Chloroquine,a lysosomotropic agent,inhibits zygote formation in yeast

The effects of solar UV-B radiation on the green flagellate, Euglena gracilis, are measured under controlled conditions. Both photoorientation and motility are drastically impaired even after short exposure times of a few hours to sunlight not filtered by an ozone cuvette. Phototactic orientation starts to deteriorate after about 90 min and is completely lost after about 5 h. The percentage of motile cells in a population decreases likewise after an exposure of about 2 h and the velocity distributions shows a reduced speed of movement after an initial photokinetic increase. The damage is irreversible: in populations exposed for >2 h no living cell was found 24 h later. The UV-B sensitivity seems to be independent of the culture age at least over three weeks: While the percentage of motile cells changes with a peak at about 8 d, the relative UV-B induced inhibition is constant and depends only on the UV dose. DNA seems not to be the primary UV-B target since UV-B inhibition could not be repaired during subsequent dark or moderate light conditions even after low doses.     

Tilorone acts as a lysosomotropic agent in fibroblasts

Tilorone, an amphiphilic cationic compound with antiviral activity perturbed the lysosomal system. In cultured fibroblasts tilorone induced storage of sulfated glycosaminoglycans, enhanced secretion of precursor forms of lysosomal enzymes, inhibited intracellular proteolytic maturation of lysosomal enzymes, and inhibited receptor-mediated endocytosis of lysosomal enzymes. In isolated lysosomes tilorone was found to increase pH and to abolish the ATP-dependent acidification. These effects suggest that tilorone acts like a weak base that accumulates in acid compartments of the cells, raises the pH therein and interferes with lysosomal catabolic activity and with receptor-mediated transport of lysosomal enzymes.     

Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester

Lysosomes are fundamental for cell growth, and thus inhibition of the lysosomal function often leads to cell death. L-Leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. LeuLeuOMe is taken up through receptor-mediated endocytosis, and then converted into (LeuLeu)_n-OMe (n>3) by dipeptidyl peptidase I (DPPI) in lysosomes, which reportedly causes rupture of the lysosomes and DNA fragmentation. In this study we examined how lysosomal damage causes DNA fragmentation in LeuLeuOMe-treated HL-60 cells. When acridine orange was employed to monitor lysosomal membrane integrity, orange or red granular fluorescence was seen in normal cells. In contrast, LeuLeuOMe-treated cells showed orange, yellow or green cellular fluorescence all over the cytoplasm, suggesting that LeuLeuOMe induced a loss of lysosomal membrane integrity. The loss was inhibited by a DPPI inhibitor, GlyPheCHN₂ (GFCHN₂), but not by a caspase-3 inhibitor, Ac-DEVD-CHO, indicating that a condensation product was responsible for the loss. LeuLeuOMe also induced the activation of caspase-3-like protease and DNA fragmentation, both of which were inhibited by either GFCHN₂ or Ac-DEVD-CHO. Therefore, the activation of caspase-3-like protease links the loss of lysosomal membrane integrity to DNA fragmentation during apoptosis induced by LeuLeuOMe.     

Arachidonic and oleic acids stimulate zygote formation in the presence of mating pheromone in the yeast,Saccharomyces cerevisiae

Effect of exogenous fatty acids on zygote formation in Saccharomyces cerevisiae was studied. Arachidonic and oleic acids considerably stimulated zygote formation, but other fatty acids tested, linoleic, linolenic, stearic and palmitic acids, did not. Pretreatment experiments with arachidonic acid showed that the stimulation of zygote formation by the fatty acid required the presence of mating pheromone.Abbreviations YPD yeast-peptone-dextrose medium - A₅₃₀ absorbance at 530 nm     

The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probe.

The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids. (J Histochem Cytochem 48:251-258, 2000)     

PHEX neutralizing agent inhibits dentin formation in mouse tooth germ

The mutation of phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) can lead to human X-linked hypophosphatemic rickets which displays hypo-mineralization in bone and dentin. To study its possible roles in teeth, PHEX antibody was injected into pregnant mice on E15 to explore its roles on the formation of enamel and dentin. Mallory trichrome staining results showed that arrangements of ameloblasts and odontoblasts were irregular after PHEX antibody treatment. Differentiation of odontoblasts and the formation of dentin were inhibited. Spatiotemporal distribution of PHEX protein was observed in various stages of tooth germ. Immunohistochemical results showed positive PHEX signals appeared in the inner enamel epithelium on E16 and became stronger on E18. Ameloblasts and odontoblasts showed much higher PHEX expression on P1 and P3. Expression of PHEX in odontoblasts decreased accordingly. However, enamel formation was only slightly affected. The findings proved that a decrease in PHEX expression could suppress dentin formation.     

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

A sigma-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6- and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.     

Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death

The growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the ‘two-in-one'' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy.     

Fluorescence properties and staining behavior of monodansylpentane, a structural homologue of the lysosomotropic agent monodansylcadaverine.

We have recently shown that monodansylcadaverine labels autophagic vacuoles. Analysis of the mechanism underlying the labeling revealed that monodansylcadaverine acts as a lysosomotropic agent, being concentrated into acidic compartments by an ion-trapping mechanism, and as a solvent polarity probe, increasing its relative fluorescence intensity by interacting with membrane lipids that are highly concentrated in the autophagic vacuoles. In this study, we synthesized three structurally related derivatives of monodansylcadaverine, replacing the primary amino group of monodansylcadaverine with a neutral (dansylamylamine; MDH), a polar (dansylaminopentanol; MDOH), or an acidic group (dansylaminovaleric acid; MDA), to replace the lysosomotropic character of the marker. Whereas MDH showed a specific staining of autophagic vacuoles, the polar and acidic derivatives did not show any staining. We further demonstrate that the MDH staining of autophagic vacuoles is independent on the acidic pH and thus on an ion-trapping mechanism, but it still shows the same preferences for autophagic membrane lipids as monodansylcadaverine. We propose that MDH can specifically interact with lamellar bodies of the autophagic type as a solvent polarity probe. Therefore, dansylated aminopentane can be used as a specific marker for autophagic vacuoles in vivo and in fixed cells.(J Histochem Cytochem 49:177-185, 2001)     

The alkalinizing,lysosomotropic agent ML-9 induces a pH-dependent depletion of ER Ca2+ stores in cellulo

ML-9 elicits a broad spectrum of effects in cells, including inhibition of myosin light chain kinase, inhibition of store-operated Ca²⁺ entry and lysosomotropic actions that result in prostate cancer cell death. Moreover, the compound also affects endoplasmic reticulum (ER) Ca²⁺ homeostasis, although the underlying mechanisms remain unclear. We found that ML-9 provokes a rapid mobilization of Ca²⁺ from ER independently of IP₃Rs or TMBIM6/Bax Inhibitor-1, two ER Ca²⁺-leak channels. Moreover, in unidirectional ⁴⁵Ca²⁺ fluxes in permeabilized cells, ML-9 was able to reduce ER Ca²⁺-store content. Although the ER Ca²⁺ store content was decreased, ML-9 did not directly inhibit SERCA's ATPase activity in vitro using microsomal preparations. Consistent with its chemical properties as a cell-permeable weak alkalinizing agent (calculated pKa of 8.04), ML-9 provoked a rapid increase in cytosolic pH preceding the Ca²⁺ efflux from the ER. Pre-treatment with the weak acid 3NPA blunted the ML-9-evoked increase in intracellular pH and subsequent ML-9-induced Ca²⁺ mobilization from the ER. This experiment underpins a causal link between ML-9's impact on the pH and Ca²⁺ dynamics. Overall, our work indicates that the lysosomotropic drug ML-9 may not only impact lysosomal compartments but also have severe impacts on ER Ca²⁺ handling in cellulo.     

Chloroquine inhibits cyclization of squalene oxide to lanosterol in mammalian cells

Chloroquine inhibits the incorporation of [14C]acetate into sterols at a concentration of 10 microM or more in mouse L cells but has no effect on fatty acid synthesis and CO2 production from the same substrate even at a 10-fold higher concentration of the drug. The site of inhibition is distal to the formation of mevalonate since chloroquine also inhibits [14C]mevalonate metabolism to sterols and does not decrease the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into the total nonsaponifiable lipids. Analyses by thin layer and high pressure liquid chromatography of the nonsaponifiable lipid fraction from cultures incubated with chloroquine show an accumulation of radioactivity in the region of squalene oxide. Identification of the radiolabeled lipid as squalene oxide has been established by: (a) its co-migration with the authentic squalene oxide standard; (b) its conversion into squalene glycol by acid hydrolysis; and (c) its further metabolism to desmosterol when chloroquine is removed from the medium. Addition of chloroquine (12.5-50 microM) to 20,000 X g supernatant fractions of mouse liver homogenates inhibits the incorporation of [14C]mevalonolactone into cholesterol and lanosterol, with corresponding increases of [14C]squalene oxides, in a concentration-dependent manner. It appears, therefore, that chloroquine inhibits the enzymatic step catalyzed by 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7). Incubation of cell cultures with chloroquine (50 microM) arrests cell growth and causes cell death after 1-3 days. However, simultaneous incubation of chloroquine with either cholesterol or lanosterol prevents cell death and permits cell growth. Uptake of chloroquine is not affected by exogenous sterols since intracellular chloroquine concentrations are the same in cells grown with or without added sterols. The cytotoxicity of chloroquine, under our experimental conditions, must, therefore, be due primarily to its inhibition of sterol synthesis. In addition to its well known effect on protein catabolism, chloroquine has been found to inhibit protein synthesis. The significance of these findings concerning the use of chloroquine in studying the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity is discussed.     

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.     

Azithromycin, a lysosomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts

The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.     

Transmission, segregation, and recombination of mitochondrial genomes in zygote clones and protoplast fusion clones of yeast

Summary The average transmission-and recombination frequencies of mitochondrial markers are similar in diploid clones derived from zygotes or from fusion of haploid protoplasts of identical mating type. The transmissional patterns of mitochondrial markers in individual fusion-or zygote-clones, however, are very different. The time needed for regeneration of cell walls of fused protoplasts is found to be mainly responsible for this difference, since delay of first cell division in zygotes leads to similar results.     

Evidence for thylakoid membrane fusion during zygote formation in Chlamydomonas reinhardtii

To understand whether fusions of thylakoid membranes from the parental chloroplasts occurred during zygote formation in Chlamydomonas reinhardtii, we performed an ultrastructural analysis of the zygotes produced by crossing mutants lacking photosystem I or II protein complexes, in the absence of de novo chloroplast protein synthesis. Thylakoid membranes from each parent could be distinguished on thin sections due to their organization in "supergrana" in mutants lacking photosystem I centers, by freeze-fracturing due to the absence of most of the exoplasmic-face (EF) particles in mutants lacking photosystem II centers, by immunocytochemistry using antibodies directed against photosystem II subunits. We demonstrate that a fusion of the thylakoid membranes occurred during zygote formation approximately 15 h after mating. These fusions allowed a lateral redistribution of the thylakoid membrane proteins. These observations provide the structural basis for the restoration of photosynthetic electron flow in the mature zygote that we observed in fluorescence induction experiments.     

Haemoprotein formation in yeast

Summary A procedure was described for the isolation of mutants affected in the regulation of catalase activity. Two such mutants, cgr 1 and cgr 2 were obtained. Both of them show catalase activity that is resistant to repression by glucose, but is sensitive to anoxia to the same extent as the wild type.