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Role of tau phosphorylation in formation of tau envelopes
Karhanová, Adéla ; Lánský, Zdeněk (advisor) ; Štěpánek, Luděk (referee)
Tau is an intrinsically-disordered microtubule-associated protein important for axonal development and a critical regulator of microtubule functions in axons. Tau activity is controlled by phosphorylation and its deregulation resulting in tau hyperphosphorylation and aggregation has been linked to multiple neurodegenerative disorders, collectively termed tauopathies. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive "envelopes" around microtubules. Tau envelopes regulate the action of other microtubule-associated proteins, such as the motility of molecular motors, and protect microtubules against degradation by microtubule-severing enzymes. How the formation, dynamics, and function of tau envelopes are regulated, however, is unknown. Here we show that tau phosphorylation impedes the formation and functioning of protective tau envelopes. Using a combination of reconstitution experiments and live cell imaging, we show that phosphorylated tau incorporates into tau envelopes and that it slows down the envelope growth. Importantly, we demonstrate that phosphorylated tau also destabilizes already existing envelopes leading to their disassembly. Together, our results demonstrate...
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Steric exclusion effects and biochemical activity of molecular motors
Brucknerová, Adéla ; Lánský, Zdeněk (advisor) ; Hendrych, Tomáš (referee)
Bachelor Thesis: Steric exclusion effects and biochemical activity of molecular motors Adéla Brucknerová, 2022 The cytoskeleton and its associated proteins, the so-called molecular motors, belong to the cellular apparatus of every eukaryotic cell. Molecular motors, including kinesin-1, have the ability to temporarily bind to the cytoskeleton and transport cargo vesicles (e.g. organelles) along it. During such movement, kinesin-1 molecules hydrolyse one ATP molecule for each step of their chemomechanical cycle. It turns out, that the chemomechanical cycle may be affected by the presence of inert macromolecules. The influence of these macromolecules on the chemomechanical cycle of kinesin-1 is, however, not entirely known. Using total internal reflection fluorescence microscopy, single molecules of kinesin-1 were tracked in vitro. Their movement along reconstituted microtubules was recorded and the average velocity was measured. The performed experiments were carried out with variety of concentrations of soluted macromolecules - polyethylene glycol. For experiments, polyethylene glycols of molecular weight 6 kg/mol, 1000 kg/mol and combination of both were used. From average velocities, biochemical activity of kinesin-1 molecules at various concentrations of polyethylene glycol was obtained. In...
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Identification and characterization of ciliary tip proteins
Gorilák, Peter ; Varga, Vladimír (advisor) ; Lánský, Zdeněk (referee) ; Dean, Samuel (referee)
The distal tip of the cilium/flagellum, also known as the ciliary tip domain (CTD), is critical for the structure and function of the eukaryotic cilium. The limited knowledge of its protein constituents hinders a better understanding of the domain. In this thesis, we set out to verify the localization of a subset of known mammalian CTD constituents and to assess the localization of candidate CTD proteins, orthologs of which localize to the tip of the flagellum of evolutionary distant protozoan Trypanosoma brucei. Using our localization pipeline, we identified two proteins that robustly localize to the CTD of the primary cilium. One of these proteins (ZC2HC1C), in addition, also localizes to stationary foci along the axoneme, positions of which coincide with sites of intraflagellar train pausing and turning. We hypothesize that these may be ends of sub-distally terminating axonemal microtubules. We further show that the protein ULK4 localizes to the CTD of motile ependymal cilia but not to the CTD of primary cilia, consistent with previously published phenotypes in ULK4 depleted mice and exemplifying differences in the composition of CTDs of the two types of cilia. Finally, we demonstrate that Expansion microscopy, a rapid and robust super-resolution technique, is well suited for ultrastructural and...
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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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Regulation of the microtubule severing activity of katanin
Podhájecký, Roman ; Lánský, Zdeněk (advisor) ; Libusová, Lenka (referee)
Cytoskeleton is a dynamic system which contributes to a variety of cellular pro- cesses, for example cell division or cell motility. Katanin is a microtubule associated protein and its key feature is the microtubule severing. The microtubule severing is important for microtubule array amplification and its organization. However, precise control of Katanin activity is needed, otherwise dysregulated microtubule severing results in severe developmental disorders. Here, we investigated Katanin localization to microtubule crossovers and consequences of tubulin post-translational modifica- tions on Katanin severing activity. We found that Katanin localization to crossovers is its inherent property and the contribution of the subunit p80 to Katanin localiza- tion to crossovers is insignificant. Our results suggest that the subunit p80 localizes to crossovers via unknown interacting partner. We also conducted characterization of Katanin binding and severing in the context of different post-translational mod- ifications and tubulin isotypes. We found that despite higher binding of Katanin to heavily-modified microtubules, its severing activity is lower. Our results em- phasize the importance of post-translational modifications and its potential role in organization of cellular processes. Key words:...
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MAP code and regulation of microtubule-based processes
Karhanová, Adéla ; Lánský, Zdeněk (advisor) ; Tomášová, Štěpánka (referee)
Microtubule associated proteins (MAPs) are considered as key regulators of molecular trafficking in cells. Even though their malfunctioning results in severe pathologies, such as neurodegenerative disorders, the regulatory roles of these proteins remain under debate. Since MAPs bind to the cytoskeleton, this structure has to be vital for the function of MAPs. Microtubules, a highly dynamic type of cytoskeletal structure, have been given extra attention due to their association with cell division and vital functions in neurons. Microtubules can undergo post-translational modifications that affect molecular motors as well as binding of other proteins, such as MAPs. Whether post-translational modifications of microtubules regulate the distribution of MAPs is so far not sufficiently documented. However, MAPs have been shown to cooperatively form cohesive envelopes on the microtubules and thereby regulate the access of motors and severing enzymes. As there are many types of MAPs and they are mutually exclusive, a hypothesis of a regulatory 'MAP code' emerged recently in the literature. Using available literature, this review will try to introduce the new model of MAP code and provide some background information on previous research on this topic.
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Quantitative fluorescence microscopy techniques to study three-dimensional organisation of T-cell signalling molecules.
Chum, Tomáš ; Cebecauer, Marek (advisor) ; Lánský, Zdeněk (referee) ; Brameshuber, Mario (referee)
10 SUMMARY Proteins represent one of the basic building blocks of all organisms. To understand their function at the molecular level is one the critical goals of current biological, biochemical and biophysical research. It is important to characterise all aspects that affect the localisation of proteins into different compartments with specific functions, the dynamic structure of proteins and their role in multiprotein assemblies, because altering these properties can lead to various diseases. Most of the proteomic studies are nowadays performed using biochemical approaches that allow us to study multicellular organism or tissue at once. The disadvantage of these methods is complex preparation of sample and the need for a large number of cells, which leads to the loss of information at the molecular level and in individual cells. On the contrary, microscopy can provide rather detailed information about proteins of interest and at the level of a single cell. A variety of fluorescence microscopy methods in combination with recombinant DNA techniques were applied to elucidate subcellular localisation of transmembrane adaptor proteins (TRAPs) in human lymphocytes and their nanoscopic organisation at the plasma membrane. Linker of activation of T lymphocytes (LAT), phosphoprotein associated with...
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Regulation of microtubule dynamics revealed by single-molecule TIRF and IRM microscopy
Zhernov, Ilia ; Lánský, Zdeněk (advisor) ; Cifra, Michal (referee) ; Varga, Vladimír (referee)
The microtubular cytoskeleton is a ubiquitous and highly diverse biopolymer network present in all eukaryotic cells. Microtubules stochastically alternate between phases of growth and shrinkage. Cells take advantage of this dynamicity to generate forces for essential processes, such as cell division, motility or morphogenesis. Regulating the microtubule dynamics enables cells to adaptively respond to a wide range of tasks and conditions. Molecular mechanisms underpinning the regulation are not fully understood. Using a bottom-up approach and the combination of single molecule total internal reflection fluorescence (TIRF) microscopy and interference reflection microscopy (IRM), we here reconstituted and explored two dynamic cytoskeletal systems. (i) Microtubule doublets, comprising incomplete B-microtubule on the surface of a complete A- microtubule, provide an essential structural scaffold for flagella. Despite the fundamental role of microtubule doublets, the molecular mechanism governing their formation is unknown. We here demonstrate an inhibitory role of tubulin C-terminus in microtubule doublet assembly. By partial enzymatic digestion of polymerized microtubules followed by the addition of free tubulin in the presence of a stabilizing agent, we assembled microtubule doublets and revealed the B-...
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