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ARP2/3 complex interaction with plant secretory pathway components
Voloshina, Mariia ; Schwarzerová, Kateřina (advisor) ; Oulehlová, Denisa (referee)
The ARP2/3 complex is an evolutionarily conserved protein complex involved in actin nucleation. In animals, the loss of function of ARP2/3 subunits is usually lethal. In contrast, arp2/3 plants are viable and exhibit certain morphological defects. One of the characteristic traits of arp2/3 plants is impaired cell adhesion. This suggests a role for the ARP2/3 complex in plant cell wall secretion, specifically pectins. However, the exact molecular mechanism of ARP2/3 function in this pathway has not been elucidated yet. Preliminary results suggest a possible interaction of ARP2/3 with the secretory pathway proteins GNL1, COPI, and COG. This work combines two methods - TIRFM/VAEM and co-immunoprecipitation - to study the interaction of ARP2/3 with selected markers. However, neither colocalization nor physical interaction was demonstrated. This work also characterises COPI, COG and GNL1 with TIRFM/VAEM and shows that there is an interaction between GNL1 and endocytic markers. Key words: ARP2/3, COPI, COG, GNL1, secretory pathway
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Nuclear Transport Signals in the proteins of the ARP2/3 complex
Němcová, Barbora ; Bellinvia, Erica (advisor) ; Cvrčková, Fatima (referee)
The intracellular movement of macromolecules such as proteins and RNA is essential for maintaining cellular homeostasis and coordinating various cellular processes. In eukaryotic cells, the transport of these molecules between the cytoplasm and the nucleus is carefully regulated. Nuclear transport signals (NTS) play a key role in facilitating the import and export of proteins across the nuclear envelope. The ARP2/3 complex, which is an important regulator of activity dynamics, has been studied mainly for its functions in the cytoplasm, such as cell movement and cell division. However, new findings suggest that the ARP2/3 complex might also have nuclear functions in plants. Plants are unique multicellular organisms that rely on precisely coordinated cellular activities for growth, development, and response to stimuli. Understanding the molecular mechanisms behind nuclear processes in plants has recently become a focus of research. The ARP2/3 complex, which consists of seven subunits, is known for its ability to branch actin filaments and thereby control cellular processes requiring actin remodeling. However, recent studies have revealed a potential link between the ARP2/3 complex and nuclear functions in plants. Proteins associated with the ARP2/3 complex have been found to localize within the plant...
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The role of the WASH complex in endolysosomal homeostasis
Beránková, Pavla ; Libusová, Lenka (advisor) ; Rösel, Daniel (referee)
The WASH (WASP and SCAR homologue) complex is an actin nucleation promoting factor essential for endosomal cargo sorting. Upon WASH complex depletion, endosomal cargoes are mislocalized and the endolysosomal system collapses. Here, we employed high-speed vesicle tracking and real-time rescue experiments to test the effect of WASH complex depletion on endolysosomal homeostasis. We found that large lysosome-like vacuoles emerge in knockout cell lines of individual WASH complex subunits, although the overall dynamics of the lysosomal network does not substantially change. Follow-up experiments revealed that the WASH complex does not act directly on the vacuoles during their rescue. Overall, the data indicate that the emergence of vacuoles in WASH complex knockouts is a secondary process that depends on the WASH complex indirectly.
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The role of ARPC2 in plant cells
Šlajcherová, Kateřina ; Schwarzerová, Kateřina (advisor) ; Klíma, Petr (referee)
ARPC2 protein localization in a plant cell Kateřina Šlajcherová 1 Abstract Actin cytoskeleton is an ubiquitous structure which plays numerous irreplacable roles. Actin nucleation is, beside formins, performed by ARP2/3 complex (actin-related protein), comprising of seven subunits (ARP2, 3, C1-C5) and activated by protein SCAR/WAVE complex. ARP2/3 complex is attached to the membrane and branches existing microfilaments, apart from nucleating them de novo. ARP2/3 mutants in most organisms show severe defects. However, plant mutants exhibit only mild phenotype, for example, Arabidopsis thaliana ARPC2 mutant (dis2-1) has deformed trichomes and leaf epidermal cells, but its viability is not impaired. The aim of the thesis is to map ARPC2 localization within the cell and broaden our understanding of ARP2/3 complex role in plant cell morphogenesis. Tobacco ARPC2 (NtArpC2) subunit was visualized in Arabidopsis plants, using the GFP fusion protein as well as imunofluorescence and anti-ARPC2 antibody. Experiments were undertaken to collocalize the subunit with actin and microtubular cytoskeleton, with mitochondrions, endosomes and other membrane organelles. The specimens were observed in confocal and TIRF microscope. The GFP-NtARPC2 protein shows as motile dots; their movement, but not their existence, is dependent...
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The effect of Arp2/3 complex on vacuolar structure, cytoplasmic streaming and dictyosome motility.
Semerák, Matěj ; Schwarzerová, Kateřina (advisor) ; Eliášová, Kateřina (referee)
In plant cells, actin filaments are nucleated in two different ways: The growth of single filaments or their bundles is enabled by various types of formins, whereas branched meshworks emerge due to Arp2/3 complex activity. Mutations in genes of these nucleators lead to various phenotypic traits. This thesis deals in the first place with influence of Arp2/3 complex subunits' dysfunction on intracellular motility (cytoplasmic streaming, stop-and-go movements of Golgi apparatus cisternae), since it had not been extensively studied before, and also attempts to quantify the already known impacts of mutations in genes for ARP2 and ARPC5 subunits on the vacuolar morphogenesis. For comparison, a few experiments with plants which carried a mutation in gene for FH1 formin were also realised when measuring the cytoplasmic streaming. The experiments were conducted with a model plant Arabidopsis thaliana. The methods particularly included transformation with fluorescent markers by Agrobacterium tumefaciens (or usage of a fluorescent dye), microscopy (both standard and confocal) and subsequent evaluation of the acquired data using a computer. During the cytoplasmic streaming research, effects of cytoskeletal drug latrunculin B were studied, too. The outputs did not prove that the Arp2/3 complex defects would manifest...
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The role of ARP2/3 complex in plant cells
Schiebertová, Petra ; Schwarzerová, Kateřina (advisor) ; Martinec, Jan (referee)
2 Abstract ARP2/3 protein complex is formed from seven proteins (ARP2, ARP3 and ARC1- ARPC5) with a relatively conserved structure. ARP2/3 complex branches and nucleates new actin filaments. This thesis focuses on the study of the role and importance of the individual subunits of the complex ARP2/3 in plants. One of the principal aims of this work is to determine whether complex ARP2/3 may at least partially maintain its role when one or more of the subunits are not available. Furthermore if the individual subunits play another, plant-specific role and if the subunits are functionally equivalent in the complex. The main way how to achieve this objective is the analysis of multiple mutants of Arabidopsis thaliana in subunits of ARP2/3 complex. After comparing several phenotypes of mutant lines it is obvious that all the subunits are functionally equivalent. A loss of ARPC5 subunit usually manifests the strongest phenotypic expression. On the contrary, loss ARPC3 and ARPC2b subunits have weak phenotypic manifestations. Because some phenotypes, such as phenotype distorted trichomes was detected only in some mutant lines, whereas the phenotype of faster roots gravitropic response or vacuolar system fragmentation that was detected in all analyzed mutants suggests, that different subunits play varying roles...
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Analysis of endomembrane markers in the cortical cytoplasm and their co-localization with Arp2/3 complex
Jelínková, Barbora ; Schwarzerová, Kateřina (advisor) ; Fendrych, Matyáš (referee)
ARP2/3 is an evolutionarily conserved heteroheptameric protein complex. Its main activity lies in the nucleation of dendritic actin filaments that are involved in membrane remodeling. ARP2/3 takes part in plasma membrane remodeling and the formation of cytoplasmic protrusions that serve in the amoeboid motion of mammalian cells and some protists and plays role in exocytosis and endocytosis of animal and yeast cells. The main objective of this work was to find a connection between the ARP2/3 complex and the regulation of the plant endomembrane system. Using TIRF microscopy we visualized the localization of the ARP2/3 complex in the cortical layer of plant cells and compared it to the localization of several endomembrane markers from the Rab family and an exocytotic marker Exo84b. In the vicinity of the plasma membrane, the ARP2/3 complex subunits localized to dynamic dots very similar to the localization of Exo84b protein. Colocalization analysis showed that a small portion of Exo84b marker and ARP2/3 complex signals colocalize and this result was seconded by the biochemical approach of coimmunoprecipitation. Key words: ARP2/3, endomembrane system, cortical layer, RabA1g, RabC1, RaD2a, Exo84b
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Actin and the ARP 2/3 complex in the nucleus
Němcová, Barbora ; Bellinvia, Erica (advisor) ; Hála, Michal (referee)
The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. Actin constitutes a wide family of proteins that are major components of the cytoskeleton. Actin is one of the most abundant proteins in living organisms. Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes. Recent studies have shown that actin is actively transported from the cytoplasm to the nucleus, where it regulates transcriptional aktivity, regulates RNA polymerases, is involved in chromatin remodeling and repair damaged DNA. The presence of typical actin filaments in the nucleus has not been demonstrated directly.but nuclear actin occurs in many forms such as actin rods, short actin polymers, actin monomers, or actin complexes with profilin or cofilin. Most eukaryotic cells also contain at least eleven actin-related proteins (ARPs). Although many ARPs are cytoskeletal, recent biochemical and genetic work has demonstrated that some ARPs function largely or entirely in the nucleus. Nuclear ARPs are recognized as novel key regulators of genome function, and affect not only the remodeling of chromatin but also the...
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The effect of induced silencing of ARP2/3 complex subunits on plant cell structure
Fišerová, Kamila ; Fischer, Lukáš (advisor) ; Oulehlová, Denisa (referee)
This thesis is focused on the ARP2/3 complex, which is a de novo actin cytoskeleton nucleator. This highly conserved complex is composed of seven subunits and regulates branching of actin filaments at a constant angle of 70 degrees. In plant and animal cells ARP2/3 is involved in various processes, which are connected with the initiation of actin polymerization; for example it participates in determining the direction and speed of cell growth and the movement of vesicles and organelles within the cell. The mutation of individual subunits is lethal for animal cells, but in plants, these mutants have only mild symptoms such as distorted trichomes or changes in epidermal cells. The aim of the presented work was to study the function of the ARP2/3 complex by the method of partial silencing of subunits using RNA interference. Specifically, it was the ARPC1 subunit of Arabidopsis thaliana and the ARPC2 subunit studied on the cellular model, the tobacco BY-2 cell line. Experimental work involved the creation of DNA constructs for induction of silencing, transformation of plant material, silencing rate analysis, and phenotype tracking in selected lines. Although lines with reduced transcript levels of the given ARP2/3 complex subunit were found, no phenotypic changes were observed in these lines. Key words...
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The effect of Arp2/3 complex on vacuolar structure, cytoplasmic streaming and dictyosome motility.
Semerák, Matěj ; Schwarzerová, Kateřina (advisor) ; Eliášová, Kateřina (referee)
In plant cells, actin filaments are nucleated in two different ways: The growth of single filaments or their bundles is enabled by various types of formins, whereas branched meshworks emerge due to Arp2/3 complex activity. Mutations in genes of these nucleators lead to various phenotypic traits. This thesis deals in the first place with influence of Arp2/3 complex subunits' dysfunction on intracellular motility (cytoplasmic streaming, stop-and-go movements of Golgi apparatus cisternae), since it had not been extensively studied before, and also attempts to quantify the already known impacts of mutations in genes for ARP2 and ARPC5 subunits on the vacuolar morphogenesis. For comparison, a few experiments with plants which carried a mutation in gene for FH1 formin were also realised when measuring the cytoplasmic streaming. The experiments were conducted with a model plant Arabidopsis thaliana. The methods particularly included transformation with fluorescent markers by Agrobacterium tumefaciens (or usage of a fluorescent dye), microscopy (both standard and confocal) and subsequent evaluation of the acquired data using a computer. During the cytoplasmic streaming research, effects of cytoskeletal drug latrunculin B were studied, too. The outputs did not prove that the Arp2/3 complex defects would manifest...
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