National Repository of Grey Literature 40 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
Vesicular roles of Arp2/3 nucleation-promoting factors
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Malínský, Jan (referee) ; Befekadu, Asfaw (referee)
F-actin is involved in key aspects of vesicular traffic, such as membrane deformation, tubulation and vesicle motion. Branching of F-actin is mediated by Arp2/3 but this complex must first be activated by so-called nucleation-promoting factors (NPFs). These factors play an essential role in the decision where and when branched actin should form on the membrane surface. The thesis focuses on the mechanisms which underlie localization and activation of NPFs, especially in terms of the phosphoinositide composition of the vesicle membranes. I show that one of the NPFs, the WASH complex, does not exclusively depend on the retromer complex for its membrane anchoring, as previously theorized. Rather, its understudied subunit SWIP enables the complex to independently bind to the membrane. I also present data showing that the WASH complex has essential roles in maintaining lysosomal function. Additionally, I elucidate the function of another NPF known as WHAMM in the ERGIC compartment, showing that it depends on the presence of myotubularin 9 for its ability to form membrane tubules. The thesis improves our understanding of the interface between the actin cytoskeleton and intracellular membrane system.
Ezrin-Radixin-Moesin (ERM) proteins: The link between tetraspanin web and cytoskeleton in gametes
Picková, Jana ; Frolíková, Michaela (advisor) ; Lánská, Eva (referee)
Ezrin, which belongs to the ERM protein family along with radixin and moesin, plays an important role in linking membrane proteins to the actin cytoskeleton. In sperm, ezrin was described so far in bovine and humans, where it probably contributes in actin polymerization during capacitation. During the acrosome reaction, actin plays a significant role in relocation of protein from the inner acrosomal membrane to the equatorial segment, which is an essential process for successful fertilization. One of the proteins that relocates to the equatorial segment, the site of initiation of gamete fusion, is protein CD9. This protein probably stabilizes protein interactions between sperm and oocyte during gamete fusion. In this diploma thesis, we focused on the role of ezrin in linking the CD9 protein to the actin cytoskeleton in mouse sperm. Using indirect immunofluorescence, we detected ezrin, CD9 and actin in acrosome intact mouse sperm. The co-localization of these proteins in the apical acrosomal region suggests their interactions. However, this was not confirmed by our co-immunoprecipitation experiments. Nevertheless, in the apical acrosomal region we also detected EWI-2 protein, which could act as linker between CD9 and ezrin. In this thesis, we also investigated changes in levels of active...
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...
Molecular mechanisms of the assembly and function of BBSome
Prasai, Avishek ; Huranová, Martina (advisor) ; Varga, Vladimír (referee) ; Bosáková, Michaela (referee)
Bardet Biedl syndrome is a genetic disorder caused by the dysfunction of the BBSome, an octameric cargo adaptor protein complex. The BBSome facilitates the transport of signaling receptors into and out of the primary cilium, a microtubule based sensory organelle of the cell. The first part of this thesis focuses on the elucidation of the assembly of the BBSome in living cells. We generated a library of human and mouse cells lines deficient in the individual BBSome subunits and transduced them with the other YFP tagged subunits. We employed biochemical assays, immunofluorescence and quantitative fluorescence microscopy techniques to analyze the individual steps in the BBSome assembly pathway. We revealed that the BBSome assembly occurs sequentially in spatially regulated steps. We showed that BBS4 nucleates the assembly of a pre-BBSome at the pericentriolar satellites. The translocation of the pre-BBSome to the ciliary base is facilitated by BBS1. We also revealed that in a BBS chaperonin deficient cell line, BBS12 KO cells, a small fraction of the BBSome and/or BBSome sub-complexes are still able to form and localize to the cilium. This could suggest that the BBS chaperonins might act later in the BBSome assembly pathway providing a means for quality control for the BBSome. Ciliary ectocytosis...
Characterization of perinuclear actin fibers and their role in cell migration
Hlaváčková, Tereza ; Vomastek, Tomáš (advisor) ; Binarová, Pavla (referee)
Cell migration is crucial for such physiological and pathological processes as wound healing, emryonal development, immune response, and methastasizing of the cancer cells. It is tightly coupled with cell polarization, nuclear traslocation, and turnover of actin cytoskeleton. Substantial, but so far poorely explored, part of actin cytoskeleton is perinuclear actin cap - dome-like structure above the nucleus costructed from perinuclear actin fibers. At the apical side of the nucleus perinuclear actin fibers are associated with LINC complex through nesprin proteins; at the edges of the cell they are anchored to focal adhesions. In the literature there were assumptions that this type of actin fibers can generate traction forces for nuclear reorientation during cell migration. The aim of this thesis is to elucidate the mechanism involved in the attachment of perinuclear actin to the LINC complex and the nucleus, thereby regulating the formation of the perinuclear actin cap. In addition, we aimed to establish a semi- automatic tool for perinuclear actin fibers quantification. Rat2 fibroblasts were used as the model cell line because they contain well-developed perinuclear actin cap. We focused on the inactivation of LINC complex components, namely Giant nesprin proteins (nesprin 1 and nesprin 2) and...
Materials for education in biochemistry at secondary school - smooth and skeletal muscle
Honomichl, Petr ; Martínek, Václav (advisor) ; Teplá, Milada (referee)
Modern society gives humanity many guarantees and many benefits. Although one of the biggest problems nowadays is that most of us spend much time sitting at computers or TVs. Another problem is the excess of an unsuitable diet, which threatens our health. This lifestyle harms our health and contributes to the outbreak of diseases in civilization, among other things. These diseases are the leading cause of death in civilized countries. It is no wonder that some parts of society are already aware of this issue, and its goal is to motivate people to spend their free time actively, for example, with regular exercise or sports. It is necessary to emphasize the relationship between health problems and a sedentary lifestyle by integrating such topics into teaching grammar school subjects. We could combine chemistry, biology and physical education with helping students find new ways to understand the importance of physical activity. The work focuses on skeletal and smooth muscle because it is closely related to movement and is also the subject of teaching in secondary schools. However, in the current teaching materials, this topic is conceived rather descriptively, with students learning many medical terms, and the interpretation of the mechanism of muscle activity is limited to skeletal muscles. This...
Cytoskeleton-membrane protein interaction network in sperm
Adamová, Zuzana ; Komrsková, Kateřina (advisor) ; Tolde, Ondřej (referee)
In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...
Actin and its regulation in clathrin endocytosis
Pešanová, Denisa ; Libusová, Lenka (advisor) ; Janštová, Vanda (referee)
4 Abstract Actin filaments and their dynamics play an important role in eukaryotic membrane remodelling. Actin regulatory proteins are required for actin dynamics control. During clathrin-mediated endocytosis actin and regulatory proteins interact with clathrin and adaptors proteins. Together they assist membrane invagination and scission of vesicles. Actin structures are fundamental for formation, internalization and movement of endocytic vesicles in yeast. In mammalian cells, actin is less involved especially in the early stage of endocytosis. Models reflecting different roles of actin in clathrin-mediated endocytosis in yeast and mammals are still lacking despite the latest imaging methods. The goal of this bachelor thesis is to offer a compact summary of controversial observations of actin regulation in endocytosis based on recent studies and simultaneously present selected mechanisms of interactions of the most important proteins, which influence actin dynamics in the clathrin-mediated endocytosis. Key words actin, clathrin-mediated endocytosis, Saccharomyces cerevisiae, regulatory proteins
Non-traditional roles of formins besides actin nucleation
Metlička, Jáchym ; Cvrčková, Fatima (advisor) ; Opatrný, Zdeněk (referee)
Formin homology 2 (FH2) domaincontaining proteins (formins) have, since their discovery in 1990, been observed in all analyzed species of eukaryotic kingdoms. Our knowledge of structure and function of the defining FH2 domain has greatly increased over the last couple of years. Its function in nucleation, polymerization and processive capping of actin filaments designates formin protein family an important cytoskeletonremodelling factor. But FH2 domain is just one part of the puzzle additional optional conserved peptide structures surrounding it, as well as concrete variation of the FH2 domain itself, greatly influence the functional properties and cellular localization of the resultant formin protein. Formins have been implicated in variety of cellular processes, which often (but not always) involve the cytoskeleton e.g. Factin network management, crosstalk of Factin filaments and microtubules or plasma membrane. They also partake in processes integral to cell division, function in conserved signalling pathways and much more. This thesis explains the structure and function of FH2 and FH1 domains, outlines the main formin phylogenetic clades in multicellular eukaryotes and reviews various roles that formins fulfill or are thought to fulfill. Such goal, however, is very bold and (considering the...
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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