|
|
The role of cytochalasins in cancer cell migration and invasiveness
Brož, Alexandr ; Brábek, Jan (advisor) ; Bašta, Miroslav (referee)
While metastasis causes the majority of cancer related deaths, purely migrastatic drugs have been neglected in clinical research. This trend has been changing in recent years, with drugs capable of preventing metastasis, migrastatics, coming into focus. Cytochalasins are a large group of actin-directed fungal metabolites, often used for cytoskeletal and anticancer research. As the actin cytoskeleton plays a major role in tumour cell invasion and migration, processes integral in metastasis, it is possible that cytochalasins could have a future as migrastatics. The majority of cytochalasin-related research pertains to cytochalasin B and the more potent cytochalasin D, making both a large focus of this thesis, though the lesser researched cytochalasins A, E, H, Q and others are mentioned as well. Cytochalasins B and D have been shown to inhibit migration and invasion of cells with liposome encapsulation reducing side effects greatly, possibly enough to alleviate one of the main concerns of any cytochalasin therapy, toxicity. Though other cytochalasins have also been shown to inhibit cell motility, any conclusions as to their migrastatic translational potential are hard to draw due to the lack of research.
|
|
|
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...
|
|
|
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...
|
|
|
Cell polarity establishment and changes during Saccharomyces cerevisiae cell cycle
Luxová, Pavla ; Malcová, Ivana (advisor) ; Šťovíček, Vratislav (referee)
Cell polarity can be defined as an asymmetric organization and distribution of biomolecules, cellular organelles and structures which are important for many cellular processes. Cell polarity establishment is essential for the proper development of all organisms. This work focuses on main mechanisms of cell polarity establishment, its maintenance and changes during Saccharomyces cerevisiae cell cycle. Budding yeast is one of the preferred model organism. Bud site selection is determined by the spatial landmarks which are accumulated at the previous division site. The spatial landmarks are recognized by Rho GTPases which act on their effectors and thus affect the actin cytoskeleton and septins. These structures are essential for polarized bud growth that is coordinated with the cell cycle. Newborn cells arising after the bud separation from the mother cell at the end of each cycle are able to undergo many more division cycles than their mothers what is a new challenge to study cell polarity in terms of cell aging.
|
guest ::
