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Kinases regulating AP2 complex phosphorylation in Caenorhabditis elegans
Zounarová, Apolena ; Macůrková, Marie (advisor) ; Vinopal, Stanislav (referee)
Heterotetrameric adaptor protein 2 complex (AP2) is a fundamental component of clathrin-coated pits playing a part in every step of clathrin-coated vesicle generation. Although the mechanism of AP2 function has been extensively studied for no less than 20 years, the exact role of the regulatory phosphorylation on T156 of its μ2 subunit remains unclear. The main kinase responsible for the T156 phosphorylation in mammals is AAK1; however, many observations suggest that other kinases collaborate on this event. The aim of this project is to shed light on the importance of AP2 phosphorylation in Caenorhabditis elegans and elucidate the function of SEL-5/AAK1 kinase. To determine the relationship between SEL-5/AAK1, AP2 phosphorylation, and the function of endocytosis, we used a combination of phenotype analysis of C. elegans transgenic lines and in vitro assays. We showed that DPY-23, the C. elegans μ2 subunit, is phosphorylated analogously to its human orthologue. We confirmed that DPY-23 phosphorylation depends on SEL-5, yet we were not able to prove the kinase activity of SEL-5 directly. Interestingly, our results further revealed that DPY-23 phosphorylation is dispensable for the endocytosis of a model cargo, and we also showed that interaction motifs located outside the kinase domain of SEL-5 are...
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Mechanical forces in asymmetric cell division
Šabata, Vojtěch ; Macůrková, Marie (advisor) ; Middelkoop, Teije Corneel (referee)
Cell division is one of the most studied topics in the field of cellular and molecular biology. In some cases, cells can exploit diverse mechanisms to alter the basic function of their division machineries to divide asymmetrically. This results in two daughter cells that differ from each other in some manner, which is an essential premise for the development and adult homeostasis of complex multicellular organisms. This work is focused on the division of Caenorhabditis elegans zygote, a classic example of asymmetric division. Using this model system, this work highlights the cellular mechanisms used to generate polarity, with emphasis on the purely mechanical aspects present. In recent years, great progress has been made in describing these fundamental pathways, which are at their core highly conserved from nematodes to humans. Deeper knowledge of processes, that are responsible for a successful cell division in general, can be beneficial for a better understanding of organismal development in health and disease.
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Molecular basis of interactions between Dishevelled 3 (Dvl3) and Protein Regulator Of Cytokinesis 1 (PRC1)
Kropáčková, Veronika ; Bařinka, Cyril (advisor) ; Macůrková, Marie (referee)
Scaffolding protein Disheveled (Dvl) is a key component of Wnt signaling cascades. Dvl participates in a number of biological processes, such as cell proliferation, differentiation and migration, determination of cell polarity, and also stem cell self-renewal. It is therefore indispensable for the correct embryo development and tissue homeostasis in adulthood. The protein regulator of cytokinesis (PRC1) is a microtubule-associated protein. PRC1 is involved in spindle midzone formation during cell division. Spindle midzone precedes the contractile ring assembly and is essential for normal cell cleavage. In our laboratory, PRC1 was identified as a putative interaction partner of DVL3. This master thesis is focused on delineation of the interaction interface between DVL3 and PRC1 using TIRF microscopy (Total Internal Reflection Fluorescence microscopy). To this end, full-length DVL and PRC1 proteins together with their truncated variants were designed, expressed and purified. It was discovered that PRC1 interacts with all three DVL isoforms and the N-terminal part of PRC1 is required for the interaction between PRC1 and DVL3. Furthermore, the DEP domain of DVL3 is likely involved in PRC1interactions. Key words: Dishevelled 3, DVL3, Protein regulator of cytokinesis 1, PRC1, interaction interface, TIRF...
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Wingless/Notch crosstalk in Drosophila melanogaster
Hajšmanová, Hana ; Mašek, Jan (advisor) ; Macůrková, Marie (referee)
Fundamental for the existence of multicellular organisms is the presence signalling pathways, complex biochemical cascades, allowing reciprocal communication amongst the cells. Both Notch and Wnt pathways are crucial for embryonic development and their deregulation leads to many severe diseases. Decades of research, largely based on model organism D. melanogaster, showed that components of these pathways do not function strictly separately but on contrary interact with each other (crosstalk). This phenomenon is explored only to a lesser extent in mammals. The aim of this thesis is to recapitulate existing findings of Wnt/ Notch crosstalk in D. melanogaster and outline where crosstalk could play a role in mammals.
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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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The role of CUP-4 protein in Wnt signalling
Žídek, Radim ; Macůrková, Marie (advisor) ; Pospíchalová, Vendula (referee)
Wnt signalling is indispensible for proper development of organisms and maintaining of adult tissue homeostasis. Its disruption often leads to disease. In nematode Caenorhabditis elegans, Wnt signalling governs vast array of developmental processes, among others also migration of the Q neuroblasts and their descendants. The sole Wnt acting in this process, EGL-20, triggers the canonical β-catenin Wnt signal transduction pathway in QL but not in QR which leads to QL remaining in the posterior while the QR migrates anteriorly. This represents a useful tool for studying Wnt signalling. Recently, mutation of gene cup-4 was found to disrupt migration of the QL neuroblast in a small proportion of the mutant population. cup-4 encodes a ligand-gated ion channel family homologue and it was shown to participate in endocytosis by coelomocytes, specialized phagocytic cells in the C. elegans body cavity. Here, I present the results of my effort to determine the place of CUP-4 action in Wnt signalling and to elucidate the mechanism of its function. I found that CUP-4 acts upstream of PRY- 1/Axin, which is involved in signal transduction in signal receiving cells, and most probably downstream of adaptin AP2, which is important for recycling of Wnt cargo receptor Wntless (Wls) in Wnt producing cell. cup-4 also...
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The role of Fam208a during mouse embryogenesis
Bhargava, Shohag ; Chawengsaksophak, Kallayanee (advisor) ; Machoň, Ondřej (referee) ; Macůrková, Marie (referee)
(in English) Post-implantation embryo development proceeds through several key morphogenetic events that are fine-tuned by epigenetic modifications. Gastrulation is one of the most crucial developmental event that occurs during early post-implantation stage resulting in the formation of the three germ layers together with the establishment of the anterior-posterior (A-P) axis. It requires a highly coordinated interaction between the embryonic and extra-embryonic regions of the developing embryo. Gastrulation initiates with the formation of the primitive streak and, during which, cells of the epiblast delaminate and ingress through the primitive streak to form the mesoderm and definitive endoderm. During early post-implantation stages, the pluripotent cell population of the epiblast undergoes very rapid cellular proliferation and extensive epigenetic programming. There are numerous studies emphasizing various signaling molecules, transcription factors and epigenetic machinery maintaining the genomic stability that drive successful gastrulation and any discrepancy or defects almost always results in embryonic lethality. One such newly highlighted silencing machinery is that of the Human silencing hub (HUSH) complex comprising of the core members; FAM208A; H3K9me3-reader, MPP8 and PPHLN that together...
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Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii
Žídek, Radim ; Kozmik, Zbyněk (advisor) ; Janečková, Lucie (referee) ; Macůrková, Marie (referee)
Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...
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The role of inactive MTMR phosphatases in mammalian cells
Sixtová, Nikola ; Macůrková, Marie (advisor) ; Klíma, Martin (referee)
Variable composition of the cellular membranes influences many cellular events such as endosomal transport, autophagy or cellular signalling. The membrane identity is significantly determined by the specific distribution of phosphoinositide derivatives. These derivatives are specifically distributed among cellular membranes and they are tightly regulated by the interplay of corresponding lipid kinases and phosphatases. Myotubularins (MTMRs) form a family of phosphatases dephosphorylating phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate at the 3rd position of the inositol ring. Similarly to their substrates, MTMRs are involved in various cellular events such as endosomal transport or autophagy. Mutations in MTMR proteins lead to dysregulation of the cellular events and manifestation of severe pathologies. Among the most studied are two hereditary diseases, X- linked myotubular myopathy and Charcot-Marie-Tooth syndrome, caused by mutations in MTM1 and MTMR2 genes, respectively. One of the specific features of the MTMR family is the presence of catalytically inactive members. These members were found to regulate protein stability, activity and localization of their active partners. MTMR10 and MTMR12 are two inactive members of the MTMR family, directly interacting with the...
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