Munehiro Kikuyama

N-acetylchitooligosaccharides, biotic elicitor for phytoalexin production, induce transient membrane depolarization in suspension-cultured rice cells

SummaryN-acetylchitooligosaccharides (fragments of chitin) elicit the production of phytoalexin in suspension-cultured rice cells. This oligosaccharide elicitor induced rapid and transient membrane depolarization at sub-nanomolar concentrations. Only the oligomers with a certain degree of polymerization were active, while deacetylated chitooligosaccharides caused no effect. Such specificity coincided well with that for the elicitor activity, suggesting possible involvement of this transient change in membrane potential as one of the initial signals in the signal transduction sequence for the activation of defense responses.

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.

Cessation of cytoplasmic streaming follows an increase of cytoplasmic Ca2+ during action potential inNitella

SummaryTaking advantage of prolonged action potential under low temperature, we studied temporal relationship among the action potential, increase of cytoplasmic Ca2+ concentration and cessation of cytoplasmic streaming inNitella. The Ca2+ concentration began to increase at a very early stage of the action potential and the cessation of streaming followed that increase.

Mechanosensitive Ion Channels in Chara: Influence of Water Channel Inhibitors, HgCl2 and ZnCl2, on Generation of Receptor Potential

Characean internodal cells generate receptor potential (ΔEm) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated ΔEm at the moment of both compression and decompression, and the amplitude of ΔEm at the moment of decompression, (ΔEm)E, was larger than that at compression. The long-lasting stimulus caused a membrane deformation (ΔDm) having two components, a rapid one, (ΔDm)rapid, at the moment of compression and a slower one, (ΔDm)slow, during the long-lasting compression. We assumed that (ΔDm)slow might have some causal relation with the larger ΔEm at (ΔEm)E. We treated internodal cells with either HgCl2 or ZnCl2, water channel inhibitors, to decrease (ΔDm)slow. Both inhibitors attenuated (ΔDm)slow during compression. Cells treated with HgCl2 generated smaller (ΔEm)E compared to nontreated cells. On the other hand, cells treated with ZnCl2 never attenuated (ΔEm)E but, rather, amplified it. Thus, the amplitude of (ΔDm)slow did not always show tight correlation with the amplitude of (ΔEm)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 (ΔEm)E. Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca2+ channel.

Membrane control of cytoplasmic streaming in characean cells

When a characean cell generates an action potential, cytoplasmic streaming transiently stops and then recovers gradually. Calcium ion is one of the most important factors mediating between membrane excitation and cessation of cytoplasmic streaming.When an internode ofNitella flexilis is subjected to transcellular osmosis, both membrane depolarization and cessation of streaming take place at the endoosmotic part of the cell. It was also found that Ca2+ plays a key role in mediating between osmosis induced hydration of the cytoplasm and the cessation of cytoplasmic streaming. The present article reviews how Ca2+ acts as a second messenger in intracellular signal transduction in controlling the cytoplasmic streaming.

Membrane Stretching Triggers Mechanosensitive Ca2+ Channel Activation in Chara

In order to confirm that mechanosensitive Ca2+ 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 Ca2+ (Δ[Ca2+]c). However, the observed Δ[Ca2+]c decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (ΔEm) and stretching or compression of the plasma membrane. Significant ΔEm 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). ΔEm 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 ΔEm 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.

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.

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca(2+) and exocytosis of cortical alveoli (CABD). The sperm-penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at </=40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6-dimethylaminopurine (6-DMAP), however, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short-lived protein kinase(s), sensitive to 6-DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Copyright 1999 Wiley-Liss, Inc.

Studies on cessation of cytoplasmic streaming under K+-induced depolarization inNitella axilliformis

Internodal cells ofNitella axilliformis had a membrane potential of about−120mV and showed active cytoplasmic streaming with a rate of about 90 μm/sec in artificial pond water (APW) at 25C. When APW was replaced with 50 mM KCl solution, the membrane potential depolarized accompanying an action potential, and the cytoplasmic streaming stopped. Soon after this quick cessation, the streaming started again, but its velocity remained very low for at least 60 min. Removal of KCl from the external medium led to repolarization of the membrane and accelerated recovery of the streaming. The change in the concentration of free Ca2+ in the cytoplasm ([Ca2+]c) was monitored by light emission from aequorin which had previously been injected into the cytoplasm. Upon application of KCl to the external medium, the light emission, i.e., [Ca2+]c, quickly increased. It then decreased exponentially and reached the original low level within 100 sec. The cause of the long-lasting inhibition of cytoplasmic streaming observed even when [Ca2+]c had returned to its low resting level is discussed based on the mechanism proposed for action potential-induced cessation of cytoplasmic streaming; inactivation of myosin by Ca2+-dependent phosphorylation or formation of cross bridge between actin filaments and myosin.

Effect of caffeine on cytosolic Ca2+ level and cytoplasmic streaming inNitella axilliformis

SummaryThe effect of caffeine on cytosolic Ca2+ level and rate of cytoplasmic streaming was studied inNitella. External application of caffeine induced both a transient rise in cytosolic Ca2+ level and a decline in the rate of cytoplasmic streaming ofNitella. When EGTA-injected cells were bathed in EGTA-containing medium, external application of caffeine inhibited cytoplasmic streaming but induced no transient rise in cytosolic Ca2+ level. These results suggest that the inhibitory effect of caffeine on cytoplasmic streaming is independent of cytosolic Ca2+ level.