Membrane cycling and macrovacuolation under the influence of cytochalasin: kinetic and morphometric studies

Fibroblasts exposed to higher doses of cytochalasin accumulate very big discrete endoplasmic vacuoles, the membrane of which is derived by internalization of plasmalemma. Morphometry confirms that the amount of surface interiorized is equal to the difference between the original cell surface area (before CD) and the reduced surface area measurable after CD-induced rounding. Correspondingly, there is a nearly two-fold increase in the activity of the ectoenzyme 5'-nucleotidase (a marker for plasma membrane) internally within the cytoplasm, after treatment with CD. Macrovacuolation increases cell volume by approximately 30%. Surface membrane is internalized as micropinocytotic vesicles at a rate measurable by the accumulation of HRP, a marker of fluid-phase pinocytosis. Uptake of HRP is shown to be enhanced at all times during exposure to CD, and is balanced by accelerated exocytic recycling of membrane except during a phase (approximately 4-8 hr) in which pinocytic uptake exceeds exocytosis. Vesicular membrane accumulated intracellularly in this period is retained in the endoplasm, and by successive fusions forms vacuoles in close approximation to microfilament aggregates. Once established, this new macrovacuolar membrane compartment is in dynamic equilibrium with the cell surface, and its membrane is cycled like the plasma membrane, in a mutual exchange of pinosomes between the several vacuoles and the cell surface. In drug-free medium vacuole membrane apparently reverts to the surface by pinocytotic recycling, and the cells recover normal characteristics 4-6 hr after withdrawal of cytochalasin.     

Myosin V-mediated vacuole distribution and fusion in fission yeast

The class V myosins are actin-based motors that move a variety of cellular cargoes [1]. In budding yeast, their activity includes the relocation of a portion of the vacuole from the mother cell to the bud [2, 3]. Fission yeast cells contain numerous (approximately 80) small vacuoles. When S. pombe cells are placed in water, vacuoles fuse in response to osmotic stress [4]. Fission yeast possess two type V myosin genes, myo51(+) and myo52(+) [5]. In a myo51Delta strain, vacuoles were distributed throughout the cell, and mean vacuole diameter was identical to that seen in wild-type cells. When myo51Delta and wild-type cells were placed in water, vacuoles enlarged by fusion. In myo52Delta cells, by contrast, vacuoles were smaller and mostly clustered around the nucleus, and fusion in water was largely inhibited. When cells containing GFP-Myo52 were placed in water, Myo52 was seen to redistribute from the cell poles to the surface of the fusing vacuoles. Vacuole fusion in fission yeast was inhibited by the microtubule drug thiabendazole (TBZ) but not by the actin inhibitor latrunculin B. This is the first demonstration of the involvement of a type V myosin, possibly via an interaction with microtubules, in homotypic membrane fusion.     

Pollen vacuoles and their significance

Vacuoles of several types can be observed in pollen throughout its development. Their physiological significance reflects the complexity of the biological process leading to functional pollen grains. Vacuolisation always occurs during pollen development but when ripe pollen is shed the extensive translucent vacuoles present in the vegetative parts in previous stages are absent. Vacuole functions vary according to developmental stage but in ripe pollen they are mainly storage sites for reserves. Vacuoles cause pollen to increase in size by water accumulation and therefore confer some degree of resistance to water stress. Modalities of vacuolisation occur in pollen in the same manner as in other tissues. In most cases, autophagic vacuoles degrade organelles, as in the microspore after meiosis, and can be regarded as cytoplasm clean-up following the transition from the diploid sporophytic to the haploid gametophytic state. This also occurs in the generative cell but not in sperm cells. Finally, vacuoles have a function when microspores are used for pollen embryogenesis in biotechnology being targets for stress induction and afterwards contributing to cytoplasmic rearrangement in competent microspores.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.     

Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora

Vacuoles are suggested to play crucial roles in plant defense-related cel death. During programmed cel death, previous live cel imaging studies have observed vacuoles to become simpler in structure and...     

Cytoskeletal organization affects cellular responses to cytochalasins: comparison of a normal line and its transformant

The relationships between cytoskeletal network organization and cellular response to cytochalasin D (CD) in a normal rat fibroblast cell line (Hmf-n) and its spontaneous transformant (tHmf-e), with markedly different cytoskeletal phenotypes, were compared (using immunofluorescence, electron microscopy, and DNAse I assay for actin content). Hmf-n have prominent, polar stress fiber (SF) arrays terminating in vinculin adhesion plaques whereas tHmf-e, which are apolar, epithelioid cells with dense plasma membrane-associated actin networks, lack SF and adhesion plaques. Hmf-n exposed to CD become markedly retracted and dendritic, SF-derived actin aggregates form large endoplasmic masses, and discrete tabular aggregates at the distal ends of retraction processes. Prolonged exposure leads to recession of process, cellular rounding, and development of large cystic vacuoles. tHmf-e cells exposed to similar doses of CD display a diagnostically different response; retraction is less drastic, cells retain broad processes containing scattered actin aggregates in discrete foci often associated with plasma membrane, large tabular aggregates are never found and processes persist throughout long exposure, vacuolation is uncommon. The CD-induced microfilamentous aggregates in Hmf-n are composed of short, kinky filament fragments forming a felt-like skein, often aggregates contain a more ordered array of roughly parallel fragments, while those of tHmf-e are very short, kinky, randomly orientated filaments imparting a distinctly granular nature to the mass. Total actin content and the amount of actin associated with detergent-resistant cytoskeletons increase following CD exposure in both cell types. Throughout exposure to CD, the actin-associated contractile proteins tropomyosin, myosin, and alpha-actinin co-localize within the actin aggregates in both cell types. Fodrin, the protein linking cortical actin to membrane, co-localizes with actin aggregates in tHmf-e cells and most, but not all, such aggregates in Hmf-n cells, consistent with their stress fiber derivation. Vinculin is lost from the tabular aggregates at the distal ends of retraction processes in Hmf-n cells concomitant with the fragmentation and contraction of SF. The aborized processes in both cells types contain strikingly similar axial cores of bundled vimentin filaments associated with passively compressed microtubules. The characteristic CD-induced distribution of actin filament aggregates and redistribution of vimentin in these cell types also occur when cells are allowed to respread from the rounded state in the presence of CD.     

An ultrastructural study of HeLa cell invasion with Salmonella typhi GIFU 10007

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium-attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair-like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane-bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane-bound vacuoles.     

Fine Structure of a Marine Ameba Associated with a Blue-Green Alga in the Sargasso Sea*,†

SYNOPSIS An ameba, bearing a fringe of scales on the plasmalemma surface, dwells among the filaments of the colonial, blue-green alga Trichodesmium thiebautii (Sournia), and preys upon bacteria growing within the colony. The cytoplasm is clearly differentiated into a fine fibrillar ectoplasm at the periphery of the cell and a central endoplasm containing most of the membranous organelles. The nucleus contains a spheroidal nucleolus which is centrally located, and a double membrane containing pores. The tubular mitochondria, microbodies, lysosomes, and endoplasmic reticulum are typical for protozoa. The Golgi apparatus consists of an array of elongate flattened cisternae. One surface is associated with a fine fibrillar layer and the opposite surface contains electron-dense vesicles (perhaps primary lysosomes) and scale-containing vesicles that appear to be the origin of the scales deposited on the plasma membrane. Three kinds of bacteria-containing vacuoles are presnt: (a) vacuoles surrounded by 3 membranes and containing bacteria that are either healthy or in an early stage of digestion, (b) singlemembrane vacuoles which are food vacuoles that become converted to digestive vacuoles, and (c) larger vacuoles resembling those in (b) which contain prey in an advanced stage of digestion. The presence of amebae within pelagic algal communities provides further evidence for the diversity of their habitats in the ocean.     

Colchicine-induced tubular,vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. I. Morphology and phosphatase cytochemistry

Treatment of rats with colchicine administered intraperitoneally at a dosage of 0.5 mg per 100 g of body weight for 6 hr induces extensive accumulations of tubular-vesicular and cisternal organelles in the absorptive cells of the small intestine. The formation of these organelle aggregates coincides with a reduction of microtubules and massive changes in the cellular organization including alterations of the Golgi apparatus and the plasma membrane. In most cases the accumulated tubules and vesicles contain a homogeneous electron-dense matrix, the cisternae often having the character of rigid lamellae. The organelle aggregates mainly occupy apical cell portions subjacent to the terminal web as well as basal cellular regions close to the basolateral plasma membrane. Tubular-vesicular as well as cisternal organelles react strongly for thiamine pyrophosphatase (TPPase), inosine diphosphatase (IDPase), acid phosphatase (AcPase) and trimetaphosphatase (TMPase). The staining pattern of TMPase differs from that of the other phosphatases in that the reaction is restricted to the colchicine-induced tubular-vesicular and cisternal aggregates, whereas TPPase, IDPase, and AcPase, respectively, also appear over Golgi stacks, multivesiculated bodies and plasma membrane. This phosphatase reactivity indicates the lysosomal character of the organelle aggregates.     

Action of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface

The projection of knobby protuberances at the cell surface (zeiosis) is a general cellular response to cytochalasin D (CD), resulting from herniation of endoplasm through undefended places of the cortex during cell contractions and displacement of microfilaments induced by CD. Zeiosis is prevented by agents that interfere with the contractile response to CD, such as inhibitors of energy metabolism or cyclic AMP. The developed protrusions, which remain relatively stable in the presence of CD, contain chiefly mono- or subribosomes, and occasionally other organelles normally resident in endoplasm; compact microfilament felt occupies their bases and extends into their proximal stalks. Protein synthesis in the knobs is less than half of that in the polyribosome-containing endoplasm residual in the main body of the cell. Knobs first protrude singly near the margin of the contracting cells and rapidly cluster into small groups in the periphery even at lower temperature. The clusters then migrate centripetally and coalesce into a large aggregate near the apex of the immobilized and retracted cell: this movement is energy- and temperature-dependent. Aggregation is more prominent and stable in cell lines of epithelial derivation than in fibroblastic or other lines in which nuclear extrusion occurs more readily. The latter is regarded as a special manifestation of zeiosis. Macromarkers, such as latex spherules, migrate like the zeiotic knobs on the cell surfaces in the presence of CD. The aggregated knobs, although persistent for days in the presence of CD, are rapidly recessed after withdrawal of the agent as ruffling is resumed and the cells spread. These movements are discussed in terms of current concepts of mobility of the cell membrane.     

The dynamic changes of tonoplasts in guard cells are important for stomatal movement in Vicia faba

Stomatal movement is important for plants to exchange gas with environment. The regulation of stomatal movement allows optimizing photosynthesis and transpiration. Changes in vacuolar volume in guard cells are known to participate in this regulation. However, little has been known about the mechanism underlying the regulation of rapid changes in guard cell vacuolar volume. Here, we report that dynamic changes in the complex vacuolar membrane system play a role in the rapid changes of vacuolar volume in Vicia faba guard cells. The guard cells contained a great number of small vacuoles and various vacuolar membrane structures when stomata closed. The small vacuoles and complex membrane systems fused with each other or with the bigger vacuoles to generate large vacuoles during stomatal opening. Conversely, the large vacuoles split into smaller vacuoles and generated many complex membrane structures in the closing stomata. Vacuole fusion inhibitor, (2s,3s)-trans-epoxy-succinyl-l-leucylamido-3-methylbutane ethyl ester, inhibited stomatal opening significantly. Furthermore, an Arabidopsis (Arabidopsis thaliana) mutation of the SGR3 gene, which has a defect in vacuolar fusion, also led to retardation of stomatal opening. All these results suggest that the dynamic changes of the tonoplast are essential for enhancing stomatal movement.     

Tyrosine storage vacuoles in insect fat body

The watery vacuoles first described from larval insect fat body (Chironomus, Voinov, 1927; Aedes, Wigglesworth, 1942; Rhodnius, Wigglesworth, 1967) have been studied in 4th and 5th stage Calpodes larvae. The vacuoles arise at the beginning (E+6–24 hr) of the 4th stadium from plasma membrane infolds that separate from the cell surface as provacuoles less than 1 μm in diameter. These provacuoles grow and fuse with one another through the intermolt until about half the volume of each fat body cell is occupied by a single, large vacuole. The vacuoles begin to disappear at molting. Their membrane is either incorporated into the plasma membrane by exocytosis or fragmented into vesicles that fuse to become lamellar bodies where the membranes are presumably digested. All the vacuoles have gone by a few hours after ecdysis.The tyrosine content of the fat body increases and decreases in proportion to the size of the vacuoles. As the vacuoles decrease at molting the titre of tyrosine in the hemolymph is transiently elevated at the time when there is most demand for phenolics for cuticle stabilization. Crystals having the form of tyrosine crystallize out from vacuoles separated from the fat body. In fat body extracts separated by thin layer chromatography, similar crystals occur only in the eluates from spots corresponding to tyrosine. The vacuoles are therefore presumed to be tyrosine stores used in cuticle stabilization at molting. They correspond to a type of aqueous storage compartment that is well known in plants but hitherto little recognized in animal cells.     

Nuclear functions in postconjugational development of Paramecium caudatum: Ability to form food vacuoles analyzed by nuclear elimination and implantation

Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc.     

Isolation and electrical characterization of tonoplast vesicles from the Kiwi fruit (Actinidia chinensis)

Nomarski interference microscopy technique showed that the cell juice of the Kiwi fruit ( Actinidia chinensis Planch.) is rich in membrane vesicles that resemble protoplasts and free vacuoles. These vesicles are obtained without enzyme or chemical treatment and probably arise from the rupture and revesiculation of the tonoplasts that limit the cytoplasmic strands of the cells. Vacuole fragmentation in situ probably causes the tonoplast to recombine around the vacuolar sap as well as around the cytoplasmic strands, which implies either original or inverse orientation of the inner face. Electrophysiological measurements in vesicles judged to have the original membrane orientation showed that their polarization was inside positive, the same as central vacuoles of protoplasts and isolated vacuoles.     

Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.     

An electron microscopic observation of conidium and hypha of Erysiphe graminis hordei

Summary The fine structure of conidia and hyphae ofErysiphe graminis hordei, attacking leaves of barley, were investigated. The cell walls of conidia and hyphae were relatively thin and consisted of two layers, the inner and outer layers. The surface of conidia was not smooth and the thickness of cell walls was irregular. A nucleus, mitochondria, endoplasmic reticula and vacuoles in plasma were identified. The vacuoles in conidia were tightly packed with fine granules. Such granules in vacuoles, however, were not observed in hyphal cells.A lamellar structure was located in conidia, but not in hyphal cells. This structure may be specific in conidia of this fungus, but its function is not yet known. Many glycogen granules were observed in endoplasm of conidia, which were scattered or congregated in groups. In hyphae, however, they were extremely few. Hyphal septa were connected directly with the inner layer of cell walls. These had simple septal pore. The Woronin bodies were detected in the endoplasm in the vicinity of hyphal septa.Contribution No. 192.     

RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell

Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin-based motility, such as CSNK2B, Listeria monocytogenes requires specific host factors, such as CSNK1A1 in order to form productive membrane protrusions and spread from cell to cell.     

A Highlights from MBoC Selection: mTOR regulates phagosome and entotic vacuole fission

Macroendocytic vacuoles formed by phagocytosis, or the live-cell engulfment program entosis, undergo sequential steps of maturation, leading to the fusion of lysosomes that digest internalized cargo. After cargo digestion, nutrients must be exported to the cytosol, and vacuole membranes must be processed by mechanisms that remain poorly defined. Here we find that phagosomes and entotic vacuoles undergo a late maturation step characterized by fission, which redistributes vacuolar contents into lysosomal networks. Vacuole fission is regulated by the serine/threonine protein kinase mammalian target of rapamycin complex 1 (mTORC1), which localizes to vacuole membranes surrounding engulfed cells. Degrading engulfed cells supply engulfing cells with amino acids that are used in translation, and rescue cell survival and mTORC1 activity in starved macrophages and tumor cells. These data identify a late stage of phagocytosis and entosis that involves processing of large vacuoles by mTOR-regulated membrane fission.     

ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.     

Identification and mitotic partitioning strategies of vacuoles in the unicellular red alga <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis>

Cyanidioschyzon merolae is considered as a suitable model system for studies of organelle differentiation, proliferation and partitioning. Here, we have identified and characterized vacuoles in this organism and examined the partitioning of vacuoles using fluorescence and electron microscopy. Vacuoles were stained with the fluorescent aminopeptidase substrate 7-amino-4-chloromethylcoumarin l-arginine amide, acidotrophic dyes quinacrine and LysoTracker, and 4′,6-diamidino-2-phenyl indole, which, at a high concentration, stains polyphosphate. Vacuoles have been shown to be approximately 500 nm in diameter with a mean of around five per interphase cell. The vacuolar H⁺-ATPase inhibitor concanamycin A blocked the accumulation of quinacrine in the vacuoles, suggesting the presence of the enzyme on these membranes. Electron microscopy revealed that the vacuoles were single membrane-bound organelles with an electron-dense substance, often containing a thick layer surrounding the membrane. Immunoelectron microscopy using an anti-vacuolar-H⁺-pyrophosphatase antibody revealed the presence of the enzyme on these membranes. In interphase cells, vacuoles were distributed in the cytoplasm, while in mitotic cells they were localized adjacent to the mitochondria. Filamentous structures were observed between vacuoles and mitochondria. Vacuoles were distributed almost evenly to daughter cells and redistributed in the cytoplasm after cytokinesis. The change in localization of vacuoles also happened in microtubule-disrupted cells. Since no actin protein or filaments have been detected in C. merolae, this result suggests an intrinsic mechanism for the movement of vacuoles that differs from commonly known mechanisms mediated by microtubules and actin filaments.