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Wei Guo - One of the best experts on this subject based on the ideXlab platform.
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endosidin2 14 targets the Exocyst complex in plants and fungal pathogens to inhibit exocytosis
Plant Physiology, 2019Co-Authors: Lei Huang, Wei Guo, Xianglin Yin, Chaojan Liao, Tesfaye Mengiste, Mingji Dai, Chunhua ZhangAbstract:The evolutionarily conserved octameric Exocyst complex tethers secretory vesicles to the site of membrane fusion during exocytosis. The plant Exocyst complex functions in cell wall biosynthesis, polarized growth, stress responses, and hormone signaling. In fungal pathogens, the Exocyst complex is required for growth, development, and pathogenesis. Endosidin2 (ES2) is known to inhibit exocytosis in plant and mammalian cells by targeting the EXO70 subunit of the Exocyst complex. Here we show that an analog of ES2, ES2-14, targets plant and two fungal EXO70s. A lower dosage of ES2-14 than of ES2 is required to inhibit plant growth, plant exocytic trafficking, and fungal growth. ES2-14 treatments inhibit appressorium formation and reduce lesion sizes caused by Magnaporthe oryzae. Inhibition of EXO70 by ES2-14 in Botrytis cinerea also reduces its virulence in Arabidopsis (Arabidopsis thaliana). Interestingly, ES2-14 did not affect EXO70 localization or transferrin recycling in mammalian cells. Overall, our results indicate that a minor change in ES2 affects its specificity in targeting EXO70s in different organisms and they demonstrate the potential of using ES2-14 to study the mechanisms of plant and fungal exocytosis and the roles of exocytosis in fungus-plant interactions.
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the Exocyst complex
Current Biology, 2018Co-Authors: Kunrong Mei, Wei GuoAbstract:The Exocyst is a multisubunit protein complex that was first identified and characterized in budding yeast. Later studies have demonstrated its conservation in eukaryotes, from plants to mammals. This complex mediates the tethering of secretory vesicles to the plasma membrane prior to fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The Exocyst has been implicated in a variety of cellular processes, such as exocytosis, cell growth, cytokinesis, cell migration, primary ciliogenesis and tumorigenesis. Recent years have seen major progress in our understanding of this complex. In this Primer, we focus on some of the basic information about the Exocyst complex, including its structure, assembly, molecular interactions, function in vesicle tethering and membrane fusion, and involvement in many physiological processes.
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the Exocyst at a glance
Journal of Cell Science, 2015Co-Authors: Wei GuoAbstract:The Exocyst is an octameric protein complex that is implicated in the tethering of secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. Spatial and temporal control of exocytosis through the Exocyst has a crucial role in a number of physiological processes, such as morphogenesis, cell cycle progression, primary ciliogenesis, cell migration and tumor invasion. In this Cell Science at a Glance poster article, we summarize recent works on the molecular organization, function and regulation of the Exocyst complex, as they provide rationales to the involvement of this complex in such a diverse array of cellular processes.
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the role of sec3p in secretory vesicle targeting and Exocyst complex assembly
Molecular Biology of the Cell, 2014Co-Authors: Guangzuo Luo, Jian Zhang, Wei GuoAbstract:During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE)-mediated membrane fusion. The Exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight Exocyst subunits was expressed on the surface of mitochondria. We find that most of the Exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the Exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of Exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the Exocyst complex. Our study helps to establish the role of the Exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.
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erk1 2 regulate exocytosis through direct phosphorylation of the Exocyst component exo70
Developmental Cell, 2012Co-Authors: Jinqi Ren, Wei GuoAbstract:The Exocyst is a multiprotein complex essential for exocytosis and plasma membrane remodeling. The assembly of the Exocyst complex mediates the tethering of post-Golgi secretory vesicles to the plasma membrane prior to fusion. Elucidating the mechanisms regulating Exocyst assembly is important for the understanding of exocytosis. Here we show that the Exocyst component Exo70 is a direct substrate of the extracellular signal-regulated kinases 1/2 (ERK1/2). ERK1/2 phosphorylation enhances the binding of Exo70 to other Exocyst components and promotes the assembly of the Exocyst complex in response to epidermal growth factor (EGF) signaling. We further demonstrate that ERK1/2 regulates exocytosis, because blocking ERK1/2 signaling by a chemical inhibitor or the expression of an Exo70 mutant defective in ERK1/2 phosphorylation inhibited exocytosis. In tumor cells, blocking Exo70 phosphorylation inhibits matrix metalloproteinase secretion and invadopodia formation. ERK1/2 phosphorylation of Exo70 may thus coordinate exocytosis with other cellular events in response to growth factor signaling.
Viktor žarský - One of the best experts on this subject based on the ideXlab platform.
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plant cytokinesis is orchestrated by the sequential action of the trappii and Exocyst tethering complexes
Developmental Cell, 2014Co-Authors: Katarzyna Rybak, Lukáš Synek, Ivan Kulich, Viktor žarský, Alexander Steiner, Susan Klaeger, Eva Facher, Gerhard Wanner, Bernhard KusterAbstract:Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and Exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the Exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including Exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.
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Exocyst complexes multiple functions in plant cells secretory pathways
Current Opinion in Plant Biology, 2013Co-Authors: Tamara Pečenková, Matyas Fendrych, Ivan Kulich, Viktor žarskýAbstract:The Exocyst is a complex of proteins mediating first contact (tethering) between secretory vesicles and the target membrane. Discovered in yeast as an effector of RAB and RHO small GTPases, it was also found to function in land plants. Plant cells and tissues rely on targeted exocytosis and this implies that the Exocyst is involved in regulation of cell polarity and morphogenesis, including cytokinesis, plasma membrane protein recycling (including PINs, the auxin efflux carriers), cell wall biogenesis, fertilization, stress and biotic interactions including defence against pathogens. The dramatic expansion of the EXO70 subunit gene family, of which individual members are likely responsible for Exocyst complex targeting, implies that there are specialized functions of different Exocysts with different EXO70s. One of these functions comprises a role in autophagy-related Golgi independent membrane trafficking into the vacuole or apoplast. It is also possible, that some EXO70 paralogues have been recruited into Exocyst independent functions. The Exocyst has the potential to function as an important regulatory hub to coordinate endomembrane dynamics in plants.
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the Exocyst complex contributes to pin auxin efflux carrier recycling and polar auxin transport in arabidopsis
Plant Journal, 2013Co-Authors: Edita Janková Drdová, Lukáš Synek, Michal Hála, John E. Fowler, Tamara Pečenková, Ivan Kulich, Angus S Murphy, Viktor žarskýAbstract:: In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. 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. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 Exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 Exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other Exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the Exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.
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visualization of the Exocyst complex dynamics at the plasma membrane of arabidopsis thaliana
Molecular Biology of the Cell, 2013Co-Authors: Matyas Fendrych, Lukáš Synek, Edita Janková Drdová, Tamara Pečenková, Juraj Sekeres, Riet De Rycke, Moritz K Nowack, Viktor žarskýAbstract:The Exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of Exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in Exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the Exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 Exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the Exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.
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evolution of the land plant Exocyst complexes
Frontiers in Plant Science, 2012Co-Authors: Fatima Cvrckova, Michal Hála, Ivan Kulich, Viktor žarský, Anamika Rawat, Michal Grunt, Radek BezvodaAbstract:Exocyst is an evolutionarily conserved vesicle tethering complex functioning especially in the last stage of exocytosis. Homologs of its eight canonical subunits -Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 - were found also in higher plants and confirmed to form complexes in vivo, and to participate in cell growth including polarized expansion of pollen tubes and root hairs. Here we present results of a phylogenetic study of land plant Exocyst subunits encoded by a selection of completely sequenced genomes representing a variety of plant, mostly angiosperm, lineages. According to their evolution histories, plant Exocyst subunits can be divided into several groups. The core subunits Sec6, Sec8 and Sec10, together with Sec3 and Sec5, underwent few, if any fixed duplications in the tracheophytes (though they did amplify in the moss Physcomitrella patens), while others form larger families, with the number of paralogs ranging typically from two to eight per genome (Sec15, Exo84) to several dozens per genome (Exo70). Most of the diversity, which can be in some cases traced down to the origins of land plants, can be attributed to the peripheral subunits Exo84 and, in particular, Exo70. As predicted previously, early land plants (including possibly also the Rhyniophytes) encoded three basal Exo70 paralogs which further diversified in the course of land plant evolution. Our results imply that plants do not have a single "Exocyst complex" – instead, they appear to possess a diversity of Exocyst variants unparalleled among other organisms studied so far. This feature might perhaps be directly related to the demands of building and maintenance of the complicated and spatially diverse structures of the endomembranes and cell surfaces in multicellular land plants.
Peter Novick - One of the best experts on this subject based on the ideXlab platform.
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The Neurospora crassa Exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth
Molecular biology of the cell, 2014Co-Authors: Meritxell Riquelme, Robert W. Roberson, Olga A. Callejas-negrete, Erin L. Bredeweg, Sarah Ludwig, Alejandro Beltrán-aguilar, Stephan Seiler, Peter Novick, Michael FreitagAbstract:Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkorper. The Exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric Exocyst complex is required for the formation of a functional Spitzenkorper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of Exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkorper, which partially coincides with the outer macrovesicular layer, whereas Exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkorper, respectively, depends on actin and microtubule cytoskeletons. The apical region of Exocyst-mediated vesicle fusion, elucidated by the plasma membrane-associated Exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.
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the synaptobrevin homologue snc2p recruits the Exocyst to secretory vesicles by binding to sec6p
Journal of Cell Biology, 2013Co-Authors: David Shen, Hua Yuan, Alex H Hutagalung, Avani Verma, Daniel Kummel, Karin M Reinisch, James A Mcnew, Peter NovickAbstract:A screen for mutations that affect the recruitment of the Exocyst to secretory vesicles identified genes encoding clathrin and proteins that associate or colocalize with clathrin at sites of endocytosis. However, no significant colocalization of the Exocyst with clathrin was seen, arguing against a direct role in Exocyst recruitment. Rather, these components are needed to recycle the exocytic vesicle SNAREs Snc1p and Snc2p from the plasma membrane into new secretory vesicles where they act to recruit the Exocyst. We observe a direct interaction between the Exocyst subunit Sec6p and the latter half of the SNARE motif of Snc2p. An snc2 mutation that specifically disrupts this interaction led to Exocyst mislocalization and a block in exocytosis in vivo without affecting liposome fusion in vitro. Overexpression of Sec4p partially suppressed the Exocyst localization defects of mutations in clathrin and clathrin-associated components. We propose that the Exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins. This could help to confer both specificity and directionality to vesicular traffic.
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the structures of Exocyst subunit exo70p and the exo84p c terminal domains reveal a common motif
Nature Structural & Molecular Biology, 2005Co-Authors: Gang Dong, Peter Novick, Alex H Hutagalung, Karin M ReinischAbstract:The Exocyst is a large complex that is required for tethering vesicles at the final stages of the exocytic pathway in all eukaryotes. Here we present the structures of the Exo70p subunit of this complex and of the C-terminal domains of Exo84p, at 2.0-A and 2.85-A resolution, respectively. Exo70p forms a 160-A-long rod with a novel fold composed of contiguous α-helical bundles. The Exo84p C terminus also forms a long rod (80 A), which unexpectedly has the same fold as the Exo70p N terminus. Our structural results and our experimental observations concerning the interaction between Exo70p and other Exocyst subunits or Rho3p GTPase are consistent with an architecture wherein Exocyst subunits are composed of mostly helical modules strung together into long rods.
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functional specialization within a vesicle tethering complex bypass of a subset of Exocyst deletion mutants by sec1p or sec4p
Journal of Cell Biology, 2004Co-Authors: Andreas Wiederkehr, Johanowen De Craene, Susan Ferronovick, Peter NovickAbstract:The Exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five Exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Δ, sec5Δ, and exo70Δ strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the Exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples Exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.
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vesicles carry most Exocyst subunits to exocytic sites marked by the remaining two subunits sec3p and exo70p
Journal of Cell Biology, 2004Co-Authors: Charles Boyd, Thomas E Hughes, Marc Pypaert, Peter NovickAbstract:Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the Exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the Exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all Exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the Exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.
Kenneth E Sawin - One of the best experts on this subject based on the ideXlab platform.
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fission yeast ndr lats kinase orb6 regulates exocytosis via phosphorylation of the Exocyst complex
Cell Reports, 2019Co-Authors: Ye Dee Tay, Marcin Leda, Christos Spanos, Juri Rappsilber, Andrew B Goryachev, Kenneth E SawinAbstract:NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the Exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for Exocyst localization to cell tips and for Exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to Exocyst function in concert with Exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.
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Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex
'Elsevier BV', 2019Co-Authors: Ye Dee Tay, Marcin Leda, Christos Spanos, Juri Rappsilber, Andrew B Goryachev, Kenneth E SawinAbstract:Summary: NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the Exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for Exocyst localization to cell tips and for Exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to Exocyst function in concert with Exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels. : NDR/LATS kinases are known primarily for their role in controlling cell and tissue proliferation and morphogenesis, e.g., via regulation of transcription in the Hippo pathway. Using fission yeast S. pombe as a model system, Tay et al. show that the NDR/LATS kinase Orb6 is a major regulator of exocytosis. Keywords: Orb6, NDR/LATS kinase, Cdc42, phosphoproteomics, exocytosis, Exocyst, Sec3, phosphorylation, fission yeast, Schizosaccharomyces pomb
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fission yeast ndr lats kinase orb6 regulates exocytosis via phosphorylation of Exocyst complex
bioRxiv, 2018Co-Authors: Ye Dee Tay, Marcin Leda, Christos Spanos, Juri Rappsilber, Andrew B Goryachev, Kenneth E SawinAbstract:NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals, but few of their substrates are known. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity via spatial regulation of Gef1, a guanine nucleotide exchange factor for the small GTPase Cdc42. Here we show that Orb6 plays a critical role as a positive regulator of exocytosis, independent of Gef1. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify several proteins involved in membrane trafficking as Orb6 targets, and we confirm Sec3 and Sec5, conserved components of the Exocyst complex, as substrates of Orb6 both in vivo and in vitro. Our results suggest that Orb6 kinase activity is crucial for Exocyst localization to actively-growing cell tips and for Exocyst activity during septum dissolution after cytokinesis. We further show that Orb6 phosphorylation of Sec3 serine-201 contributes to Exocyst function in parallel with Exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.
Tamara Pečenková - One of the best experts on this subject based on the ideXlab platform.
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microtubule dependent targeting of the Exocyst complex is necessary for xylem development in arabidopsis
New Phytologist, 2017Co-Authors: Lukáš Synek, Tamara Pečenková, Nemanja Vukasinovic, Yoshihisa Oda, Přemysl Pejchar, Anamika Rawat, Juraj SekeresAbstract:Summary Cortical microtubules (MTs) play a major role in the patterning of secondary cell wall (SCW) thickenings in tracheary elements (TEs) by determining the sites of SCW deposition. The EXO70A1 subunit of the Exocyst secretory vesicle tethering complex was implicated to be important for TE development via the MT interaction. We investigated the subcellular localization of several Exocyst subunits in the xylem of Arabidopsis thaliana and analyzed the functional significance of Exocyst-mediated trafficking in TE development. Live cell imaging of fluorescently tagged Exocyst subunits in TE using confocal microscopy and protein–protein interaction assays were performed to describe the role of the Exocyst and its partners in TE development. In TEs, Exocyst subunits were localized to the sites of SCW deposition in an MT-dependent manner. We propose that the mechanism of Exocyst targeting to MTs involves the direct interaction of Exocyst subunits with the COG2 protein. We demonstrated the importance of a functional Exocyst subunit EXO84b for normal TE development and showed that the deposition of SCW constituents is partially compromised, possibly as a result of the mislocalization of secondary cellulose synthase in Exocyst mutants. We conclude that the Exocyst complex is an important factor bridging the pattern defined by cortical MTs with localized secretion of the SCW in developing TEs.
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Exocyst complexes multiple functions in plant cells secretory pathways
Current Opinion in Plant Biology, 2013Co-Authors: Tamara Pečenková, Matyas Fendrych, Ivan Kulich, Viktor žarskýAbstract:The Exocyst is a complex of proteins mediating first contact (tethering) between secretory vesicles and the target membrane. Discovered in yeast as an effector of RAB and RHO small GTPases, it was also found to function in land plants. Plant cells and tissues rely on targeted exocytosis and this implies that the Exocyst is involved in regulation of cell polarity and morphogenesis, including cytokinesis, plasma membrane protein recycling (including PINs, the auxin efflux carriers), cell wall biogenesis, fertilization, stress and biotic interactions including defence against pathogens. The dramatic expansion of the EXO70 subunit gene family, of which individual members are likely responsible for Exocyst complex targeting, implies that there are specialized functions of different Exocysts with different EXO70s. One of these functions comprises a role in autophagy-related Golgi independent membrane trafficking into the vacuole or apoplast. It is also possible, that some EXO70 paralogues have been recruited into Exocyst independent functions. The Exocyst has the potential to function as an important regulatory hub to coordinate endomembrane dynamics in plants.
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the Exocyst complex contributes to pin auxin efflux carrier recycling and polar auxin transport in arabidopsis
Plant Journal, 2013Co-Authors: Edita Janková Drdová, Lukáš Synek, Michal Hála, John E. Fowler, Tamara Pečenková, Ivan Kulich, Angus S Murphy, Viktor žarskýAbstract:: In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. 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. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 Exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 Exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other Exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the Exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.
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visualization of the Exocyst complex dynamics at the plasma membrane of arabidopsis thaliana
Molecular Biology of the Cell, 2013Co-Authors: Matyas Fendrych, Lukáš Synek, Edita Janková Drdová, Tamara Pečenková, Juraj Sekeres, Riet De Rycke, Moritz K Nowack, Viktor žarskýAbstract:The Exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of Exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in Exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the Exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 Exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the Exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.
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The Arabidopsis Exocyst Complex Is Involved in Cytokinesis and Cell Plate Maturation
The Plant Cell, 2010Co-Authors: Matyas Fendrych, Lukáš Synek, Hana Toupalová, Rex A. Cole, Edita Janková Drdová, Miroslava Šedinová, Michal Hála, Tamara Pečenková, Jana Nebesařova, John E. FowlerAbstract:Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric Exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant Exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein–tagged Exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the Exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.