|
|
Characterization of Arabidopsis thaliana FLOTILLINs and HYPERSENSITIVE INDUCED RESPONSE proteins - dynamics, interactions and functions
Daněk, Michal ; Martinec, Jan (advisor) ; Baluška, František (referee) ; Ovečka, Miroslav (referee)
This work is a collection of three research articles and one review article focused on flotillins (FLOTs) and hypersensitive induced reaction proteins (HIRs) in Arabidopsis thaliana. FLOTs and HIRs are closely related membrane-associated proteins forming two subfamilies both belonging to SPFH domain superfamily. While FLOTs are present in organisms of all evolutionary lineages HIRs are plant specific proteins. The review article sums up the knowledge gained on FLOTs and HIRs from different organisms in terms of cellular localization, interaction with cellular membranes and with other proteins, and physiological functions. The research articles were targeted at three aspects of AtFLOTs and AtHIRs: involvement in response to exogenous stimuli; determination of protein interactors; and subcellular localization and dynamics. The first aspect was approached by transcription measurement of AtFLOTs and phenotypic screen of single loss-of-function mutants of AtFLOTs upon various treatments covering biotic and abiotic stress and phytohormone application. Although we observed changes in transcription none of the treatments provoked a phenotype manifestation in any of AtFLOT mutants. In the second article we focused on interactome of AtFLOT2 and performed co- immunoprecipitation followed by mass spectrometry...
|
|
|
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.
|
|
|
Evolutionary-developmental study of membrane proteins
Vosolsobě, Stanislav ; Schwarzerová, Kateřina (advisor) ; Baluška, František (referee) ; Štorchová, Helena (referee)
Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...
|
|
|
Characterization of Arabidopsis thaliana FLOTILLINs and HYPERSENSITIVE INDUCED RESPONSE proteins - dynamics, interactions and functions
Daněk, Michal ; Martinec, Jan (advisor) ; Baluška, František (referee) ; Ovečka, Miroslav (referee)
This work is a collection of three research articles and one review article focused on flotillins (FLOTs) and hypersensitive induced reaction proteins (HIRs) in Arabidopsis thaliana. FLOTs and HIRs are closely related membrane-associated proteins forming two subfamilies both belonging to SPFH domain superfamily. While FLOTs are present in organisms of all evolutionary lineages HIRs are plant specific proteins. The review article sums up the knowledge gained on FLOTs and HIRs from different organisms in terms of cellular localization, interaction with cellular membranes and with other proteins, and physiological functions. The research articles were targeted at three aspects of AtFLOTs and AtHIRs: involvement in response to exogenous stimuli; determination of protein interactors; and subcellular localization and dynamics. The first aspect was approached by transcription measurement of AtFLOTs and phenotypic screen of single loss-of-function mutants of AtFLOTs upon various treatments covering biotic and abiotic stress and phytohormone application. Although we observed changes in transcription none of the treatments provoked a phenotype manifestation in any of AtFLOT mutants. In the second article we focused on interactome of AtFLOT2 and performed co- immunoprecipitation followed by mass spectrometry...
|
|
|
Role of formins in the organization and dynamics of intracellular structures in Arabidopsis thaliana
Rosero Alpala, Elvia Amparo ; Cvrčková, Fatima (advisor) ; Baluška, František (referee) ; Malcová, Ivana (referee)
On the basis of detailed phenotypic examination of fh1 and fh2 mutants we observed that the main housekeeping Arabidopsis thaliana formin AtFH1 (At3g25500) and its closest relative, AtFH2 (At2g43800) are involved in both actin filaments and microtubule dynamics. fh1 mutants showed increased sensitivity to the actin polymerization inhibitor Latrunculin B (LatB). Formin mutants had cotyledon pavement cells which exhibited more pronounced lobes compared to the wild type, and alterations in vascular tissue patterning were found. The double fh1 fh2 homozygote was not obtained, suggesting that at least one functional formin gene is required for proper gametophyte development. Methods used to observe and quantify both architecture and dynamics of the cortical cytoskeleton from confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM) were standarized and allowed to find that mutants exhibited more abundant but less dynamic F- actin bundles and more dynamic microtubules than wild type seedlings, fh1 mutant phenotype observed in roots was further aggravated by a (heterozygous) fh2 mutation. The formin inhibitor SMIFH2 mimicked the alterations observed in fh1 mutants in plants, it has been the first report of this inhibitor in plants. Defects in membrane trafficking were...
|
|
|
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.
|
|
|
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...
|
|
|
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...
|
|
|
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....
|
guest ::
