Metabolomics of a single vacuole reveals metabolic dynamism in an alga Chara australis.

Metabolomics is the most reliable analytical method for understanding metabolic diversity in single organelles derived from single cells. Although metabolites such as phosphate compounds are believed to be localized in different organelles in a highly specific manner, the process of metabolite compartmentalization in the cell is not thoroughly understood. The analysis of metabolites in single organelles has consequently presented a significant challenge. In this study, we used a metabolomic method to elucidate the localization and dynamics of 125 known metabolites isolated from the vacuole and cytoplasm of a single cell of the alga Chara australis. The amount of metabolites in the vacuole and the cytoplasm fluctuated asynchronously under various stress conditions, suggesting that metabolites are spatially regulated within the cell. Metabolite transport across the vacuolar membrane can be directly detected using the microinjection technique, which may reveal a previously unknown function of the vacuole.

Membrane stretching triggers mechanosensitive Ca2+ channel activation in Chara.

In order to confirm that mechanosensitive Ca(2+) channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca(2+) (Delta[Ca(2+)](c)). However, the observed Delta[Ca(2+)](c) decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (DeltaE(m)) and stretching or compression of the plasma membrane. Significant DeltaE(m) values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). DeltaE(m) appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large DeltaE(m) values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara.

Mechanosensitive ion channels in chara: influence of water channel inhibitors, HgCl2 and ZnCl2, on generation of receptor potential.

Characean internodal cells generate receptor potential (DeltaE (m)) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated DeltaE (m) at the moment of both compression and decompression, and the amplitude of DeltaE (m) at the moment of decompression, (DeltaE (m))(E), was larger than that at compression. The long-lasting stimulus caused a membrane deformation (DeltaD (m)) having two components, a rapid one, (DeltaD (m))(rapid), at the moment of compression and a slower one, (DeltaD (m))(slow), during the long-lasting compression. We assumed that (DeltaD (m))(slow) might have some causal relation with the larger DeltaE (m) at (DeltaE (m))(E). We treated internodal cells with either HgCl(2) or ZnCl(2), water channel inhibitors, to decrease (DeltaD (m))(slow). Both inhibitors attenuated (DeltaD (m))(slow) during compression. Cells treated with HgCl(2) generated smaller (DeltaE (m))(E) compared to nontreated cells. On the other hand, cells treated with ZnCl(2) never attenuated (DeltaE (m))(E) but, rather, amplified it. Thus, the amplitude of (DeltaD (m))(slow) did not always show tight correlation with the amplitude of (DeltaE (m))(E). Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (DeltaE (m))(E). Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca(2+) channel.

AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles.

A blue light (BL) receptor was discovered in stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae). Two homologs were identified in Vaucheria; each has one basic region/leucine zipper (bZIP) domain and one light-oxygen-voltage (LOV)-sensing domain. We named these chromoproteins AUREOCHROMEs (AUREO1 and AUREO2). AUREO1 binds flavin mononucleotide via its LOV domain and forms a 390-nm-absorbing form, indicative of formation of a cysteinyl adduct to the C(4a) carbon of the flavin mononucleotide upon BL irradiation. The adduct decays to the ground state in approximately 5 min. Its bZIP domain binds the target sequence TGACGT. The AUREO1 target binding was strongly enhanced by BL treatment, implying that AUREO1 functions as a BL-regulated transcription factor. The function of AUREO1 as photoreceptor for BL-induced branching is elucidated through RNAi experiments. RNAi of AUREO2 unexpectedly induces sex organ primordia instead of branches, implicating AUREO2 as a subswitch to initiate development of a branch, but not a sex organ. AUREO sequences are also found in the genome of the marine diatom Thalassiosira pseudonana (Bacillariophyceae), but are not present in green plants. AUREOCHROME therefore represents a BL receptor in photosynthetic stramenopiles.

Ionic mechanism of mechano-perception in Characeae.

Characean internodal cells generate receptor potential in response to mechanical stimuli. We studied responses of internodal cells to a long-lasting stimulus and the results were as follows. (i) The cell generated receptor potential at the moment of both compression and decompression. (ii) The receptor potential (DeltaE (m)) was significantly larger at the moment of decompression than at compression. (iii) The longer the duration of the stimulus, the larger was the magnitude of DeltaE (m) at the moment of decompression. (iv) Aequorin studies revealed that the increase in [Ca(2+)](c) (Delta[Ca(2+)](c)) took place at the moment of both compression and decompression. (v) The amplitude of Delta[Ca(2+)](c) was larger at the moment of decompression than at compression, as was the case for DeltaE (m). It was suggested that the amplitude of the receptor potential had a tight correlation with the degree of membrane deformation. We discussed the ionic mechanism of mechano-perception under a long-lasting stimulus in relation to mechanosensitive activation of Ca(2+) channels at the plasma membrane.

Possible involvement of mechanosensitive Ca2+ channels of plasma membrane in mechanoperception in Chara.

When an internodal cell of Chara corallina was stimulated with a mechanical pulse of various amplitudes lasting for 0.1 s (mechanical stimulus), the cell generated a receptor potential, which was highly dependent not only on the strength of the stimulus but also on the extracellular Cl- concentration. Extracellular Ca2+ was indispensable for generating receptor potential, since removal of Ca2+ reversibly inhibited generation of the receptor potential. The cytoplasmic Ca2+ level transiently rose upon mechanical stimulation. The stronger the mechanical stimulus, the larger was the increase in the cytoplasmic level of Ca2+. It is proposed that the first step of receptor potential is an activation of mechanosensitive Ca2+ channels at the plasma membrane.

Is Ca2+ release from internal stores involved in membrane excitation in characean cells?

An action potential in characean cells is accompanied by an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) which subsequently causes cessation of cytoplasmic streaming. Two Ca(2+ )origins are postulated for the increase in [Ca(2+)](c), extracellular and intracellular ones. For the extracellular origin, a Ca(2+) influx through voltage-dependent Ca(2+)-permeable channels is postulated. For the intracellular origin, a chain of reactions is assumed to occur, involving phosphoinositide-specific phospholipase C (PI-PLC) activation, production of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-dependent Ca(2+) release from internal stores [Biskup et al. (1999) FEBS Lett. 453: 72]. The hypothesis of the intracellular Ca(2+) origin was tested in three ways: injection of IP(3) into the streaming endoplasm, application of inhibitors of PI-PLC (U73122 and neomycin) and application of an inhibitor of IP(3)-receptor (2-aminoethoxydiphenyl borate; 2APB). Injection of 1 mM IP(3) into Chara cells did not change the rate of cytoplasmic streaming. Both U73122 (20 micro M) and neomycin (200 micro M) did not affect the generation of the action potential, cessation of cytoplasmic streaming and the increase in [Ca(2+)](c) caused by electric stimulus even 20-30 min after application. 2APB depolarized the membrane and inhibited the excitability of the plasma membrane. The results are not consistent with the data obtained by Biskup et al. (1999) who found inhibition of the excitatory inward current by neomycin and U73122. The hypotheses of internal and external Ca(2+) origins are discussed in the light of the present results.

Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs-An Experiment with Acetone-Treated Eggs.

To investigate whether or not causal relationship exists between the increase in intracellular Ca2+ and other cortical reactions at fertilization in the medaka, Oryzias latipes, intracellular Ca2+ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca2+ . Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca2+ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca2+ and exocytosis.

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes: (aequorin/calcium/oocyte maturation/1-methyladenine/starfish).

A transient increase in intracellular Ca2+ upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca2+ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca2+ transient followed by GVBD. Even when the transient increase of Ca2+ was inhibited by injecting EGTA into the oocyte, 1-MeAde treatment for a long period induced GVBD. These facts indicate that the Ca2+ increase is neither necessary nor sufficient for maturation of the starfish oocyte. When the oocyte, which had been treated with 1-MeAde for 1 min at a limited area around the animal pole, was treated again with 1-MeAde for 10 min starting about 15 min after the first treatment, a Ca2+ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1-MeAde at an area around the vegetal pole, neither Ca2+ transient nor GVBD was induced by the second treatment with 1-MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.