Kei Inouye

Oscillations of membrane potential in L cells. I. Basic characteristics.

SummaryThe membrane potentials and resistances of L cells were measured using a standard electrophysiological technique. The values obtained in physiological media were around −15 mV and 37 MΩ, respectively. Almost all the large nondividing L cells (giant L cells) showed spontaneous oscillations of the membrane potential between around −15 and −40 mV. Application of an appropriate electrical or mechanical stimulus was also capable of eliciting responses but such were usually induced only once. The total membrane conductance increased significantly and in parallel with such a hyperpolarizing response. Cooling of the cells and application of metabolic inhibitors to the cells completely blocked the spontaneous oscillation despite the fact that the electrically induced hyperpolarizing response remained. Intracellular K+, Na+ and Cl− concentrations were measured by means of a flame photometer and a chloridometer, and the equilibrium potential for each ion was estimated.

Analytical studies on migrating movement of the pseudo-plasmodium ofDictyostelium discoideum

SummaryMigrating movement of a pseudoplasmodium (slug) of the cellular slime mouldDictyostelium discoideum was analyzed using a time-lapse video tape recorder. Since slugs usually migrated with repeated interruptions of advance, migrating velocities were measured only within a period of forward movement. On the basis of some known facts and assumptions, a dynamical model for slug movement was formulated, which consists of motive force generated by slug cells against their intrinsic resistance and resistance of slime sheath at the tip. The migrating velocity of a slug depended neither on its width nor its volume, but solely on its length. Under any experimental conditions tested, a linear relationship always held between reciprocals of the two variables. The results were in good agreement with predictions of the model. Quantitative analyses of experimental results by the use of the model lead to the conclusions that a decrease in velocity at a low temperature is due to an increase in resistance of slime sheath at the tip, but that a decrease in velocity during prolonged migration is due to a decrease in motive force of constituent cells. An anterior isolate dissected from a slug migrated at a velocity greater than that of an intact slug of the same length. This was interpreted by the model to be due to the fact that the anterior cells have greater motive forces and intrinsic resistances than the posterior cells. The heterogeneous distributions of the two variables in the cell mass is discussed in reference to the mechanism of sorting out of cells.

Talin B is required for force transmission in morphogenesis of Dictyostelium

Talin plays a key role in the assembly and stabilisation of focal adhesions, but whether it is directly involved in force transmission during morphogenesis remains to be elucidated. We show that the traction force of Dictyostelium cells mutant for one of its two talin genes talB is considerably smaller than that of wild‐type cells, both in isolation and within tissues undergoing morphogenetic movement. The motility of mutant cells in tightly packed tissues in vivo or under strong resistance conditions in vitro was lower than that of wild‐type cells, but their motility under low external force conditions was not impaired, indicating inefficient transmission of force in mutant cells. Antibody staining revealed that the talB gene product (talin B) exists as small units subjacent to the cell membrane at adhesion sites without forming large focal adhesion‐like assemblies. The total amount of talin B on the cell membrane was larger in prestalk cells, which exert larger force than prespore cells during morphogenesis. We conclude that talin B is involved in force transmission between the cytoskeleton and cell exterior.

Correlations between prestalk-prespore tendencies and cAMP-related activities in Dictyostelium discoideum.

Amoebae of the cellular slime mould Dictyostelium discoideum (strain Ax2) grown in axenic medium containing 86 mM glucose [G(+) cells] or no glucose [G(−) cells] were examined for the characteristics of aggregation, cAMP secretion rate, cAMP phosphodiesterase activity and cell surface cAMP-binding activity. (When G(+) and G(−) cells are mixed, G(+) cells preferentially differentiate into prespore cells and sort out to the posterior region of a slug.) Under the same conditions, G(−) cells aggregate later than G(+) cells and the formation of stable streams by G(−) cells was particularly delayed. The movements of G(−) cells during aggregation were less organized compared with the ordered behaviour of G(+) cells, yet G(−) cells seemed to be more sensitive to chemotactic signals. Furthermore, the cAMP-binding activity of G(−) cells was considerably higher than that of G(+) cells, a difference that is probably due to the difference in the number of the cAMP receptor sites. Also G(−) cells, compared to G(+) cells, secreted slightly more cAMP and showed higher activity of cell-bound phosphodiesterase activity at the aggregation stage, whereas the extracellular phosphodiesterase activity was lower, although these differences were minor.

Talin couples the actomyosin cortex to the plasma membrane during rear retraction and cytokinesis

Contraction of the cortical actin cytoskeleton underlies both rear retraction in directed cell migration and cytokinesis. Here, we show that talin, a central component of focal adhesions, has a major role in these processes. We found that Dictyostelium talin A colocalized with myosin II in the rear of migrating cells and the cleavage furrow. During directed cell migration, talin A-null cells displayed a long thin tail devoid of actin filaments, whereas additional depletion of SibA, a transmembrane adhesion molecule that binds to talin A, reverted this phenotype, suggesting a requirement of the link between actomyosin and SibA by talin A for rear retraction. Disruptions of talin A also resulted in detachment of the actomyosin contractile ring from the cell membrane and concomitant regression of the cleavage furrow under certain conditions. The C-terminal actin-binding domain (ABD) of talin A exhibited a localization pattern identical to that of full-length talin A. The N-terminal FERM domain was found to bind phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] in vitro. In vivo, however, PtdIns(4,5)P2, which is known to activate talin, is believed to be enriched in the rear of migrating cells and the cleavage furrow in Dictyostelium. From these results, we propose that talin A activated by PtdIns(4,5)P2 in the cell posterior or cleavage furrow links actomyosin cytoskeleton to adhesion molecules or other membrane proteins, and that the force is transmitted through these links to retract the tail during cell migration or to cause efficient ingression of the equator during cytokinesis.

Origin and function of the stalk-cell vacuole in Dictyostelium.

Large vacuoles are characteristic of plant and fungal cells, and their origin has long attracted interest. The cellular slime mould provides a unique opportunity to study the de novo formation of vacuoles because, in its life cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly develops a single large vacuole and cellulosic cell wall to become plant-like cells (stalk cells). Here we describe the origin and process of vacuole formation using live-imaging of Dictyostelium cells expressing GFP-tagged ammonium transporter A (AmtA-GFP), which was found to reside on the membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate from acidic vesicles and autophagosomes, which fuse to form autolysosomes. Their repeated fusion and expansion accompanied by concomitant cell wall formation enable the stalk cells to rapidly develop turgor pressure necessary to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which are rich in H(+)-ATPase as in plant vacuoles, remained separate from these vacuoles. We further argue that AmtA may play an important role in the control of stalk-cell differentiation by modulating the pH of autolysosomes.

A voltage- and K+-dependent K+ channel from a membrane fraction enriched in contractile vacuole of Dictyostelium discoideum.

We obtained a membrane fraction enriched in the contractile vacuole by aqueous-polymer two-phase partitioning and its channel activities were analysed by incorporating it into artificial planar lipid bilayers. In asymmetrical KCl solutions (cis, 300 mM/100 mM, trans), we observed single-channel currents of a highly K(+)-selective channel with slope conductance of 102 pS and reversal potential of -20.4 mV, which corresponded to PK+/PCl- = 7. They showed bursts separated by infrequent quiescent periods. At 0 mV the mean open time was 2.0 ms. Among monovalent cations, Na+ and Li+ were impermeable, whereas Rb+ showed permeability equivalent to that of K+, although the unitary conductance was apparently reduced when the current flowed from the Rb+ containing side, suggesting that Rb+ is a permeant blocking ion. The open probability within bursts remained constant at approx.0.6 as long as the holding potential was positive on the cis side with respect to the trans side, but it decreased to 0 at negative potential. This channel was blocked by submillimolar concentrations of quinine and 30 mM TEA+. The open probability-voltage relationship showed a striking dependency on the KCl concentration on either side. This channel may play a role in water transport in this organelle.

SunB, a novel Sad1 and UNC‐84 domain‐containing protein required for development of Dictyostelium discoideum

A gene, sunB, encoding a novel class of Sad1 and UNC‐84 (SUN) domain, was isolated from a cDNA screen for suppressors of a mutation in Dd‐STATa – a Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription). The SunB protein localized in the area around the nucleus in growing cells, but in the multicellular stages it was predominantly found in prespore vacuoles (PSVs). A disruptant of sunB was multinucleated in the vegetative phase; during development it formed mounds with multiple tips and failed to culminate. The mutation was cell autonomous, and showed reduced expression of the prespore marker gene pspA and elevated expression of marker genes for prestalk AB cells. Interestingly, the level of SunB was abnormally high in the prestalk cells of Dd‐STATa mutants, which are defective in culmination. We conclude that SunB is essential for accurate prestalk/prespore differentiation during Dictyostelium development and that its cell‐type dependent localization is regulated by a Dd‐STATa‐mediated signaling pathway.

Wing colors based on arrangement of the multilayer structure of wing scales in lycaenid butterflies (Insecta: Lepidoptera)

Male wing colors and wing scale morphology were examined for three species of lycaenid butterflies: Chrysozephyrus ataxus, Favonius cognatus and F. jezoensis. Measurement of spectral reflectance on the wing surface with a spectrophotometer revealed species‐specific reflection spectra, with one or two peaks in the ultraviolet and/or green ranges. Observations of wing scales using an optical microscope revealed that light was reflected from the inter‐ridge regions, where transmission electron microscopy revealed a multilayer structure. Based on the multilayer dimensions obtained, three models were devised and compared to explain the measured reflectance spectrum. The results showed that the best fit is a model in which thicknesses of thin films of the multilayer system are not constant and air spaces between cuticle layers are more or less packed with cuticle spacers. This suggests that the specific wing colors of the species examined are produced by the species‐specific arrangement of the multilayer structure of wing scales.

Calcium-binding of synaptosomes isolated from rat brain cortex, III. Binding with some divalent heavy metal ions and calcium-binding sites.

SummaryFree ion concentration of some divalent heavy metal ions such as Mn2+, Co2+, Ni2+, Cd2+ and Zn2+ in the synaptosomal suspension was measured to determine binding with synaptosomes isolated from rat brain cortex. A dual wavelength spectrophotometer was utilized to monitor the absorbance changes of murexide raised by stepwise addition of these ions (as chloride salts). Such titration experiments of the synaptosomal suspension revealed that a part of the added divalent cation such as Mn2+, Co2+ or Ni2+ was almost instantaneously bound to synaptosomes in isotonic NaCl media. Our previous study (Kamino, Uyesaka & Inouye,J. Membrane Biol.17∶13, 1974) demonstrated that raised external K+ resulted in a specific noncompetitive inhibition of synaptosomal Ca-binding. Just like the Ca-binding, Mn-, Co- or Ni-binding was almost completely depressed by high external K+ or ruthenium red when the free concentration of the cations was 10 μm or less, while at higher concentrations the binding was not affected. The present results indicate that tested divalent cations bind with both “Ca-binding sites” and “non-Ca-binding sites” of synaptosomal membrane, the nature of the binding sites of both being quite different: the former is sensitive to high external K+ and to ruthenium red but the latter is not.