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Mechanisms of establishment and maintenance of PIN polarity in Arabidopsis
Glanc, Matouš ; Friml, Jiří (advisor) ; Grebe, Markus (referee) ; Luschnig, Christian (referee)
Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...
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Endocytosis in plants
Svitáková, Lenka ; Krtková, Jana (advisor) ; Hála, Michal (referee)
Endocytosis is a process typical for eukaryotic cells. It is a mechanism that ensures invagination of the plasma membrane which leads to the creation of a membrane vesicle with specifically selected cargo. The vesicle is then internalized by the cell. Endocytosis has a crucial role in plant growth and development not only thanks to the reception of nutrients from the extracellular space but also by having a huge impact on the membrane composition which influences cellular signalling. There are few types of endocytosis known in plants. They differ from each other by the molecular machinery which ensures their process. The most studied type in plants is the clathrin-mediated endocytosis. Clathrin-coated vesicle formation proceeds through five stages. However, there are less details known about it in plant cells than in animal cells. In the first part of my bachelor thesis I describe recent knowledge of this topic and the most important participants in this process. I also mention the mechanisms of regulations that plant cells use to coordinate clathrin-mediated endocytosis. In the second part I summarize recently discovered types of endocytosis in plants that are independent from clathrin. These are endocytosis associated with microdomains and fluid- phase endocytosis. The details of these endocytic...
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Functions of RAB GTPases and SNARE proteins in post-Golgi trafficking pathways in response to abiotic stress in plants
Moulík, Michal ; Hála, Michal (advisor) ; Mašková, Petra (referee)
Plants as sessile organisms are strongly affected by abiotic and biotic stress factors. Thus, they have developed an array of morfological, biochemical and physiological adaptations to reduce the negative effects of these factors. The membrane trafficking, among others, plays very important role in adaptation to abiotic stress. In my bachelor thesis I have focused on two important protein families involved in this trafficking, namely on RAB GTPases and SNARE proteins. In the first part, the phenomenon of stress is characterized and the strategies how plants cope with the effect of stressors are described, especially the production of reactive oxygen species (ROS) and autophagy. Following chapter deals with an introduction to the membrane trafficking in plants. In the rest of the thesis, I characterize RAB GTPases and SNARE proteins and provide contemporary insight in the mechanism of their function. The aim of the key parts of these chapters is to summarize current knowledge of RAB GTPases' and SNARE proteins' functions in post-Golgi trafficking pathways during response to abiotic stressors or secondary oxidative stress. Key words: plants, abiotic stress, membrane trafficking, secretion, endocytosis, vacuole, RAB GTPases, SNARE proteins
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Effect of pH on initiation of viral infection
Vomáčka, Petr ; Španielová, Hana (advisor) ; Šmahelová, Jana (referee)
In organisms, acidic pH occurs either in the intracellular or extracellular environment. Intracellularly an acidic pH is found in particular cellular compartments, such as early and late endosomes or lysosomes. In the extracellular space, the acidic pH is found in hypoxic tissues, or in tissues where inflammation or tumour growth occurs. The value of the pH is usually in the range of approximately from 5.5 up to 6.5. This work focuses on the description of the conformation changes of viral proteins, which occur due to a decreased pH and lead to escape of viral components from membrane compartments. Changes in pH in organelles of endocytic pathways as well as changes in extracellular pH affect significantly the infection of many viruses. Research on the role of pH during the viral can bring important information for the development of antiviral and anticancer treatment. Key words: virus, conformation, pH, receptor, endocytosis
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Trafficking of purinergic P2X receptor in eukaryotic cell
Pražák, Šimon ; Tvrdoňová, Vendula (advisor) ; Kolář, David (referee)
Purinergic receptors are membrane ion channels activated by extracellular ATP. In vertebrates, seven genes encoding P2X subunits was found. These subunits are designated as P2X1 - 7. Every P2X receptor subunit consists of two transmembrane domains, extracellular domain and intracellular N- and C- termini. P2X receptors fold to homo- or heterotrimers. P2X receptors have a wide distribution in the organism, functional receptors are found in neurons, glial cells, muscle cells and also in nonexcitable tissues as epithelial, endothelial, and in hemopoietic tissue. Purinergic signalling plays an important role in pain transmission, CNS injury and immune processes. P2X receptors are synthesized on the rough endoplasmic reticulum and are transported to the plasma membrane after post-translational modifications in the Golgi apparatus. The distribution and transport of P2X receptors is subunit specific and dependent on the cell type in which they are expressed. P2X receptors can be divided into three groups according to the way they are moved in the cell, which differ in transport speed, plasma membrane accumulation rate and rate of internalization.
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Application on nanoparticles in bio-medicine.
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Beranová, Jana (referee)
During last few years nanoparticles of different origin have been used in biomedicine. Their interactions with cells are however a big point of concern and so further research is needed to be done regarding their properties, internalization into cells and their fate within the cell. All of this is needed for correct selection of proper nanoparticles for bioimaging and also for future use in human medicine. One of the main parts of research is deep understanding of interactions of nanodiamonds and silicon based nanoparticles with cells, because of their highly unique properties (autofluorescence), biocompatibility and in case of silicon nanoparticles also their degradability.
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Phosphoinositides and their effectors in regulation of the Wnt signalling pathway
Knop, Filip ; Macůrková, Marie (advisor) ; Krausová, Michaela (referee)
Phosphoinositides (PIs) make up only a small proportion of overall amount of lipids in cell membranes. However, their function mediated through protein effectors is indispensable for cell signaling, vesicular trafficking, cell movement and other important aspects of cellular life. In this bachelor thesis function of PIs is described in relation to Wnt signaling pathway. Proper execution of several steps of the Wnt signaling pathway requires the presence of PIs. Retrograde transport of Wntless (Wls) from the plasma membrane (PM) back to the Golgi apparatus (GA) in Wnt producing cells or internalization of Wnt receptors in Wnt receiving cells are only two examples. All processes are tightly regulated and malfunction of enzymes processing PIs can cause their deregulation resulting in disruption of the Wnt signaling pathway. As deregulated Wnt signaling is a known cause of serious diseases including cancer, understanding the crosstalk between PIs and Wnt signaling could help in designing novel strategies for therapeutic intervention.
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Cytoplasmic membrane and tonoplast dynamics during closing and opening of stomata
Röder, Matěj ; Žárský, Viktor (advisor) ; Albrechtová, Jana (referee)
Stomata are epidermal structures mediating regulated contact of plant apoplast with surrounding environment via stomatal opening. Change of turgor plays crucial part in initiation of stomatal opening or closure. During stomatal movement, guard cell undergo considerable and repetitive changes in cell volume and consequently surface area over a period of minutes. Alteration in the surface of membrane must occurs due to limited stretching capability of the plasma membrane. It can be achieved through membrane invaginations and endocytosis of small vesicles. Microscopy and electrophysiology techniques have proven that both processes are happening in guard cells. These processes are controlled and managed by complex web of signal pathways in which actin and microtubular cytoskeletons, SNARE proteins, ion channels and others molecules have crucial parts. The aim of this work is to summarize current knowledge on the processes and mechanisms of these membranes surface changes and their molecular principle.
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Adenylate-cyclase toxin of Bordetella pertussis as a marker for the study of the complement receptor CD11b/CD18 endocytosis.
Chvojková, Věra ; Bumba, Ladislav (advisor) ; Černý, Jan (referee)
Bordetella pertussis is an important human pathogen that causes an infection disease called whooping cough. This gram-negative bacterium produces an adenylate cyclase toxin (CyaA) that recognizes an integrin receptor CD11b/CD18 present on the surface of myeloid phagocytes and delivers an adenylate cyclase (AC) domain into the cell cytosol. This thesis deals with the endocytic machinery of CyaA and its potential use as a specific marker for endocytosis of the CD11b/CD18 receptor molecule. Detoxified mutant of CyaA, CyaA-AC- , that has the capacity to promote calcium influx as well the potassium efflux, was shown to trigger activation of the integrin receptor CD11b/CD18 followed with endocytic uptake by clathrin-dependent pathway. On the other side, the inactive mutant CyaA-KP-AC- that is unable to provoke integrin activation was endocytosed by clathrin-independent pathway. These results suggest that the various endocytic pathways of the CD11b/CD18 are determined by different conformational states of the receptor molecule.
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