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Regulatory mechanisms of microtubule reorganization in activated mast cells
Rubíková, Zuzana ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
Microtubules (MTs) are highly dynamic structures essential for the spatio-temporal intracellular organization and transport, signal propagation, cell differentiation, motility and division. To perform these roles, MTs create arrangements capable of fast and precise adaptation to various signals. MTs are under the control of many factors regulating MT nucleation, stability and dynamics. Bone marrow-derived mast cells (BMMCs) are important immune system cells, which can cause serious diseases if their functions are deregulated. Although MT reorganization during BMMC activation is well established, the molecular mechanisms that control their remodelling are largely unknown. In the presented thesis we functionally characterised GIT1/βPIX signalling proteins, PAK1 kinase, and Ca2+ signalling in the regulation of MT nucleation in BMMCs and other cell types. We also elucidated the function of miltefosine (hexadecylphosphocholine), a promising candidate for the treatment of mast cell-driven diseases. We found that GIT1/βPIX signalling proteins are γ-tubulin-interacting proteins associating with centrosomes in BMMCs. MT nucleation is positively regulated by GIT1 and Ca2+ , whereas βPIX is a negative regulator of MT nucleation in BMMCs. Cytosolic Ca2+ affects γ-tubulin properties and stimulates the...
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Cell signaling pathways controlling meiotic maturation of mammalian oocytes
Šolc, Petr ; Motlík, Jan (advisor) ; Petr, Jaroslav (referee) ; Dráber, Pavel (referee)
4 2 Summary in English The female germ cells called oocytes arise from the primordial germ cells during embryogenesis. They are essential for the reproduction. Already during embryogenesis oocytes enter meiosis, however, they arrest at the dictyate stage of prophase I. After onset of sexual maturity luteinizing hormone induces the resumption of meiosis of follicle enclosed oocytes (GV stage) in animals (in vivo) but removing of oocytes from follicles and culture in a suitable medium allows the spontaneous resumption of meiosis in vitro. Nuclear envelope break down (NEBD or GVBD) is the first visible mark of the meiosis resumption. Later after GVBD, the metaphase I (MI) spindle forms and after all chromosome bivalents are correctly attached to microtubules (MTs) anaphase I occurs. Following meiosis I completion, oocytes enter directly meiosis II and arrest at metaphase II (MII). These oocytes are fertilizable and sperm trigger meiosis II completion. The development from GV to MII oocytes is governed mainly by meiosis promoting factor (MPF) that consists of cyclin dependent kinase 1 (CDK1) and cyclin B (CCNB). On the mouse oocytes, we have shown using functional studies (RNA interference, mRNA microinjection) that phosphatases CDC25A and B cooperate in the induction of CDK1 activity and resumption of meiosis....
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Uloha kataninu, ATPázy štěpící mikrotubuly, při modulaci buněčné motility a proliferace glioblastomových buněk.
Uhlířová, Jana ; Dráber, Pavel (advisor) ; Libusová, Lenka (referee)
Glioblastomas are the most common and the deadliest types of brain tumours. Due to their highly invasive behaviour, they are incurable by convencial therapeutical strategies. It was shown that some components of microtubules, namely class III β-tubulin, γ-tubulin and microtubule severing protein spastin are overexpressed in glioblastoma cell lines as well as glioblastomas. This diploma thesis is focused on the expression, subcellular distribution and function of katanin, another microtubule-severing enzyme, in gliobastoma cell lines. Katanin is formed by catalytic (p60) and regulatory (p80) subunits. Expression and cellular localization of both katanin subunits was studied in panel of human glioblastoma cell lines isolated form adults (T98G, U87MG, U118MG and U138 MG) and child (KNS42). Data presented in this thesis demonstrated that katanin subunits were overexpresed both on transcript and protein levels in T98G, U87MG and KNS42 cell lines, but not in U138MG and U118MG cell lines when compared to normal non- transformed human astrocytes. Immunofluorescence microscopy revealed that both katanin subunits were diffusively distributed in cytoplasm and concentrated on spindle poles of mitotic cells and on leading edges of migrating cells. Examination of cell motility revealed that velocities in...
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New regulatory mechanisms of microtubule nucleation
Černohorská, Markéta ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
MT nucleation from γ-tubulin complexes, located at centrosome, is an essential step in the formation of MT cytoskeleton. In mammalian cells, -tubulin is encoded by two genes. We functionally characterized two γ-tubulin proteins and have found that both are functionally equivalent. γ-Tubulin 2 is able to substitute for γ-tubulin 1 in MT nucleation. However, we revealed that unlike TUBG1, TUBG2 expression is downregulated in mouse preimplantation development. Mast cells represent effectors of the allergy reaction. Their activation by antigen induces number of cellular processes such as degranulation, proliferation and cytoskeleton rearrangements. The regulatory mechanisms of MT reorganization during mast cell activation are unknown. We identified new signaling proteins, GIT1 and PIX that interact with - tubulin. Depletion of GIT1 or PIX leads to changes in MT nucleation. GIT1 is phosphorylated on tyrosine and associates with γ-tubulin in a Ca2+ -dependent manner. Our data suggested a novel signaling pathway for MT rearrangement in mast cells where tyrosine kinase-activated GIT1 and βPIX work in concert with Ca2+ signaling to regulate MT nucleation. We tested the capability of GIT1 and PIX to influence -tubulin function in more cell types. We found out that GIT1/βPIX signaling proteins together...
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Characteristics of novel protein interactions of gamma-tubulin and their roles with microtubules and in cell division
Kohoutová, Lucie ; Binarová, Pavla (advisor) ; Kubelka, Michal (referee) ; Dráber, Pavel (referee)
Spatial and temporal regulation of microtubule nucleation and dynamics is required for formation of specific microtubular arrays that react to internal and external signals and change accordingly. Microtubules are nucleated from microtubule-organizing centres such as centrosomes in animal cells or spindle pole bodies in fungi. All higher plants lack centrosomes and thus present a model for study of acentrosomal cell division and microtubule nucleation and organization. γ-Tubulin is a conserved protein from tubulin superfamily with a central role in microtubule nucleation. It also regulates microtubule dynamics and organization including mitotic spindle positioning. Moreover, γ-tubulin functions in cell cycle regulation, checkpoints control, regulation of transcription, and coordination of late mitotic events. We aimed to characterize protein interactions of γ-tubulin and their functions in Arabidopsis. We identified Arabidopsis homologue of putative centrososomal protein RanBPM. Our data showed that AtRanBPM is a member of CTLH complexes. Our finding of CTLH complexes in plants confirmed their conservation in eukaryotic cells. We found that NITRILASE1 is a cell cycle regulator in Arabidopsis that is important for maintenance of genome stability and proper cell division. We studied a role of AtTPX2,...
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Kinázová signalizace v meióze I savčích oocytů
Brzáková, Adéla ; Šolc, Petr (advisor) ; Dráber, Pavel (referee)
PLK1 belongs to the extended family of serine/threonine kinases controlling the cell cycle. It is well known for its role in the control of mitosis and contributes also to the regulation of meiotic division. On a basis of Live Cell Imaging (LCI) experiments we can describe the phenotype of the oocytes with PLK1 inhibited by small molecular inhibitor BI2536. PLK1 inhibition leads to delayed nuclear envelope breakdown (NEBD) and chromatin condensation (CC) and also causes desynchronization of NEBD and CC; in contrast to control oocytes, PLK1 inhibited oocytes break down their nuclear envelope with chromatin almost fully condensed. Also duration of these two early nuclear events is prolonged in oocytes with inhibited PLK1. In contrast to somatic cells, PLK1 inhibition in mouse oocytes does not prevent assembly of spindle with two distinct poles but affects the final spindle volume. Similar to somatic cells, mouse oocytes with PLK1 inhibited from the beginning of the meiotic maturation stay arrested in metaphase I but in the case of mouse oocytes, this block is not dependent on Spindle Assembly Checkpoint (SAC) persisting activity. When mouse oocytes are synchronized on metaphase I/anaphase I transition by proteasome inhibition and then PLK1 kinase activity is inhibited, about 2/3 of the oocytes stay arrested...
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The role of microtubules splitting proteins in regulation of cytoskeleton.
Uhlířová, Jana ; Dráber, Pavel (advisor) ; Libusová, Lenka (referee)
Microtubules are highly dynamic cytoskeletal structures, which secure cell shape, intracellular transport and organization of cellular organelles. They also participate in cell migration and in formation of mitotic spindle. Microtubules create a highly organized network which changes its organisation in response to the immediate need of the cell. They are formed of αβ-tubulin dimers and a large number of associated proteins. Microtubules are a polar structures with (+) and (-) ends, that differ in the rate of assembly. For fast regulation of microtubule structure are important microtubule-severing proteins (spastin, katanin and fidgetin), that belong to the AAA ATPases. These proteins associate with microtubules and consequently sever microtubules into two parts creating new (+) and (-) ends. In this way, they regulate many cellular processes, such as maintaining of cell shape, cell motility, proliferation and differentiation. Beside microtubule-severing activity, spastin and fidgetin are involved in processes in cell nucleus. Spastin is also associated with membranes and participates in modulation of endoplasmic reticulum and generation of endosomal vesicles. Mutations of microtubule-severing proteins cause embryonal development disorders, nervous system defects or infertility. The latest research...
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Characterization of gamma-tubulin protein interactions and their functions in acentrosomal cells
Doskočilová, Anna ; Binarová, Pavla (advisor) ; Dráber, Pavel (referee) ; Bíšová, Kateřina (referee)
The long term goal of the Laboratory of Functional Cytology is to characterize the roles of γ-tubulin in acentrosomal plant model. Besides characterizing γ-tubulin interactions with plant homologues of proteins of γ-tubulin complexes (GCPs) and determination of their canonical role in microtubule nucleation, we also collect the evidence for γ-tubulin association with proteins that are not directly involved in microtubule nucleation. These γ-tubulin interacting proteins have been of high interest since their characterization might provide a platform for understanding the novel, yet not fully understood functions of γ-tubulin. The major aim of the laboratory is to determine signalling that regulates γ-tubulin complexes and their role in multiple processes in plants. Specifically, the cell cycle regulatory signalling in regulation of microtubular cytoskeleton nucleation and organization is of high interest.
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Tubuline isotypes in cancerogenesis
Hořejší, Barbora ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Opatrný, Zdeněk (referee)
The long-term goal of the Laboratory of Biology of Cytoskeleton is to explain in molecular terms the role of microtubules in cellular processes and to characterize proteins associated with microtubules and its organizing centers (MTOCs). The main attention of our research is focused on -tubulin and its role in microtubule nucleation. We have shown previously that besides its localization on centrosomes, -tubulin is found on celullar membranes (Macůrek et al., 2008), plant kinetochores (Dryková et al., 2003) and marginal bend of embryonal chicken erytrocytes (Linhartová et al., 2002). g-Tubulin also interacts with protein tyrosine kinases, that can regulate microtubule nucleation (Sulimenko et al., 2006; Macůrek et al. 2008). There are growing evidence that centrosomal proteins can affect genetic stability of the cell. g-Tubulin is involved in the regulation of the cell cycle (Vardy et al., 2002) and can participate in activation of checkpoint mechanism controlling the integrity of DNA. Recently, it has been proved its interaction with nuclear protein ATR (Zhang et al., 2007) and Rad51 (Lesca et al., 2005). That's why g-tubulin has been connected with the process of cancerogenesis. Cancer cells also display the changes in expression of b-tubulin isotypes (Katsetos et al., 2003). Moreover, increased level of...
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