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Characterization of Euglena gracilis and their role in metaboly movement
Šmída, Adam ; Hampl, Vladimír (advisor) ; Pergner, Jiří (referee)
Euglena gracilis is a free-living freshwater flagellate from the class Euglenida (Euglenozoa, Discoba, Excavata). The synapomorphy of Euglenida group is their pellicle, a firm layer underneath the surface of the cell which is composed of proteinaceous stripes (pellicle strips). The strips are locked into each other and run along the cell`s surface underneath the plasma membrane. In E. gracilis these strips can move by parallel sliding, therefore contracting the cell and changing its surface which results in a characteristic peristaltic euglenoid movement called metaboly. The mechanism behind this movement is still unresolved, however, it is expected that the force is generated by the cytoskeletal structures beneath the pellicle strips which consist of microtubules and other unknown cytoskeletal fibers. This thesis tests a hypothesis which proposes that centrins are involved in the pellicle contraction. Centrins are small proteins capable of binding calcium ions, they are very conserved throughout the eukaryotic tree and serve many purposes in the cell, of which one is to form contractile structures in ciliates species e.g., Vorticella or Paramecium. Sequences of centrins were searched for in the transcriptome of E. gracilis and after comparison with the available proteomic dataset of the isolated...
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Cellular determinants of the distribution of PIN auxin transporters in the plasma membrane
Stelate, Ayoub ; Petrášek, Jan (advisor) ; Mravec, Jozef (referee) ; Nodzynski, Tomasz (referee)
Asymmetric localization of auxin carriers has always attracted the attention of many scientists around the world. However, to address this topic while focusing on the plasma membrane (PM), requires advanced microscopy techniques and knowledge of biophysics and biology. This doctoral work bridges the two disciplines to contribute to our understanding of the dynamics and distribution of PIN-FORMED auxin carriers from tobacco (NtPINs) within the PM. I have developed a novel correlative light electron microscopy (CLEM) method using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). To my knowledge, this is the first effort to achieve a correlation between immunofluorescence and electron microscopy imaging of plant integral PM proteins. As I have shown, individual NtPINs are organized differently within the PM. Dynamic analyses that quantify individual nanodomains' diffusion rates allowed me to show that NtPINs have constraints behavior with different diffusion rates. I have investigated the role of the cell wall and cytoskeleton in the organization and dynamics of NtPINs. Using pharmacological treatments, I have shown that they differentially affect the mobility and organization of NtPINs within the PM. Complete removal of the...
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LINC complex: The link between chromatin integrity and sperm motility
Šanovec, Ondřej ; Komrsková, Kateřina (advisor) ; Lánská, Eva (referee)
The LINC complex (Linker of the Nucleoskeleton and Cytoskeleton) is a protein structure located in the nuclear membrane that connects the cytoskeleton with the nucleoskeleton. This complex can be found in every mammalian cell including the gametes. However, here the LINC complex is more diverse and less studied than in the somatic cells. In this thesis, the LINC complex and its role in spermiogenesis have been studied in wild-type and Protamine 2 knockout (Prm2-/- ) mice. Protamines are small proteins that replace histones during spermiogenesis. The mouse model generated by the group of prof. Hubert Schorle has a deletion in Prm2 in exon 1 and its sperm possess a surprising phenotype including complete loss of motility. Therefore, it was hypothesized that the LINC complex might be responsible for miscommunication between the sperm head and tail which leads to the loss of sperm motility. Results from this study suggest that the LINC complex is not influenced by Prm2 deletion, however, actin dynamics, cytoskeletal motor proteins and tubulin acetylase/ histone deacetylase activity might be impaired. Prm2-/- sperm have a significantly higher abundance of β-actin compared to the wild type. Next, Prm2-/- sperm also show a different pattern of acetylation of α-tubulin but no change in the abundance of...
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The mechanism of impaired formation of head-tail coupling apparatus in centrobin deficient acephalic spermatozoa
Vlčková, Monika ; Frolíková, Michaela (advisor) ; Krejčová, Tereza (referee)
Acephalic sperm syndrome (ASS) is a rare form of teratozoospermia that is probably genetic in origin. The sperm of individuals with this syndrome have a damaged apparatus connecting the head and flagellum (HTCA), which leads to the formation of acephalic sperm, i.e. sperm with separate heads from flagella. Individuals affected by ASS are almost exclusively infertile. One of the proteins whose mutation causes ASS is SUN5. Together with KASH proteins, SUN proteins are part of the so-called LINC complex, which ensures the connection of the nucleoskeleton with the cytoskeleton. Centrobin is a protein involved in centriole duplication and assembly of the dividing spindle. Rats carrying a defective gene for centrobin have reduced fertility and exhibit an ASS phenotype. For that reason, this model organism was used in this work to study the possible interrelationships of proteins involved in the formation of ASS. Given the similar phenotype of centrobin and SUN5 mutated spermatozoa, there is a possibility that centrobin interacts with one of the LINC complex proteins and together they participate in the formation of the head-flagella junction. Sperm from rats with a defective centrobin gene also show significant damage to the flagella. Decreased Odf1 protein expression has previously been reported in ASS...
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The role of actin cytoskeleton in the targeting of auxin carriers to the plasma membrane.
Kebrlová, Štěpánka ; Petrášek, Jan (advisor) ; Pernisová, Markéta (referee)
Auxin plays an important morphogenic role in plant development, mainly through its effect on gene expression, but also through a number of faster processes that are directly dependent on its concentration. Therefore, in many plant tissues, directional auxin transport using specific transporters in the plasma membrane, is important for the coordination of morphogenic stimuli. The amount of auxin carriers in the plasma membrane directly affects the resulting auxin concentration inside the cell. Although the localization of auxin transporters and their abundance in the plasma membrane could be determined primarily by the actin cytoskeleton and its involvement in vesicle transport processes, this relationship is currently still unclear. Therefore, in this study, we were interested in how the localization and function of auxin transporters is affected when the function of the actin cytoskeleton is affected in a given cell type. To this end, the localization of the auxin transporters PIN3, PIN4, PIN7, and AUX1 was studied in epidermal cells of cotyledons in young seedlings of Arabidopsis thaliana whose morphogenesis was affected by mutations in subunits of the actin nucleation complex ARP2/3. Crosses of mutants in the ARP2/3 complex subunits with marker lines carrying fluorescently labeled auxin carriers...
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Roles of tubulin post-translational modifications in regulation of microtubule-based processes
Šliková, Pavlína ; Novák, Petr (advisor) ; Libusová, Lenka (referee)
Microtubular cytoskeleton plays crucial roles during diverse cellular processes, such as intracellular transport, cell motility and chromosome segregation during cytokinesis. Tubulin, the building block of microtubules, undergoes numerous post-translational modifications which affect microtubular dynamics and organization as well as their interaction with associated proteins. Understanding the role post-translational modifications play in the diversification of functions and properties of microtubules is key for our comprehension of the dynamics of the complex microtubule cytoskeleton. However, mechanisms behind the effect of post-translational modifications on microtubule cytoskeleton are not fully understood. In this work, we focus on the influence of post-translational modifications on microtubule polymerization and interaction with molecular motor kinesin-1. Using total internal fluorescence and interference reflection microscopy techniques, we here show that high levels of post-translational modifications on microtubules decrease the time of microtubule-kinesin interaction whereas binding affinity and median velocity are not significantly different on modified and unmodified microtubules. Further, we show that the absence of polyglutamylation on tubulin isotypes leads to a faster microtubule...
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Structural and functional studies of signaling molecules in axon guidance
Knapp, Kryštof ; Rozbeský, Daniel (advisor) ; Novák, Petr (referee)
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical, biophysical, and bioinformatical approaches. MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes, as they facilitate the reorganization of the actin cytoskeleton. MICAL-1 has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments. However, recent findings indicate that MICAL-1 can also serve as a sig- naling molecule, using localized hydrogen peroxide production to regulate other downstream effectors. Despite the consensus that MICAL-1 activity must be strictly regulated, the exact molecular mechanism of this regulation has not yet been described. In this work, we provide a novel model of MICAL-1 autoinibiton mechanism based on a comparison of steady-state kinetic experiments and molecular dynamics simulations between full-length MICAL-1 from Coturnix japonica and its truncated form lacking the C-terminal domain. In our model, we conclude that changes in MICAL-1 activity are the result of intramolecular protein interac- tions between the C-terminal and the monooxygenase domain. Furthermore, we rule out the role of MICAL-1 oligomerization in its activity...
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Mechanisms of symmetry breaking during embryogenesis
Ždímalová, Michaela ; Lánský, Zdeněk (advisor) ; Búran, Peter (referee)
Left-right symmetry breaking is crucial for correct body development of many organisms, including humans. The fact that the left-right asymmetry is established consistently in all healthy individuals of given species fascinates researchers for a long time. Although several models offering a mechanistic insight to this phenomenon were proposed or already accepted, they lack a sufficient molecular description or do not explain all cases. A model of acto-myosin flows - intracellular counter-rotating flows driven by an active torque generation in acto-myosin cortex - leading to the left-right symmetry breaking during embryogenesis is a topic of particular interest in current research. This thesis introduces the problematics of acto-myosin flows in a context of the previous research related to the left-right symmetry breaking. Since the left-right asymmetry is tightly associated with chirality at different scales, this thesis also discusses the current knowledge about possible processes of propagating chirality of molecules to the larger scale. 1
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Non-neuronal cell outgrowth in Caenorhabditis elegans model system
Smidžárová, Lucie ; Macůrková, Marie (advisor) ; Strouhalová, Kateřina (referee)
1 Abstract Oriented cell growth is an important process for regular tissue development, in which regulated changes in cell shapes occur. An important role in this mechanism is played by remodelling of cytoskeletal elements in the growing parts of the cell, which is regulated by many extracellular and intracellular factors. This bachelor's thesis is focused on oriented non-neuronal cells growth in the model organism Caenorhabditis elegans, specifically the growth of the excretory cell and the anchor cell invasion. The growth mechanism of the excretory canal cell is in many aspects similar to growth of neurons, however it also involves a unique mechanism of growth through extension of an intracellular lumen. In case of the anchor cell the growth mechanisms support the basement membrane disruption and invasive behavior reminiscent of metastatic cancer cells. Regulation of growth of these two cells thus could serve as a good model for human pathologies such as cystic kidney disease or cancer cell metastasis, and therefore further study in this area could lead to new insights into these diseases. At the end of the thesis, other non- neuronal cells with oriented growth in the model organism C.elegans are briefly described. Key words: cell outgrowth, excretory canal cell, anchor cell, signalling, cytoskeleton
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Computational Simulation of Mechanical Behaviour of Endothelial Cells
Jakka, Veera Venkata Satya ; Gumulec, Jaromír (referee) ; Majer, Zdeněk (referee) ; Matsumoto,, Takeo (referee) ; Burša, Jiří (advisor)
Ateroskleróza je v rozvinutém světě hlavní příčinou úmrtí a finančně zatěžuje zdravotnické systémy po celém světě. Převládající hemodynamické působení spolu s lokální koncentrací mechanického napětí hrají důležitou roli v lokální povaze aterosklerózy a jejím rozvoji ve specifických oblastech lidských cév. Endotel v krevních cévách je tvořen tenkou vrstvou buněk, ležící na rozhraní mezi krevním řečištěm a cévní stěnou. Dysfunkce endoteliálních buněk se podílí na hlavních patologiích. Například ateroskleróza se rozvíjí, když jsou narušeny bariérové a protizánětlivé funkce endotelu, což umožňuje akumulaci cholesterolu a dalších materiálů v arteriální stěně. U rakoviny je klíčovým krokem v růstu nádoru jeho vaskularizace a proces migrace endoteliálních buněk. Mechanické zatížení endoteliálních buněk hraje klíčovou roli v jejich funkci a dysfunkci. Počítačové modelování může zlepšit porozumění buněčné mechanice a tím přispět k poznání vztahů mezi strukturou a funkcí různých typů buněk v různých stavech. K dosažení tohoto cíle jsou v této práci navrženy konečnoprvkové modely endoteliálních buněk, tj. model buněk plovoucích v roztoku a model buněk přilnutých k podložce, které objasňují reakci buňky na globální mechanické zatížení, jako je tah a tlak, jakož i model buňky s jeho přirozeným tvarem uvnitř endoteliální vrstvy. Zachovávají hlavní principy tensegritních struktur, jako je předpětí a spolupůsobení jednotlivých součástí, ale prvky se mohou organizovat vzájemně nezávisle. Při implementaci nedávno navržené bendo-tensegritní koncepce uvažují tyto modely namáhání mikrotubulů nejen v tahu/tlaku, ale i ohybu a také zohledňují vlnitost intermediálních filament. Modely umožňují, že jednotlivé komponenty cytoskeletu mohou změnit svůj tvar a uspořádání bez zhroucení celé buněčné struktury, dokonce i když jsou odstraněny, a umožňují nám tak vyhodnotit mechanický přínos jednotlivých cytoskeletálních složek k buněčné mechanice. Navržené modely jsou validovány porovnáním jejich křivek síla-posunutí s experimentálními výsledky. Model plovoucí buňky realisticky popisuje silově-deformační odezvu buňky při tahu a tlaku a obě reakce ilustrují nelineární zvýšení tuhosti s mechanickým zatížením. Je simulována také tlaková zkouška ploché endoteliální buňky a porovnána s testem přilnuté buňky a jeho simulací. Poté se simuluje smykový test ploché buňky, aby se vyhodnotilo její chování při smykovém zatížení vyskytujícím se v cévní stěně v důsledku proudění krve. Poté byla zkoumána mechanická odezva ploché buňky ve vrstvě endotelu za fyziologických podmínek v arteriální stěně. Později byla zkoumána buněčná odezva při odtrhování od položky během cyklických úseků pomocí 3D simulací metodou konečných prvků. Navrhované modely poskytují cenné poznatky o vzájemných souvislostech mechanických vlastností buněk, o mechanické roli jednotlivých cytoskeletálních složek i jejich synergii a o deformaci jádra za různých podmínek mechanického zatížení. Proto by práce měla přispět k lepšímu pochopení cytoskeletální mechaniky, zodpovědné za chování buněk, což může zase pomoci při zkoumání různých patologických stavů souvisejících s buněčnou mechanikou, jako je rakovina a vaskulární onemocnění.
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