Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

Formation and reversion ofSchizosaccharomyces pombe cells with apical protoplast protuberances

Lytic enzymes from the hepatopancreas ofHelix pomatia do not induce a uniform digestion of the cell wall ofSchizosaccharomyces pombe over the entire cell surface. Perforations are formed in the growth zones through which a protoplast can locally protrude. Conditions were found under which the frequency of formation of apical protoplast protuberances is higher than 90% cells with such protuberances can reverse to normally multiplying cells. Translated by J. Spížek     

K+ fluxes in Schizosaccharomyces pombe

All living cells accumulate high concentrations of K+ in order to keep themselves alive. To this end they have developed a great diversity of transporters. The internal level of K+ is the result of the net balance between the activities of the K+ influx and the K+ efflux transporters. Potassium fluxes have been extensively studied and characterized in Saccharomyces cerevisiae. However, this is not the case in the fission yeast and, in addition, the information available indicates that both yeasts present substantial and interesting differences. In this paper we have reviewed and summarized the information on K+ fluxes in Schizosaccharomyces pombe. We have included some unpublished results recently obtained in our laboratory and, in particular, we have highlighted the significant differences found between the well-known yeast S. cerevisiae and the fission yeast Sch. pombe.     

Electron microscopy study of cell structures and their changes during growth and regeneration ofSchizosaccharomyces pombe protoplasts

The submicroscopic structure of growing and regeneratingSchizosaccharomyces pombe protoplasts cultivated in solid and liquid medium was studied by means of ultrathin sectioning. The protoplasts regenerate within 24 hours. Shortly before growth commences, rudiments of the new cell wall can be identified on the protoplast surface. Simultaneously, a large number of dictyosomes appears in the cytoplasm and decreases as synthesis of the new wall progresses. An increase occurs in the number of endoplasmic reticulum membranes some of which are arranged parallel with the cytoplasm membrane of the protoplast. Throughout the whole time of regeneration the protoplasts contain only one nucleus. The nucleo-cytoplasm ratio of growing and regenerating protoplasts is lower than in intact cells. The number of mitochondria falls at the outset of regeneration and does not rise again until towards the end.     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

A method for estimating ΔΦ generated by the H+-ATPase ofSchizosaccharomyces pombe in reconstituted plasma membrane vesicles

This work was supported by the NATO Linkage Grant HTECH.LG 930686 and by grant from theDeutsche Forschungsgemeinschaft     

Periodic forcing of a K+ channel at various temperatures.

The random sequence of openings and closings of single ion channels and the channel conductances have been the object of intense study over the past two decades with a view toward illuminating the underlying kinetics of the channel protein molecules. Channels that are sensitive to voltage, such as many K(+)-selective channels, have been particularly useful, because the kinetic rates can be manipulated by changing the membrane voltage. Most such studies have been performed under stationary conditions and usually at a single temperature. Here we report the results of experiments with sinusoidal modulation of the membrane potential performed at several temperatures. Dwell time and cycle histograms, objects not normally associated with ion channel experiments, are herein reported. From the last, the transition probability densities for channel opening and closing events are obtained. A new and unusual phase anticipation is observed in the cycle histograms, and its temperature dependence is measured.     

K+ binding sites and interactions between permeating K+ ions at the external pore mouth of an inward rectifier K+ channel (Kir2.1).

The arginine at position 148 is highly conserved in the inward rectifier K+ channel family. Increases of external pH decrease the single-channel conductance in mutant R148H of the Kir2.1 channel (arginine is mutated into histidine) but not in the wild type channel. Moreover, in 100 mM external K+, varying external pH induced biphasic changes of open channel noise, which peaks at around pH 7.4 in the R148H mutant but not in the wild type channel. The maximum single-channel conductances are higher in the wild type channel and R148H mutant at pH 6.0 than those in the R148H mutant at pH 7.4. However, the maximal conductance is achieved with much lower external [K+] for the latter. Interestingly, the single-channel conductances and open channel noise of the wild type channel at pH 6. 0 and the R148H mutant at pH 6.0 and 7.4 become the same in [K+] = 10 mM. These results indicate that the residue at position 148 is accessible to the external H+ and probably is involved in the formation of two K+ binding sites in the external pore mouth. Effective repulsion between permeating K+ ions in this area requires a positive charge at position 148, and such K+-K+ interaction is the essential mechanism underlying high K+ conduction rate through the Kir2.1 channel pore.     

Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel

We investigated the effects of changing extracellular K⁺ concentrations on block of the weak inward-rectifier K⁺ channel Kir1.1b (ROMK2) by the three intracellular cations Mg²⁺, Na⁺, and TEA⁺. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K⁺ in the inside (cytoplasmic) solution and 11 mM K⁺ in the outside (extracellular) solution, these three cations blocked K⁺ currents with a range of apparent affinities (K_i (0) = 1.6 mM for Mg²⁺, 160 mM for Na⁺, and 1.8 mM for TEA⁺) but with similar voltage dependence (zδ = 0.58 for Mg²⁺, 0.71 for Na⁺, and 0.61 for TEA⁺) despite having different valences. When external K⁺ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg²⁺ but not for Na⁺ or TEA⁺. In contrast, the N152Y mutation increased the affinity for block by TEA⁺ but not for Na⁺ or Mg²⁺. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg²⁺ but had a small effect on that by Na⁺. TEA⁺ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K⁺ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K⁺ are explained by shifts in the occupancy of sites within the selectivity filter by K⁺ ions.     

Ultrastructure and cell wall composition in cell division cycle mutants ofSchizosaccharomyces pombe deficient in septum formation

A number of temperature-sensitive cdc^- mutants ofSchizosaccharomyces pombe that are affected in septum formation were analyzed with respect to their ultrastructure and the composition of their cell wall polymers. One mutant strain, cdc 16–116, has a cell wall composition similar to the wild type (strain 972 h^-). However two other mutants, cdc 4 and cdc 7, show a higher galactomannan content and a lower -glucan content. In all the mutants tested, total glucose incorporation, protein, RNA and DNA synthesis increased similarly to wild type over 3 1/2 h. After 2–3 h of incubation at the non permissive temperature-35°C-, cell numbers remained constant although, increases in optical densities at 600 nm were observed. According to scanning electron microscopy, the mutants had aberrant shapes after 5h of incubation at 35°C. Transmission electron microscopy showed that cdc 3 is unable to complete septum formation. cdc 4 showed the most varied morphological shapes and aberrant depositions of cell wall material. cdc 8 exhibited a deranged plasma membrane and cell wall regions near of cell poles; an abnormal septum and several nuclei. cdc 7 showed elongated cells with several nuclei and with an apparently normal cell wall completely lacking in septum and septal material. cdc 16 showed more than one septum per cell.     

Calcium influx mediated by the inwardly rectifying K+ channel Kir4.1 (KCNJ10) at low external K+ concentration

COS-1 cells with heterologeous expression of the Kir4.1 (KCNJ10) channel subunit, possess functional Kir4.1 channels and become capable to generating cytosolic Ca2+ transients, upon lowering of the extracellular K+ concentration to 2 mM or below. These Ca2+ transients are blocked by external Ba2+ (100 microM). Acute brain stem slices from wild-type mice (second post-natal week), which were loaded with the fluorescent Ca2+ indicator Oregon Green BAPTA-1-AM, were exposed to 0.2 mM K+. Under these conditions astrocytes, but not neurons, responded with cytosolic Ca2+ elevations in wild-type mice. This astrocyte-specific response has previously been used to identify astroglial cells type [R. Dallwig, H. Vitten, J.W. Deitmer, A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28 (2000) 247-259]. In Kir4.1 knock-out (Kir4.1-/-) mice, the number of responding cells was dramatically reduced and the Ca2+ transients in responding cells were significantly smaller than in wild-type mice. Our results indicate that Kir4.1 channels are the molecular substrate for the observed Ca2+ influx in astrocytes under conditions of low external K+-concentration.     

Insulin-induced changes in membrane potential and 3-O-methylglucose uptake at various external K concentrations in frog skeletal muscle

Insulin induced a hyperpolarization of the membrane and stimulated the 3-O-methylglucose (3-O-MG) uptake in frog skeletal muscle. In the present study, the relationship between the insulin-induced changes in the membrane potential and the 3-O-MG uptake was investigated. The stimulatory action of insulin on the 3-O-MG uptake was mediated by two different mechanisms. One of them was dependent on the change in the membrane potential and the other was independent of the change in the membrane potential. Both values of the insulin-induced changes in the membrane potential and the 3-O-MG uptake were diminished by increasing the external K concentration. One of the causes for the diminution of the 3-O-MG uptake with a rise of the external K concentration would be the decrease in the magnitude of the insulin-induced hyperpolarization.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Internal and external K+ help gate the inward rectifier.

Recent investigations have demonstrated substantial reductions in internal [K+] in cardiac Purkinje fibers during myocardial ischemia (Dresdner, K.P., R.P. Kline, and A.L. Wit. 1987, Circ. Res. 60: 122-132). We investigated the possible role these changes in internal K+ might play in abnormal electrical activity by studying the effects of both internal and external [K+] on the gating of the inward rectifier iK1 in isolated Purkinje myocytes with the whole-cell patch-clamp technique. Increasing external [K+] had similar effects on the inward rectifier in the Purkinje myocyte as it does in other preparations: increasing peak conductance and shifting the activation curve in parallel with the potassium reversal potential. A reduction in pipette [K+] from 145 to 25 mM, however, had several dramatic previously unreported effects. It decreased the rate of activation of iK1 at a given voltage by several-fold, reversed the voltage dependence of recovery from deactivation, so that the deactivation rate decreased with depolarization, and caused a positive shift in the midpoint of the activation curve of iK1 that was severalfold smaller than the associated shift of reversal potential. These changes suggest an important role of internal K+ in gating iK1 and may contribute to changes in the electrical properties of the myocardium that occur during ischemia.