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Functional analysis of plant Arp2/3 complex subunits
Kukla, Jakub ; Schwarzerová, Kateřina (advisor) ; Honys, David (referee)
1. Abstract ARP2/3 complex is well studied in case of animals, it plays key roles in motility of cells and intracellular organels. It's malfunctions result in severe growth disorders and even lethality of affected cells. On the contrary, plant cells do not exhibit such dramatic phenotype of ARP2/3 complex mutations like it is by animals. It is possible that just the different life strategies of plants and animals contribute to differences in a way how animal and plant cells use their cytoskeleton, where ARP2/3 complex is it's part as well. It is highly conserved 7 protein complex from yeast to human. His main functions are creation of new "de novo" actin filaments, actin branched filaments network. Some of the parasite organisms are capable of missusing its nucleator activity to actively move inside of host cell. Because of the plant cells are sourounded by the cell wall, which give them support in creating various shapes and also hinders active movement of the whole cell body, it is likely that ARP 2/3 complex could be possibly involved in novel plant specific functions as well. If we think about the different life strategy of plants and animals we can not ignore all the things these two kingdoms have in common regarding to cytoskeleton processes. That is the need both for vesicular transport and...
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The role of tubulin transport between the nucleus and the cytoplasm
Bokvaj, Pavel ; Schwarzerová, Kateřina (advisor) ; Zažímalová, Eva (referee)
Tubulin is the basic building block of microtubules which ensure intracellular transport, morphological changes, the connection of proteins of metabolic pathways, the segregation of chromosomes during mitosis and many other essential processes in plant cells. The localization of tubulin regarding nucleus is strictly controlled and during the interphase, tubulin is usually not present in the nucleus. As we previously showed, one of the exceptions of the presence of tubulin in the nucleus is the cold stress condition. Also, we discovered several plant-specific nuclear export sequences (NES) in tubulin molecules: one in α-tubulin and two in β-tubulin. In this work I found that double mutation of both functional β-tubulin NESes cause slight accumulation of the protein in the nuclei. However, the phenotype of the transgenic plants expressing β-tubulin with mutations is probably not affected by the presence of the mutated protein. The effect of the expression of the mutated β-tubulins was observed in Nicotiana tabacum BY-2 cells as well. The expression of β-tubulin carrying a single NES3 mutation or double mutation of both NESes bring changes of the division activity of the cells, while the NES2 mutation does not have any effect. Furthermore, it was discovered that the 0řC cold tr eatment does not cause...
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Actin isoforms in the plant cell
Šlajcherová, Kateřina ; Schwarzerová, Kateřina (advisor) ; Fišerová, Jindřiška (referee)
The thesis deals with a question of redundancy in plant actin isotypes, especially in a case of Arabidopsis thaliana. Available proofs both confirm and disprove the redundancy of actin isotypes. Similarities and differences in gene structure are considered, as well as gene and protein sequence, expression, expression regulation and function. Despite conserved features as the gene structure, protein sequence, or higher similarity among ortologues than paralogues, there are differences in actin gene families such as the regulation of genes expression, the structure and function of non-coding sequences, or expression patterns. Moreover, actin isotypes divergetion times correlate with important events in plant evolution history. The thesis also deals with diverse functions of actin isotypes in plants. In addition, directions for future research are provided as well.
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Actin nucleation in plant cell
Schiebertová, Petra ; Schwarzerová, Kateřina (advisor) ; Cvrčková, Fatima (referee)
The ability of plant cells to nucleate actin is important especially for dynamic of intracellular movements of organelles and vesicles, cell structure and growth organization and coordination of endo- and exocytosis . Actin nucleation means the development of new actin filaments from G-actin. Actin associated proteins - Arp2/3 complex and formins serve for this purpose. Arp2/3 complex polymerizes new "daughter " filament from the side of the "parent" filament at an angle of 70 degrees and after the nucleation remains at minus end of filaments, thus inducing branching of filaments. Nucleation by Arp2/3 complex further requires nucleation promoting factors - NPFs. Deletion of subunits of Arp2/3 complex is often lethal in animal cells. On the other hand, deletion results in rather mild phenotype in plants. Formins catalyze the formation of direct actin fibers and remain on the plus end of the fiber after nucleation. There are multiple isoforms of formins in plants than in animals, which suggest an important role of formins in plants. New actin nucleators Cobl, Lmod, Spire, JMY and APC containing WH2 (WASP homology 2) domain were identified in non-plant cells. This type of actin nucleation was not described in plants. Kew words: Actin, Arp2/3 complex, formins, Scar/WAVE, Cobl, Lmod, Spire
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Aluminum toxicity in plant cell
Schiebertová, Lucie ; Schwarzerová, Kateřina (advisor) ; Tylová, Edita (referee)
Aluminium being the third most abundant metal in the earth's crust is in its toxic form a serious threat for crop productivity in acid soils, which comprise almost half of the arable land. As the most phytotoxic form is considered free ion Al3+ , which affects root growth by acting in the root apical zone, resulting in growth inhibition in a very short time at micromolar concentrations. At cellular and molecular level, many cell components are affected by aluminium toxicity including cell wall, plasma membrane, signal transduction pathways, calcium homeostasis, DNA and numerous cytoplasmic enzymes. Although it is difficult to distinguish the primary targets from the secondary effects so far, understanding of the target sites of aluminium toxicity is helpful to elucidate the mechanisms by which aluminium exerts its deleterious effects in root growth. On the other hand, some plant species have evolved mechanisms to cope with aluminium toxicity. In the future, the attention should be paid to basic mechanisms of aluminium toxicity and our understanding of the current problem should be unified. Key words: Al toxicity, Al resistance/tolerance, phytotoxicity, Al stress, acid soils, root elongation
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The role of the cytoskeleton in morphogenesis of plant cells
Havelková, Lenka ; Schwarzerová, Kateřina (advisor) ; Hašek, Jiří (referee) ; Malínská, Kateřina (referee)
6. Conclusions ► The capability of ARPC2 protein (subunit of ARP2/3 complex) to bind microtubules was described in this study. Our results indicated that ARP2/3 complex may mediate direct interaction between microfilaments and microtubules in processes where the coordination of microtubule reorganization and actin mediated growth was required. ► We showed that cell-malforming effect of growth retardant ancymidol was based on its inhibitory action on cellulose synthesis. Microtubules played a passive role in this process and were not directly responsible for changes of the cell shape induced by ancymidol. ► Meristematic and suspensors cells in maturating somatic embryos differed in sensitivity to latrunculin B, probably due to different composition of actin isoforms in these cells. Application of low doses of latrunculin B resulted in selective death of suspensor cells and thus contributed to the development of high-quality embryos. ► Aluminium caused rapid cessation of root growth of Arabidopsis thaliana seedlings. We showed that the immediate reason of root growth cessation was plasma membrane rigidization and loss of endocytosis in root cells. Aluminium stabilized cortical microtubules within minutes and induced their loss within hours of treatment. However, the effect of aluminium on microtubules was not...
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Characterization of membrane protein DREPP
Vosolsobě, Stanislav ; Schwarzerová, Kateřina (advisor) ; Cvrčková, Fatima (referee)
Proteins of DREPP family (20-25 kDa, syn. PCaP1 in Arabidopsis thaliana) first appeared in ferns and we have shown that several independent duplications of DREPP protein occurred during evolution of large families (Poaceae, Brassicaceae, Solanaceae and Asteraceae) and in group Coniferophyta. Secondary losses of one paralogue occurred in subfamilies Pooideae and Solanoideae.We have also detected two large-scale modification of DREEP protein in Asparagales and Brassicaceae (this divergent paralogue was previously described as MAP18 protein). We have examined colinearity of chromosome fragments in vicinity both PCaP1 and MAP18 paralogues in Arabidopsis thaliana and we hypothesize that MAP18 gene arose during genome duplication on the origin of Brassicaceae family. DREPP protein was previously identified in detergent-resistant membrane microdomain fraction and a myristyl anchor was shown to be necessary for their membrane localization. Membrane association was shown to be modified by the interaction of unique N-terminal domain with PtdInsPs, which was inhibited by binding of Ca-calmodulin (Nagasaki et al., 2008). The mutation of Gly2 by Ala in the myristilation site, or C-terminal GFP-fusion (GFP-DREPP), affect membrane association in Arabidopsis thaliana (Nagasaki et al., 2008). Several DREPP paralogues in...
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The role of actin dynamics in auxin transport
Stillerová, Lenka ; Cvrčková, Fatima (advisor) ; Schwarzerová, Kateřina (referee)
Phytohormones are signalling molecules directing physiological and developmental processes in plants. One of them, auxin, is involved in the diverse regulation of plant processes, e.g. embryogenesis, organogenesis, vascular tissue formation and tropisms. Auxin transport is polar. Auxin isdistributed via the phloem, utilizing specialized membrane transport proteins; small amount diffuse also through the membrane. Aux1/Lax transporters mediate auxin entry into the cell, auxin efflux is mediate mostly by PIN transporters, which are the crucial factors in determining the directionality of auxin flow. Asymmetric localization of membrane PIN proteins depends on vesicle transport from Golgi to the plasma membrane. Vesicles are transported along actin filaments which are dynamically rebuilted by regulators. They are maintaining asymmetric cellular localization of the auxin transport proteins. PIN proteins are cycling between endosomes and plasma membrane. Cycling is regulated by ARF-GEF proteins and serin/threonin kinase (PID, PINOID). Newly synthesized PIN proteins are equally distributed in the plasma membrane, afterwards they are asymmetrically redistributed. Regulation of actin filaments formation and remodelling is the crucial factor for transport of vesicles with PIN proteins. Many proteins which regulate...
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Nuclear transport: its regulation and role in plant cells
Bokvaj, Pavel ; Fišerová, Jindřiška (referee) ; Schwarzerová, Kateřina (advisor)
The nucleocytoplasmic transport represents a crucial checkpoint of all signal pathways leading to the gene expression, the cell cycle maintenance, and RNA export processes. It is mediated by nuclear pore complexes (NPC) anchored in double nuclear membrane. The NPC structure and the basic architecture of the transport regulations are evolutionarily highly conserved across eukaryotic kingdoms; however, some significant differences and specifics exist in plants. In this thesis, I describe the contemporary level of our knowledge of the nucleocytoplasmic transport regulation and its actors in animalia generally and in planta in detail.
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