Dissertations / Theses on the topic 'Exocysts'

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and to the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multi-subunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in other trafficking pathways. Contrary to these other pathways, our lab previously suggested that the exocyst subunit Sec6, a component of the exocytosis-specific tethering complex, inhibited Sec9:Sso1 SNARE complex assembly due to interactions in vitro with the SNARE protein Sec9 (Sivaram et al., 2005). My goal for this project was to test the hypothesis that Sec6 inhibited SNARE complex assembly in vivo. I therefore chose to generate Sec6:Sec9 loss-of-binding mutants, and study their effect both in vitro and in vivo. I identified a patch of residues on Sec9 that, when mutated, are sufficient to disrupt the novel Sec6-SNARE interaction. Additionally, I found that the previous inhibitory role for Sec6 in SNARE assembly was due to a data mis-interpretation; my re-interpretation of the data shows that Sec6 has a mild, if any, inhibitory effect on SNARE assembly. My results suggest a potential positive role for Sec6 in SNARE complex assembly, similar to the role observed for other tether-SNARE interactions.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and to the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multi-subunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in other trafficking pathways. Contrary to these other pathways, our lab previously suggested that the exocyst subunit Sec6, a component of the exocytosis-specific tethering complex, inhibited Sec9:Sso1 SNARE complex assembly due to interactions in vitro with the SNARE protein Sec9 (Sivaram et al., 2005). My goal for this project was to test the hypothesis that Sec6 inhibited SNARE complex assembly in vivo. I therefore chose to generate Sec6:Sec9 loss-of-binding mutants, and study their effect both in vitro and in vivo. I identified a patch of residues on Sec9 that, when mutated, are sufficient to disrupt the novel Sec6-SNARE interaction. Additionally, I found that the previous inhibitory role for Sec6 in SNARE assembly was due to a data mis-interpretation; my re-interpretation of the data shows that Sec6 has a mild, if any, inhibitory effect on SNARE assembly. My results suggest a potential positive role for Sec6 in SNARE complex assembly, similar to the role observed for other tether-SNARE interactions.

The Exocyst is a hetero-octameric complex involved in tethering of post-Golgi vesicle transport to sites of membrane expansion. In budding yeast, the Exocyst targets vesicles to bud site resulting in bud emergence and abscission of the daughter cell. Mammalian Exocyst is recruited to developing lateral membranes after cadherin mediated adhesion and then is segregated to adherens junctional complexes (AJC). In polarized epithelia, the Exocyst is required for basal-lateral transport of LDL receptor. Additional Exocyst subunit localizations and functions have been identified. It is not known whether these supplementary roles can be attributed to the Exocyst or other unidentified Exocyst subcomplexes. Sec3, an Exocyst subunit, is hypothesized to be a landmark of polarization in yeast. In polarized epithelia, GFP tagged Sec3 remained cytosolic in polarized epithelia unlike Sec6/8. Sec3-GFP was recruited to lateral membranes only after dual over expression of heterologous GLYT1. Little is known about endogenous mammalian Sec3. Our work suggests Sec3 defines an Exocyst subcomplex that is required for desmosome integrity. Sec3 and additional subunits (Sec6, Sec8, Sec15, Exo70, and Exo84) were present at desmosomes, but Sec3 failed to localize to AJC. Only antibodies to Sec6 and Sec8 labeled AJC. Reduction of Sec3 protein expression resulted in the impairment of desmosome morphology and function with no detrimental effect on adherens junctions. These data suggest the existence of functionally different Exocyst subcomplexes. Sec3-exocyst recruited minus-end directed microtubule motor KifC3 to desmosomes. KifC3 was previously shown to be recruited with a microtubule anchoring complex to basal-lateral membrane. This suggests Sec3 may recruit KifC3 to organize microtubules at desmosomes. This would establish a pathway to efficiently transport newly synthesized basal-lateral cargo. These results suggest a novel mechanism of the Exocyst to regulate post-Golgi vesicular transport and intercellular adhesion.

La dissémination des cellules cancéreuses et la formation de métastases sont des étapes cruciales dans la progression tumorale et constituent une cause majeure des décès dus au cancer. La métalloprotéase transmembranaire MT1-MMP est un acteur clé impliqué dans le franchissement des barrières tissulaires et le remodelage de la matrice extracellulaire (ECM) par les cellules cancéreuses. MT1-MMP est présente dans des vésicules intracellulaires, appelées endosomes, via lesquels elle est adressée à la membrane plasmique (PM) afin d'y dégrader la ECM. Des travaux menés au laboratoire ont identifié le complexe exocyste (CE) comme un acteur important pour la formation d'invadopodes dans la lignée d'adénocarcinome mammaire MDA-MB-231. Ce complexe multiprotéique (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 et Exo84) est impliqué dans l'arrimage des vésicules intracellulaires à la PM. Des cribles double-hybride ont identifiés la protéine WASH comme partenaire potentiel du CE (via les sous-unités Exo84 et Sec3). WASH est capable d'induire la polymérisation de l'actine en activant le complexe Arp2/3. In vitro, nous avons montré que les complexes WASH et exocyste interagissent physiquement et coordonnent le trafic intracellulaire et l'adressage de MT1-MMP à la PM. Ces résultats mettent en évidence une étroite collaboration entre le cytosquelette d'actine et les mécanismes d'exocytose lors des étapes précoces de dégradation de la ECM ainsi que dans l'invasion tumorale
Cancer cell invasion is a prerequisite to tumor progression and metastasis. In order to disseminate, tumor cells must degrade and remodel the extracellular matrix (ECM) in a process that requires the trans-membrane matrix metalloproteinase MT1-MMP, which is a key component of the ECM remodeling apparatus of cancer cells. MT1-MMP overexpression in cancers is associated with increased invasion and metastasis. Many cellular proteins are involved in the transport and delivery of MT1-MMP-containing vesicles to the PM. Previous work from the laboratory identified the exocyst complex (EC) as a key component required for matrix proteolysis and invasion of cancer cells. This multiprotein complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) plays essential roles in docking secretory vesicles at the PM for exocytosis. To better characterize this complex, a yeast two-hybrid screen was performed, identifying the protein WASH as a potential partner of Exo84 and Sec3. WASH is a Nucleation Promoting Factor (NPF) able to activate the actin nucleating Arp2/3 complex. Results of the present study showed that WASH and the exocyst complexes interact and localize on MT1-MMP-positive endosomes in MDA-MB-231 breast cancer cells. This study highlight a direct implication of WASH and exocyst complex in ECM degradation by cancer cells through the docking and exocytosis of MT1-MMP-containing endosomes at the PM through connections between these compartments and the extracellular medium. This WASH- and exocyst-dependent MT1-MMP exocytosis mechanism is required for degradation of adjacent tissue by cancer cells during tumour cell invasion

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

In polarized epithelial cells, vectorial protein trafficking is important for transporting specific membrane proteins to generate distinct apical and basolateral membrane protein compositions. The Exocyst is a conserved hetero-octameric protein complex, which regulates different aspects of protein trafficking, including tethering of the Golgi-derived vesicles to target membranes. Two of the Exocyst subunits, Sec5 and Exo84, competitively bind to the small GTPases, RalA and RalB, in a GTP-dependent manner. Although Ral GTPases have been proposed to mediate assembly of Exocyst holocomplexes, we hypothesize that they actually serve to allosterically regulate Exocyst functions by promoting association or disassociation of additional factors. Previous studies have shown that active RalA, but not RalB, accelerated basolateral exocytosis of E-cadherin. In contrast, knockdown of RalB, but not RalA, disrupts endocytosis of E-cadherin. However, mechanisms by which association of Ral GTPases with Sec5 and Exo84 regulate basolateral protein trafficking remain unclear. Here we investigate roles of Ral GTPases and the Exocyst in regulating basolateral protein trafficking using Madin Darby canine kidney (MDCK) cells and RNA interference (RNAi) technology. We show that RalA, but not RalB, is required for basolateral exocytosis of vesicular stomatitis virus glycoprotein (VSV-G) in the MDCK cells. We combined immunofluorescent labeling and surface biotinylation assays to demonstrate that RalA regulates VSV-G trafficking through the distinct interactions with Sec5 and Exo84. We also show that a Ral-uncoupled Sec5 mutant, but not a Ral-uncoupled Exo84 mutant, inhibits E-cadherin exocytosis. These results suggested that RalA and the Exocyst are required for basolateral exocytosis, and that RalA-Sec5 and RalA-Exo84 interactions play different roles during this process. Our study may provide new insights into mechanisms regulating protein trafficking in epithelial cells, and potentially lead to development of new therapeutic targets for the treatment of diseases in which exocytosis is impaired, such as Polycystic kidney disease and diabetes.

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicles are targeted to sites of exocytosis on the plasma membrane in part by a conserved multi-subunit protein complex termed the exocyst. In addition to tethering vesicles to the plasma membrane, the exocyst complex and components therein may also add a layer of regulation by directly controlling assembly of the SNARE complex, which is required for membrane fusion, as well as other regulatory factors such as Sec1p. In the past, we have shown that Sec6p interacts with Sec9p in vivo and that that interaction retards binary SNARE complex formation in a SNARE assembly assay. Though many interactions have been mapped using in vitro methods, confirming them in vivoand placing them into the context of a complete model that accounts for all observed interactions (and lack of interactions) has proven difficult. In order to address these problems, I have studied the interactions between Sec6p and other factors involved in exocytosis at the plasma membrane via in vivo methods. My hypothesis was that Sec6p interaction with Sec9p and subsequent inhibition of SNARE complex assembly in vitro was an intermediate state and Sec6p was part of a set of cofactors that accelerated SNARE complex assembly in vivo. To test this hypothesis I showed that the interaction between the plasma membrane t-SNARE Sec9p and the yeast exocyst subunit Sec6p can be observed in vivoand designed point mutations to disrupt that interaction. Interestingly, I also showed that Sec6p:Sec9p interaction involves the free pool of Sec6p rather than the exocyst bound fraction of Sec6p. Point mutations in the N-terminal domain of Sec6p result in temperature sensitive growth and secretion defects, without loss of Sec6p-Sec9p interaction. However, at the non-permissive temperature, the exocyst subunits Sec5p, Sec10p and Sec15p are mislocalized and are absent from the exocyst complex. The resulting subcomplex, containing Sec3p, Sec8p, Exo70p and Exo84p, remains stably assembled and localized at sites of polarized secretion. This subcomplex is likely due to disruption of interaction between Sec6p and Sec5p, and may be similar to that observed at restrictive temperatures in the sec6-54temperature sensitive mutant. Additionally, one of the sec6 temperature sensitive mutants displays a loss of binding to the yeast regulatory protein Sec1p. In vitro binding studies indicate a direct interaction between Sec1p and the free pool of the wild-type Sec6p protein, suggesting close interplay between Sec6p and Sec1p in the regulation of SNARE complexes. A coherent model which incorporates all these interactions has continued to be elusive. However, the results I have found do suggest several hypotheses which should prove testable in the future.

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.

HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunnelling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. In this dissertation, we developed and characterized a lentiviral vector-based system to express Nef in T-cell lines and primary human peripheral blood mononuclear cells, and then used this system to perform a proteomic screen to identify Nef-associated host cell factors and better understand how Nef hijacks the T-cell machinery to maximize HIV production and dissemination. Bioinformatic and cell-based analysis of the resulting host factors revealed a mechanism by which Nef enhances nanotube formation. To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). Wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Together, our findings identify the exocyst complex as a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Furthermore, linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.

The exocyst is an evolutionarily conserved, hetero-octameric protein complex proposed to serve as a multi-subunit tethering complex for exocytosis, although it remains poorly understood at the molecular level. The classification of the exocyst as a multisubunit tethering complex (MTC) stems from its known interacting partners, polarized localization at the plasma membrane, and structural homology to other putative MTCs. The presence of 8 subunits begs the questions: why are so many subunits required for vesicle tethering and what are the contributions of each of these subunits to the overall structure of the complex? Additionally, are subunit or subcomplex dynamics a required feature of exocyst function? We purified endogenous exocyst complexes from Saccharomyces cerevisiae, and showed that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. This conclusion contrasts starkly with current models suggesting that the yeast exocyst tethers vesicles by transient assembly of subcomplexes at sites of exocytosis. Using a combination of biochemical and auxininduced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as an octameric complex in yeast with a stably assembled, elongated structure. This is the first complete structure of a CATCHR family MTC and it differs greatly from the EM structures available for the partial COG and Dsl1 complexes. Future work will be necessary to determine whether exocyst conformational changes are a required feature of vesicle tethering and how such changes are regulated. These architectural insights are now informing the design of the first in vitro functional assay for the exocyst complex. We developed methodology for attaching fluorescently-labeled exocyst complexes to glass slides and monitoring the capture of purified, endogenous secretory vesicles by single molecule TIRF microscopy. By this approach, we can monitor tethering events in real time and determine the required factors and kinetics of exocytic vesicle tethering.

The exocyst is an evolutionarily conserved, hetero-octameric protein complex proposed to serve as a multi-subunit tethering complex for exocytosis, although it remains poorly understood at the molecular level. The classification of the exocyst as a multisubunit tethering complex (MTC) stems from its known interacting partners, polarized localization at the plasma membrane, and structural homology to other putative MTCs. The presence of 8 subunits begs the questions: why are so many subunits required for vesicle tethering and what are the contributions of each of these subunits to the overall structure of the complex? Additionally, are subunit or subcomplex dynamics a required feature of exocyst function? We purified endogenous exocyst complexes from Saccharomyces cerevisiae, and showed that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. This conclusion contrasts starkly with current models suggesting that the yeast exocyst tethers vesicles by transient assembly of subcomplexes at sites of exocytosis. Using a combination of biochemical and auxininduced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as an octameric complex in yeast with a stably assembled, elongated structure. This is the first complete structure of a CATCHR family MTC and it differs greatly from the EM structures available for the partial COG and Dsl1 complexes. Future work will be necessary to determine whether exocyst conformational changes are a required feature of vesicle tethering and how such changes are regulated. These architectural insights are now informing the design of the first in vitro functional assay for the exocyst complex. We developed methodology for attaching fluorescently-labeled exocyst complexes to glass slides and monitoring the capture of purified, endogenous secretory vesicles by single molecule TIRF microscopy. By this approach, we can monitor tethering events in real time and determine the required factors and kinetics of exocytic vesicle tethering.

L'acquisition d'une polarité cellulaire est essentielle à la définition de domaines basolatéraux et apicaux fonctionnellement distincts au sein des cellules épithéliales, ainsi qu'à la localisation polarisée des déterminants cellulaires au cours des divisions asymétriques. Le but de mon travail de thèse était l'identification de nouveaux intervenants et de régulateurs des complexes déjà impliqués dans les processus de maintien de la polarité apico-basale des cellules épithéliales et, dans l'établissement de la polarité planaire lors de la division asymétrique de la cellule pi, précurseur des organes sensoriels externes, sur le thorax dorsal de la drosophile. Mon étude de la division asymétrique de la cellule pi a permis de mettre en évidence l'importance de la protéine Lethal giant larvae (Lgl). Lgl régule le destin cellulaire en contrôlant la localisation corticale de Pon, la localisation asymétrique des déterminants cellulaires Numb et Neuralized et la localisation à la membrane plasmique de Sanpodo. De plus, mes résultats montrent que la fonction de Lgl est inhibée par phosphorylation par DaPKC. Le complexe E-Cadhérine-Caténines joue un rôle crucial dans l'adhésion cellulaire, la polarisation et la morphogenèse. J'ai étudié l'implication de l'exocyste dans le mécanisme de localisation de la DE-Cadhérine dans les cellules épithéliales du thorax dorsal de la drosophile. La perte de fonction des composants de l'exocyste, sec5, sec6 ou sec15, entraîne une accumulation de DE-Cadhérine dans des compartiments de recyclage marqués par Rab11 et inhibe l'adressage de la DE-Cadhérine à la membrane. Ces résultats permettent de proposer un modèle dans lequel l'exocyste régule le trafic de la DE-Cadhérine depuis les endosomes de recyclage jusqu'à la membrane cellulaire. Cette étude constitue la première description de la fonction de l'exocyste dans la polarisation des cellules épithéliales chez la drosophile
Cell polarity is essential to define the apical and the basolateral domains of epithelial cells and is necessary to polarise the localisation of cell fate determinants during asymmetric divisions. The aim of my thesis was to identify new partners and regulators of protein complexes which had been implicated in apico-basal polarity and in the establishment of planar polarity during the asymmetric division of the pi cell, the external sensorial organs precusrsors, on the dorsal thorax of Drosophila melanogaster. My study of the pi cell asymmetric division highlights the importance of the protein Lethal giant larvae (Lgl). Lgl regulates cellular fate by controlling the cortical localisation of Pon, the asymmetric localisation of the cell fate determinants Numb and Neuralized and the membrane localisation of Sanpodo. Moreover, my results show that Lgl function is inhibited by DaPKC phosphorylation. The E-Cadherin-Catenins complex is essential in cell adhesion, polarisation and morphogenesis. I studied the implication of the exocyst complex in the mechanism of DE- Cadherin localisation in epithelial cells of the drosophila dorsal thorax. The loss of function of the exocyst components sec5, sec6 or sed5 led to an accumulation of DE-Cadherin in recycling compartiments regulated by Rab11 and inhibited DE-Cadherin transport to the plasma membrane. This result led me to propose a model in which the exocyst complex regulates DE-Cadherin trafficking from recycling endosomes to plasma membrane. This study is the first description of exocyst function in Drosophila epithelial cell polarity

Host colonization is mediated through the secretion of effector proteins in order to neutralize host immune responses. However, the mechanism of the effector delivery during biotrophic invasion is not well defined in M. oryzae. In this thesis, I define the role of the exocyst complex, an evolutionarily conserved octameric protein complex involved in vesicle docking to the plasma membrane (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84), during infection-related development in M. oryzae. Like other filamentous fungi, M. oryzae, exocyst components localize to the vegetative hyphal tip distinct from the Spitzenkörper. However, at the initial stage of infection-related development all the exocyst components localise as a ring at the cortex of the appressorium and re-assembles around the appressorium pore in an actin-dependent manner in mature appressoria. I report that the septin network is required for the transition of exocyst ring from periphery to the appressorium pore. Deletion of Exo70 and Sec5 showed significant reduction in protein secretion and plant infection. I show that Sec6 is required for the exocyst assembly around the appressorium pore and effector secretion from the appressorium. I report that, during biotrophic invasion, effectors are secreted through a distinct pathway. Apoplastic effectors, Bas4 and Slp1 are secreted via a Golgi-dependent pathway while secretion of cytoplasmic effectors, Pwl2 and Bas1 meditates through a Golgi-independent pathway in which exocyst components Exo70 and Sec5 are involved.

Cell growth and division requires delivery of new membrane and remodelling factors to the cell surface. In budding yeast, this involves actin-dependent transport of secretory vesicles to sites of growth, followed by vesicle fusion to the plasma membrane. Rho-type GTPases regulate actin polarization and vesicle fusion, but how they signal to diverse effectors controlling these separate processes is not well understood. Here, we show that the cortical proteins Boi1 and Boi2 work together with Rho GTPase signalling to regulate the exocyst, a complex that mediates the tethering of secretory vesicles to the plasma membrane. Cells lacking both Boi proteins show normal actin polarity but are defective in bud emergence, bud growth, and fusion of Bgl2-containing vesicles to the plasma membrane. A gain-of-function version of Exo70, a component of the exocyst and effector of Cdc42 and Rho3, restores growth of boi1 boi2 mutant cells. Furthermore, hyperactivation of Rho-GTPase signalling rescues boi defects, suggesting that the essential function of Boi proteins is to mediate Rho-dependent activation of the exocyst during cell growth. Finally, we find that Boi1 and Boi2-dependent inhibition of abscission in cells with chromatin bridges, via their function in the NoCut checkpoint, is bypassed by expression of gain-of-function EXO70, suggesting the NoCut pathway also involves regulation of the exocyst.
Tant el creixement com la divisió cel·lular requereix el transport de membrana i altres factors a la superfície cel·lular. En cèl·lules de S. cerevisiae, aquests processos necessiten el transport de vesícules de secreció a través dels cables d’actina fins a les zones de creixement actiu, on es fusionen. Les Rho GTPases regulen la polarització de l’actina i la fusió de vesícules, però es desconeix com les GTPases senyalitzen els diversos efectors i com regulen els dos tipus de funcions. En aquest estudi, demostrem que les proteines Boi1 i Boi2 treballen conjuntament amb la senyalització de les Rho GTPases, per tal de regular la funció del complexe “exocyst” que media els contactes inicials entre vesícules de secreció i la membrana plasmàtica. Cèl·lules sense Boi1/2 tenen el citoesquelet d’actina polaritzat, però la cèl·lula filla no pot emergir ni créixer. Un al·lel d’Exo70, una subunitat de l’”exocyst”, que és efector de les GTPases Rho3 i Cdc42, restaura el creixement de cel·lules defectuoses en la funció de Boi1/2. A més a més, l’hiperactivació de GTPases rescata defectes dels mutants de Boi1/2, suggerint que la funció essencial de Boi1 i Boi2 és promoure l’activació de l’”exocyst”, depenent de Rho, durant el creixement cel·lular. Finalment, hem demostrat que la inibició de la divisió cel·lular que controlen via NoCut els efectors Boi1/2 en cèl·lules amb defectes en la segregació de cromosomes, es rescata amb l’al·lel d’Exo70 descrit anteriorment. Aquestes observacions suggereixen que NoCut podria funcionar també a través de la regulació de l’”exocyst”.

Although much progress has been made in understanding the events that lead to successful cell division, many details of this process remain a mystery. This dissertation presents findings which help to explain events that occur in the latest stages of cytokinesis, with an emphasis on the role of centrosome proteins. The first chapter introduces the novel centrosome protein centriolin. We show that this protein is localized specifically to the subdistal appendages of the maternal centriole in interphase, and it localizes to the midbody during cytokinesis. Disruption of this protein results in a unique cytokinesis defect in which cleavage furrow formation and ingression appear normal, but the cells remain connected by a thin intracellular bridge for extended periods of time. These results lead us to the conclusion that centriolin has an important function in cytokinesis. The second chapter describes our attempt to identify centriolin interacting partners. A yeast two hybrid screen was performed, and the results of this screen revealed an interaction between centriolin and proteins involved in vesicle target specificity and fusion. Further studies of these proteins revealed a novel localization to the midbody in cycling cells and a novel function in the final stages of cytokinesis, similar to centriolin. The third chapter discusses my attempts to clone and characterize a novel GTPase Activating Protein (GAP), which was also discovered in the screen for centriolin interacting proteins.

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.

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.

Cilia are conserved organelles that extend from the surface of most eukaryotic cells. During development cilia play key roles in force generation and perception of the extracellular environment. Ciliary defects cause a broad class of human diseases called ciliopathies characterized by pleiotropic symptoms including cystic kidneys, retinal degeneration, cardiac malformations and skeletal deformations. Perception of the environment relies on specific proteins being localized to the ciliary membrane compartment. The mechanism for sorting and trafficking membrane proteins to the cilium is poorly understood. To address this question, I developed a fluorescence-based pulse-chase assay to measure the transport kinetics of ciliary membrane proteins. This assay was used to determine the importance of candidate proteins to the delivery of fibrocystin, polycystin-2, and smoothened to cilia. Using this assay, I found that ciliary delivery of fibrocystin and polycystin-2 requires IFT20, GMAP210 and the exocyst while smoothened delivery is largely independent of these proteins. In addition, I determined that polycystin-2, but not smoothened or fibrocystin require the biogenesis of lysosome related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with a requirement for BLOC-1 in ciliary transport of polycystin-2, BLOC-1 mutant mice have cystic kidney disease. BLOC-1 functions in endosomal sorting and I find that disrupting the recycling endosome also reduced ciliary polycystin-2 and causes its accumulation in the recycling endosome. This is the first demonstration of a role for BLOC-1 in ciliary biogenesis and highlights the complexity of trafficking pathways to the cilium.

Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins. This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures. With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells. In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor. Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient.
(Faculty of Medicine)

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but are poorly understood. Here we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented towards the distal wing tip. They are constructed by a hexagonal array of wing epithelial cells. We find that wing epithelial cells are irregularly arranged throughout most of development but become hexagonally packed shortly before hair formation. During the process, individual cell junctions grow and shrink, resulting in local neighbor exchanges. These dynamic changes mediate hexagonal packing and require the efficient delivery of E-cadherin to remodeling junctions; a process that depends on both the large GTPase Dynamin and the function of Rab11 recycling endosomes. We suggest that E-cadherin is actively internalized and recycled as wing epithelial cells pack into a regular hexagonal array. Hexagonal packing furthermore depends on the activity of the Planar Cell Polarity proteins. The Planar Cell Polarity group of proteins coordinates complex and polarized cell behavior in many contexts. No common cell biological mechanism has yet been identified to explain their functions in different tissues. A genetic interaction between Dynamin and the Planar Cell Polarity mutants suggests that the planar cell polarity proteins may modulate Dynamin-dependent trafficking of E-cadherin to enable the dynamic remodeling of junctions. We furthermore show that the Planar Cell Polarity protein Flamingo can recruit the exocyst component Sec5. Sec5 vesicles also co-localizes with E-cadherin and Flamingo. Based on these observations we propose that during the hexagonal repacking of the wing epithelium these proteins polarize the trafficking of E-cadherin-containing exocyst vesicles to remodeling junctions. The work presented in this thesis shows that one of the basic cellular functions of planar cell polarity signaling may be the regulation of dynamic cell adhesion. In doing so, the planar cell polarity pathway mediates the acquisition of a regular packing geometry of Drosophila wing epithelial cells. We identify polarized exocyst-dependent membrane traffic as the first basic cellular mechanism that can explain the role of PCP proteins in different developmental systems.

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...

The mechanical strength of plant tissues and organs can be attributed to specific properties of the cell wall. In many cases, in order to establish their final shape, cells deposit various cell wall materials in a localized manner. This is achieved by highly organized action of the endomembrane system which is essential for biosynthesis and secretion of cell wall proteins and polysaccharides. The exocyst complex is a conserved tethering complex in eukaryotes and it is involved in tethering of secretory vesicles to the sites of secretion at the plasma membrane. In this study, we address several aspects of the plant exocyst complex architecture and cell wall development using molecular biology techniques and advanced confocal microscopy. We demonstrated that two SEC10 exocyst subunits are present in Arabidopsis thaliana and share redundant functions. We also showed that the architecture of the plant exocyst complex shares several structural features with the yeast one. We demonstrated the importance of the functional EXO84b exocyst subunit for normal tracheary element development and showed that the main constituents of the secondary cell walls are deposited normally in exocyst mutants. We described a clear difference in the exocyst microtubule-independent dynamics in epidermal cells vs. cell type...

胞吐作用定義為囊運小泡將物質運輸到質膜或細胞外空間的轉運過程。其中關鍵的一步發生在同源SNARE 蛋白介導的膜融合之前，即將胞吐囊泡瞄向並靶定在適當的質膜位點。先前在酵母和哺乳動物中的研究表明，一個名為exocyst 的蛋白質複合體在這一關鍵步驟發揮作用。exocyst 蛋白複合體最早在酵母發現，之後這個複合體也在哺乳動物中被發現。這個複合體包含8 個不同的亞基：SEC3，SEC5，SEC6，SEC8，Sec10，Sec15，Exo70 和Exo84。Exocyst 同源蛋白也已在植物中發現。相比酵母和動物，exocyst 在植物體內的功能還鮮為人知，尤其是在胞吐運輸過程中的作用 。通過瞬時表達熒光蛋白標記的擬南芥同源的exocyst 蛋白Exo70：AtExo70E2 以及使用這個同源物的特異抗體，我們在擬南芥和煙草BY－2 懸浮培養細胞中發現了一種新的細胞器，並命名為exocyst 陽性細胞器（EXPO）。這種細胞器分別位於質膜或是細胞質中。由於它未能與任何傳統的細胞器標記物重合，或是被布雷菲爾德菌素A，渥曼青黴素和刀豆素A 影響，以及不能與FM4-64 重合，我們判斷這些細胞器不定位於常規的分泌或胞吞途徑中。對於快速冷凍樣本進行的免疫電子顯微鏡顯示EXPO 的雙膜性質，同時也發現了陽性標記的位於質膜外的單膜囊泡的存在。與此同時，在野生型細胞中也發現了同樣結構的細胞器。EXPO和自噬體非常相似， 都有兩層膜。然而，EXPO 不能被的自噬標記物（AtAtg8e）所標記。同時，在營養脅迫條件下，EXPO 的數量也沒有增加。因此，EXPO 代表著植物所特有的一種非常規分泌形式。
此外，通過在擬南芥原生質體內進行瞬時表達，我進一步證實在AtExo70E2 存在的條件下， 一些exocyst 成員可以被招募到EXPO 。AtExo70E2 的旁系同源物AtExo70A1 是在這方面物法取代AtExo70E2 的作用。蛋白蛋白相互作用分析證實了AtSec10 或AtSec6 與AtExo70E2 之間的相互作用。 AtExo70E2，而不是它在酵母或是動物中的同源蛋白，可以誘導EXPO 在動物細胞中的形成。反之，人或是酵母Exo70 同源蛋白都不能誘導EXPO 在植物細胞中的形成。這些結果表明AtExo70E2 在EXPO 形成過程中的特定的以及至關重要的作用。
Exocytosis defines the process in which vesicles transport substances to the plasma membrane (PM)/extracellular space of the cell. One key step of exocytosis is the targeting and docking of the exocytic vesicles to the appropriate PM sites, which is prior to membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). Previously studies have demonstrated that a protein complex called exocyst complex is involved in this key step in yeast and mammals. The exocyst complex, containing eight different subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, was first identified in yeast and subsequently in mammals. Exocyst homologs have also been found in plants. In comparison to its yeast and animal counterparts, little is known about the function of exocyst proteins in plants especially in the process of exocytosis. By using both antibodies specific for one of the orthlogs of exocyst protein: AtExo70E2 as well as transiently-expressed fluorescently-tagged constructs for this exocyst subunit, a novel organelle termed exocyst-positive organelle (EXPO) was identified in suspension cultured Arabidopsis and tobacco BY-2 cells. These organelles were located to both the plasma membrane and cytosol. Based on their failure to overlap with any conventional organelle markers or response to brefeldin A (BFA), wortmannin or concanamycin A (ConcA) treatments, as well as their inability to take up the endocytic dye FM4-64, these organelles were thus not lie on the conventional secretory or endocytic pathways of plant cells. Immunogold electron microscopy (EM) of cryofixed samples revealed the double membrane nature of EXPO and also produced labeling of large single-membrane bound vesicles outside of the PM. These structures were also identified in wild type cells. EXPO and autophagosomes are similar in that both have two boundary membranes. However, EXPO did not label positively with YFP-AtAtg8e, a standard marker for autophagosomes, nor did the number of EXPO increase when the cells were subjected to nutrient stress. Therefore, EXPO represents a form of unconventional secretion unique to plants.
Further studies demonstrated that a number of exocyst subunits can be positively recruited to EXPO in the presence of AtExo70E2 by performing transient expression in Arabidopsis protoplasts. The paralog AtExo70A1 is unable to substitute for AtExo70E2 in this regard. Protein-protein interaction assay have confirmed the interaction between AtExo70E2 and AtSec6 and AtSec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in animal cells. Inversely, neither human nor yeast Exo70 homologs are able to cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Ding, Yu.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 101-118).
Abstracts also in Chinese.
Abstract --- p.i
摘要 --- p.iii
Acknowledgements --- p.v
Table of Contents --- p.vii
List of Tables --- p.x
List of Figures --- p.xi
List of Abbreviations --- p.xiv
Chapter CHAPTER 1 --- p.1
General Introduction --- p.1
Chapter 1.1 --- The secretory system in eukaryotic cells --- p.2
Chapter 1.2 --- Exocytosis and exocyst complex --- p.6
Chapter 1.3 --- Project Objectives --- p.7
Chapter CHAPTER 2 --- p.9
Exocyst-positive organelles (EXPOs) mediate unconventional protein secretion in plant cells --- p.9
Chapter 2.1 --- Abstract --- p.10
Chapter 2.2 --- Introduction --- p.11
Chapter 2.3 --- Materials and Methods --- p.12
Chapter 2.4 --- Results --- p.20
Chapter 2.4.1 --- Expression pattern of different AtExo70 paralogs with fluorescent tag in Arabidopsis protoplasts --- p.20
Chapter 2.4.2 --- The organelles labeled by AtExo70E2 are distinct from well known endomembrane markers --- p.23
Chapter 2.4.3 --- The AtExo70E2 positive organelles do not lie on the secretory or endocytic pathways --- p.27
Chapter 2.4.4 --- Arabidopsis Exo70E2-specific antibodies confirm identity of AtExo70E2-positive organelles --- p.31
Chapter 2.4.5 --- AtExo70E2 positive organelles are true and novel double membrane organelles --- p.33
Chapter 2.4.6 --- EXPO are not autophagosomes but sequester cytosolic proteins to release them into the apoplast --- p.41
Chapter 2.5 --- Discussion --- p.53
Chapter 2.5.1 --- EXPO: novel organelles labeled by exocyst --- p.53
Chapter 2.5.2 --- EXPO and autophagosome: same or not? --- p.55
Chapter 2.5.3 --- EXPO: the evidence of unconventional secretion in plant cells --- p.56
Chapter 2.6 --- Perspectives --- p.56
Chapter CHATER 3 --- p.58
AtExo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals --- p.58
Chapter 3.1 --- Abstract --- p.59
Chapter 3.2 --- Introduction --- p.60
Chapter 3.3 --- Materials and Methods --- p.62
Chapter 3.4 --- Results --- p.70
Chapter 3.4.1 --- AtExo70E2 is required for the membrane recruitment of a number of exocyst subunits --- p.70
Chapter 3.4.2 --- AtExo70E2 is required for the recruitment of some other, but not all, AtExo70 subunits --- p.74
Chapter 3.4.3 --- AtExo70A1 is unable to recruit other exocyst subunits --- p.74
Chapter 3.4.4 --- FRET and BiFC confirm interactions between AtExo70E2 and other exocyst subunits --- p.80
Chapter 3.4.5 --- Arabidopsis Exo70E2 can also induce EXPO formation in animal cells --- p.84
Chapter 3.4.6 --- Neither human nor yeast Exo70 can induce EXPO in plant protoplasts --- p.84
Chapter 3.4.7 --- EXPO induced by AtExo70-GFP expression in HEK cells do not colocalize with standard organelle markers --- p.87
Chapter 3.4.8 --- Electron microscopy confirms the presence of EXPO-like, double membrane structures in HEK cells after expression of AtExo70E2-GFP --- p.87
Chapter 3.5 --- Discussion --- p.91
Chapter 3.5.1 --- Plant exocyst and the discovery of EXPO --- p.91
Chapter 3.5.2 --- AtExo70E2 is a key player in exocyst recruitment onto EXPO --- p.93
Chapter 3.5.3 --- AtExo70E2 expression as a signal for EXPO formation --- p.96
Chapter 3.6 --- Perspectives --- p.100
References: --- p.101
Chapter List of publications derived from this Ph.D. thesis research --- p.119

The exchange of gases between the plant and the environment is mediated by guard cells, which, by changing their volume, regulate the size of the stomatal pore where the exchange takes place. Unique properties of stomata are given by a specifically designed cell wall, allowing asymmetric cell expansion. During the opening of stomata, there is a large increase in turgor pressure, accompanied by the uptake of considerable amount of water. For this reason, it is necessary to quickly change the inner surface of the guard cell. This process is ensured by the dynamic movement of the membranes inside the cell. During these processes, the vesicles are removed from the plasma membrane and reintegrated back during stomatal closure. One of the important regulators of the polarized transport of vesicles within the cell is a multiprotein complex exocyst. Due to the multiplication of exocyst subunits, the exocyst complex functions in plants are extensively diversified and involved in many cellular processes. In this thesis the effect of mutation in subunits exo70B1, exo70B2 and its combination exo70B1/exo70B2, on stomatal dynamics is studied. Both of these mutations affect the opening of the stomata during increased light intensity. The mutation of exo70B1 is in this case affected in greater manner. These...

Exocyst is a protein complex conserved in yeast, animals and plants. It mediates tethering of a secretory vesicle to the plasma membrane in the semifinal step of exocytosis. Several roles of exocyst in the processes of cell polarization in plant cells have been implied, including polarized growth of polen tubes and root hairs, cytokinesis, deposition of seed coat pectin and possibly autophagy. One of the most recent roles of exocyst includes also a response to bacterial and fungal pathogens. Exo70B2 and Exo70H1 subunits were shown to play prominent roles in this respect, with Exo70H1 being responsible for mediating defense against bacterial (Pseudomonas syringae) and Exo70B2 defense against both bacterial and fungal (Blumeria graminis) pathogens. Recently, new data appeared indicating the interaction between Exo70B1 and RIN4 and Exo70A1 and NOI6, respectively. RPM-1 interacting protein 4 (RIN4) is a well known negative regulator of both basal and effector-triggered resistance. This thesis shows interaction between NOI6 and several exocyst subunits, confirming previous data. I show here that exocyst subunints interact specifically with N terminus of NOI6 protein and that this interaction is lost in the shorter version of NOI6 mimicking AvrRpt2 cleavage. Since AvrRpt2 is an effector protein from...

Trichomes are fine epidermal outgrowths covering aerial organs of most land plants. Although unicellular trichomes of Arabidopsis thaliana have long been used as a model system in plant cell and developmental biology, surprisingly little is known about the processes involved in cell wall biogenesis during the last stage of trichome maturation. A role of EXO70H4, a putative subunit of the vesicle tethering complex exocyst, in trichome maturation has recently been identified in our laboratory. Image analysis, histochemical detection and FT-IR spectroscopy methods were used in this study to analyze cell wall defects of the exo70H4 LOF mutant, revealing the mutation causes altered deposition of pectins and possibly also lignins and hemicelluloses. Transgenic lines with EXO70 paralogues driven by the EXO70H4 promoter were prepared and their analysis revealed that the closest paralogue EXO70H3, unlike EXO70A1 and EXO70B1, can complement the exo70H4 mutation. Based on the results, questions concerning trichome cell wall composition, the role of EXO70H4 in trichome maturation and functions of the plant exocyst complex are discussed. Keywords: Arabidopsis, trichome, cell wall, secretory pathway, exocyst complex, EXO70H4, FT-IR spectroscopy

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...

Wang, Juan.
Thesis Ph.D. Chinese University of Hong Kong 2015.
Includes bibliographical references (leaves 70-79).
Abstracts also in Chinese.
Title from PDF title page (viewed on 16, November, 2016).

Exocyst is an octameric protein complex, conserved across all Eukaryotes. Its role, originally described in yeast, resides in a tethering of the secretory vesicles to the plasma membrane prior to the membrane fusion of the two membranes. Subunits SEC3 and EXO70 are believed to be spatial landmarks for the vesicles delivery. While yeast genome encodes single EXO70, we find dozens of them in land plants (23 in Arabidopsis). This work is focused at a role of the exocyst complex in plant cells. Its first part documents, that exocyst is essential for delivery of the cell wall components, namely pectins, but also for pathogen induced secondary cell wall thickening. Second part reveals an unconventional role of EXO70B1 subunit harboring exocyst subcomplex at an autophagic pathway to the vacuole and raises many questions about plant secretory pathway.

Stomata are structures in plant epidermis which regulate contact between inner and outer environment of the plant by mediating their stomatal aperture. Many inner and outer signals contribute to the ontogenesis of the stomatal pattern. Guard cells undergo significant change of volume and surface during stomatal movement. This change of surface must be compensated by intracellular trafficking of membrane material because biological membrane has limited elasticity. Most of this trafficking takes place between plasma membrane and endosomal compartments. Complex exocyst is protein complex that ensures proper targeting of secretory vesicles to their destination on the plasma membrane. Function of this complex is essential for many cellular processes that require precise targeting of secretion. Mutation in gene Exo70B1 causes different development of the stomatal pattern. Plants with mutated Exo70B1 differ in stomatal size depending on the cultivation conditions more than wild type plant. Protein EXO70B1 is also directly involved in stomatal dynamics because mutants exo70B1 have retarded stomatal opening in response to light. This direct connection can be observed on the fluorescently labeled protein EXOB1 which significantly changes its localization during stomatal movements. None of these observed phenotypes is...

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014
Os cílios primários são organelos sensoriais expostos à superfície da maioria das células eucarióticas. Estes funcionam como estruturas semelhantes a antenas, detectando e transmitindo sinais químicos provenientes do ambiente extracelular para o interior da célula. A sua correcta formação e funções sensoriais são fundamentais para o adequado desenvolvimento embrionário e pós-natal, bem como para a homeostase dos tecidos no adulto. De facto, diversas doenças humanas, conhecidas por ciliopatias, devem-se a defeitos no cílio primário. Este organelo é constituído por um axonema que se forma a partir do corpo basal, originado pelo centríolo-materno da célula, e uma membrana ciliar que encerra uma pequena porção do citoplasma designado por ciliopasma. A membrana ciliar e a membrana plasmática são topologicamente contínuas mas diferem na sua composição lipídica. A membrana ciliar contém vários receptores de sinalização celular, canais iónicos e proteínas de transporte que tornam este organelo numa autêntica antena celular. Até ao momento, ainda não é totalmente conhecido o conjunto de proteínas que regula o processo de montagem do cílio. Várias pequenas proteínas G da superfamília Ras foram implicadas neste processo. Estas proteínas são conhecidas por regularem as vias de tráfego intracelular, assegurando o transporte correcto de moléculas entre compartimentos delimitados por membranas. O cílio primário é um desses compartimentos, e é sabido que as proteínas ciliares são transportadas do citoplasma até ao cílio através destas vias de tráfego intracelular, uma vez que este organelo não possui maquinaria para realizar a síntese proteica. Em particular, proteínas da família “Arf-like” (Arl) tais como a Arl3, Arl6 e Arl13b foram associadas ao cílio primário. Mutações nestas proteínas causam distrofia e disfunção renal, obesidade, polidactilia, entre outros defeitos característicos de ciliopatias. O nosso grupo descobriu recentemente que a Arl13b tem um papel na regulação de tráfego endocítico de reciclagem. Por outro lado, mutações do gene que codifica a Arl13b provocam síndrome de Joubert, uma ciliopatia caracterizada por defeitos neurológicos. Em ratinhos, a mutação de Arl13b causa letalidade embrionária devido a falhas na via de sinalização “Sonic Hedgehog”, presumivelmente causadas por anomalias na estrutura dos cílios primários. Dado que uma das vias que as proteínas ciliares utilizam para chegar ao cílio depende do tráfego endocítico de reciclagem, os nossos resultados que sugerem um papel da Arl13b na regulação desta via poderão contribuir para explicar os defeitos no cílio primário que advêm de mutações na Arl13b. Sendo assim, é possível que a Arl13b tenha um papel no endereçamento/ancoragem de vesículas endocíticas de reciclagem, transportando proteínas ciliares para a região periciliar. Para além disso, é sabido que as proteínas G quando estão no seu estado activo ou ligadas a GTP efectuam as suas funções ligando-se a proteínas designadas por efectores. O nosso grupo obteve evidências sólidas de que Arl13b interage com o exocisto, através da sua subunidade Sec8. O exocisto é um complexo formado por oito subunidades que participa na ligação e ancoragem de vesículas provenientes da rede trans-Golgi e do compartimento endocítico de reciclagem. Recentemente, a subunidade Exo84 do exocisto foi identificada como mutada em pacientes de síndrome de Joubert e através da subunidade Sec10 foi comprovado que o exocisto está envolvido na formação de cílios. Sendo assim, colocámos como hipótese que o papel da Arl13b no tráfego de proteínas ciliares para a região periciliar ocorra através da sua interação com o exocisto. Desta forma, o nosso objectivo é estudar o papel da interacção entre Arl13b e o exocisto na ciliogénese e no tráfego ciliar. O nosso primeiro objectivo foca-se na caracterização da interação entre Arl13b e Sec8. O segundo objectivo concentra-se no papel da interação Arl13b-exocisto na formação de cílios. Os nossos resultados provam que o Sec8 é uma efector da Arl13b, uma vez que a ligação entre as proteínas aumenta na presença de GTP, em linhas celulares de mamífero e que esta interação é independente da presença de cílio. Mostramos também através da síntese in vitro de Arl13b-FLAG e Sec8-Myc que a interação entre estas proteínas é directa e que não necessita da presença das outras subunidades do complexo. Para além disso, observamos também que tanto o domínio N-terminal como o C-terminal da Arl13b são necessários para garantir que esta interação ocorra, uma vez que o domínio N-terminal é responsável pela ligação ao efector e o domínio C-terminal é responsável por estabilizar a proteína e garantir a alteração entre a forma activa e inactiva da mesma garantido a troca entre GTP e GDP. São necessários mais estudos para determinar que região da Arl13b é responsável pela viabilidade da interação com o exocisto, sendo para isso necessário produzir novos plasmídeos de Arl13b com deleções mais pequenas e com mutações pontuais para que a conformação da proteína quando expressa nas células não sofra alterações tão drástica e seja possível determinar a região de interação. Vimos também que outra subunidade do exocisto, o Exo70, é co-immunoprecipitada com Arl13b. Este resultado levanta uma nova hipótese em que o Sec8 medeia a interação entre a Arl13b e todo o complexo exocisto. Por ultimo, mostramos também pela primeira vez que o Sec8 é necessário para a correta ciliogénese, uma vez que ao silenciarmos o Sec8 obtemos uma redução na percentagem de células ciliadas . Este fenótipo não é tão acentuado como o observado aquando do silenciamento da Arl13b mas permite especular que as duas proteínas possam estar envolvidas na mesma via de transporte de proteínas necessárias para a correta formação do cílio. Este resultado reforça a ideia de que a Arl13b é uma peça chave na formação e manutenção da correta biologia ciliar. Um colaborador deste projeto observou uma interação genética sinérgica entre a Arl13b e Sec10 indicando que estas proteínas participam num via comum ou paralela. Em conclusão, com os resultados apresentados neste trabalho, com as informações obtidas a partir do nosso colaborador e com o facto de que Arl13b regula o tráfego endocítico de reciclagem, nós propomos um modelo onde a Arl13b e o exocisto estão envolvidas no transporte de proteínas ciliares originárias do compartimento endocítico de reciclagem até ao interior do cílio. No nosso modelo, o processo baseia- se em dois passos: o Sec8 numa primeira fase é responsável pela seleção e transporte de vesículas contendo Arl13b até à região periciliar; numa segunda fase Exo70 é responsável pela fusão dessas vesículas com a membrana ciliar permitindo a libertação das proteínas ciliares no cilioplasma. Mais estudos são necessários para testar esta hipótese e comprovar o modelo proposto. Os resultados aqui apresentados e os estudos futuros propostos irão fornecer a base para melhor compreender e interpretar a pleiotropia fenotípica e etiologia do síndrome de Joubert.
Primary cilia are sensory organelles present on nearly every eukaryotic cell. Arl13b belongs to the Arf family of small G proteins. Mutations in this protein were identified in patients with Joubert syndrome, a ciliopathy characterized by neurological defects combined with polydactyly and cystic kidneys. Nevertheless, the precise role of Arl13b in ciliogenesis remains elusive. We found that Sec8, a subunit of the exocyst, interacts with Arl13b. The exocyst is an octameric complex involved in the tethering of post-Golgi vesicles at their site of fusion, and it has been shown to be required for cilia formation. Interestingly, it was identified in a family with Joubert syndrome amutation in one of the subunit of the exocyst. Furthermore we show that Sec8 is a bona fide effector of Arl13b, since it binds only to the activated form of Arl13b, and that the interaction is direct. We proved that both the N- and C- terminal of Arl13b are required for the interaction with Sec8. Moreover, we found that Exo70, another subunit of the exocyst, also co-immunoprecipitates with Arl13b, suggesting that Sec8 mediates the interaction of Arl13b with the exocyst complex. Regarding the ciliogenesis defects, we show for the first time that silencing of Sec8 causes an abrogation in cilia formationTogether, these results suggest that Arl13b and exocyst function together in the same pathway during ciliarelated processes leading to functional cilia. Since we and others found that Arl13b are involved in endocytic recycling trafficking form the endocytic recycling compartment, it is possible that both proteins work together in the transport of recycling vesicles containing ciliary proteins from the endocytic recycling compartment toward primary cilia.

Exocyst is protein complex evolutionary conserved in yeasts, animals and plants, which plays a role in control of cell morphogenesis and polarity. It is a tethering complex whose function is to attach secretory vesicles to specifi c foci on plasma membrane. Complex exocyst is formed by eight subunits. Subunit EXO70 is encoded by 23 paralogue genes in genome of Arabidopsis thaliana. Mutation in paralogue subunit EX070H4 causes defect in trichome maturation. Mutant trichomes have thin, not reinforced cell wall, making them soft and elastic. Transcription of EXO70H4 gene is induced by UV radiation, therefore observations of plants cultivated on UV-B radiation were done. Analysis of mutants cultivated on UV-B radiation revealed hyperaccumulation of vesicules in cytoplasm, which were visible by light microscope. Hyperaccumulation was not observed in control plants cultivated on UV-B radiation, but thickening of cell wall was induced. Th is reaction to UV in trichomes hasn't been described yet. Analysis of cellular localization made with YFP tagged constructs revealed that EXO70H4 localizes into mobile corpuscules associating with Golgi apparatus. It was found with yeast two hybrid system that EXO70H4 interacts with TRS120, subunit of tethering complex TRAPPII which is active in Golgi apparatus....

The final step of secretion termed exocytosis is mediated by the exocyst complex. The exocyst is an evolutionary conserved protein complex that tethers secretory vesicle to the target membrane and consists of eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo84, and Exo70. Sec15 exocyst subunit was previously shown to connect the rest of the exocyst complex with a secretory vesicle in yeast, mammals and fruit fly via interaction with Rab GTPase and GEF of Rab GTPase. Here, I show that plant SEC15B potentially functions in evolutionary conserved manner. First, two mutant lines of Arabidopsis thaliana sec15b mutant were tested in characteristics typical for other exocyst mutants. Although some characteristics reach certain level of plasticity, both sec15b-1 and sec15b-2 show similar tendencies, which are mostly consistent with defects with other mutants in exocyst subunits. sec15b-1 has been determined as a stronger allele that is defective in formation of seed coat, elongation of etiolated hypocotyl, growth of stem and primary root, establishment of axillary branches and lateral roots, diameter of rosette and, unexpectedly, growth of pollen tubes. Phenotype of sec15b-1 was rescued by insertion of SEC15B gene under SEC15B promotor. Second, complementation test showed that SEC15B and SEC15A are...

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...

During the course of evolution the early land plants gained extensive innovations that can be seen in modern day plants. The polar growth is an ancient feature of eukaryotic cells and is one of preadaptations that helped plants in successful colonization of land. The polar growth in plants regulates not only the direction of cell expansion and structural properties of cell wall but especially also the orientation of cell division, and is governed by various factors, including the exocyst complex. The exocyst is a well conserved vesicle tethering multi-subunit complex involved in tethering of secretory vesicles to the target membrane. The essential role of the exocyst complex in regulation of various cellular processes in Angiosperms is now well documented. Here I present results of a doctoral project that contributed to phylogenetic analyses of the land plant exocyst complex and especially to uncovering functions of three moss exocyst subunits, namely EXO70 (isoform PpEXO70.3d), SEC6 and SEC3 (isoforms PpSEC3A and PpSEC3B) in the model organism Physcomitrella patens. Various knock-out (KO) mutants in several moss exocyst subunits (Ppexo70.3d, Ppsec6, Ppsec3a and Ppsec3b) show pleiotropic defects directly or indirectly linked to the cell polarity regulation. Cell elongation and differentiation,...

In the recent years, there has been a growing number of publications indicating at the involvement of plant secretory pathway in defense against phytopathogens. Specifically, roles of plant exocyst complex have been explored in deeper detail in current research. Yet, exactly how exocyst- mediated exocytosis contributes to secretion of antimicrobials and cell wall-based defense remains unclear. In the presented Dissertation, I provide both experimental evidence and devise further hypotheses on selected exocyst's subunits in plant immune reactions. Particularly, I show that EXO70B1 exocyst subunit interacts with immunity-related RIN4 protein. Cleavage of RIN4 by AvrRpt2 Pseudomonas syringae effector protease releases both RIN4 fragments and EXO70B1 from the plasma membrane when transiently expressed in Nicotiana benthamiana leaves. I speculate on how this might have an implication in regulation of polarized callose deposition. In a co-authored opinion paper, we also hypothesize that EXO70B1-mediated autophagic degradation of TN2 resistance protein prevents its hyperactivation and lesion mimic phenotype development. In addition, in collaboration with my colleagues, I present data on EXO70H4's engagement in PMR4 callose synthase secretion, required for silica deposition. Representing a possible...

Exocyst is a protein complex involved in tethering of secretory vesicles to cytoplasmic membrane before SNARE-mediated fusion event. Its presence and function in secretory pathway has been confirmed in yeasts, animals and plants. This thesis describes some properties of Sec5, one of the exocyst subunits, in plant model Arabidopsis thaliana. Microscopic methods, including VAEM/TIRF microscopy, were used to study subcellular localization and dynamics of Sec5-GFP fusion protein. Sec5 is cytoplasmic protein that also localizes to cytoplasmic membrane, particulary in cells with high secretory activity. It strongly localizes to maturating cell plates during late cytokinesis and its localization to cytoplasmic membrane partially depends on actin cytoskeleton. Generally, obtained results are in agreement with corresponding observations of behavior of other exocyst subunits in plant cells, suggesting, that Sec5 executes its function as part of the exocyst complex.

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,...

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.

Plants have an artillery to defend themselves. The plant surface is protected by water- resistant cuticle and mechanically strong cell wall. Then each plant cell has tools to recognize and to answer to a pathogen threat. In an extreme case, the answer is programmed cell death. Plant immunity is a complex process integrating these passive and active mechanisms in an effort to overstay a pathogen attack. When the plant cell is attacked by a pathogen, the metabolic resources are redirected towards immunity reaction which results in growth restriction. Both the immunity reaction and the growth are dependent on the efficient polarized secretion of various cargoes. Exocyst complex mediates tethering of a secretory vesicle with a target membrane and SNARE complex orchestrates the subsequent steps of vesicle docking and fusion. Exocyst and SNAREs are regulated by various proteins. In my work, I focused on identifying the exocyst interaction partners in plant immunity. In cooperation with my colleagues, we found the direct association between Qa-SNARE SYP121 involved in plant penetration resistance and EXO70B2 exocyst subunit. Moreover, we confirmed the relevance of their interaction for the formation of epidermal defensive structures, papillae and haustorial encasements in plant defence against non-adapted...

Cytokinesis is the last step of cell cycle when two individual daughter cells separate in process called abscission. This process involves various cellular membrane structures such as endoplasmic reticulum or trans-Golgi network. Moreover, recent investigation has also highlighted an important role of recycling endosomes. The membrane dynamics appear to be important during cell division especially for the formation of new plasma membrane between two daughter cells. Numerous studies suggest that cytokinesis is tightly linked with highly sophisticated transmembrane shuttle that is controlled by Ras-superfamily members such as Rab and Ral proteins. Moreover, during last years has also been revealed the involvement of tethering factors which mediate the fusion of intracellular vesicles with the target plasma membrane. The best known tethering factor is the evolutionary conserved exocyst complex found in all eukaryotic cells. This protein complex is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) and was found to interact with members of Ras- superfamily suggesting its involvement in the regulation of cytokinesis. Although the exact mechanism remains shrouded in fog this work suppose the possible interactions among Ras- like proteins and exocyst members which may...

Plant cell morphogenesis is largely dependent on the coordination of cytoskeletal elements, plasma membrane, and vesicle trafficking. Formin proteins are nucleators of the actin cytoskeleton. Plant Class I family formins are integral membrane proteins and thus have the ability to coordinate cytoskeletal dynamics with the plasma membrane localization. We identified Arabidopsis thaliana formin AtFH4 as a microtubule associated protein. The binding is conferred by a novel domain located between the transmembrane domain and the formin homology 1 domain. The protein associated with actin in in vitro conditions. Overexpressed AtFH4 accumulated in the endoplasmic reticulum, and induced coalignment of endoplasmic reticulum membranes with microtubules. Together, these data suggest that the combination of plant-specific and conserved domains enables AtFH4 to function as an interface between membranes and both major cytoskeletal networks . Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plant cells. Prior to their fusion with the plasma membrane, secretory vesicles are tethered at exocytic sites by the exocyst, an octameric protein complex. We analysed the mutant in the EXO84b exocyst subunit, and discovered that the mutant plants were dwarfed and exhibited...