The RopGEF KARAPPO Is Essential for the Initiation of Vegetative Reproduction in Marchantia polymorpha

1. Download : Download high-res image (177KB)
2. Download : Download full-size image

     

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid‐rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB‐suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB‐suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.     

Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.Mast cells play pivotal roles in inflammation and immediate-type allergic reactions by secreting biologically active substances including histamine, eicosanoids, proteolytic enzymes, cytokines, and chemokines. The antigen-induced aggregation of the high affinity IgE receptor (FcϵRI)² expressed on the cell surface triggers the degranulation of mast cells. FcϵRI has a tetrameric structure comprised of an IgE binding α-chain, a β-chain, and a disulfide-linked γ-chain dimer (1). The aggregation of FcϵRI by means of multivalent antigen-IgE complexes activates cytosolic Src protein-tyrosine kinases, such as Fyn and Lyn, which then regulate the activation of mast cells (2). Fyn kinase plays a key role in mast cell degranulation and in cytokine production by regulating Gab2 and phosphatidylinositol 3-kinase (3). Phosphorylated Lyn activates immunoreceptor tyrosine-based activation motifs of the β- and γ-chains, and the phosphorylated immunoreceptor tyrosine-based activation motifs of the γ-chain phosphorylate Syk kinase. Thereafter, a number of other signaling and adaptor molecules, such as phospholipase Cγ and protein kinase C (PKC), are phosphorylated (4). Phospholipase Cγ catalyzes the generation both of inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate is an inducer of intracellular Ca²⁺ mobilization, which is critical for degranulation, and diacylglycerol is an activator of PKC (5). Activated PKC is translocated from the cytosol to the plasma membrane fraction. PKC regulates many functions of mast cells, including leukotriene generation, cytokine synthesis, and degranulation (6, 7).Many studies have provided evidence that lipid rafts are involved in the activation of intracellular signaling molecules mediated by FcϵRI, the T cell receptor, the B cell receptor, and other cell surface receptors (8, 9). Lipid rafts are originally defined as microdomains in terms of their resistance to solubilization by non-ionic detergents such as Triton X-100, and are enriched in sphingolipids and cholesterol (10). Because numerous cell surface receptors and palmitoyl-anchored signaling molecules, including Src family tyrosine kinases, are associated with lipid rafts, it has been suggested that lipid rafts function as platforms regulating the induction of signaling pathways. Aggregated, but not non-aggregated, FcϵRIs are localized in lipid rafts fractionated by sucrose density gradient ultracentrifugation of detergent-treated cells (11, 12). The translocation of FcϵRI to lipid rafts is the key event that initiates the degranulation.Carotenoids are a class of widespread natural pigments that have multiple functions (13). Dietary carotenoids have been associated with a decreased risk for certain types of immune diseases, such as asthma and atopic dermatitis. Consumption of β-carotene suppresses the production of specific IgE and IgG₁ and decreases antigen-induced anaphylactic responses due to an improvement of the T_h1-T_h2 balance (14). Furthermore, β-carotene blocks nuclear translocation of the NF-κB p65 subunit, which correlates with the prevention of IκBα phosphorylation and degradation (15). It has been reported that fucoxanthin, a major carotenoid of edible brown algae, shows an anti-inflammatory effect on endotoxin-induced uveitis by decreasing the production of prostaglandin E₂ and tumor necrosis factor-α (16). Astaxanthin, found in the red pigment of crustacean shells and salmon, also has anti-inflammatory effects due to its suppression of NF-κB activation (17, 18). It has been assumed that these anti-inflammatory activities of carotenoids are due to their antioxidant activity. However, there is no information to date about the direct effect of carotenoids on the degranulation of mast cells.In the present study, we investigated the effects of carotenoids on antigen-induced degranulation of RBL-2H3 cells and mouse bone marrow-derived mast cells. In addition, to elucidate the mechanism of the modulation of degranulation by carotenoids, we focused on FcϵRI-mediated signaling in mast cells.     

Identification and Characterization of the Na+/H+ Antiporter Nhas3 from the Thylakoid Membrane of Synechocystis sp. PCC 6803

Na⁺/H⁺ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na⁺/H⁺ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na⁺/H⁺ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na⁺ concentrations. NhaS3 had no K⁺/H⁺ exchange activity itself but enhanced K⁺ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO₂ to high CO₂ conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO₂ consumption caused by the ceasing of O₂-evolving photosynthesis. This is the first report of a Na⁺/H⁺ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H⁺, Na⁺, and K⁺ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.Na⁺/H⁺ antiporters are integral membrane proteins that transport Na⁺ and H⁺ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na⁺ concentrations and influence proton or sodium motive force across the membrane (1, 2). In Escherichia coli, three Na⁺/H⁺ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4). The triple mutant was shown to be hypersensitive to extracellular Na⁺. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na⁺/H⁺ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5, 6). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (6–8). By heterologous expression in E. coli, Na⁺/H⁺ exchange activities could be shown for NhaS1–5 (5, 6). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5, 6). It has been reported that in these mutants the concentration of Na⁺ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9, 10). Therefore the Na⁺ extrusion by Synechocystis Na⁺/H⁺ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress.In contrast to the case in E. coli, Na⁺ is an essential element for the growth of some cyanobacteria (11, 12). Interestingly, the Na⁺/H⁺ antiporter homolog NhaS4 was identified as an uptake system for Na⁺ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na⁺ concentrations (7). The requirement of a Na⁺ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b₆f, and c₆) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na⁺/H⁺ antiporters therefore may coordinate both H⁺ and Na⁺ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11). It remains to be determined whether the six Na⁺/H⁺ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO₂ concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis.     

Identifying New Components Participating in the Secondary Cell Wall Formation of Vessel Elements in Zinnia and Arabidopsis

Xylem vessel elements are hollow cellular units that assemble end-to-end to form a continuous vessel throughout the plant body; the xylem vessel is strengthened by the xylem elements'' reinforced secondary cell walls (SCWs). This work aims to unravel the contribution of unknown actors in xylem vessel differentiation using the model in vitro cell culture system of Zinnia elegans differentiating cell cultures and the model in vivo system of Arabidopsis thaliana plants. Tracheary Element Differentiation-Related6 (TED6) and TED7 were selected based on an RNA interference (RNAi) screen in the Zinnia system. RNAi reduction of TED6 and 7 delayed tracheary element (TE) differentiation and co-overexpression of TED6 and 7 increased TE differentiation in cultured Zinnia cells. Arabidopsis TED6 and 7 were expressed preferentially in differentiating vessel elements in seedlings. Aberrant SCW formation of root vessel elements was induced by transient RNAi of At TED7 alone and enhanced by inhibition of both TED6 and 7. Protein–protein interactions were demonstrated between TED6 and a subunit of the SCW-related cellulose synthase complex. Our strategy has succeeded in finding two novel components in SCW formation and has opened the door for in-depth analysis of their molecular functions.     

A Mobile Secretory Vesicle Cluster Involved in Mass Transport from the Golgi to the Plant Cell Exterior

Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.     

Exo- and endocytotic trafficking of SCAMP2

Exo- and endocytotic membrane trafficking is an essential process for transport of secretory proteins, extracellular glycans, transporters and lipids in plant cells. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco BY-2 cells as a model system, we recently demonstrated that SCAMP2 positive structures containing secretory materials are transported from the Golgi apparatus to the plasma membrane (PM) and/or cell plate. This structure is consisted with clustered vesicles and was thus named the secretory vesicle cluster (SVC). Here, we have utilized the reversible photoswitching fluorescent protein Dronpa1 to trace the movement of SCAMP2 on the PM and cell plate. Activated SCAMP2-Dronpa fluorescence on the PM and cell plate moved into the BY-2 cells within several minutes, but did not spread around PM. This is consistent with recycling of SCAMP2 among endomembrane compartments such as the TGN, PM and cell plate. The relationship between SVC-mediated trafficking and exo- and endocytosis of plant cells is discussed taking into account this new data and knowledge provided by recent reports.Key words: SVC, secretory vesicle cluster, secretory carrier membrane protein 2, SCAMP2, exocytosis, endocytosis, dronpa, trans-Golgi network, Golgi apparatus, pectin, secretory protein, plasma menbrane, endosome, endomembrane systemExo- and endocytosis are essential events for cellular division and expansion. During exocytosis, lipids, proteins and polysaccharides are synthesized and/or modified in the Golgi apparatus and sorted into secretory vesicles at the trans-Golgi network (TGN) for transport to the PM² or extracellular space. Secretory carrier membrane proteins (SCAMPs) are a group of transmembrane proteins that plays vesicle trafficking between Golgi apparatus and PM in higher eukaryotic cells.³ Recently it was reported that in BY-2 cells, the rice SCAMP1 is localized to the PM and clathrin-coated tubularvesicular structures that were likely the early endosomal compartment.⁴ The same protein is also targeted to the cell plate in dividing cells.⁵ We have recently reported that another member of the SCAMP family, SCAMP2 from tobacco, is localized to the TGN, PM, cell plate and previously uncharacterized SVC organelles, which are an intermediate organelle between the TGN and PM.⁶Both SCAMP1 and SCAMP2 appear to be recycled between the PM and intracellular compartments. This was suggested by data using stelyl dye FM4-64 as an endocytotic marker, fluorescent-tagged SCAMP proteins and protein trafficking inhibitors such as brefeldin A and 2,3-butanedione monoxime. We reported that SCAMP2 is exported to the PM from dotted structures in the cells, and back from the PM via the acto-myosin pathway but do not transport FM4-64 positive early endosome.⁶ As SCAMP2 did not localize on multivesicular bodies, endocytic vesicles may be directly transported to TGN or Golgi.⁶ However, this data was obtained using inhibitors that disrupt the trafficking system, and thus we have now investigated the endocytotic transport in the absence of inhibitors.Dronpa is a reversible photo-switching fluorescent protein. Using 488 and 405 nm laser light this protein can be converted between fluorescent and non-fluorescent forms within milliseconds.¹ In order to test whether SCAMP2 returned to internal compartments from the PM, and to characterize the initial compartment of endocytosis, we expressed Dronpatagged SCAMP2 (SCAMP2-Dronpa) in tobacco BY-2 cells. The fluorescence of SCAMP2-Dronpa was similar to that for SCAMP2-YFP and -mRFP fusions⁶ (Fig. 1A, upper part). To visualize the endocytic transport of SCAMP2-Dronpa, we first erased the majority of Dronpa fluorescence by illumination with 488 nm laser and then activated the protein at a part of the PM by 405 nm illumination using confocal laser scanning microscope (LSM) (Fig. 1A, upper right part). The fluorescence was then traced by 30 minutes interval up to 90 minutes (Fig. 1A, lower pictures). SCAMP2 signals at the PM did not spread laterally in the PM and decreased over the time. In parallel, signals were detected in the cytosol and some of them appeared as puncta (Fig. 1A, arrowheads). This observation is consistent with our proposal that SCAMP2 is recycled back into the intracellular compartment from the PM, possibly through the TGN without passing through the early endosome.⁶Open in a separate windowFigure 1Time-lapse images of BY-2 cells expressing ScamP2-Dronpa. Fluorescence of Dronpa (mBL) tagged ScamP2 in the cells was erased by 488 nm laser and then a spot of Pm (a) or cell plate (B) was activated by 405 nm diode laser. these data were obtained by LSm510 meta, 63x oil lens, Argon laser with 488-nm excitation and a 505 nm LP filter (Zeiss). Arrowheads indicate dotted structures. Bar = 20 μm.During cytokinesis, cell wall materials and membrane proteins accumulate in the cell plate.^7–9 It has been shown that clathrin-coated vesicles (CCVs) and their constituents such as adapter proteins and dynamins are associated with cell plate membrane.¹⁰ However, it is not clear whether these molecules on the cell plate are re-used in daughter cells or are degraded at the cell plate. We thus investigated the movement of SCAMP2-Dronpa fluorescence on the cell plate during cytokinesis. Fluorescence of SCAMP2-Dronpa within late metaphase cells was first erased, followed by activation of SCAMP2-Dronpa specifically on the cell plate (Fig. 1B). Following a 15 min of incubation, SCAMP2-Dronpa associated fluorescence on the cell plate moved into intracellular structures within daughter cells. This confirmed our previous observation that SCAMP2 was transported to the trans-Golgi/TGN or intracellular structures from the cell plate during the cytokinesis.⁶Transmission electron microscope and LSM studies have revealed that CCVs are present in cell plates.¹⁰ Recent tomographic observation suggested that early- and late TGNs having CCVs exist not only in the cell plate region but also other places of the plant cell.¹¹ We found that immature SVCs, which might be identical to late TGN, are converted to mature SVCs by budding CCVs.⁶ Therefore, transport from the Golgi apparatus located inside of the cells to the PM or cell plate is mediated by SVCs, which are generated as immature SVCs from the TGN and converted to mature SVCs by budding CCVs during transport. Eventually, the mature SVC fuses with the PM and/or expanding cell plate (Fig. 2, left), after which CCVs are generated from the expanded cell plate to recycle SCAMPs and other molecules back to the daughter cells.Open in a separate windowFigure 2A model of the exocytotic pathway and SCAMP2 trafficking in plant cells. From the Golgi apparatus or tGn, at least two distinct compartments, such as maSc and SVc are generated for secretion. ScamP2 locates in the SVc and is transported to the Pm or cell plate. thereafter, SCAMP2 is recycled back to the TGN via clathrin-mediated endocytosis.     

Analysis of postextrasystolic relaxation response in the human heart

Postextrasystolic potentiation is the phenomenon in which ventricular contractile force is strengthened by a preceding premature beat. However, the response of diastolic function after an extrasystole is unknown. We studied 58 patients with chronic heart failure (CHF) and two control subjects to evaluate the response of relaxation following extrasystole. At cardiac catheterization, from the derivative of the left ventricular (LV) pressure, the ratio of LV peak negative dP/dt (–dP/dt) of a postextrasystole to a basal beat was calculated and defined as the postextrasystolic relaxation response (PRR). PRR was compared with parameters of left ventriculography: LV end-diastolic volume index (EDVI), LV end-systolic volume index (ESVI), and LV ejection fraction (EF). The PRRs of the two control subjects were 0.80 and 0.84. The mean PRR of the CHF patients was 0.99 ± 0.15. In all subjects, including patients and controls, correlation analysis between (EDVI, ESVI, and EF) and PRR yielded the following: (a) EDVI vs. PRR: R = 0.273, p = 0.036; (b) ESVI vs. PRR: R = 0.446, p < 0.001; and (c) EF vs. PRR: R = –0.520, p < 0.001. Thus, normal or non-failing human hearts showed a decline of –dP/dt in postextrasystole compared with the basal beats, but failing hearts had potentiated relaxation following an extrasystole.