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Functional characterization of plant EXO70 exocyst subunit isoforms and their membrane targeting mechanisms
Sekereš, Juraj ; Potocký, Martin (advisor) ; Jaillais, Yvon (referee) ; Ischebeck, Till (referee)
Vesicle tethering complex exocyst is one of the key regulators of the cell polarity and morphogenesis in eukaryotes. The complex interacts with the secretory vesicle, as well as plasma membrane, and facilitates formation of cis SNARE complex leading into fusion of the vesicle with target destination. Two of the eight exocyst subunits, the SEC3 and EXO70 are known to bind plasma membrane via protein and lipid interactors in Opisthokont model organisms. Genomes of angiosperm plants encode a surprisingly wide repertoire of EXO70 isoforms with over 20 present in both Arabidopsis and diploid tobacco genome. It has been proposed that different EXO70 isoforms would form parts of functionally distinct subtypes of the plant exocyst complex driving membrane trafficking to various membrane domains. Specific interactions of peripheral membrane proteins with particular membrane phospholipids largely contribute to targeting of cellular components to subcellular compartments and membrane domains. This thesis focuses on role of protein-lipid interactions in regulation of plant cell polarity and contributes to functional analysis of the plant EXO70 family diversity. We introduce the topic with the theoretical reviews summarizing role of protein-lipid interactions in establishing plant cell membrane domains at...
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Phenotypic characterization of transgenic tobacco lines in functional analysis of plant genes
Srba, Miroslav ; Fischer, Lukáš (advisor) ; Ovečka, Miroslav (referee) ; Potocký, Martin (referee)
Plant cell lines represent useful models in plant cell biology. They allow simple analysis of the effects of various factors including modulated gene expression at cellular and subcellular level. Tobacco BY-2 cell line is a favored model due to its high proliferation rate, capability of effective synchronization and accessibility to transformation. Relatively high uniformity of BY-2 cultures allows morphological phenotyping and assessment of growth parameters like mitotic index, viability or cell density. Presented thesis summarizes established methods and up-to-date experience with tobacco BY-2 cell lines. Selected results of two research projects focused on hybrid proline-rich proteins and heterotrimeric G-proteins are included. These projects are treated as case studies of cell line phenotyping and evaluation of cytological parameters. Protocols and general experimental suggestions that were optimized during the solving of the projects are described and discussed.
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The role of signaling phospholipids in the regulation of plant polar growth
Pleskot, Roman ; Potocký, Martin (advisor) ; Testerink, Christa (referee) ; Spiwok, Vojtěch (referee)
Polar cell growth is one of the most fundamental processes in plant development. Pollen tubes represent excellent experimental system to study basic rules of this phenomenon. Polar growth is governed by a precise spatiotemporal coordination of various molecules, such as small GTPases, actin cytoskeleton, protein kinases and reactive oxygen species, to achieve its establishment and maintenance. In this study, we utilized a combination of diverse experimental approaches together with advanced computational methods to investigate the role of two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphophate (PIP2) in the polar growth. We described the involvement of PA in the regulation of actin cytoskeleton, which dynamics is essential for the proper pollen tube growth. We found direct interaction of actin with the PA-producing enzyme, phospholipase D (PLDβ)1 and we showed that actin affects the activity of PLDβ. We further described structural details of the PA inhibition of actin-capping protein and we proposed the model of positive- feedback loop of the actin dynamics regulation by PA and PLDβ1. To get insight into the PA localization in pollen tubes, we prepared PA-binding domain of Spo20p as YFP-fusion protein and we used it as PA-biosensor. We found that PA is enriched...
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Are there any sequence determinants of functional divergence of GTPases?
Kraus, Ondřej ; Novotný, Marian (advisor) ; Potocký, Martin (referee)
Small GTPases are important proteins that affect many cellular processes. In my work I compare the five most important protein families of small GTPases - Arf, Rab, Ran, Ras and Rho to identify amino acids responsible for major functional differences between different protein families. To compare them, I have used the structural data from the PDB database and sequences from the UniProt database. I have discovered previously undescribed groups of amino acids specific for each protein family of small GTPases with the help of programs ConSurf and Sca5. I also carried out a pilot study of the applicability of B-factors as indicators of bond strength in the protein structure on the example of small GTPases. The first results are not entirely conclusive, but they do not exclude the applicability of B-factors as indicators of bond strength either. Powered by TCPDF (www.tcpdf.org)
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