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Following the Arp2/3-based processes in plant cells
Fišerová, Jindřiška ; Opatrný, Zdeněk (advisor) ; Binarová, Pavla (referee) ; Dráber, Pavel (referee)
1. ABSTRACT An actin cytoskeleton comprises an essential cytoskeletal structure that organizes a cytoplasm during number of processes occurring in animal and plant cells, such as cell division and growth, formation of membrane protrusions and cellular movement or cytoplasmic streaming. A dynamic actin net and an exact spatial and temporal actin filament organization is, therefore, necessary for correct growth processes and development of multicellular organism. Depolymerization and re-establishment of actin arrays is mediated by actin associated proteins. Among them, an Arp2/3 complex (actin related protein 2 and 3) plays an important role as a catalyst of the actin filament nucleation and, thus, enables the formation of dynamic actin network. In non-plant cells, Arp2/3 complex-dependent polymerization of actin filaments is required for movement of whole cells (fibroblasts or keratocytes), it drives organels and pathogens throughout the cell or mediates endocytic processes. The role of the Arp2/3 complex in non-motile plant cells is despite the considerable effort over last three years far from being understood. Unlike animals, where loss-of-function mutations in the Arp2/3 complex are lethal, only limited number of cell types show significant phenotype in Arabidopsis mutants. The evidence exists that the...
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Molecular characterization of γ -tubulin interactions with signalling molecules
Macůrek, Libor ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Svoboda, Augustin (referee)
52 V. CONCLUSIONS The results of presented PhD thesis can be summarized as follows: For the first time it has been demonstrated that γ-tubulin forms complexes with αβ-tubulin dimers in brain tissue as well as in other models of neuronal differentiation. Two forms of γ- tubulin have been identified in complexes of various sizes. It has been shown that γ-tubulin is posttranslationally modified. One of the identified posttranslational modifications of γ-tubulin is phosphorylation that appears to depend on Src family kinase activity. It has been proposed that posttranslational modifications of γ-tubulin may regulate interactions of γ-tubulin with αβ-tubulin heterodimers or other associated proteins during neurogenesis. It has been shown that γ-tubulin associates with protein tyrosine kinases involved in signal transduction events. γ-Tubulin interaction with Src family kinases significantly increased after long-term RA-activation embryonal carcinoma P19 cells. A similar increase has been observed after rapid activation of mast cells, indicating that this regulatory mechanism is not restricted to a particular model system. In both models, Src family kinases bound to γ-tubulin are active and phosphorylate proteins present in γ-tubulin complexes. Fyn kinase interacts with γ-tubulin through its SH2 domain in a...
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Regulatory mechanisms of microtubule reorganization in activated mast cells
Rubíková, Zuzana ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
Microtubules (MTs) are highly dynamic structures essential for the spatio-temporal intracellular organization and transport, signal propagation, cell differentiation, motility and division. To perform these roles, MTs create arrangements capable of fast and precise adaptation to various signals. MTs are under the control of many factors regulating MT nucleation, stability and dynamics. Bone marrow-derived mast cells (BMMCs) are important immune system cells, which can cause serious diseases if their functions are deregulated. Although MT reorganization during BMMC activation is well established, the molecular mechanisms that control their remodelling are largely unknown. In the presented thesis we functionally characterised GIT1/βPIX signalling proteins, PAK1 kinase, and Ca2+ signalling in the regulation of MT nucleation in BMMCs and other cell types. We also elucidated the function of miltefosine (hexadecylphosphocholine), a promising candidate for the treatment of mast cell-driven diseases. We found that GIT1/βPIX signalling proteins are γ-tubulin-interacting proteins associating with centrosomes in BMMCs. MT nucleation is positively regulated by GIT1 and Ca2+ , whereas βPIX is a negative regulator of MT nucleation in BMMCs. Cytosolic Ca2+ affects γ-tubulin properties and stimulates the...
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Role of exocyst complex in growth and development of moss Physcomitrella patens
Rawat, Anamika Ashok ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee) ; Fendrych, Matyáš (referee)
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,...
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Gamma-Tubulin forms and functions in microtubule organization and beyond
Kourová, Hana ; Binarová, Pavla (advisor) ; Šolc, Petr (referee) ; Bíšová, Kateřina (referee)
Microtubules are highly dynamic structures forming complex arrays changing accordingly to cellular requirements. In most eukaryotes, microtubules are nucleated from defined organizing centres like centrosomes or spindle pole bodies. Despite conservation of the major cytoskeletal components in plants and animals, plant cells lack centrosome-like structures and have evolved a unique mechanism to control microtubule assembly and organization. γ-Tubulin is an essential component of microtubule organizing centres, highly conserved in all eukaryotes with a prominent role in microtubule nucleation. However, growing body of evidence suggests γ-tubulin as a multifunctional protein. Next to microtubule nucleation, γ-tubulin controls microtubule plus-end dynamics, regulates gene expression, associates with DNA repair proteins and is implemented in mitotic and cell cycle regulation. We focused on γ-tubulin complexes and aimed to characterize interactors of γ-tubulin and their function in Arabidopsis. Next to characterizing microtubule dependent functions, we analyzed cell cycle and division. We also studied DNA damage response and involvement of E2F/RBR (retinoblastoma related) pathway and possible link to γ-tubulin. We characterized NodGS protein as a novel interactor of γ-tubulin in Arabidopsis and proposed...
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New regulatory mechanisms of microtubule nucleation
Černohorská, Markéta ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
MT nucleation from γ-tubulin complexes, located at centrosome, is an essential step in the formation of MT cytoskeleton. In mammalian cells, -tubulin is encoded by two genes. We functionally characterized two γ-tubulin proteins and have found that both are functionally equivalent. γ-Tubulin 2 is able to substitute for γ-tubulin 1 in MT nucleation. However, we revealed that unlike TUBG1, TUBG2 expression is downregulated in mouse preimplantation development. Mast cells represent effectors of the allergy reaction. Their activation by antigen induces number of cellular processes such as degranulation, proliferation and cytoskeleton rearrangements. The regulatory mechanisms of MT reorganization during mast cell activation are unknown. We identified new signaling proteins, GIT1 and PIX that interact with - tubulin. Depletion of GIT1 or PIX leads to changes in MT nucleation. GIT1 is phosphorylated on tyrosine and associates with γ-tubulin in a Ca2+ -dependent manner. Our data suggested a novel signaling pathway for MT rearrangement in mast cells where tyrosine kinase-activated GIT1 and βPIX work in concert with Ca2+ signaling to regulate MT nucleation. We tested the capability of GIT1 and PIX to influence -tubulin function in more cell types. We found out that GIT1/βPIX signaling proteins together...
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Analysis of Rpg1/eIF3a mutation in Saccharomyces cerevisiae.
Luxová, Pavla ; Malcová, Ivana (advisor) ; Binarová, Pavla (referee)
Rpg1/Tif32/eIF3a is an essential and the largest subunit of translation initiation factor eIF3 in yeast Saccharomyces cerevisiae. Besides interactions within the eIF3 complex it has been shown to interact with microtubules. Preliminary data of the laboratory obtained using strains of the W303 genetic background indicated that there is a synthetic phenotype between rpg1-2 mutant and microtubule inhibitor nocodazole. Aim of this work to elucidate this "microtubule phenotype" of the rpg1-2 mutant and its dependency on used genetic background. I confirmed that independently on genetic background (W303, BY, SEY) all mutants rpg1-1, rpg1-2 and rpg1-3 were temperature- sensitive. I found that in contrast to published data on rpg1 mutants of the W303 background these mutants of the BY and the SEY backgrounds do not arrest the cell cycle in G1 phase during cultivation at the restrictive temperature (37řC, 4 hours). In addition, all three mutants did not show an increased sensitivity to benomyl and none of them affects microtubule rearrangement after a release of cells from the nocodazole treatment. I constructed new strains with a combination of the BUB1 gene deletion with the particular rpg1 mutation. Phenotypic analyses of new double mutants revealed that simultaneous dis-function of Bub1 and Rpg1 results...
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Specificity of selected exocyst subunits in trichome development
Glanc, Matouš ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
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
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Characteristics of novel protein interactions of gamma-tubulin and their roles with microtubules and in cell division
Kohoutová, Lucie ; Binarová, Pavla (advisor) ; Kubelka, Michal (referee) ; Dráber, Pavel (referee)
Spatial and temporal regulation of microtubule nucleation and dynamics is required for formation of specific microtubular arrays that react to internal and external signals and change accordingly. Microtubules are nucleated from microtubule-organizing centres such as centrosomes in animal cells or spindle pole bodies in fungi. All higher plants lack centrosomes and thus present a model for study of acentrosomal cell division and microtubule nucleation and organization. γ-Tubulin is a conserved protein from tubulin superfamily with a central role in microtubule nucleation. It also regulates microtubule dynamics and organization including mitotic spindle positioning. Moreover, γ-tubulin functions in cell cycle regulation, checkpoints control, regulation of transcription, and coordination of late mitotic events. We aimed to characterize protein interactions of γ-tubulin and their functions in Arabidopsis. We identified Arabidopsis homologue of putative centrososomal protein RanBPM. Our data showed that AtRanBPM is a member of CTLH complexes. Our finding of CTLH complexes in plants confirmed their conservation in eukaryotic cells. We found that NITRILASE1 is a cell cycle regulator in Arabidopsis that is important for maintenance of genome stability and proper cell division. We studied a role of AtTPX2,...
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Functions of actin and myosin 1c in the cell nucleus and in the cytoplasm
Kalendová, Alžběta ; Hozák, Pavel (advisor) ; Binarová, Pavla (referee) ; Forstová, Jitka (referee)
Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...
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