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Molecular mechanisms of formation and development of colonies of wild Saccharomyces cerevisiae strains
Šťovíček, Vratislav ; Palková, Zdena (advisor) ; Hašek, Jiří (referee) ; Heidingsfeld, Olga (referee)
Yeasts are capable of forming a wide range of multicellular communities, which enable the survival in harmful and changing environment. Surface associated biofilms, often connected with infections in human body, and colonies can serve as an example of such populations. This work investigates formation and development of complex structured colonies of Saccharomyces cerevisiae, which can be considered as a distinctive feature of yeast strains isolated from the wild. Architecture and properties of such colonies are fundamentally different from the spatially undifferentiated colonies of most of laboratory strains and resemble in many ways rather natural biofilms of pathogenic yeasts. Yeast populations use specific developmental processes induced by communication mechanisms to synchronize the early stages of their development. Formation of specific three-dimensional colony architecture is enabled by the presence of extracellular matrix and adhesive protein Flo11p which provide stability and integrity of the whole structure. Protection of the colonies is accomplished by spatially differentiated cell subpopulations using various mechanisms such as expression of efflux pumps capable of removing toxic substances or production of extracellular matrix functioning also as selectively permeable barrier. Phenotypic...
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Functional characterization of selected microtubule regulatory
Vinopal, Stanislav ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
Microtubules (MTs) play crucial roles in intracellular organization and transport, cell polarity, motility, signalling, division and differentiation. MTs form complex arrays, which are, due to their highly dynamic nature, capable of rapid reorganization in response to cellular requirements. Dynamics, stability and spatial organization of MTs are regulated by many factors including MT regulatory proteins. In the presented study we functionally characterized three selected MT regulatory proteins: Ca2+ -sensor STIM1, MT severing protein spastin and γ-tubulin that is essential for MT nucleation. We found out that activation of bone marrow mast cells (BMMCs) leads to the formation of plasma membrane protrusions containing MTs. Formation of these MT protrusions is dependent on an influx of extracellular Ca2+ regulated by protein STIM1, located in endoplasmic reticulum. STIM1 associates with MTs and its depletion prevents formation of MT protrusions. This indicates that Ca2+ ions might be involved in MT regulation. Since STIM1 depletion also causes defects in chemotaxis, we propose that MT protrusions might be involved in sensing of external signals recognized by BMMCs. Glioblastoma multiforme is the most common and most aggressive malignant primary brain tumor in humans. We demonstrated that MT severing...
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Cytoskeleton and endocytosis, a dynamic system for the localization of auxin efflux carriers
Jelínková, Adriana ; Petrášek, Jan (advisor) ; Nick, Peter (referee) ; Hašek, Jiří (referee)
Thanks to its sessile life strategy, the polarity of plant body reflects the polarity of single cells. The polarity is maintained by asymmetric distribution of various molecules downstream from extra and intracellular signals. Directional transport of auxin plays an important role in the pattern formation, morphogenesis, and directional growth responses. The positioning of PIN auxin efflux transporters has been shown to be crucial in the setting of auxin gradients. It is dependent on the plasma membrane deposition of membrane vesicles and their constitutive cycling between plasma membrane and endosomal space. Although some evidences support the idea of differential actin and microtubular cytoskeleton dependence of PIN protein trafficking, there is a significant lack of the information on the role of cytoskeleton in this process. In this paper we use combination of live cell imaging and immunofluorescence techniques to search for the molecular players of actin filaments (AFs) and proteins proteins associated with AFs in the mechanisms of endocytosis and directed PIN1 protein targeting. Seedlings of Arabidopsis thaliana carrying mutations in actin genes (ACT1, ACT2, ACT7, ACT11), Arp 2/3 complex genes (ARP2, ARP3, ARPC2, ARPC5), WAVE complex components genes (BRK1, NAP1, SRA1) and actin monomer...
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The role of the cytoskeleton in morphogenesis of plant cells
Havelková, Lenka ; Schwarzerová, Kateřina (advisor) ; Hašek, Jiří (referee) ; Malínská, Kateřina (referee)
6. Conclusions ► The capability of ARPC2 protein (subunit of ARP2/3 complex) to bind microtubules was described in this study. Our results indicated that ARP2/3 complex may mediate direct interaction between microfilaments and microtubules in processes where the coordination of microtubule reorganization and actin mediated growth was required. ► We showed that cell-malforming effect of growth retardant ancymidol was based on its inhibitory action on cellulose synthesis. Microtubules played a passive role in this process and were not directly responsible for changes of the cell shape induced by ancymidol. ► Meristematic and suspensors cells in maturating somatic embryos differed in sensitivity to latrunculin B, probably due to different composition of actin isoforms in these cells. Application of low doses of latrunculin B resulted in selective death of suspensor cells and thus contributed to the development of high-quality embryos. ► Aluminium caused rapid cessation of root growth of Arabidopsis thaliana seedlings. We showed that the immediate reason of root growth cessation was plasma membrane rigidization and loss of endocytosis in root cells. Aluminium stabilized cortical microtubules within minutes and induced their loss within hours of treatment. However, the effect of aluminium on microtubules was not...
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New signaling pathways involved in mast cell activation and cell membrane repair
Shaik, Gouse Mohiddin ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Hašek, Jiří (referee)
RESULTS 54 RESULTS AND DISCUSSION Vacuolin-1-induced changes in mast cells morphology In pilot experiments we determined the effect of vacuolin-1 on mast cell morphology. Incubation of RBL-2H3 cells or BMMCs with 10 μM vacuolin-1 in complete culture media for 3 h resulted in appearance of numerous vacuoles in cytosolic compartment of the cells (Fig 1A-D). Size of the vacuolin-1-treated cells rose (Fig 1E and F) as reflected by mean increase in diameter of RBL-2H3 cells from 14.8 ± 0.2 μm (mean ± S.D., n = 3) to 18.8 ± 0.3 μm, and BMMCs from 14.6 ± 0.2 μm to 17.3 ± 0.2 μm. To decide whether formation of vacuoles depends on specific metabolic pathways we examined cells exposed to various concentrations of pharmacological inhibitors and 10 μM vacuolin-1. Data presented in Table 1 show that most of the drugs tested, including Cl- and/or K+ channel blockers [indanyloxyacetic acid 94 (IAA-94), (dihydroindenyl)oxy/alkanoic acid (DIOA), 5- Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'- disulphonate (DIDS) and glybenclamide], protein kinase C (PKC) inhibitor (tamoxifen), phosphoinositid-3 kinase (PI3K) inhibitor (wortmannin), Src and Syk family kinase inhibitors (PP2 and piceatannol) and non-muscle myosin (NMM) II ATPase activity inhibitor (blebbistatin)...
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Ultra low frequency yeast cells electric activity
Cifra, Michal ; Pokorný, Jiří ; Jelínek, František ; Hašek, Jiří ; Šimša, Jan
This paper presents results from the measurement of the electrical activity cold sensitive beta-tubulin mutant tub2-401 of yeast cells in frequency region 0.4 - 1.6 kHz. 25 measurements of both synchronized and nonsynchronized cells show that the synchronized cells have higher electrical activity what corresponds to the findings of Pohl et al. Mechanical oscillations of electrically polar structures in cell may give rise to electric oscillations we measured.
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