9 results on '"ŽÁRSKÝ V."'
Search Results
2. Exocyst functions in plants: secretion and autophagy.
- Author
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Žárský V
- Subjects
- Animals, Autophagy, Cell Membrane metabolism, Plants genetics, Plants metabolism, Exocytosis physiology, Vesicular Transport Proteins metabolism
- Abstract
Tethering complexes mediate vesicle-target compartment contact. Octameric complex exocyst initiate vesicle exocytosis at specific cytoplasmic membrane domains. Plant exocyst is possibly stabilized at the membrane by a direct interaction between SEC3 and EXO70A. Land plants evolved three basic membrane-targeting EXO70 subfamilies, the evolution of which resulted in several types of exocyst with distinct functions within the same cell. Surprisingly, some of these EXO70-exocyst versions are implicated in autophagy, as is animal exocyst, and are involved in host defense, cell wall fortification and transport of secondary metabolites. Interestingly, EXO70Ds act as selective autophagy receptors in the regulation of cytokinin signaling pathway. Secretion of double membrane autophagy-related structures formed with the contribution of EXO70s to the apoplast suggests the possibility of secretory autophagy in plants., (© 2022 Federation of European Biochemical Societies.)
- Published
- 2022
- Full Text
- View/download PDF
3. SEC6 exocyst subunit contributes to multiple steps of growth and development of Physcomitrella (Physcomitrium patens).
- Author
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Brejšková L, Hála M, Rawat A, Soukupová H, Cvrčková F, Charlot F, Nogué F, Haluška S, and Žárský V
- Subjects
- Bryopsida genetics, Genes, Plant genetics, Germ Cells, Plant, Mutation, Plant Proteins genetics, Bryopsida growth & development, Exocytosis genetics, Plant Proteins physiology
- Abstract
Spatially directed cell division and expansion is important for plant growth and morphogenesis and relies on cooperation between the cytoskeleton and the secretory pathway. The phylogenetically conserved octameric complex exocyst mediates exocytotic vesicle tethering at the plasma membrane. Unlike other exocyst subunits of land plants, the core exocyst subunit SEC6 exists as a single paralog in Physcomitrium patens and Arabidopsis thaliana genomes. Arabidopsis SEC6 (AtSEC6) loss-of-function (LOF) mutation causes male gametophytic lethality. Our attempts to inactivate the P. patens SEC6 gene, PpSEC6, using targeted gene replacement produced two independent partial LOF ('weak allele') mutants via perturbation of the PpSEC6 gene locus. These mutants exhibited the same pleiotropic developmental defects: protonema with dominant chloronema stage; diminished caulonemal filament elongation rate; and failure in post-initiation gametophore development. Mutant gametophore buds, mostly initiated from chloronema cells, exhibited disordered cell file organization and cross-wall perforations, resulting in arrested development at the eight- to 10-cell stage. Complementation of both sec6 moss mutant lines by both PpSEC6 and AtSEC6 cDNA rescued gametophore development, including sexual organ differentiation. However, regular sporophyte formation and viable spore production were recovered only by the expression of PpSEC6, whereas the AtSEC6 complementants were only rarely fertile, indicating moss-specific SEC6 functions., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)
- Published
- 2021
- Full Text
- View/download PDF
4. EXO70A2 Is Critical for Exocyst Complex Function in Pollen Development.
- Author
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Marković V, Cvrčková F, Potocký M, Kulich I, Pejchar P, Kollárová E, Synek L, and Žárský V
- Subjects
- Gene Expression Regulation, Plant, Genes, Plant, Genetic Variation, Genotype, Phylogeny, Arabidopsis genetics, Arabidopsis growth & development, Exocytosis genetics, Exocytosis physiology, Pollen Tube genetics, Pollen Tube growth & development
- Abstract
Pollen development, pollen grain germination, and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to ovules for fertilization. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we demonstrate that Arabidopsis ( Arabidopsis thaliana ) EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in the male gametophyte, governing the conventional secretory function of the exocyst, analogous to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination, and pollen tube growth. GFP:EXO70A2 was localized to the nucleus and cytoplasm in developing pollen grains and later to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for EXO70A1 function in the sporophyte, but not vice versa, indicating partial functional redundancy of these two closely related isoforms and higher specificity of EXO70A2 for pollen development-related processes. Phylogenetic analysis revealed that the ancient duplication of EXO70A, one of which is always highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in Arabidopsis pollen that is required for efficient plant sexual reproduction., (© 2020 American Society of Plant Biologists. All Rights Reserved.)
- Published
- 2020
- Full Text
- View/download PDF
5. Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes.
- Author
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Sekereš J, Pejchar P, Šantrůček J, Vukašinović N, Žárský V, and Potocký M
- Subjects
- Amino Acid Sequence, Base Sequence, Chromatography, High Pressure Liquid methods, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Microscopy, Confocal, Phylogeny, Plant Proteins classification, Plant Proteins metabolism, Pollen Tube growth & development, Pollen Tube metabolism, Protein Isoforms genetics, Protein Isoforms metabolism, Proteomics methods, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Spectrometry, Mass, Electrospray Ionization, Nicotiana metabolism, Cell Membrane metabolism, Exocytosis genetics, Plant Proteins genetics, Pollen Tube genetics, Nicotiana genetics
- Abstract
The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco ( Nicotiana tabacum ) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell., (© 2017 American Society of Plant Biologists. All Rights Reserved.)
- Published
- 2017
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6. Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth.
- Author
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Bloch D, Pleskot R, Pejchar P, Potocký M, Trpkošová P, Cwiklik L, Vukašinović N, Sternberg H, Yalovsky S, and Žárský V
- Subjects
- Amino Acid Sequence, Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis metabolism, Arabidopsis Proteins classification, Arabidopsis Proteins genetics, Base Sequence, Binding Sites genetics, Cell Membrane metabolism, Gene Expression Profiling methods, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Microscopy, Confocal, Molecular Dynamics Simulation, Mutation, Phosphatidylinositol 4,5-Diphosphate metabolism, Phylogeny, Plants, Genetically Modified, Pollen genetics, Pollen growth & development, Pollen metabolism, Pollen Tube genetics, Pollen Tube growth & development, Protein Binding, Protein Isoforms genetics, Protein Isoforms metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Time-Lapse Imaging methods, Vesicular Transport Proteins classification, Vesicular Transport Proteins genetics, Arabidopsis Proteins metabolism, Exocytosis, Phosphatidylinositols metabolism, Pollen Tube metabolism, Vesicular Transport Proteins metabolism
- Abstract
Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP
2 ) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes., (© 2016 American Society of Plant Biologists. All Rights Reserved.)- Published
- 2016
- Full Text
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7. Membrane targeting of the yeast exocyst complex.
- Author
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Pleskot R, Cwiklik L, Jungwirth P, Žárský V, and Potocký M
- Subjects
- Cell Membrane metabolism, Kinetics, Molecular Dynamics Simulation, Mutation, Phosphatidylinositol 4,5-Diphosphate chemistry, Phosphatidylinositol 4,5-Diphosphate metabolism, Protein Binding, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Secretory Pathway, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, rho GTP-Binding Proteins chemistry, rho GTP-Binding Proteins genetics, rho GTP-Binding Proteins metabolism, Cell Membrane chemistry, Exocytosis, Saccharomyces cerevisiae Proteins chemistry, Vesicular Transport Proteins chemistry
- Abstract
The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
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8. Exocytosis and cell polarity in plants - exocyst and recycling domains.
- Author
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Žárský V, Cvrčková F, Potocký M, and Hála M
- Subjects
- Amino Acid Sequence, Molecular Sequence Data, Plant Proteins chemistry, Plants ultrastructure, Secretory Vesicles ultrastructure, Cell Polarity, Endocytosis, Exocytosis, Plant Cells, Plant Proteins metabolism
- Abstract
In plants, exocytosis is a central mechanism of cell morphogenesis. We still know surprisingly little about some aspects of this process, starting with exocytotic vesicle formation, which may take place at the trans-Golgi network even without coat assistance, facilitated by the local regulation of membrane lipid organization. The RabA4b guanosine triphosphatase (GTPase), recruiting phosphatidylinositol-4-kinase to the trans-Golgi network, is a candidate vesicle formation organizer. However, in plant cells, there are obviously additional endosomal source compartments for secretory vesicles. The Rho/Rop GTPase regulatory module is central for the initiation of exocytotically active domains in plant cell cortex (activated cortical domains). Most plant cells exhibit several distinct plasma membrane domains, established and maintained by endocytosis-driven membrane recycling. We propose the concept of a 'recycling domain', uniting the activated cortical domain and the connected endosomal compartments, as a dynamic spatiotemporal entity. We have recently described the exocyst tethering complex in plant cells. As a result of the multiplicity of its putative Exo70 subunits, this complex may belong to core regulators of recycling domain organization, including the generation of multiple recycling domains within a single cell. The conventional textbook concept that the plant secretory pathway is largely constitutive is misleading.
- Published
- 2009
- Full Text
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9. Multiple Exocytotic Markers Accumulate at the Sites of Perifungal Membrane Biogenesis in Arbuscular Mycorrhizas.
- Author
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Genre, A., Ivanov, S., Fendrych, M., Faccio, A., Žárský, V., Bisseling, T., and Bonfante, P.
- Subjects
VESICULAR-arbuscular mycorrhizas ,FUNGAL membranes ,PLANT-soil relationships ,CELL membrane formation ,SOIL fungi ,HOST plants ,GREEN fluorescent protein ,TRANSMISSION electron microscopy ,EXOCYTOSIS - Abstract
Arbuscular mycorrhizas (AMs) are symbiotic interactions established within the roots of most plants by soil fungi belonging to the Glomeromycota. The extensive accommodation of the fungus in the root tissues largely takes place intracellularly, within a specialized interface compartment surrounded by the so-called perifungal membrane, an extension of the host plasmalemma. By combining live confocal imaging of green fluorescent protein (GFP)-tagged proteins and transmission electron microscopy (TEM), we have investigated the mechanisms leading to the biogenesis of this membrane. Our results show that pre-penetration responses and symbiotic interface construction are associated with extensive membrane dynamics. They involve the main components of the exocytotic machinery, with a major participation of the Golgi apparatus, as revealed by both TEM and in vivo GFP imaging. The labeling of known exocytosis markers, such as v-SNARE proteins of the VAMP72 family and the EXO84b subunit of the exocyst complex, allowed live imaging of the cell components involved in perifungal membrane construction, clarifying how this takes place ahead of the growing intracellular hypha. Lastly, our novel data are used to illustrate a model of membrane dynamics within the pre-penetration apparatus during AM fungal penetration. [ABSTRACT FROM PUBLISHER]
- Published
- 2012
- Full Text
- View/download PDF
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