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Development of ultrastructural methods and their application in studies on the cell nucleus
Filimonenko, Anatoly ; Hozák, Pavel (advisor) ; Nebesářová, Jana (referee) ; Lanctôt, Christian (referee)
Despite the capabilities of molecular-biological methods in deciphering the interplay of different biological molecules and molecular complexes, the understanding of respective functions in living cells requires application of in situ methods. Obviously, these methods should provide maximal resolution and the best possible preservation of the biological object in a native state, as well as correct statistical evaluation of the spatial characteristics of detected molecular players. Transmission electron microscopy provides the highest possible resolution for analysis of biological samples. The simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. To analyze this, we have developed a complex stereological method for statistical evaluation of immunogold clustering and colocalization patterns of antigens on ultrathin sections, including a user-friendly interface. Functional microarchitecture of DNA replication and transcription sites has been successfully characterized using the developed stereological tools. Our data demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view...
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Localization matters: function of paxillin and phopholipids in the cell nucleus
Marášek, Pavel ; Hozák, Pavel (advisor) ; Půta, František (referee) ; Žárský, Viktor (referee)
(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...
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U2 snRNA targeting to Cajal bodies
Roithová, Adriana ; Staněk, David (advisor) ; Mašek, Tomáš (referee)
In the cell we can find a lot of small noncoding RNAs, which are important for many processes. Among those RNAs are small nuclear RNA uridin rich, which with proteins create U snRNP.These particles play important role in pre-mRNA splicing. In this process are noncoding sequences (introns) removed and coding sequences (exons) are joined. It is catalyzed by spliceosome. The core of this spliceosome is created by U1, U2, U4, U5 and U6 snRNP. They are essential for this process. Some steps of U snRNP biogenesis proceed in nuclear structures called Cajal bodies (CB). In my thesis I focused on factors, which are important for targeting U snRNA into CB. I used U2 snRNA like a model. With the aid of microinjection of fluorescently labeled U2 snRNA mutants I found, that the Sm binding site on U2 snRNA is essential for targeting to CB. Knock down of Sm B/B'showed us, that Sm proteins are necessary for transport U2 snRNA to CB. Sm proteins are formed on U2 snRNA by SMN complex. Deletion of SMN binding site on U2 snRNA had the same inhibition effect. From these results we can see, that Sm proteins and SMN complex are important for U2 snRNA biogenesis espacially for targeting into CB. Key words: U snRNP, Cajal body, U snRNA, cell nucleus
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Factors important for Cajal body formation
Roithová, Adriana ; Staněk, David (advisor) ; Valentová, Anna (referee)
This research describes the structure and function of nuclear domains called Cajal bodies (CB). CB contain proteins and factors involved in assembly and modification of snRNPs. These bodies are found in vertebrates and invertebrates and even plants. Not all cell types contain CB. Their number and size depends on the transcription activity of cell and cell cycle phase. This paper discusses the factors that affect the CB formation. One of the most important factors is the level of snRNPs and transcription activity. Recently shows that an important role in CB formation has coilin and other components phosphorylation. Other works show the influence of the environment. There is also discussion regulation of CB biogenesis, witch is not yet fully understood. Key words: Cajal bodies, coilin, cell nucleus, snRNP, pre-mRNA splicing, transcription
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Cells II
Berger, Josef
Cells II (Proceedings of the Conference on cell biology. KOPP esk Bud jovice, 1-189.Full papers: K. Smetana: Nucleoli in not-neoplastic and malignant cells, G. Vollmer et al.:Cellular functions and molecular mechanisms of action of xeno- and phytoestrogens, J. V vrov et al.: Therapeutic induction of programmed cell death, R. Janisch: Cytoskeleton - its role in the life of Protozoa, P. Fedoro ko: Cytokines and haematopoietic cell proliferation. Extended abstracs of Educational programme. Abstracts from symposia on Malignant cells, Gene engineering, toxicity and carcinogen monitoring, Maturation and differentiation, Cell death, Intercellular communication, biomembranes, Cytoskeleton, Nucleus, cell cycle, and Cell biology methods
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