial investigation of tight junction assembly found that both RalA and RalB antagonistically affect paracellular permeability. Knockdown of RalA and RalB resulted in decreased and increased incorporation of components into assembling tight junctions, respectively. Furthermore, both RalA and RalB engaged the Exocyst in order to mediate tight junction assembly. I next examined the role of RalA-Sec5 and RalA-Exo84 interactions during tumor cell migration and invasion. Both interactions were necessary for invasion and single cell migration, although disruption of each interaction affected different aspects of migration. Furthermore, significant differences in cytoskeleton organization occurred in response to disruption of RalA-Sec5 and RalA-Exo84 interactions. Finally, I investigated the effects of RalA and RalB knockdown on growth of primary cilia and cyst formation. RalA decreased primary cilia growth and reduced average cilia length, while RalB increased cilia length. Knockdown of RalA and RalB also affected lumen formation during cystogenesis, as RalA knockdown prevented lumen formation and RalB knockdown caused formation of multiple lumens. Taken together, data presented here show that Ral engages the Exocyst to mediate distinct processes during tight junction assembly and cell migration, and implicates Ral GTPases in several different aspects of cell polarity.