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Plant microtubule-organizing centers
Škrdlová, Iveta ; Schwarzerová, Kateřina (advisor) ; Libusová, Lenka (referee)
In eukaryotic cells, microtubules are nucleated and organized by protein structures called microtubule- organizing centers. There is a great diversity in structure and morphology of these structures in eukaryotic organisms. Animal centrosome, yeast spindle pole body and basal bodies of flagellar apparatus are the best known types of so called focused microtubule-organizing centers, where microtubules are nucleated from one or two concrete sites in the cell. These focused microtubule- organizing centers are replaced by diffuse centers in higher plant cells, which means that microtubules are nucleated from dispersed nucleation sites. Focused centers are rarely found in land plant cells. These focused centers comprise bicentrioles and blepharoplasts, which give rise to the locomotory apparatus of motile spermatozoid of bryophytes, pteridophytes, cycads and Ginkgo. Another types of focused microtubule-organizing centers in bryophytes are polar organizers, and the nuclear and the plastid envelopes in mitotic and meiotic cells. Powered by TCPDF (www.tcpdf.org)
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Searching for mechanisms and functions of microtubular interactions with other plant cell structures
Krtková, Jana ; Schwarzerová, Kateřina (advisor) ; Vaňková, Radomíra (referee) ; Ovečka, Miroslav (referee)
Microtubular cytoskeleton is involved in many processes in plant cells, including cell division, growth and development. Other proteins enable its functions by modulation of its dynamics and organization and by mediation of functional and structural interaction with other cell structures. Identification of the mediating proteins and the functions of these interactions under specific conditions were the main aims of the thesis. Membrane proteins interacting with microtubules were identified using biochemical methods. Surprisingly, the identified proteins co-sedimenting with microtubules were not members of the "classical" microtubule associated proteins (MAPs). There were enzymes, chaperones and plant specific proteins among them. For further studies, the identified microtubule-associated heat-shock protein 90 (Hsp90_MT) was chosen. Recombinant Hsp90_MT binds directly to microtubules and tubulin dimers in vitro. The ATP-binding pocket is not responsible for this association. In BY-2, Hsp90_MT co-localizes with phragmoplast and cortical microtubules and is involved in microtubule recovery after their depolymerization during cold treatment. In plants, Hsp90 is involved in cell cycle progression, its inhibition causes cell-cycle arrest in G1 phase. Based on literature search for animal proteins...
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Role of formins in the organization and dynamics of intracellular structures in Arabidopsis thaliana
Rosero Alpala, Elvia Amparo ; Cvrčková, Fatima (advisor) ; Baluška, František (referee) ; Malcová, Ivana (referee)
On the basis of detailed phenotypic examination of fh1 and fh2 mutants we observed that the main housekeeping Arabidopsis thaliana formin AtFH1 (At3g25500) and its closest relative, AtFH2 (At2g43800) are involved in both actin filaments and microtubule dynamics. fh1 mutants showed increased sensitivity to the actin polymerization inhibitor Latrunculin B (LatB). Formin mutants had cotyledon pavement cells which exhibited more pronounced lobes compared to the wild type, and alterations in vascular tissue patterning were found. The double fh1 fh2 homozygote was not obtained, suggesting that at least one functional formin gene is required for proper gametophyte development. Methods used to observe and quantify both architecture and dynamics of the cortical cytoskeleton from confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM) were standarized and allowed to find that mutants exhibited more abundant but less dynamic F- actin bundles and more dynamic microtubules than wild type seedlings, fh1 mutant phenotype observed in roots was further aggravated by a (heterozygous) fh2 mutation. The formin inhibitor SMIFH2 mimicked the alterations observed in fh1 mutants in plants, it has been the first report of this inhibitor in plants. Defects in membrane trafficking were...
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Microtubule-active drugs: mechanism of action and resistance
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Rösel, Daniel (referee)
Microtubular cytoskeleton represents a target for a myriad of diverse chemical compounds, referred to as microtubule-active drugs. Produced by certain plants, animals or microbes, the substances often effectively elicit cell death - especially in animals and also in plants to a certain extent, but never in species which produce them to defend against their predators. Nowadays, several microtubule-active substances constitute hallmarks of anti-cancer treatment and agricultural weed control. There is an enormous sum of knowledge about the action of paclitaxel (taxol), vinca alkaloids and colchicine, three best-known microtubule active compounds used in medicine, and new research often challenges the previously accepted theories. This work investigates the mechanism of action of microtubule-active drugs from the angle of biochemistry and cell biology, as well as from the physiological standpoint. Effects on microtubule levels and dynamics and the path towards the cell death are reviewed. In the last chapter, attention is given to drug activity in both animal and plant bodies and, finally, to drug-producing plant species which often show substantial resistance.
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Induction and course of programmed cell death in cancer cells after taxane application."
Kábelová, Adéla ; Jelínek, Michael (advisor) ; Gemperle, Jakub (referee)
The taxanes are a class of commonly used anticancer agents, which are very effective in treatment of breast, ovarian, prostate or lung cancer. Taxanes bind to the β-tubulin subunit of microtubules and lead to their stabilization and inhibition of depolymerization. Such microtubules lose their function to form mitotic spindle, thus arresting cells in G2/M phase and resulting in apoptosis. Unfortunately some cells are able to escape from taxanes-induced apoptosis by developing various mechanisms of resistance including alteration in taxanes target microtubules or upregulation of specific transporters that pump the drug out of cells. Other types of resistance are connected with process of programmed cell death (PCD), especially with proteins that after taxane application participate in its successful progress. Taxanes can directly or indirectly modify the activity of Bcl-2-family proteins that control mitochondrial and endoplasmic reticulum integrity, thus regulating the initiation of PCD. Caspases are executioners of PCD and caspase-2 activated by cytoskeletal disruption seems to be especially important in taxanes- induced apoptosis. In some cases can taxane treatment also result in caspase-independent cell death. Special role has protein p53 that seems to be involved only in apoptosis caused by low taxanes...
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Molecular base of plant HSP90-MT interaction
Benáková, Martina ; Krtková, Jana (advisor) ; Malcová, Ivana (referee)
Microtubules (MTs) are one of the essential cell structure that participate in a number of key events in the plant cells and their properties and functions are influenced and modified by many other proteins. These proteins belong to a group of microtubule- associated proteins (MAPs, microtubule-associated proteins). One of the MAPs, the molecular chaperone Hsp90, examines and fulfills a large number of different functions in the cell. Its colocalization with MTs has been demonstrated previously by Freudenreich and Nick (1998) and Petrášek et al. (1998). However, direct interaction with MTs was described only recently using cosedimentation assay. The specific cytosolic isoform of tobacco Hsp90 bound to MTs was called Hsp90_MT due to its ability to bind MTs. It has been also found that the binding to MTs is independent on the activity of ATP (Krtková et al., 2012). The authors also described a positive effect of Hsp90_MT on MT recovery after their exposure to cold stress. Although MT cytoskeleton dynamics is influenced by a large number of MAPs, it is surprising that the molecular mechanism of MAPs interaction with MTs and their MT-binding domains have not been described yet. Therefore, we decided to determine the tobacco Hsp90_MT MT-binding domain by production of a set of recombinant proteins...
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Cytoskeletal organization of senescent cell
Kolářová, Věra ; Hodný, Zdeněk (advisor) ; Hock, Miroslav (referee)
This bachelor thesis discusses the phenomenon of cellular senescence in the context of cytoskeleton organization. Differences in the organization of cytoskeleton be- tween normal proliferative cells and senescent cells are being compared. Cellular cytoskele- ton is a very dynamic structure and influences the function of the cell within a tissue. This thesis gathers current evidence about senescence and cytoskeleton and indicates possible directions for future research. Keywords: cellular senescence, antitumour barrier, cell migration, cytoskeleton, microtubules, cancer 1
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Role of cytoskeleton in plant cell morphogenesis
Miklánková, Pavlína ; Schwarzerová, Kateřina (advisor) ; Sekereš, Juraj (referee)
The cells are able to acquire variety of shapes, in which cytoskeleton plays an important role. Cytoskeleton influences deposition of cell wall materials, regulates vesicle movement in cell, participates in exocytosis and endocytosis. Cortical microtubules affect celulose accumulation in cell wall and determine direction of cell expansion, although the exact connection between microtubules and cellulose remains unclear. Actin promotes growth and contributes to its spatial regulation in both tip ang diffuse growing cells. Actin is important for secretion in expanding cells but its exact functions in cell growth regulation are not explained yet. Analysis of mutants, spectroscopic methods, cytoskeletal drugs, fluorescence proteins and other methods are used to better understand how actin and microtubule cytoskeleton are integrated during plant cell morphogenesis. Epidermal and trichome cells of Arabidopsis thaliana are a good model of research and they are used for most studies.
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Characterization of the PTEN domain of selected Arabidopsis class II formins
Přerostová, Sylva ; Cvrčková, Fatima (advisor) ; Havelková, Lenka (referee)
Formins are proteins facilitating formation of actin filaments. They affect structure of cytoskeleton and participate in cytokinesis and tip growth. There are 2 classes of formins in Arabidopsis thaliana, which include FH1 and FH2 (Formin Homology 1 and 2) domain. Formins of the class I have usually a transmembrane domain on N-terminus. Due to this fact they can interact with membranes. Some formins from the class II include PTEN domain (Phosphatase and Tensin Homolog) derived from sequences of PTEN proteins which has lost the function of phosphatase. It is assumed this domain can bind on a membrane via the phosphatase section or C2 domain. This thesis was focused on the formin AtFH13 from the class II in Arabidopsis thaliana and on its PTEN domain. There were analyzed differences between mutants and wild-types in length of roots in seedlings and in size of seeds and seed coats, and observed the effect of dexamethasone on the length of roots on AtFH13. PTEN domain of the formin was isolated from cDNA, cloned to a vector and fused with YFP. The tagged protein was visualized by the method of transient expression in epidermal cells in the leaves of Nicotiana benthamiana. No big differences were observed between plants mutant in the gene AtFH13 and wild-type in choice parameters. Dexamethasone did't influence...
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Microtubule-associated proteins in plants
Benáková, Martina ; Krtková, Jana (advisor) ; Vinopal, Stanislav (referee)
1. Abstract and key words MTs are one of the basic cellular protein structure. Their features and function are influenced and modified by group of other proteins, i.e. microtubule-associated proteins (MAPs). In the last decades, an extensive research on MAPs and their wide range of functions has been carried out. Therefore we are aware of the involvement of some of the MAPs in MT dynamics, other have been shown to have rather structural function. They bundle MTs with various cell structures, such as the other MTs, proteins, organelles, actin cytoskeleton or plasma membrane. Many described MAPs are homologous in the whole eukaryotic domain, for example MAP65 or EB1 (END BINDING 1) family, therefore it is interesting to follow if and how the functions of plant MAPs differ from their animal counterparts. On the other hand, there are many specific MAPs with unique functions in plants, e.g. ATK5 or SPR1 (SPIRAL 1). This Bachelor thesis is a survey on current knowledge of plant MAPs and it makes an effort to present their characteristic and functions in plant cell and organism. Key words: cytoskeleton, microtubules, microtubule-associated proteins, plant cell, growth and development
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