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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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Functions of actin and myosin 1c in the cell nucleus and in the cytoplasm
Kalendová, Alžběta ; Hozák, Pavel (advisor) ; Binarová, Pavla (referee) ; Forstová, Jitka (referee)
Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...
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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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Nuclear myosin 1 and its role in the regulation of plasma membrane tension
Petr, Martin ; Hozák, Pavel (advisor) ; Malínský, Jan (referee)
Myosin 1c (Myo1c) is a molecular motor involved in regulation of tension-gated ion channels, exocytosis, endocytosis, motility and other membrane-related events. Moreover, it acts as a dynamic linker between the cell membrane and cortical actin network, contributing to the maintenance of plasma membrane tension. In contrast, nuclear myosin 1 (NM1), an isoform of Myo1c, has been described only in the nucleus where it participates in various nuclear processes, including transcription or chromatin remodeling. However, although traditionally regarded as exclusively cytoplasmic or nuclear, all myosin 1c isoforms participate in nuclear functions and they are present in the cytoplasm as well. The main focus of this study was to characterize the functional significance of NM1 in the cytoplasm. We have found that NM1 localizes to plasma membrane and shows a uniform punctuated distribution with a high concentration at the cell periphery. Moreover, atomic force microscopy measurements of mouse NM1 KO fibroblasts revealed a significant increase in an overall plasma membrane elasticity in comparison to WT cells, indicating a disruption in the regulation of plasma membrane tension caused by the loss of NM1. Since a higher membrane elasticity and deformability is a characteristic marker of cancer cells,...
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Myosin I and actin binding proteins in cell
Krásna, Hana ; Hozák, Pavel (advisor) ; Motlík, Jan (referee) ; Pěknicová, Jana (referee)
Many studies have established the presence and essenciality of actin in the nucleus. Recently, actin has been associated with processes in the nucleus ranging from chromatin remodeling to transcription, splicing or nuclear transport. To ensure the dynamics of the nuclear processes, actin is coupled with one of the main motor protein such as myosin. This study demonstrates a structural role of actin and the nuclear myosin I (NMI) take in the transcription of ribonuclear genes by RNA polymerase I (Pol l). We suppressed the transcription Pol I in vitro by microinjections of antibodies anti actin and anti MNI. The series of in vitro experiments confirm transcript Pol I inhibition after applying antibodies anti actin and MNI on pure DNA as well as on pre-assembled chromatine template. The co-immunoprecipitation experiments reveal direct bound between actin, NMI and rRNA genes and transcription complex Pol I. As actin binds to the primer and elonged Pol I molecule, NMI interacts with subunit of Pol I and is capable of assembling into productive initiation complex by binding up to TIF-IA, transcriptional factor responsible for regulation rRNA synthesis. There are known number of hypothesis on the form of nuclear actin. Recent research suggests actin exists in equilibrium between its monomeric and...
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Myosin 1c isoforms and their functions in the cell nucleus and in the cytoplasm
Venit, Tomáš ; Hozák, Pavel (advisor) ; Černý, Jan (referee) ; Hašek, Jiří (referee)
Nuclear myosin 1 (NM1) was the first myosin described in the cell nucleus. From its discovery, it has been found to function in processes of Pol I and Pol II transcription, chromatin remodeling, and chromosomal movements. However, direct mechanisms of how NM1 works in the cell nucleus were still missing. We therefore decided to prepare NM1 knock-out mice to answer questions about phyiological functioning of this protein. Myo1c is an isoform of NM1 protein, previously described in the cytoplasm. The only difference between these isoforms is 16 amino-acids at the N-terminus of NM1, which were thought to be the nuclear localization signal. However, we discovered that the nuclear localization signal is located in the neck domain of myosin, and therefore it is able to direct both isoforms to the nucleus. Moreover, we found that the ratio between both proteins is nearly the same in the nucleus and deletion of NM1 does not cause compensatory overexpression of Myo1c. NM1 KO mice are fully viable with minor changes in bone mineral density and red blood cells size. We found that the function of NM1 in processes such as Pol I transcription can be fully covered by Myo1c protein, suggesting redundancy and interchangeability of these two isoforms in the cell nucleus. We also found that PIP2, a phosphoinositol...
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Myosin-PIP2 interaction in the cell nucleus
Yildirim, Sükriye ; Hozák, Pavel (advisor) ; Černý, Jan (referee) ; Forstová, Jitka (referee)
Even though nuclear myosin 1 (NM1) and myosin 1C (Myo1C) are the products of the same gene, NM1 has additional 16 amino acids at the N-terminus due to alternative start of transcription. Studies claim that NM1 and Myo1C are nuclear and cytoplasmic proteins, respectively. Therefore, researchers thought that NM1 translocates into nucleus via nuclear localization signal (NLS) in its N-terminal extension. However, here we show that NLS is placed within second IQ domain where calmodulin (CaM) binds in a calcium- dependent manner. Since both NM1 and Myo1C have identical neck domains where NLS resides, we have confirmed that both myosin isoforms localize to nucleus. Based on findings indicating Myo1C binding to phosphatidylinositol 4,5-bisphosphate (PIP2) via its tail domain, we tested if NM1 and Myo1C can interact with PIP2 in the nucleus. We show that both isoforms can bind to PIP2 via their tail domains, and interactions with PIP2 can recruit other nuclear proteins into this lipo-protein complex. PIP2 makes complex with a subset of Pol I transcription and processing machinery proteins and modulate their functions in the nucleolus. Moreover, PIP2 depletion results in a dramatic loss of Pol I transcription activity. NM1 and actin were already shown to promote Pol I transcription. Here, we show that...
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Nuclear dynamics and interactions of myosin 1c
Dzijak, Rastislav ; Hozák, Pavel (advisor) ; Hašek, Jiří (referee) ; Kaňka, Jiří (referee)
1. ABSTRACT Myosins are proteins that convert chemical energy stored in ATP into mechanical force that is applied on an actin filament. Nuclear myosin 1 (NM1) was the first myosin detected in the cell nucleus. Together with nuclear actin they were shown to play important roles in DNA transcription and chromatin remodeling. However, the molecular details of the NM1 functions are largely unknown. To expand our knowledge about this molecular motor we studied tissue expression, mechanism of nuclear localization and molecular interactions of this myosin motor. In the first part we examined the expression pattern of NM1 in various mouse tissues. We demonstrated that NM1 is present in cell nuclei of all mouse tissues examined except for cells in terminal stages of spermatogenesis. Quantitative PCR and western blots demonstrated that the expression of NM1 in tissues varies, with the highest levels in the lungs. NM1 is a nuclear isoform of earlier identified myosin 1c (Myo1c), which was described initially as a cytosolic, and plasma membrane associated protein. The only known difference between these two proteins was the presence of additional 16 amino acids at the N-terminus of NM1. Next we focused on the influence of NM1 domains, including the N-terminus, on the subcellular localization of this protein. We found...
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Promoter-terminator interactions in eukaryotic RNA polymerase II transcription
Petr, Martin ; Hozák, Pavel (advisor) ; Vaňková Hausnerová, Viola (referee)
Gene loops are chromatin structures formed by juxtaposition of distal genomic regions. Since these regions are often involved in transcription cycle control, gene loops therefore provide another mechanism of regulation of gene expression. This thesis summarizes recent findings about gene loops, focusing specifically on loops formed by interactions between promoter and terminator regions of genes transcribed by the eukaryotic RNA polymerase II. Different cases of gene loops discovered in several yeast genes, the mammalian BRCA1 tumor suppressor and the HIV-1 integrated provirus are described, including mechanisms that possibly lead to the formation of these structures. Since gene loops and interactions between promoter and terminator in yeast have been linked to the transcriptional memory, their involvement in this phenomenon is discussed. Finally, as BRCA1 and HIV-1 are directly linked to serious human diseases, the potential significance of alterations of gene loops in the development of various pathological conditions is presented. Keywords: gene loops, chromatin loops, chromatin conformation, transcriptional memory, transcription, gene expression, regulation
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