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Study of the organization and dynamics of the membraneless cell compartments
Blažíková, Michaela ; Heřman, Petr (advisor) ; Cmarko, Dušan (referee) ; Hašek, Jiří (referee)
of Doctoral Thesis Title: Study of the organization and dynamics of the membraneless cell compartments Author: Michaela Blažíková Institute: Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Supervisor: Doc. RNDr. Petr Heřman, CSc., Charles University in Prague, Faculty of Mathematics and Physics, Institute of Physics of Charles University Abstract Eukaryotic cells contain many organelles and specific bodies. Beside the membrane delimited organelles such as nucleus, mitochondria or Golgi apparatus there are other structurally and functionally distinct membraneless structures in the cells. In this work we studied the self-organization processes, i.e. the processes that do not require specific interactions, of membraneless structures in nuclei, cytoplasm and plasma membrane of mammalian cells and yeast. The research was focused on the formation of nucleoli and Cajal bodies in mammalian cell nulei and processing bodies (P- bodies) in the cytoplasm of mammalian cells. The organization of MCC domains in the yeast plasma membrane (Membrane compartment of Can1) was studied as well. It was shown that nonspecific interactions as the result of macromolecular crowding could be one of the main driving forces in formation and stabilization of these...
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Conventional and Novel Functions of the Exocyst Complex in Plants
Kulich, Ivan ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Exocyst is an octameric protein complex, conserved across all Eukaryotes. Its role, originally described in yeast, resides in a tethering of the secretory vesicles to the plasma membrane prior to the membrane fusion of the two membranes. Subunits SEC3 and EXO70 are believed to be spatial landmarks for the vesicles delivery. While yeast genome encodes single EXO70, we find dozens of them in land plants (23 in Arabidopsis). This work is focused at a role of the exocyst complex in plant cells. Its first part documents, that exocyst is essential for delivery of the cell wall components, namely pectins, but also for pathogen induced secondary cell wall thickening. Second part reveals an unconventional role of EXO70B1 subunit harboring exocyst subcomplex at an autophagic pathway to the vacuole and raises many questions about plant secretory pathway.
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Regulation of mast cell activation at the level of the high-affinity IgE receptor and STIM1
Bugajev, Viktor ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Hašek, Jiří (referee)
(EN) This thesis is focused on two important gate-keepers of mast cell signaling. The first is the complex of the high-affinity receptor for immunoglobulin E (IgE) (FcεRI) associated with Lck/Yes- related novel tyrosine kinase (Lyn), which is involved in acquired immune responses and the second is the stromal interaction molecule (STIM)1, which senses calcium levels in endoplasmic reticulum (ER) and upon depletion of ER Ca2+ stores participates in opening of the plasma membrane Ca2+ release- activated Ca2+ (CRAC) channels. Although the structure of FcεRI is known for many years and numerous molecules associated with the receptor have been described, the exact molecular mechanism of initiation and termination of the FcεRI signaling is elusive. Therefore, we evaluated the current knowledge on the molecular mechanisms of FcεRI phosphorylation with emphasis on the newly described model according to which cross-talk between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) sets the threshold for FcεRI tyrosine phosphorylation (PTK-PTP interplay model). Furthermore, we extended the knowledge about topography of active phosphatases which are prone to oxidation within the clusters of transmembrane adaptor proteins non-T cell activation linker (NTAL) and linker for activation of T...
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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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The characteristics of stress granules in yeast Saccharomyces cerevisiae
Slabá, Renata ; Hašek, Jiří (advisor) ; Binarová, Pavla (referee)
9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...
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Regulatory functions of the transmembrane adaptor protein NTAL in activation of mast cells
Tůmová, Magda ; Dráber, Petr (advisor) ; Vyklický, Ladislav (referee) ; Hašek, Jiří (referee)
Mast cells originate from hematopoietic pluripotent stem cells present in bone marrow. They do not circulate, but are spread throughout a body and reside in all vascularized tissues. Mast cells as a part of innate immune system are also able to influence adaptive immune system and are effectors in IgE-mediated allergic and inflammatory diseases. Mast cells possess high affinity receptor for IgE (FcεRI). Aggregation of the receptor triggers signaling cascades which lead to the release of preformed and de-novo synthesized effector molecules. Early phases of mast cell activation include tyrosine phosphorylation of numerous substrates and formation of signaling assemblies (also called signalosomes). Important structural components of the signalosomes are transmembrane adaptor proteins. One of them is the transmembrane adaptor protein NTAL (Non-T cell Activation Linker), which becomes rapidly tyrosine- phosphorylated upon FcεRI triggering and serves as a positive or a negative regulator of FcεRI signaling. To be able to study fine topography of NTAL and other plasma membrane components in mouse bone marrow-derived mast cells (BMMCs), we first developed a new method of plasma membrane sheets isolation from non-adherent cells. The method is based on adsorption of the cells to ultraclean glass cover-slips,...
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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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Kortikální cytoskelet, exocytický komplex exocyst a jejich role v morfogenzi rostlinných buněk
Fendrych, Matyáš ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Plant cell morphogenesis is largely dependent on the coordination of cytoskeletal elements, plasma membrane, and vesicle trafficking. Formin proteins are nucleators of the actin cytoskeleton. Plant Class I family formins are integral membrane proteins and thus have the ability to coordinate cytoskeletal dynamics with the plasma membrane localization. We identified Arabidopsis thaliana formin AtFH4 as a microtubule associated protein. The binding is conferred by a novel domain located between the transmembrane domain and the formin homology 1 domain. The protein associated with actin in in vitro conditions. Overexpressed AtFH4 accumulated in the endoplasmic reticulum, and induced coalignment of endoplasmic reticulum membranes with microtubules. Together, these data suggest that the combination of plant-specific and conserved domains enables AtFH4 to function as an interface between membranes and both major cytoskeletal networks . Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plant cells. Prior to their fusion with the plasma membrane, secretory vesicles are tethered at exocytic sites by the exocyst, an octameric protein complex. We analysed the mutant in the EXO84b exocyst subunit, and discovered that the mutant plants were dwarfed and exhibited...
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The secretory vesicles tethering complex exocyst and the auxin transport polarization
Janková Drdová, Edita ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
The polarization of exocytosis in yeast and animals is assisted by the exocyst - an octameric vesicle tethering complex and an effector of Rab and Rho GTPases. Recently, the exocyst was described as a functional complex involved in morphogenesis also in plants. Hála et al. (2008) described involvement of exocyst complex in pollen tube growth and hypocotyls elongation in dark grown seedlings, Fendrych et al. (2010) uncovered key role of exocyst in cell plate formation, Kulich et al. (2010) emphasized the participation of exocyst in seed coat generation and Pečenková et al. (2011) described the contribution of exocyst subunits in plant defense towards the pathogens. All these processes are intimately linked to polarized secretion. Here we show involvement of exocyst in auxin efflux carriers PINs recycling. Using direct auxin transport measurement and GFP-tagged proteins, we showed that the exocyst is involved in recycling and polarization of PIN proteins and polar auxin transport regulation. Rootward polar auxin transport is compromised in loss-of-function mutants in exocyst subunits EXO70A1. On the cellular level we have detected small portion of PIN2:GFP in the "BFA-like" FM4-64 labelled compartments distinct from VHAa1 labeled endosoms. Moreover recycling of PIN1 and PIN2 is retarded in roots of...
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Exocyst subunit AtSEC15b: its role in plant cell morphogenesis and characterization of its Rab interacting partner
Toupalová, Hana ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Organization of endomembrane compartments in all eukaryotic cells is dependent on continuous transport of membrane vesicles. Major part of the core regulators of intracellular membrane transport is represented by small GTPases from the Rab family. Rab GTPases cycle between the GTP-bound "active" and GDP-bound "inactive" forms. In their active form, they are able to interact with specific effectors and perform their functions. Exocyst is an octameric complex involved in regulation of secretion. It functions as an effector of Rab GTPases in yeast and mammals and tethers secretory vesicles to the plasma membrane prior to the actual membrane fusion. Using publicly available expression data, we have identified candidates from Rab GTPase family for the interaction with exocyst subunit AtSEC15b in plants and demonstrated that AtSEC15b specifically interacts with AtRABA4a GTPase. We also showed that, like in yeast and mammals, Arabidopsis Sec15b binds Rab GTPase also probably in GTP-dependent manner, implying that this interaction is well conserved throughout the eukaryotic kingdoms. We also successfully demonstrated the complementation of yeast thermo-sensitive mutant strain, sec15-1. Based on this observation we concluded that AtSEC15b is able to substitute the function of yeast SEC15 and restore the phenotype....
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