Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Calmodulin Localization and its Effects on Endocytic and Phagocytic Membrane Trafficking in Paramecium multimicronucleatum

ABSTRACT. In ciliates, calmodulin (CaM), as in other cells, has multiple functions, such as activation of regulatory enzymes and modulating calcium‐dependent cellular processes. By immunogold localization, CaM is concentrated at multiple sites in Paramecium. It is seen scattered over the cytosol, but bound to its matrix, and is concentrated at the pores of the contractile vacuole complexes and with at least three microtubular arrays. It was localized peripheral to the nine‐doublet microtubules of the ciliary axonemes. The most striking localization was on the akinetic side only of the cytopharyngeal microtubular ribbons opposite the side where the discoidal vesicles, acidosomes and the 100‐nm carrier vesicles bind and move. CaM was also present at the periphery of the postoral microtubular bundles along which the early vacuole moves and was associated with the cytoproct microtubules that guide the spent digestive vacuoles to the cytoproct. It was not found on the membranes of, or in the interior of nuclei, mitochondria, phagosomes, and trichocysts, and was only sparsely scattered over the cytosolic sides of discoidal vesicles, acidosomes, lysosomes, and digestive vacuoles. Together the associations with specific microtubular arrays and the effects of trifluoperazine and calmidazolium indicate that CaM is involved (i) in vesicle transport to the cytopharynx area for vacuole formation and subsequent vacuole acidification, (ii) in early vacuole transport along the postoral fiber, and (iii) in transporting the spent vacuole to the cytoproct. Higher CaM concentrations subjacent to the cell's pellicle and close to the decorated tubules of the contractile vacuole complex may support a role for CaM in ion traffic.     

A New Entodiniomorphid Ciliate,Troglocorys cava n. g., n. sp., from the Wild Eastern Chimpanzee (Pan troglodytes schweinfurthii) from Uganda

ABSTRACT. Troglocorys cava n. g., n. sp. is described from the feces of wild eastern chimpanzee, Pan troglodytes schweinfurthii, in Uganda. This new species has a spherical body with a frontal lobe, a long vestibulum, a cytoproct located at the posterior dorsal side of the body, an ovoid macronucleus, a contractile vacuole near the cytoproct, and a large concavity on the left surface of the body. Buccal ciliature is non‐retractable and consists of three ciliary zones: an adoral zone surrounding the vestibular opening, a dorso‐adoral zone extending transversely at the basis of the frontal lobe, and a vestibular zone longitudinally extending in a gently spiral curve to line the surface of the vestibulum. Two non‐retractable somatic ciliary zones comprise arches over the body surface: a short dorsal ciliary arch extending transversely at the basis of the frontal lobe and a wide C‐shaped left ciliary arch in the left concavity. Because of the presence of three ciliary zones in the non‐retractable buccal ciliature, the present genus might be a member of the family Blepharocorythidae, but the large left concavity and the C‐shaped left ciliary arch are unique, such structures have never been described from other blepharocorythids.     

The cell structure of the reticulopodial amoeba <Emphasis Type="Italic">Filoreta marina</Emphasis> Bass et Cavalier-Smith (Cercozoa)

The cell structure of a reticulopodial amoeba, Filoreta marina Bass et Cavalier-Smith, is described. The cell is covered by a unitary membrane; glycostyles are absent. The life cycle comprises the uninucleate stage, multinucleate plasmodium, and spherical uninucleate cysts. The microtubules inside pseudopodia and the flagella are absent. The vesicular nuclei, endoplasmic reticulum, and Golgi apparatus are of a typical structure. The plasmodium produces a branched network of narrow anastomosing (reticulopodia) and wide pseudopodia. Thin unbranched micropseudopodia have also been observed. Oval mitochondria with a size of 0.3 × 0.6 μm contain the tubular cristae. A bidirectional motion of the cytoplasm inside the reticulopodia has been detected. Extrusomes (extrusive organelles) have not been found. The contractile vacuole is absent. F. marina feeds on bacteria. A similarity of this amoeba to other filose and reticulopodial amoebas is discussed.     

Observations on the Ultrastructure of Uronema spp., Marine Scuticociliates*

SYNOPSIS. Observations of the ultrastructure of marine scuticociliatids, tentatively assigned to the genus Uronema, were made by light, transmission electron, and scanning electron microscopy. Giant, cortically oriented mitochondria filled the subpellicular, intermeridional areas, and were in close association with the epiplasm immediately under the inner alveolar sac membranes. Reconstructions of serial sections of the posterior poles of ciliates indicated that the intermeridional mitochondria could fuse at that point and the entire chondriome might at times be a single organelle. A system of tubules was observed to be intimately associated with the mitochondria in the posterior region. The tubules anastomosed and were directed posteriorly into the region of the nephridial-contractile vacuole system. The outer surfaces were coated with projections arranged in helical patterns. The system may be regarded as a fluid segregation organelle. The tripartite nature of the polar basal body complex observed by silver impregnation was confirmed by transmission electron microscopy. The 3 structures were the basal body of the caudal cilium and 2 parasomal sacs. A prominent ring around the caudal cilium was observed by scanning electron micrcscopy; it is probably responsible for the silver deposition surrounding the polar basal body complex that can be seen by light microscopy of silver-impregnated specimens. The ultrastructure of the nonmotile caudal cilium and its kinetosome was unremarkable, being like that of the motile, somatic cilia. The micronuclear and macronuclear outer membranes were continuous at several sites. Such interconnections explain the intimate physical relationship between the nuclei during interphase in many ciliates, and could be a structural basis for chemical communication between the 2 nuclear types. Within the cytoplasm surrounding the opening of the cytoproct, numerous clear vesicles were observed. Their position and appearance suggested that the cytoproct may be involved in the elimination of solutions as well as solids. Food vacuoles, cortical microtubules, lamellar vesicles, disc-shaped vesicles, mucocysts, and a contractile vacuole and its pore were also observed.     

Ultrastructural Aspects of the Somatic Cortex and Contractile Vacuole of the Ciliate,Ichthyophthirius multifiliis Fouquet1

The trophont stage in the life cycle of Ichthyophthirius multifiliis was studied in the electron microscope. Surface ridges contain up to 24 ridge microtubules, disposed as a ribbon. Kinetosomes show the classic morphology of 9 triplets of microtubules. Associated with each kinetosome is a kinetodesmal fibril, originating in proximity to triplets 5, 6, and 7, and having a 30 nm periodicity; 3 to 5 postciliary microtubules, originating between triplets 8 and 9; and up to 3 transverse microtubules, originating at triplet 4, as well as a parasomal sac. Each cell is partially enclosed by a system of 3 “unit” membranes: the outer limiting membrane, and the outer and inner alveolar membranes. The last two membranes define the alveolar sac. Mucocysts, each with a dense core, are present in large numbers. The contractile vacuole system includes the contractile vacuole, associated tubules and vesicles, injection canals, a discharge canal, and a pore. Microtubules abound in the walls of the contractile vacuole, injection and discharge canals, and in the region of the pores, where both ring and radial microtubular arrangements are noted. The ultrastructure suggests that I. multifiliis is more closely related to Tetrahymena pyriformis than to Paramecium aurelia.     

Light and Electron Microscopic Description of a Colorless Euglenoid,Serpenomonas costata n. g., n. sp.1

ABSTRACT. A new genus of rigid, colorless, phagotrophic euglenoid is described from a standing pond on a salt marsh. The cell body measures 21–25 μm long, is about 18 μm wide, has a slight dorso-ventral flattening, and is marked by distinct pellicular ridges. The organism, described as Serpenomonas costata, has two unequal, antapically inserted, heterodynamic flagella. The shorter flagellum is anteriorly directed during swimming and the longer one trails posteriorly. Cells move along the substrate with a creeping motion. The ingestion apparatus is composed of separate ribs extending the length of the cell and is incorporated into the ventral pellicular ridge. The apparatus is independent of the canal and reservoir and is not protrusible. The taxonomic affinities of Serpenomonas with other euglenoids are discussed.     

Ultrastructure of the marine predatory flagellate <Emphasis Type="Italic">Metromonas simplex</Emphasis> Larsen et Patterson, 1990 (Cercozoa)

The ultrastructure of the marine predatory flagellate Metromonas simplex Larsen et Patterson was studied. The cell is surrounded by a low-contrast fibrous layer composed of thin hairs covered by a thin bilayer membrane and an outer layer of thin short fibers. The plasmalemma lies under these layers. The predator captures whole cells of the prey, usually bodonids or chrysomonads. The cytostome as a cell pocket is undetectable. The long flagellum bears very thin mastigonemes (hairs) with lengths of 0.8–1.0 μm; the short flagellum is naked and reduced in length. The transitional zone lacks spirals or other additional elements. The transversal plate is elevated on the cell surface. The flagellar root system is very simple and has one microtubular band which originates near the kinetosomes. The latter are parallel to each other and interconnected by fibrous bridges. The vesicular nucleus, Golgi apparatus, and endoplasmic reticulum are of typical structures. The oval mitochondria of 0.6–2.5 μm contain lamellar cristae. The cylindrical extrusomes (trichocysts) found in the cytoplasm have lengths of 1.0–1.4 μm and diameters of 0.12–0.08 μm. The trichocysts have a wheel-shaped structure with 13 spokes visible in cross-sections. The contractile vacuole is absent. The similarity that M. simplex shares with Metopion fluens Larsen et Patterson, cryothecomonads, and other predatory flagellates is discussed.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

Observations on Askenasia volvox (Claparède & Lachmann, 1859)

Specimens of Askenasia volvox from southwestern Indiana have a variable body shape and a size range of 29 × 22 μ m to 42 × 27 μ m. The pectinelles, membranelles, and cirri each number 47–48, and respectively have a length of 8–10, 20, and 30 μ m. The pectinelles arise from ciliary rows consisting of ～10 kinetosomes, and every membranelle originates from 2 adjacent rows of ～14–17 kinetosomes. Different algae as well as Bodo and certain other protozoa are ingested. The macronucleus, micronucleus, the cytoplasm and its inclusions, trichites, the function of the contractile vacuole, and movements and behavior are described.     

Membrane Dynamics of the Contractile Vacuole Complex of Paramecium1

ABSTRACT. Membrane dynamics of the contractile vacuole complex of Paramecium were investigated using conventional electron microscopy of cells so that the vacuoles were serial-sectioned longitudinally and transversely. During systole, vacuolar membrane collapses first into flattened cisternae which undergo further modification into a mass of interconnected small membrane tubules. These tubules retain their connections with the radiating microtubular ribbons; consequently they are found only in the poleward hemisphere. Permanent connections between ampullae and the collapsed vacuole membrane could not be verified nor was a sphincter-like mechanism for closing such a junction observed. Membranes of the ampullae and the collecting canals also collapse to varying extents into arrays of tubules that remain bound to microtubular ribbons during diastole. Thus vacuole, ampullae, and collecting canal membranes all assume tubular forms when internal volume is at a minimum. Having failed to observe a microfilamentous encasement of the vacuole, we suggest that an alternative mechanism for the “contractile” function should be sought. One such is based on fluid volume increase and fluid flow within transiently interconnected tubular membrane systems that cycle between a tubular and a planar membrane form as internal volume is periodically increased and reduced. The driving force for this mechanism might best be sought in the molecular structure of the membranes of the contractile vacuole complex.     

Fine structural and immunohistochemical detection of collar enamel in the teeth of <Emphasis Type="Italic">Polypterus senegalus</Emphasis>, an actinopterygian fish

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middle-region- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.     

Ultrastructure of the Spermathecae of Parafabricia ventricingulata and Three Species of Oriopsis (Polychaeta: Sabellidae)

Parafabricia ventricingulata females have a pair of spermathecae located in the radiolar crown anterio-dorsal to the buccal opening. The spermathecae have three regions; an entrance, 7 μm across, leading into a ciliated ‘atrium’ that is approximately 50 μm long; a connecting piece, 2–5 μm across and 25 μm long, leading from the ‘atrium’ to the sperm receptacle. The sperm receptacle is heavily pigmented and spherical. The sperm lie in a large mass in the receptacle with no particular orientation. Oriopsis bicoloris females have a pair of unpigmented spermathecae in the collar behind the radiolar crown. Each spermatheca is a simple blind duct 100 μm long, with a lumen 8 μm in diameter. Between 30 and 40 sperm lie in the lumen of each spermatheca. Oriopsis brevicollaris females have a pair of spermathecae located in the radiolar crown above the buccal opening. From the opening, 10 μm across, a blind duct runs for 90 μm. Sperm are stored in the distal region of the duct. Sperm lie along the margins of the duct in close contact with microvilli. Up to 10 sperm were found in each spermatheca. Oriopsis mobilis females have a pair of spermathecae located in the radiolar crown above the buccal opening. The opening, 3 μm across, leads into a blind duct that runs for 30 μm. Sperm are stored in the distal region of the spermathecae where they are embedded in spermathecal cells. Between 10 and 20 sperm were found in each spermatheca. Oriopsis dentata was found not to have spermathecae. The homologies of the spermathecae found within the Sabellinae and Fabriciinae (Sabellidae) and the Spirorbinae (Serpulidae) are discussed, but cannot be resolved on present evidence.     

ONTOGENY,HISTOCHEMISTRY AND FINE STRUCTURE OF CELLULAR INCLUSIONS IN VEGETATIVE CELLS OF ANTITHAMNION DEFECTUM (CERAMIACEAE,RHODOPHYTA)1

The ultrastructure and histochemistry of developing and mature cell inclusions in vegetative cells of Antithamnion defectum Kylin were examined. Those studied were chloroplast inclusions, cytoplasmic crystals and spherical bodies within the vacuole. Chloroplasts of mature vegetative cells contain an interthylakoidal, apparently noncrystalline deposit of undetermined chemical identity. The bodies are parallel to the long axis of the plastid, are square (0.13 μm) in cross-section, and up to 3 μm long. Spherical vacuolar bodies (0.5–1.5 μum diam) are formed during early stages of vacuole formation by accumulation of protein deposits in swelling endoplasmic reticulum (ER) cisternae. Swelling of smooth ER contiguous to the ER containing the deposits results in the vacuole enclosing the spherical bodies. In mature cells, vesicles appear to be secreted into the preformed vacuole. Cytoplasmic proteinaceous crystalloids develop without a bounding membrane and may serve as protein reserves.     

THE CONTRACTILE VACUOLE AS AN ENDOCYTIC ORGANELLE OF THE CHLAMYDOMONAD FLAGELLATE GLOEOMONAS KUPFFERI (VOLVOCALES,CHLOROPHYTA)1

The endomembrane system of the chlamydomonad flagellate, Gloeomonas kupfferi Skuja, consists of a complex network of endoplasmic reticulum, Golgi bodies, and various vacuoles. One of the more distinct vacuolar components is the contractile vacuole (CV) complex, which consists of two anterior contractile vacuoles that expand/contract approximately every 30 s. In this study, experimental cytochemical labeling was performed to help elucidate possible endocytic/membrane recycling mechanisms in Gloeomonas and the possible role of the contractile vacuole in this process. When incubated with 0.5 mg · mL^?1 cationic ferritin for short periods of time (2–60 min), labeling follows this route: inner membrane of CV, globular deposits in the CV and associated vesicles, and ultimately the terminal trans face cisternae of the Golgi apparatus (GA). Similar incubations with Lucifer yellow and concanavalin A—gold conjugates support distinct uptake of exogenous ligands by the CV and associated vesicles. Our results suggest that the contractile vacuole may be a site of endocytosis and that the trans GA loci may be a key site of membrane recycling.     

The Fine Structure of the Colonial Kinetoplastid Flagellate Cephalothamnium cyclopum Stein

Cell structure, cell adhesion, and stalk formation have been examined by electron microscopy in the colonial flagellate, Cephalothamnium cyclopum. Each cell is obconical or spindle-shaped, pointed posteriorly and truncated anteriorly. The cell membrane is underlain by epiplasm 0.1 μm thick in the posterior region, but bands of microtubules support the anterior region which is differentiated into a flagellar pocket, oral apparatus and contractile vacuole. Each of 2 flagella, joined a short way above their bases by an interflagellar connective, has a paraxial rod and mastigonemes. One flagellum is free and is important in food gathering while the other is recurrent and lies in a shallow groove on the ventral cell surface but projects posteriorly into the stalk. The basal bodies of these flagella are bipartite structures connected by a pair of striated rootlets with accessory microtubular fibers. The oral apparatus consists of a funnel-shaped buccal cavity and cytostome. It is supported by helical and longitudinal microtubules and also has nearby striated and microtubular fibers. Possible roles of associated oral vesicles in relation to ingestion are discussed. A reticulate mitochondrion houses a massive kinetoplast which has a fibrillar substructure resembling that of dinoflagellate chromosomes. Adjacent flagellates adhere by laminate extensions of their posterior regions and attach by their recurrent flagella to a communally secreted stalk composed of finely fibrillar material. This study indicates that Cephalothamnium belongs in the order Kinetoplastida, and has many features in common with members of the family Bodonidae.     

Morphology and morphogenesis of Epistylis plicatilis ehrenberg, 1831 (Ciliophora,Peritrichia) from Wuhan,China

A limnetic peritrichous ciliate, Epistylis plicatilis Ehrenberg, 1831, was collected from a freshwater ditch beside Moshan Hill, Wuhan, China. Its morphology, infraciliature, and morphogenesis were investigated based on specimens examined in vivo, following staining with protargol and by scanning electron microscopy. The characteristics of the Wuhan population of E. plicatilis are as follows: 1) colonial, each colony typically comprising 30–50 individuals, with a dichotomously branched, noncontractile stalk; 2) fully expanded zooids measure 90–155 × 30–50 µm in vivo; 3) a series of 6 or 7 conspicuous folds appear in the posterior region of the zooid when it contracts; 4) single horseshoe‐shaped macronucleus oriented transversely; 5) single contractile vacuole located in peristomial region on dorsal wall of infundibulum; 6) myoneme system comprises 20–24 longitudinal fibers, peristomial disk fibers as a wreath‐like net and peristomial ring fibers; 7) narrowly spaced transverse striations on the surface of the body; 8) infundibular polykineties 1 and 2 are three‐rowed, infundibular polykinety 3 is two‐rowed; and 9) stomatogenesis is of the buccokinetal type; in the new oral apparatus, infundibular polykineties 2 and 3, the haplokinety, and the germinal kinety all originate from the germinal kinety of the parental oral apparatus whereas the polykinety and infundibular polykinety 1 originate from the parental haplokinety. An improved diagnosis of E. plicatilis is supplied. J. Morphol. 275:882–893, 2014. © 2014 Wiley Periodicals, Inc.     

FOOD VACUOLE MEMBRANE GROWTH WITH MICROTUBULE-ASSOCIATED MEMBRANE TRANSPORT IN PARAMECIUM

Evidence from a morphological study of the oral apparatus of Paramecium caudatum using electron microscope techniques have shown the existence of an elaborate structural system which is apparently designed to recycle digestive-vacuole membrane. Disk-shaped vesicles are filtered out of the cytoplasm by a group of microtubular ribbons. The vesicles, after being transported to the cytostome-cytopharynx region in association with these ribbons, accumulate next to the cytopharynx before they become fused with the cytopharyngeal membrane. This fusion allows the nascent food vacuole to grow and increase its membrane surface area. The morphology of this cytostome-cytopharynx region is described in detail and illustrated with a three-dimensional drawing of a portion of this region and a clay sculpture of the oral apparatus of Paramecium. Evidence from the literature for the transformation of food vacuole membrane into disk-shaped vesicles both from condensing food vacuoles in the endoplasm and from egested food vacuoles at the cytoproct is presented. This transformation would complete a system of digestive vacuole membrane recycling.     

A Phagomyxa-like Endoparasite of the Centric Marine Diatom Bellerochea malleus: A Phagotrophic Plasmodiophoromycete1

In September 1993 the marine centric diatom, Bellerochea malleus (Brightwell) Heurck, collected in the Wadden Sea near List/Sylt, was parasitized by a Phagomyxa algarum-like organism. Karling (1944) reported Phagomyxa algarum Karling in North Carolina as a parasite of the filamentous brown algae Ectocarpus and Pylaiella. The Bellerochea parasite develops an endocytoplasmic Plasmodium and incorporates host cytoplasm into a large, central digestion vacuole, by a form of phagocytosis. Later on, the Plasmodium cleaves to form a zoosporangiosorus. Each zoosporangium is surrounded by a thin wall. It releases zoospores (2.5 × 4 μm) with two unequal flagella, an anterior (4 μm long) and a posterior (8 μm long). Cystosori and cysts could not be detected. The ultrastructure of the zoosporangia and zoospores was investigated, with particular attention to the flagellar apparatus and its rearrangement during zoospore release. This process is very similar to that recorded for zoospores of the plasmodiophoromycete Polymyxa graminis Ledigham (Barr and Allan, 1982). The Bellerochea parasite is closely related to or identical with Phagomyxa algarum. The taxonomic position of Phagomyxa is discussed. In spite of its phagotrophic nutrition and the possible lack of cystosori and cysts, Phagomyxa should be regarded as a member of the Plasmodiophoromycota (or Plasmodiophorida) but not included in a separate order Phagomyxida as proposed by Cavalier-Smith (1993a).     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane