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Reorganization of in vitro reconstituted actin-based networks
Sabó, Ján ; Lánský, Zdeněk (advisor) ; Varga, Vladimír (referee) ; Cifra, Michal (referee)
The cytoskeleton provides living organisms with machinery to move. On the molecular scale, the same cytoskeletal components undergo constant reorganization to contribute to distinct cellular processes, such as the navigation of neuronal growth cones in brain development or the creation of chiral actin flows during cellular division. During brain development, neuronal growth cones navigate by employing cytoskeletal crosstalk between actin filament networks and microtubules. Crosstalk in the growth cones between these cytoskeletal components was linked with known microtubule polymerase human cytoskeleton associated protein 5 (CKAP5; homolog of XMAP215, MSPS, Zyg9), while the details of the direct molecular mechanism of CKAP5-provided remodeling of both microtubules and actin networks are lacking. Therefore, we used total internal reflection fluorescence (TIRF) microscopy together with state- of-the-art in vitro reconstituted assays combining microtubules, actin filament networks, and recombinant proteins to understand cytoskeleton-provided molecular mechanisms underlying cellular motion. In this thesis, we show that CKAP5 alone bundles both supposedly randomly oriented and parallel actin filaments, crosslinks actin filaments to microtubules regardless of their polarity, positions prevailing actin...
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The role of CKAP5 in mediating crosstalk between actin and microtubule cytoskeleton
Dujava Ždímalová, Michaela ; Lánský, Zdeněk (advisor) ; Middelkoop, Teije Corneel (referee)
Dynamic changes in cytoskeletal architecture are essential for many crucial events throughout lives of all cells. One of the examples is neuronal pathfinding, ensured by specialized axonal structures enriched in actin and microtubule cytoskeleton, known as neuronal growth cones. Growth cones act as motile sensors and navigators, as the tight regulation of their actin and microtubule cytoskeleton results in directed axonal outgrowth guided towards the proper targets, which is crucial for processes such as synaptogegnesis or regeneration. Recently, protein CKAP5 (previously described as a microtubule + tip polymerase) has been proposed to facilitate actin-microtubule crosstalk in growth cones, indispensable for their proper functioning. Here, we combine the power of in vitro reconstitution assays with total internal reflection fluorescence microscopy to explore the underlying mechanism of CKAP5 actions. Our findings confirm that CKAP5 also associates with actin filaments and indeed, recruits them to the microtubule lattice. Furthermore, we describe a remarkable behavior of dynamic system containing CKAP5 and both, microtubules and actin filaments, wherein actin bundles are templated along dynamic microtubules by CKAP5. Importantly, upon microtubule depolymerization, the corresponding actin bundle can...
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Tau proteins cooperatively assemble into cohesive envelopes that protect microtubules against severing enzymes
Siahaan, Valerie ; Braun, Marcus (advisor) ; Middelkoop, Teije Corneel (referee) ; Piliarik, Marek (referee)
Tau is a microtubule-associated protein that is preferentially found in the neuronal axons. In neu- rodegenerative diseases, collectively termed tauopathies, malfunction of tau and its detachment from axonal microtubules, often associated with abnormal phosphorylation of tau, are correlated with axonal degeneration and loss of microtubule mass (Kneynsberg et al., 2017). Tau can protect microtubules from microtubule-degrading enzymes such as katanin (Qiang et al., 2006) and regulate transport by molecular motors along the microtubule (Vershinin et al., 2007; Dixit et al., 2008). However, how tau carries out these regulatory functions is still unclear. Using in vitro re- constitution and TIRF microscopy, we show that tau molecules can bind to microtubules in two distinct modes: either as (i) single tau molecules independently diffusing on the microtubule sur- face, or (ii) cooperatively-bound tau that form a cohesive tau "envelope" enclosing the microtubule lattice (Siahaan et al., 2019; Tanetal., 2019; Siahaan et al., 2022). We found that tau envelope formation alters the spacing of tubulin dimers within the microtubule lattice, where envelope for- mation compacted the underlying lattice, and lattice extension induced tau envelope disassembly (Siahaan et al., 2022). Tau envelopes form a selectively...
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The role of anillin in the growth cone of neurons
Tomášová, Štěpánka ; Libusová, Lenka (advisor) ; Vinopal, Stanislav (referee)
During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...
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Role of NAV3 in glioblastoma cells invasiveness
Legátová, Anna ; Brábek, Jan (advisor) ; Libusová, Lenka (referee)
The invasion of tumor cells from the primary lesion and the formation of metastases are the main reasons for the severe impact of cancer diseases. An option for dealing with this poor impact is the development of drugs (so-called migrastatics) that would target these processes and thus limit the spread of tumor cells from the site of the primary tumor. However, to develop such drugs, it is essential to clarify the molecular mechanisms that control or promote cell migration. One of the possible strategies for migrastatics development is the targeting of cytoskeletal structures, which play an indispensable role in cell migration. This work is focused on Neuron navigator 3 (NAV3), a protein that binds to + ends of microtubules (MTs), participates in their stabilization, and is able to mediate crosstalk between MTs and the actin network. The function of NAV3 is important for directing MTs into growing axons and proper neurite outgrowth, which is necessary for brain development. The results of this thesis suggest that NAV3 could act as a pro-tumor factor, which localizes not only to the + ends of MTs, but also to the cell protrusions, and whose presence supports the cell expansion and increases the invasive potential of glioblastoma cell lines. Key words: neuron navigator 3, microtubules, cancer,...
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Tubulin post-translational modifications and microtubule associated proteins in neural development and disease.
Belyaeva, Polina ; Balaštík, Martin (advisor) ; Siahaan, Valerie (referee)
Microtubules (MTs) are essential components of the cytoskeleton in all eukaryotic cells. Their function is particularly important in neurons where MTs stabilize their long processes and are responsible for the precisely regulated anterograde and retrograde, intra-axonal and intra- dendritic transport over long distances. MTs are essential also during development of the vertebrate brain and all its major steps: neurogenesis, neuronal migration and neuronal differentiation. MTs are regulated at multiple levels, but two seem to be particularly important: 1. posttranslational modifications of tubulin (PTMs) have been shown to control several MT properties as stability or MT-based transport. 2. microtubule-associated proteins (MAPs) that bind soluble MT subunits, MT lattice as well as MT ends and control MT-based transport and MT dynamics by either stabilizing, destabilizing or severing MTs. Consequently, deregulation of either tubulin PTMs or MAPs may induce severe changes in neuronal cytoskeleton. Bachelor's thesis summarizes current knowledge on how PTMs (especially polyglutamylation) and MAPs (especially microtubule cleaving proteins such as spastin) regulate MT and neuronal development and degeneration. Keywords: Microtubules, tubulin post-translational modifications, polyglutamylation,...
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Cytoskeletal orchestration of early mammalian development
Novotný, Jan ; Libusová, Lenka (advisor) ; Mašek, Jan (referee)
Structures of the cytoskeleton play a critical role in early mammalian development. Microtubules, intermediate filaments and actin microfilaments provide structural and mechanical basis for major developmental transitions of preimplantation development. Microvilli supported by bundles of actin filaments become restricted to one pole of the blastomere during the process of polarisation. All components of the cytoskeleton network undergo apparent reorganisation in embryo compaction. Cells flatten and adhere to each other thanks to the formation of cytoskeleton-linked intercellular junctions and oscillatory contractions generated by actomyosin complexes. Filopodia, actin based protrusions, are debated for their potential role in blastomere apposition and cell signalling. The first process of cell line specification in mammalian development is that which defines outer cells of morula as presumptive trophectoderm and inner cells as presumptive inner cell mass. Proteins and structures of cytoskeleton are asymmetrically distributed between these two populations helping to determine the fate of each blastomere. The aim of the thesis is to summarise the roles of cytoskeleton in blastomere polarisation, embryo compaction and specification of inner and outer cell populations in mammalian preimplantation...
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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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The role of cytoskeleton in movement and navigation of neuronal growth cones
Olekšák, Adam ; Libusová, Lenka (advisor) ; Weissová, Romana (referee)
Growth cone is a dynamic structure localized at the tips of growing neurites. It detects guidance cues in the extracellular environment and enables growing neurite to properly respond to them. Cytoskeleton plays a key role in navigation of growing neurite. Microtubules and microfilaments enable the motion of the growth cone by generating force acting on the cytoplasmic membrane as well as on the substrate on which the neuron grows. Microfilaments are located mainly in the periphery of the growth cone. They are more dynamic than microtubules, and their polymerization and depolymerization enable formation of dynamic processes on the growth cone. These are used by neurons to gain information about their surroundings and for formation of adhesions on permissive substrates. Adhesions facilitate the growth of neurites. Direction, timing and speed of formation of the dynamic processes are regulated by a number of actin-associated proteins. These proteins are common targets of signalling pathways that are induced by activation of growth cones' cytoplasmic membrane-bound receptors by guidance cues. Along the microfilament bundles, microtubules enter the periphery of the growth cone aided by crosslinking and motor proteins. Microtubules are necessary for reducing the growth cone dynamics, the first step in...
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Hafnium Chloride, an Alternative Staining Reagent for Biological Electron Microscopy
BARANYI, Magdalena Victoria
This study focuses on the staining pattern of the non-radioactive heavy metal EM stain HfCl4, used during freeze substitution specimen preparation. HfCl4 was investigated as an alternative for the prominent uranyl acetate, since uranium based materials have been placed under heavy restrictions and bans worldwide. We have found a strong HfCl4 staining pattern of microtubules in myoepithelial cells of Ixodes ricinus salivary glands. Additionally, in several samples, HfCl4 was found to completely fill the cytoplasm of the myoepithelial cells. Nonetheless, artefact formation around granulated cells was also experienced.
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