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The role of cytoskeleton in endosomal fusion and fission
Získalová, Tereza ; Libusová, Lenka (advisor) ; Tolde, Ondřej (referee)
Cytoskeleton plays a key role in endocytic process. Vesicules move along microtubules to target membranes. Microtubules also partake in the formation of endosomal tubules, from which recyclated vesicules are splitted off. Actin network has in endocytosis multi-ple effect as well. In the case of membrane fusion is its role both, positive and negative, for it creates mechanical force which facilitates the fusion in last stage. By contrast, in the first stage, it acts as a physical barrier, which needs to be removed. Actin also actively participates in fission of vesicules. Actin network and microtubules are thus interconnected with endocytic pathway in time and space. Right functional connection of the cytoskeleton with dynamics of endocytic vesicles is driven by many regulatory proteins. Among important regulators of actin network belong for example proteins of Arp2/3, WASH complex, WASP or Rab and Rho proteins. Powered by TCPDF (www.tcpdf.org)
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Role of translational elongation factors in dynamics of stress granules.
Hlaváček, Adam ; Hašek, Jiří (advisor) ; Janderová, Blanka (referee)
eIF5A seems to be involved in both, translation initiation and elongation. It was also reported to affect assembly of P-bodies. Given similarities of P-bodies with stress granules (SGs) we decided to test the role of eIF5A in dynamics of heat-induced SGs and its implications for the cell recovery. For the evaluation of eIF5A function in SGs formation was used the temperature- sensitive (ts) mutant eIF5A-3 (C39Y/G118D) cultivated under permissive temperature 25řC and Rpg1-GFP fusion protein as a marker of SGs. The cells were exposed to robust heat shock at 46řC for 10 minutes. The ability of the mutant cells to recover was tested by propidium iodine staining and colony forming units plating. We found that the eIF5A-3 mutant forms heat-induced SGs more loosely aggregated, indicating that the fully functional eIF5A is necessary for SGs assembly. However, it does not seem to affect the rate of SGs dissolution. Survival tests indicate that eIF5A-3 mutant cells are susceptible to dying in a similar way as WT cells; nevertheless, their ability to resume proliferation is significantly better. We also observed a loss of the ts phenotype of the eIF5A-3 mutant. This loss cannot be explained by reversion of mutated eIF5A sequence into normal. Probable cause lies in the adaptive evolution. Our results indicate role of...
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Invasive structures of cancer cells in 3D environment
Lyková, Dominika ; Tolde, Ondřej (advisor) ; Libusová, Lenka (referee)
The ability of cells to migrate through tissue barriers plays an important role in physiological and pathological processes including immune response or invasiveness of cancer cells. The cells generate cytoplasmic protrusions called podosomes and invadopodia, collectively known as invadosomes or podosome-type adhesions (PTA), which are thought to be the key structures of cell invasion, especially of cancer cells during metastasis. Invadosomes are F-actin rich cell-matrix contacts with capability to degrade extracellular matrix components and are observed both in normal cells (such as monocytic cells, endothelial cells and smooth muscle cells) and in cancer cells. This bachelor thesis is focused on those in cancer cells, their initiation, regulation, function and morphology in 3D and in vivo and their requirement for tumor metastasis.
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Molecular mechanisms of cell polarity and morphogenesis in moss Physcomitrella patens
D'Agostino, Viktoria ; Žárský, Viktor (advisor) ; Soukup, Aleš (referee)
Plant cells are able to establish polarity and expand by tip growth. Polarized cells often embrace functions important for plant viability. The process of tip growth requires actin cytoskeleton in collaboration with a number of accessory proteins. The position of the intensively expanding region is provided by microtubules and the function of signalling proteins. Polarized secretion regulates the structural properties and subsequently the shape of the cell wall. Some components of the secretory and signalling pathways are highly conserved among eukaryotes, others are found exclusively in the plant kingdom. Though much has been discovered in yeast and animal cells, many mechanisms in plants are yet to be revealed. Model systems performing tip growth, such as root hairs, pollen tubes and protonema cells, enable comparison and thus a complementary overview of the various processes.
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Interplay of cytoskeleton and secretory pathway during exocytosis in plant cells
Aldorfová, Klára ; Sekereš, Juraj (advisor) ; Vosolsobě, Stanislav (referee)
Cytoskeleton is known to participate in exocytosis of yeast and animal cells. The role of plant cytoskeleton during exocytosis has not been fully understood yet. However, both actin and microtubules evidently contributes to the secretion of specific cargo proteins or cell wall components. Plant cytoskeleton influences the dynamics of exocytosis through various functions. First, secretory vesicles are delivered near the plasma membrane. Second, microtubules were shown to mark the place of exocytosis. Third, cytoskeleton is able to prevent membrane fusion by simple separation of compartments. Fourth, cytoskeleton potentially mediates the interaction between molecules of secretory apparatus. Secretion of certain cargo molecules appears to be dependent on different cytoskeleton types and the exocytosis seems to be specifically regulated in each tissue. This thesis aims to describe interplay of cytoskeleton and secretory pathway on the example of tip growth and to predict future direction of research on secretory pathway based on cellulose synthase secretory data.
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Specific functions of ARP2/3 complex subunits
Fišerová, Kamila ; Fischer, Lukáš (advisor) ; Oulehlová, Denisa (referee)
Actin is one of the most abundant proteins in living organisms. Regulation of the actin cytoskeleton is provided by many mechanisms, one of the regulators of actin dynamics in plants and animals is highly conserved - Arp2/3 complex. In organisms it consists of two large subunits (Arp2 and Arp3) and five small subunits (ArpC1 - ArpC5). Arp2/3 complex controls actin filament branching at an angle of 70o . This thesis describes the functions of individual subunits with a special emphasis on those which are specific for individual subunits. This summarize exceeds the boundaries of the plant kingdom, and it also discusses animals and yeast, in which the complex is actively studied, and it is a lot of information available about the mechanisms of its regulation. The paper summarizes the interactions between the subunits and their interactions with regulators of Arp2/3 complex and other proteins. Some of the subunits are in some organisms encoded by, more than one gene in such cases, these isoforms may have different functions as well. Arp2/3 complex is for animals necessary for living, but in plants mutations in the subunits of the complex have moderate symptoms. In plants the Arp2/3 complex is used primarily in fast and oriented growth, mutations of subunits showing typical distorted trichomes. Key...
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Early embryonal development and morphogenesis of selected organ systems of the rediae and cercariae of Fascioloides magna.
Pankrác, Jan ; Kašný, Martin (advisor) ; Macůrková, Marie (referee)
Fascioloides magna (giant liver fluke) is a digenetic trematode with two-host life cycle and high veterinary importancy. Typical definitive host is a deer (Cervidae), but many other species from different families can be accidentally infected, for example sheep, goat or cattle. Very important role in the life cycle of F. magna has the first host - fresh water snail of the family Lymnaeidae. Three different life stages of F. magna, two of them with ability of reproduction - sporocysts and rediae develop in the body of snail. The third stage - cercaria is produced by rediae. Cercariae are able to escape from the snail, encyst and become infective for the definitive host. Since the second half of the 19th century many researchers studied the development of particular stages in the first intermediate host, but many characteristics of this process are still not fully understood. This thesis should reveal some of unanswered questions concerning to the reproduction and ontogenetic development of trematodes, which is presented on the examples of three organ systems - muscles, nerves and excretory system of rediae and cercariae of F. magna.
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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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Localization matters: function of paxillin and phopholipids in the cell nucleus
Marášek, Pavel ; Hozák, Pavel (advisor) ; Půta, František (referee) ; Žárský, Viktor (referee)
(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...
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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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