Contractile Vacuole Research Articles

Taxonomic studies on a new marine ciliate, Apocoleps magnus gen. nov., spec. nov. (Ciliophora, Colepidae), isolated from Qingdao, China

The morphology and infraciliature of a new marine colepid ciliate, Apocoleps magnus gen. nov., spec. nov., are described based on living observations and silver impregnations. The new genus Apocoleps is characterized by having 8 (vs. 6 in most other related genera) armour tiers, spines at both ends of the cell, 3 adoral organelles and plates with 4 reniform uni-windows. Apocoleps magnus spec. nov. is defined by the following features: body elongated and slightly curved, about 100–120µm× 35–45 µm in vivo; anterior tertiary tier plate with four uni-windows, most secondary and main tier plates with four uni-windows, posterior tertiary tier plate with two uni-windows; left plate margin slightly serrated; on average 23 transverse and 22 longitudinal ciliary rows; one terminal contractile vacuole; marine habitat.

The First Report of the Chrysophyte AlgaOchrostylon(Chromulinales: Chromulinaceae) from North America and a Description of a New Species,O. thompsonii

Ochrostylon thompsonii, a member of the golden-brown algal order Chromulinales, is described from North America (Quivira National Wildlife Refuge, Kansas). Each organism consists of a basal disk and an elastic stalk at the end of which sits the markedly dorsoventral protoplast. The strongly concave heteroflagellate cell contains a single plastid and contractile vacuoles. The cell further contains a large chrysolaminarin (leucosin) droplet and numerous small droplets.

AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion ofDictyosteliumContractile Vacuoles

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane-coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.

The Dictyostelium type V myosin MyoJ is responsible for the cortical association and motility of contractile vacuole membranes

The contractile vacuole (CV) complex in Dictyostelium is a tubulovesicular osmoregulatory organelle that exhibits extensive motility along the actin-rich cortex, providing a useful model for investigating myosin-dependent membrane transport. Here, we show that the type V myosin myoJ localizes to CV membranes and is required for efficient osmoregulation, the normal accumulation of CV membranes in the cortex, and the conversion of collapsed bladder membranes into outwardly radiating cortical CV tubules. Complementation of myoJ-null cells with a version of myoJ containing a shorter lever arm causes these radiating tubules to move at a slower speed, confirming myoJ's role in translocating CV membranes along the cortex. MyoJ-null cells also exhibit a dramatic concentration of CV membranes around the microtubule-organizing center. Consistently, we demonstrate that CV membranes also move bi-directionally on microtubules between the cortex and the centrosome. Therefore, myoJ cooperates with plus and minus end–directed microtubule motors to drive the normal distribution and dynamics of the CV complex in Dictyostelium.

Myosin drives bladder movements

A myosin motor dictates the localization and movement of an organelle that expels excess water from Dictyostelium , report [Jung et al.][1] ![Figure][2] Contractile vacuoles (red) accumulate in the center of the cell in the absence (right) of MyoJ (green). To counteract the osmotic stress

Description of Eurystomatella sinica n. gen., n. sp., with establishment of a new family Eurystomatellidae n. fam. (Protista, Ciliophora, Scuticociliatia) and analyses of its phylogeny inferred from sequences of the small-subunit rRNA gene

Recently, an undescribed marine ciliate was isolated from China. Investigation of its morphology and infraciliature revealed it as an undescribed species representing a new genus, Eurystomatella n. gen., the type of the new family Eurystomatellidae n. fam. The new family is defined by close-set, apically positioned oral membranelles and a dominant buccal field that is surrounded by an almost completely circular paroral membrane. The new genus is defined by having a small oral membranelle 1 (M1), bipartite M2 and well-developed M3, a body surface faintly sculptured with a silverline system in a quadrangular, reticulate pattern and a cytostome located at the anterior third of a large buccal field. The type species of the new genus, Eurystomatella sinica n. sp., is a morphologically unique form that is defined mainly by the combination of a conspicuously flattened body, several caudal cilia, extremely long cilia associated with the buccal apparatus and a contractile vacuole located subcaudally. According to phylogenetic analyses of small-subunit (SSU) rRNA gene sequences, Eurystomatella clusters with the genus Cyclidium, as a sister group to the family Pleuronematidae. The great divergence in both buccal and somatic ciliature between Eurystomatella and all other known scuticociliates supports the establishment of a new family for Eurystomatella.

Taxonomic Study of Poorly-known Marine Pleurostomatid Ciliates of Litonotus paracygnus and L. pictus (Ciliophora: Pleurostomatida) from Korea

Two poorly known and often confused pleurostomatid ciliates, Litonotus paracygnus Song, 1994 and L. pictus Gruber, 1884, were collected from the coastal waters of Yeonggeumjeong and Bongpo-port, Gangwondo in the East Sea and from the Iwon tide embankment near Ganwol-do, Chungcheongnam-do in the Yellow Sea, Korea. These species were described based on live observations, the protargol-impregnation and morphometrics of the species. Also provided are their diagnoses. The small subunit ribosomal DNA (SSU rDNA) sequences of these species were compared with previously known sequences of related species. The diagnostics of the two Litonotus species are as follows. L. paracygnus: 150-300 μm long in vivo, strongly contractile neck region, two ellipsoid macronuclei (Ma) and one micronucleus (Mi), 7 left (LSK) and 11-14 right somatic kineties (RSK), 2-4 contractile vacuoles (CV) located on the posterior end, extrusemes (Ex) distributed on the anterior region of the ventral margin only. L. pictus: about 200-600 μm long in vivo, extremely contractile, beautiful body color with rows of yellow to yellow-brownish cortical pigment granules, 12-21 Ma arranged in moniliform pattern, infrequently vermiform, 7-11 LSK and 18-26 RSK, several CV located on both margins, Ex distributed on the anterior region of the ventral margin only. In this study, this genus was firstly recorded in Korea.

Three marine haptorid ciliates from northern China: Paraspathidium apofuscum n. sp., Trachelotractus entzi (Kahl, 1927) Foissner, 1997 and Apotrachelotractus variabialis Long, Song and Warren, 2009 (Protozoa, Ciliophora)

Three haptorid ciliates, Paraspathidium apofuscum n. sp., Trachelotractus entzi (Kahl, 1927) Foissner, 1997 and Apotrachelotractus variabialis Long, Song and Warren, 2009, isolated from sandy beaches in Qingdao, China, were morphologically investigated using both in vivo observations and silver impregnation methods. The new species, P. apofuscum, is highly contractile and has a terminally-located contractile vacuole, approximately 40 somatic kineties, and a dikinetid perioral kinety that does not form a closed circle. Redescriptions of A. variabialis and T. entzi are also provided based on the Qingdao populations.

Structural organization of Trypanosoma cruzi

Since the initial description of Trypanosoma cruzi by Carlos Chagas in 1909, several research groups have used different microscopic techniques to obtain detailed information about the various developmental stages found in the life cycle of this intracellular parasite. This review describes the present knowledge on the organization of the most important structures and organelles found in the protozoan, such as the cell surface, flagellum, cytoskeleton, kinetoplast-mitochondrion complex, glycosome, acidocalcisome, contractile vacuole, lipid inclusions, the secretory pathway, endocytic pathway and the nucleus.

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

A database search of the Paramecium genome reveals 34 genes related to Ca(2+)-release channels of the inositol-1,4,5-trisphosphate (IP(3)) or ryanodine receptor type (IP(3)R, RyR). Phylogenetic analyses show that these Ca(2+) release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP(3)Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP(3)Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca(2+) stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP(3) channel, a group II member (P. tetraurelia IP(3)R(N)-1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP(3)R(N) affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca(2+) from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca(2+) as signaling molecule.

The Role of Acidocalcisomes in Parasitic Protists1

Acidocalcisomes are acidic organelles with a high concentration of phosphorus present as pyrophosphate (PP(i)) and polyphosphate (poly P) complexed with calcium and other cations. The acidocalcisome membrane contains a number of pumps (Ca(2+)-ATPase, V-H(+)-ATPase, H(+)-PPase), exchangers (Na(+)/H(+), Ca(2+)/H(+)), and channels (aquaporins), while its matrix contains enzymes related to PP(i) and poly P metabolism. Acidocalcisomes have been observed in pathogenic, as well as non-pathogenic prokaryotes and eukaryotes, e.g. Chlamydomonas reinhardtii, and Dictyostelium discoideum. Some of the potential functions of the acidocalcisome are the storage of cations and phosphorus, the participation of phosphorus in PP(i) and poly P metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation. In addition, acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. For example, we found that platelet dense granules, which are LROs, are very similar to acidocalcisomes. They share a similar size, acidic properties, and both contain PP(i), poly P, and calcium. Recent work that indicates that they also share the system for targeting of their membrane proteins through adaptor protein 3 reinforces this concept. The fact that acidocalcisomes interact with other organelles in parasitic protists, e.g. the contractile vacuole in Trypanosoma cruzi, and other vacuoles observed in Toxoplasma gondii, suggests that these cellular compartments may be associated with the endosomal/lysosomal pathway.

Porosome: The Secretory Portal in Cells

Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles dock and fuse to release intravesicular contents to the outside during cell secretion. The porosome opening to the outside ranges from 150 nm in diameter in acinar cells of the exocrine pancreas to 12 nm in neurons. In the past decade, the composition of the porosome, its structure and dynamics at nanometer resolution in real time, and its functional reconstitution into an artificial lipid membrane have been described. Discovery of the universal secretory machinery in cells, the porosome, came as no surprise since porosome-like "canaliculi" structures for secretion from human platelets, the secretory machinery in single-cell organisms like the secretion apparatus in bacteria and Toxoplasma gondii, and the contractile vacuole in paramecium have been demonstrated. In this review, the discovery of the porosome complex and the molecular mechanism of its function and how this information provides a new understanding of cell secretion are discussed.

Mechanisms involved in the photosensitized inactivation ofAcanthamoeba palestinensistrophozoites

To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine-photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments. We followed the activities of selected marker enzymes as well as we performed fluorescence and transmission electron microscopy investigations of the alterations induced by the photoprocess in the fine structure of subcellular compartments. RLP068 is preferentially located in the contractile vacuole: the fluorescence in that site is particularly evident in the unirradiated cells and becomes more diffused after irradiation. Electron microscopic analysis of photosensitized A. palestinensis cells clearly shows that the swelling of trophozoites and the appearance of vacuoles spread throughout the cytoplasm after phototreatment. The activity of a typical cytoplasmic enzyme, such as lactate dehydrogenase, underwent a 35% decrease as a consequence of the photoprocess, reflecting the photodamage induced by migrating phthalocyanine molecules in their micro-environment. The presence of multiple targets for the phthalocyanine-photosensitized process is of utmost importance because this pattern of cell damage makes it unlikely that photoresistant A. palestinensis strains are gradually selected or mutagenic phenomena are developed as a consequence of the photoinduced damage. Photosensitization via phthalocyanines appears to represent an efficient and safe approach for achieving a close control of the population of a potentially pathogenic protozoan such as A. palestinensis, opening new perspectives for the disinfection of microbiologically polluted waters.

The System of Contractile Vacuoles in the Green Alga Mesostigma viride (Streptophyta)

The contractile vacuole (CV) is an osmoregulatory organelle which is found in many protists. We have investigated the structure and function of the CV in the green alga Mesostigma viride by light (video) and serial section electron microscopy. Mesostigma is the only known flagellate streptophyte (charophycean green algae and land plants) and therefore of great importance for our understanding of the evolution of streptophytes. The entire CV system of Mesostigma has been reconstructed 3-dimensionally for three cells. Based on light microscopy cells contain an average of 8 CVs. The maximal diameter of a CV in Mesostigma was 1.5 microm and the expulsion interval 24.1s in 10 mosM medium. Video microscopy revealed the system of CVs to be very dynamic with individual CVs connecting temporarily or fusing completely with each other. Electron microscopy confirmed these observations and showed coated vesicles to be predominantly associated with large CVs. No expulsion pore was observed by electron microscopy. Instead we encountered close contact zones of approximately 150 nm diameter, which we propose to be the site of water expulsion. A model for the function of CVs in Mesostigma is presented.

TAXONOMIC STUDY OF TWO NEW GENERA OF FUSIFORM GREEN FLAGELLATES, TABRIS GEN. NOV. AND HAMAKKO GEN. NOV. (VOLVOCALES, CHLOROPHYCEAE)(1).

On the basis of LM, we isolated strains of two species of fusiform green flagellates that could be assigned to former Chlorogonium (Cg.) Ehrenb. One species, "Cg."heimii Bourr., lacked a pyrenoid in its vegetative cells and required organic compounds for growth. The other was similar to Cg. elongatum (P. A. Dang.) Francé and "Cg."acusNayal, but with slightly smaller vegetative cells. Their molecular phylogeny was also studied based on combined 18S rRNA, RUBISCO LSU (rbcL), and P700 chl a-apoprotein A2 (psaB) gene sequences. Both species were separated from Chlorogonium emend., GungnirNakada and RusalkaNakada, which were formerly assigned to Chlorogonium. They were accordingly assigned to new genera, TabrisNakada gen. nov. and Hamakko (Hk.) Nakada gen. nov. as T. heimii (Bourr.) Nakada comb. nov. and Hk. caudatus Nakada sp. nov., respectively. Tabris is differentiated from other genera of fusiform green flagellates by its vegetative cells, which only have two apical contractile vacuoles and lack a pyrenoid in the chloroplast. Hamakko, on the other hand, is distinguishable by the fact that its pyrenoids in vegetative cells are penetrated by flattened thylakoid lamellae.

Redescription and neotypification of a poorly known freshwater ectocommensal ciliate, Larvulina variabilis ( ), and consideration of its systematic position

The morphology and infraciliature of Larvulina variabilis collected from freshwater Gammarus near Boise, Idaho (USA) were investigated using live observation and silver impregnation. Larvulina variabilis was collected from three sites in Idaho and one site in Michigan, indicating a wide distribution. Larvulina variabilis is a small (21–25×14–19 μm in vivo) hymenostome ectocommensal on freshwater amphipods. Distinguishing morphologic features include an inflexible cortex with a prominent notch in the left posterior margin, a single globular macronucleus, a single posterior contractile vacuole, a somatic ciliature with 10 kineties all consisting of ciliated dikinetids, a “6”-shaped paroral membrane of the stichodyad type, long (∼20 μm), immobile cilia arising from three adoral membranelles, a discrete dorsal thigmotactic ciliary field consisting of five kineties, distinctive extrusomes and a coarsely polygonal silverline pattern. The forward-projecting anterior cilium emphasized in the original description is an inconstant finding not essential for identification. An improved diagnosis is provided for the genus and the species. Because no type specimen exists and prior descriptions were conflicting, a specimen from the Idaho population is designated as the neotype. The systematic position of this unusual ciliate remains obscure. Possibly, it belongs to the Pleuronematida or the Thigmotrichida.

A new freshwater peritrichous ciliate from Harbin, China, Epistylis lalinensis n. sp. (Sessilida: Epistylididae)

A freshwater peritrich ciliate, Epistylis lalinensis n. sp., was collected from Lalin River, Harbin, China, and its morphology, infraciliature and silverline system were investigated based on living and silver-impregnated specimens. The new species measured 125-187 × 31-47 μ m in vivo and is characterized by having green-coloured endoplasm containing symbiotic algae, single apically located contractile vacuole, one-layer peristomial collar and slender body shape. The Cshaped macronucleus is transversely located. Three equally kineties in polykinety 3 parallel to each other. Number of silverlines between peristome and aboral trochal band, 117-145; between aboral trochal band and scopula, 50-68. Comparing most other congeners, circular fibers of this species composed of internal and external layers.

Subcellular localization of ammonium transporters in Dictyostelium discoideum.

BackgroundWith the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists.ResultsUsing RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters.ConclusionUnlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not the excretion function that is important for coupling ammonia levels to the slug versus culmination choice, but rather a sensor and/or signaling function of these proteins that is important.

Morphology and infraciliature of a new marine ciliate, Cinetochilum ovale n. sp. (Ciliophora: Oligohymenophorea)

The morphology and infraciliature of a new oligohymenophorean ciliate, Cinetochilum ovale n. sp., isolated from the littoral sediment of the Bohai Sea, northern China, were investigated using live observations and silver impregnations. This new species as follows: size about 20–30 × 15–25 µm in vivo, body oval in outline, with 12–13 bipolar somatic kineties; scutica consisting of two short rows of kinetosomes, positioned near the end of somatic kinety 1; the anterior-most row of its membranelle 1 is distinctly detached from the remaining two rows; three postoral kinetofragments and a cyrtos-like structure at the deep portion of the buccal cavity always present; one macronucleus and one micronucleus; single contractile vacuole terminally located. A key to all known Cinetochilum species is updated.

Trichopodiella faurei n. sp. (Ciliophora, Phyllopharyngea, Cyrtophoria): Morphological Description and Phylogenetic Analyses Based on SSU rRNA and Group I Intron Sequences

A new marine cyrtophorian ciliate Trichopodiella faurei n. sp., which belongs to the order Dysteriida, family Hartmannulidae, was investigated at the morphological and molecular levels. A combination of morphological features of the organism including the oval body shape, 2-3 contractile vacuoles, 22-28 nematodesmal rods in the cytopharyngeal basket, and 31-39 somatic kineties, distinguishes it from all other known congeners. In reconstructed small subunit (SSU) rRNA phylogenies, T. faurei groups with Isochona, a representative genus of the subclass Chonotrichia. The similarity of the infraciliature between hartmannulids and several chonotrichian examples also suggests that these taxa should be closely related. A new S943 intron belonging to group IC1 was identified in the SSU rRNA gene of this species. This intron is phylogenetically related to the S891 introns previously found in the suctorians Acineta sp. and Tokophrya lemnarum, and their internal guide sequences share four nucleotides, indicating that these introns were vertically inherited from a common phyllopharyngean ancestor and that reverse splicing might have been involved in the transposition.