Is cytoplasmic pH involved in the regulation of cell cycle in plants?

Modifications of cytoplasmic pH has biological significance in animal and plant cell development. Many observations suggest an important function of cytopiasmic pH in mitotic signalling in animal ceils. In Bidens pilosa cultivated under white light, acidification of cytoplasm, observed after mechanical trauma, is associated with an inhibition of DNA synthesis and a decrease in mitotic frequency. In contrast, in Bidens pilosa cultivated under blue light, mechanical stimulation induces an increase of cytoplasmic pH and stimulation of DNA duplication and mitotic activity. A correlation has been established between transient variations of cytoplasmic pH and rapid modification in cell development. The critical role of cytoplasmic pH in the regulation of the cell cycle in plants is discussed.     

pH regulation in apoplastic and cytoplasmic cell compartments of leaves

 The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO₂, HCl or NH₃ differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO₂ either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO₂ shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO₂ was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO₂-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO₂ under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO₂ stress increased in the presence of oxygen with the length of previous exposure to high CO₂. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO₂ was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO₂, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO₂ stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m² leaf area)⁻¹ s⁻¹ or less. Proton export under acid stress into the vacuole was about 1200 nmol m⁻² s⁻¹. The kinetics of pH responses to the addition or withdrawal of CO₂ indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast. Received: 19 July 1999 / Accepted: 29 December 1999     

Changes in the apoplastic pH are involved in regulation of xyloglucan breakdown of azuki bean epicotyls under hypergravity conditions

Hypergravity inhibited elongation growth of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls by decreasing the mechanical extensibility of cell walls via the increase in the molecular mass of xyloglucans [Soga et al. (1999) Plant Cell Physiol. 40: 581]. Here, we report that the pH value of the apoplastic fluid in epicotyls increased from 5.8 to 6.6 by hypergravity (300 x g) treatment. When the xyloglucan-degrading enzymes extracted from cell walls of the 1 x g control epicotyls were assayed in buffer at pH 6.6 and 5.8, the activity at pH 6.6 was almost half of that at pH 5.8. In addition, when enzymically active cell wall preparations obtained from 1 x g control epicotyls were autolyzed in buffer at pH 5.8 and 6.6 and then xyloglucans were extracted from the autolyzed cell walls, the molecular mass of xyloglucans incubated at pH 5.8 decreased during the autolysis, while that at pH 6.6 did not change. Thus, the xyloglucans were not depolymerized by autolysis at the pH value (6.6) observed in the hypergravity-treated epicotyls. These findings suggest that in azuki bean epicotyls, hypergravity decreases the activities of xyloglucan-degrading enzymes by increasing the pH in the apoplastic fluid, which may be involved in the processes of the increase in the molecular mass of xyloglucans, leading to the decrease in the cell wall extensibility.     

Screening of genes involved in cell migration in Dictyostelium

A single cell of wild-type Dictyostelium discoideum forms a visible colony on a plastic dish in several days, but due to enhanced cell migration, amiB-null mutant cells scatter over a large area and do not form noticeable colonies. Here, with an aim to identify genes involved in cell migration, we isolated suppresser mutants of amiB-null mutants that restore the ability to form colonies. From REMI (restriction enzyme-mediated integration)-mutagenized pool of double-mutants, we identified 18 responsible genes from them. These genes can be categorized into several biological processes. One cell line, Sab16 (Suppressor of amiB) was chosen for further analysis, which had a disrupted phospholipase D pldB gene. To confirm the role of pldB gene in cell migration, we knocked out the pldB gene and over-expressed gfp-pldB in wild-type cells. GFP-PLDB localized to plasma membrane and on vesicles, and in migrating cells, at the protruding regions of pseudopodia. Migration speed of vegetative pldB-null cells was reduced to 73% of that of the wild-type. These results suggest that PLDB plays an important role in migration in Dictyostelium cells, and that our screening system is useful for the identification of genes involved in cell migration.     

Initial cell type choice in Dictyostelium

Much remains to be understood about how a group of cells break symmetry and differentiate into distinct cell types. The simple eukaryote Dictyostelium discoideum is an excellent model system for studying questions such as cell type differentiation. Dictyostelium cells grow as single cells. When the cells starve, they aggregate to develop into a multicellular structure with only two main cell types: spore and stalk. There has been a longstanding controversy as to how a cell makes the initial choice of becoming a spore or stalk cell. In this review, we describe how the controversy arose and how a consensus developed around a model in which initial cell type choice in Dictyostelium is dependent on the cell cycle phase that a cell happens to be in at the time that it starves.     

Measurement of the cytoplasmic pH of Dictyostelium discoideum using a low light level microspectrofluorometer

Pyranine was employed as a sensitive pH indicator in a low light level microspectrofluorometer. The in vivo and in vitro standard curves of the 460/410-nm fluorescence excitation ratio of pyranine as a function of pH are identical. Therefore, pyranine is specifically sensitive to cytoplasmic pH in Dictyostelium. The cytoplasmic pH of single cells in a population of Dictyostelium discoideum amoebae was obtained for the first time. The median cytoplasmic pH of vegetative amoebae was 7.19. Carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler and a protonophore, lowered the median cytoplasmic pH to 6.12 when the extracellular pH was 6.1. This result is in accord with the protonophore activity of carbonyl cyanide m-chlorophenylhydrazone. Interest in the cytoplasmic pH of Dictyostelium has been greatly stimulated by the theory that cytoplasmic acidification promotes development of pre-stalk cells, while cytoplasmic alkalinization favors the pre-spore pathway (Gross, J. D., J. Bradbury, R. R. Kay, M. J. Peacey. 1983. Nature (Lond.). 303:244-245). The theory postulates that diethylstilbestrol (DES), an inducer of stalk cell differentiation and a plasma membrane proton translocating ATPase inhibitor, should cause acidification of the cytosol. Previous measurements of the effects of stalk cell inducers including DES on intracellular pH using 31P nuclear magnetic resonance measurements have failed to confirm the predictions of the theory, and have suggested that significant modification of the model may be required. Using pyranine as the pH indicator, we find that the median cytoplasmic pH in cells treated with 10 microM DES dropped from 7.19 to pH 6.02. This effect is consistent with the pharmacological action of DES and with the proposal that DES, a stalk cell inducer, should acidify the cytosol. These results provide direct support for the theory that cytoplasmic pH is an essential regulator of the developmental pathway in Dictyostelium.     

Cytoplasmic pH and glycolysis in the Dictyostelium discoideum cell cycle

Lactate production measurements during the cell cycle of synchronized populations of Dictyostelium discoideum cells reveal cyclic variations in glycolysis which correspond with pH_i oscillations which were discovered by us previously [(1985) Cell, in press]. Aerobic lactate production varies about 6-fold during the cell cycle and the lactate maxima correlate with (~ 0.25 pH unit) cyclic increases in pH. However, artificially altering pH_i using weak acids or bases does not influence the rate of lactate production in asynchronous cell populations. This result suggests that the cyclic variations in pH_i and those in glycolytic rate are not causally related events.     

Mechanisms of cytoplasmic pH regulation in alkaliphilic strains of Bacillus

The central challenge for extremely alkaliphilic Bacillus species is the need to establish and sustain a cytoplasmic pH that is over two units lower than the highly alkaline medium. Its centrality is suggested by the strong correlation between the growth rate in the upper range of pH for growth, i.e., at values above pH 10.5, and the cytoplasmic pH. The diminishing growth rate at extremely high pH values correlates better with the rise in cytoplasmic pH than with other energetic parameters. There are also general adaptations of alkaliphiles that are crucial prerequisites for pH homeostasis as well as other cell functions, i.e., the reduced basic amino acid content of proteins or segments thereof that are exposed to the medium, and there are other challenges of alkaliphily that emerge from solution of the cytoplasmic pH problem, i.e., reduction of the chemiosmotic driving force. For cells growing on glucose, strong evidence exists for the importance of acidic cell wall components, teichuronic acid and teichuronopeptides, in alkaliphily. These wall macromolecules may provide a passive barrier to ion flux. For cells growing on fermentable carbon sources, this and other passive mechanisms may have a particularly substantial role, but for cells growing on both fermentable and nonfermentable substrates, an active Na1-dependent cycle is apparently required for alkaliphily and the alkaliphile's remarkable capacity for pH homeostasis. The active cycle involves primary establishment of an electrochemical gradient via proton extrusion, a secondary electrogenic Na⁺/H⁺ antiport to achieve net acidification of the cytoplasm relative to the outside pH, and mechanisms for Na⁺ re-entry. Recent work in several laboratories on the critical antiporters involved in this cycle has begun to clarify the number and characteristics of the porters that support active mechanisms of pH homeostasis. Received: August 1, 1997 / Accepted: August 5, 1995     

Streptococcus faecalis mutants defective in regulation of cytoplasmic pH.

We have isolated two acid-sensitive mutants of Streptococcus faecalis (ATCC 9790), designated AS13 and AS25, which grew at pH 7.5 but not at pH below 6.0. The ionophore gramicidin D, which collapsed the pH gradient between the cytoplasm and the medium, had little effect on the growth of these mutants, indicating that growing cells maintain only a small pH gradient. In the presence of gramicidin D the growth rates of the parent and mutant strains were identical over a range of pH values. When glucose was added to a cell suspension at pH 6.4, the parent strain generated a pH gradient of 1.0 unit, interior alkaline; AS13 generated a pH gradient of only 0.5 units, and AS25 generated no measurable pH gradient. The proton permeability of the mutant strains was the same as that of the parent strain. These results suggest that a cytoplasmic pH of around 7.5 is required for the growth of the cells and that the mutant strains are unable to establish a neutral cytoplasmic pH in acidic medium because of damage to the regulatory system of the cytoplasmic pH. Mutant strains also have a reduced capacity to extrude protons and take up potassium. Therefore, it is likely that these cation transport systems are involved in the regulation of cytoplasmic pH.     

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum.     

14-3-3 proteins are involved in the regulation of mammalian cell proliferation

Summary. The 14-3-3 proteins are a family of abundant, widely expressed acidic polypeptides. The seven isoforms interact with over 70 different proteins. 14-3-3 isoforms have been demonstrated to be involved in the control of positive as well as negative regulators of mammalian cell proliferation. Here we used the approach of inactivating 14-3-3 protein functions via overexpression of dominant negative mutants to analyse the role of 14-3-3 proteins in mammalian cell proliferation. We found 14-3-3 dominant negative mutants to downregulate the proliferation rates of HeLa cells. Overexpression of these dominant negative mutants triggers upregulation of the protein levels of the cyclin-dependent kinase inhibitor p27, a major negative cell cycle regulator. In addition, they downregulate the protein levels of the important cell cycle promoter cyclin D1. These data provide new insights into mammalian cell proliferation control and allow a better understanding of the functions of 14-3-3 proteins.     

Microtubule arrays in fucoid zygotes are sensitive to cytoplasmic pH

Regulation of microtubule (MT) arrays and embryo‐genesis by cytoplasmic pH (pH_c) was investigated in zygotes of the brown alga Pelvetia compressa (J. Agardh) De Toni. pH_c was clamped to (set to) acidic values using a weak acid, propionic acid (PA), and to alkaline values using a weak base, methylamine (MA). Acidification of pH_c from the normal value of 7.4–7.5 to about 7.0 caused disruption of microtubule arrays. The nucleating activity was delocalized from the centrosomes and dispersed over the nuclear envelope, the number of MTs decreased, and MTs failed to extend into the cell cortex. Alkalinization to about pH 8.0 also caused dispersal of nucleating activity, but distinct centrosomes remained. MTs coursed in various directions following MA treatment, giving the array a disorganized appearance. Two MT‐dependent processes, rhizoid morphogenesis and cell division, were found to be perturbed by small changes in pH_c.     

Two regions of cadherin cytoplasmic domains are involved in suppressing motility of a mammary carcinoma cell line

E-cadherin has been termed an "invasion suppressor," yet the mechanism of this suppression is not known. In contrast, several reports indicate N-cadherin does not suppress but, rather, promotes cell motility and invasion. Here, by characterizing a series of chimeric cadherins we defined a previously uncharacterized region consisting of the transmembrane domain and an adjacent portion of the cytoplasmic segment that is responsible for the difference in ability of E- and N-cadherin to suppress movement of mammary carcinoma cells, as quantified from time-lapse video recordings. A mutation in this region enabled N-cadherin to suppress motility, indicating that both E- and N-cadherin can suppress, but the activity of N-cadherin is latent, presumably repressed by binding of a specific inhibitor. To define regions common to E- and N-cadherin that are required for suppression, we analyzed a series of deletion mutants. We found that suppression of movement requires E-cadherin amino acids 699-710. Strikingly, beta-catenin binding is not sufficient for and p120ctn is not involved in suppression of these mammary carcinoma cells. Furthermore, the comparable region of N-cadherin can substitute for this required region in E-cadherin and is required for suppression by the mutant form of N-cadherin that is capable of suppressing. Variations in expression of factors that bind to the two regions we have identified may explain previously observed differences in response of tumor cells to cadherins.     

Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation

Four isoforms of the Na+/H+ exchanger (NHE6-NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid- to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.     

Roles for cytoplasmic polyadenylation in cell cycle regulation

Polyadenylation of eukaryotic mRNAs in the nucleus promotes their translation following export to the cytoplasm and is an important determinant of mRNA stability. An additional level of control of gene expression is provided by cytoplasmic polyadenylation, which activates translation of a number of mRNAs important in orchestrating cell cycle events in oocytes. Recent studies indicate that cytoplasmic polyadenylation may be a mechanism of translational activation that is more widespread in eukaryotic cells. Here we discuss the roles of a recently identified family of nucleotidyl transferases (encoded by the cid1 gene family) in cell cycle regulation. To date, this family has been characterised mainly in yeasts, but it is conserved throughout the eukaryotes. Biochemical studies have indicated that a subset of members of this family function as cytoplasmic poly(A) polymerases targeting specific mRNAs for translation. This form of translational control appears to be particularly important for cell cycle regulation following inhibition of DNA synthesis.     

A developmental shift in cell volume regulation in Dictyostelium discoideum

Abstract. We describe developmentally modulated volume regulation phenomena in Dictyostelium discoideum amebae. Transfer of cells to a medium of different osmolarity leads initially to volume shifts in the expected direction (cells shrink in concentrated media, swell in dilute media). Subsequently, the cells may slowly return to the original volume over 30–90 min. The capacity for volume regulation appears and disappears during development. A mutant isolated for lack of regulation shows abnormalities in developmental timing. Mutant enrichment according to volume regulatory properties may thus be useful in some lines of work.     

A cytoplasmic cAMP-binding protein in Dictyostelium discoideum

A cytoplasmic cAMP-binding protein from Dictyostelium discoideum was purified about 1200-fold. The binding protein is relatively specific for cAMP, but also binds some other adenine derivatives; it has a molecular weight of approximately 185,000 and an apparent K_D of 1 μM cAMP. The highest level of cytoplasmic cAMP-binding activity is found in amoebae which have been starved for 0–2 hr. Amoebal extracts contain inhibitors of cAMP binding which are removed by chromatography through Sephacryl S200.     

Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH

Lysosomes labeled by uptake of extracellular horseradish peroxidase display remarkable changes in shape and cellular distribution when cytoplasmic pH is experimentally altered. Normally, lysosomes in macrophages and fibroblasts cluster around the cell center. However, when the cytoplasmic pH is lowered to approximately pH 6.5 by applying acetate or by various other means, lysosomes promptly move outward and accumulate in tight clusters at the very edge of the cell, particularly in regions that are actively ruffling before acidification but become quiescent. This movement follows the distribution of microtubules in these cells, and does not occur if microtubules are depolymerized with nocodazole before acidification. Subsequent removal of acetate or the other stimuli to acidification results in prompt resumption of ruffling activity and return of lysosomes into a tight cluster at the cell center. This is correlated with a rebound alkalinization of the cytoplasm. Correspondingly, direct application of weak bases also causes hyperruffling and unusually complete withdrawal of lysosomes to the cell center. Thus, lysosomes appear to be acted upon by microtubule-based motors of both the anterograde (kinesin) type as well as the retrograde (dynein) type, or else they possess bidirectional motors that are reversed by changes in cytoplasmic pH. During the outward movements induced by acidification, lysosomes also appear to be smaller and more predominantly vesicular than normal, while during inward movements they appear to be more confluent and elongated than normal, often becoming even more tubular than in phorbol-treated macrophages (Phaire-Washington, L., S. C. Silverstein, and E. Wang. 1980. J. Cell Biol. 86:641-655). These size and shape changes suggest that cytoplasmic pH also affects the fusion/fission properties of lysosomes. Combined with pH effects on their movement, the net result during recovery from acidification is a stretching of lysosomes into tubular forms along microtubules.