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...

Exocyst is a binding protein complex, which is evolutionary conserved in yeast, animal and in plant cells. It has crucial role in regulation of cell morfogenesis and cell polarity. The function of the exocyst complex is binding of secretoric vesicle to the proper side on plasma membrane in penultimate step of exocytosis. This process is essecial for function and survival of cell. Another process crucial for the cell is autophagy. In plants autophagy plays important role in the responses to nutrient starvation, senescence, abiotic and biotic stress. RabG3b are small GTPases, which have positive role in autophagy. In this work I described the interaction between RabG3b and some of subunits of exocyst complex: Exo70B1, Exo70B2 and Exo84b. I also studied changes in morfogenesis of tonoplast by induction and inhibition of authophagy and induction of anthocyans synthesis in Arabidopsis thaliana.

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

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,...

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.

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.

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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...

The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...

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...