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Subunits of exocyst complex in the development of Arabidopsis epidermis
Vojtíková, Zdeňka ; Žárský, Viktor (advisor) ; Soukup, Aleš (referee)
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....
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Cytoplasmic membrane and tonoplast dynamics during closing and opening of stomata
Röder, Matěj ; Žárský, Viktor (advisor) ; Albrechtová, Jana (referee)
Stomata are epidermal structures mediating regulated contact of plant apoplast with surrounding environment via stomatal opening. Change of turgor plays crucial part in initiation of stomatal opening or closure. During stomatal movement, guard cell undergo considerable and repetitive changes in cell volume and consequently surface area over a period of minutes. Alteration in the surface of membrane must occurs due to limited stretching capability of the plasma membrane. It can be achieved through membrane invaginations and endocytosis of small vesicles. Microscopy and electrophysiology techniques have proven that both processes are happening in guard cells. These processes are controlled and managed by complex web of signal pathways in which actin and microtubular cytoskeletons, SNARE proteins, ion channels and others molecules have crucial parts. The aim of this work is to summarize current knowledge on the processes and mechanisms of these membranes surface changes and their molecular principle.
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Funkční charakterizace podjednotky Sec5 komplexu exocyst u rostlin.
Sekereš, Juraj ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
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.
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Kortikální cytoskelet, exocytický komplex exocyst a jejich role v morfogenzi rostlinných buněk
Fendrych, Matyáš ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
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...
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The secretory vesicles tethering complex exocyst and the auxin transport polarization
Janková Drdová, Edita ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
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...
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Exocyst subunit AtSEC15b: its role in plant cell morphogenesis and characterization of its Rab interacting partner
Toupalová, Hana ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Organization of endomembrane compartments in all eukaryotic cells is dependent on continuous transport of membrane vesicles. Major part of the core regulators of intracellular membrane transport is represented by small GTPases from the Rab family. Rab GTPases cycle between the GTP-bound "active" and GDP-bound "inactive" forms. In their active form, they are able to interact with specific effectors and perform their functions. Exocyst is an octameric complex involved in regulation of secretion. It functions as an effector of Rab GTPases in yeast and mammals and tethers secretory vesicles to the plasma membrane prior to the actual membrane fusion. Using publicly available expression data, we have identified candidates from Rab GTPase family for the interaction with exocyst subunit AtSEC15b in plants and demonstrated that AtSEC15b specifically interacts with AtRABA4a GTPase. We also showed that, like in yeast and mammals, Arabidopsis Sec15b binds Rab GTPase also probably in GTP-dependent manner, implying that this interaction is well conserved throughout the eukaryotic kingdoms. We also successfully demonstrated the complementation of yeast thermo-sensitive mutant strain, sec15-1. Based on this observation we concluded that AtSEC15b is able to substitute the function of yeast SEC15 and restore the phenotype....
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Secretory pathway in plant cell wall polysaccharide biosynthesis and FT-IR spectroscopy methods in plant cell wall composition analysis
Glanc, Matouš ; Žárský, Viktor (advisor) ; Votrubová, Olga (referee)
All plant cells are encapsulated in a cell wall that determines the cells' shape and size and is essential to many of their vital processes. The cell wall of streptophyte plants is composed mainly of polysaccharides of high molecular weight. Cellulose, the main constituent of the plant cell wall, is synthesized by protein complexes bound to the PM, while hemicelluloses and pectins are synthesized in the Golgi apparatus. Several proteins that participate in the synthesis of cell wall polysaccharides have been identified, but hundreds of them remain to be discovered. Secretory pathway plays an important role in plant cell wall biogenesis as it transports cellulose synthase complexes and noncellulosic polysaccharide molecules to the cell surface. Some regulatory mechanisms that might be involved in cell wall material secretion, such as actin cytoskeleton, Ca2+ gradient or PIP kinases have been proposed; however, the regulation of this process is very complex and far from being understood. FT-IR spectroscopy is a method that can detect molecular vibrations and provide information about chemical composition of virtually all substances. It has been used successfully in screens for cell wall mutants, the study of interactions between cell wall polymers, as well as other areas of cell wall biology. FT-IR...
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Mechanisms of epidermal cells polarization in plants.
Vojtíková, Zdeňka ; Žárský, Viktor (advisor) ; Soukup, Aleš (referee)
Plant epidermal cells form contact area of the plant, they protect it from impacts of surrounding environment and they mediate the communication with its neighbourhood. In the epidermis there are evenly distributed several cell types with quite specialized morphology (pavement cells, trichomes and guard cells) due to the polarization mechanisms. The cytoskeleton and signal molecules of ROP GTPase family promote the polarized growth. Thanks to polarized growth the cells reach their shapes. The cytoskeleton responds to the signal by expanding the cell, helps with targeting of the secretion to the sites of active growth and mediates polarized formation of the cell wall. On the upper side of the epidermis the cuticle and layer of epicuticular waxex is secreted. The secretion of cuticular components is baso-apicaly polarized. This work summarizes the mechanisms of the polarization in plant epidermal cells discoverd untill now.
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The role of the exocyst in development and maintaining of cell migration structures
Vaškovičová, Katarína ; Žárský, Viktor (referee) ; Brábek, Jan (advisor)
The exocyst is a hetero-octameric protein complex which mediates tethering secretory vesicles to specific sites of plasma membrane for polarized exocytosis. The exocyst was long known to contribute to processes such as yeast budding, cytokinesis, epithelia polarization and neurite outgrowth. Recently, the role of the exocyst in regulation of actin cytoskeleton and cell migration was discovered. It was shown, that the exocyst is important for formation of cell migration structures such as lamellipodia and filopodia in motile cells and invadopodia in invasive cancer cells. These structures are all actin-based membrane protrusions and the exocyst can through its Exo70 subunit interact with the Arp2/3 complex, the activator of actin nucleation. By binding and activating the Arp2/3 complex, the exocyst mediates actin polymerization resulting in formation of these membrane protrusions. Furthermore, the exocyst probably targets the Arp2/3 complex to specific sites of plasma membrane that are intended to become membrane protrusions. In addition, the exocyst mediates secretion of matrix metalloproteinases (MMPs) in invadopodia. MMPs are important for degradation of the extracellular matrix, an essential process in cancer cell invasion. The exocyst seems to be part of the cascade downstream of cytokines...
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