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Roberto Docampo - One of the best experts on this subject based on the ideXlab platform.

  • Rab11 Regulates Trafficking of Trans-sialidase to the Plasma Membrane through the Contractile Vacuole Complex of Trypanosoma cruzi
    2016
    Co-Authors: Sayantanee Niyogi, Juan Mucci, Oscar Campetella, Roberto Docampo
    Abstract:

    Trypanosoma cruzi is the etiologic agent of Chagas disease. Although this is not a free-living organism it has conserved a Contractile Vacuole complex (CVC) to regulate its osmolarity. This obligate intracellular pathogen is, in addition, dependent on surface proteins to invade its hosts. Here we used a combination of genetic and biochemical approaches to delineate the contribution of the CVC to the traffic of glycosylphosphatidylinositol (GPI)-anchored proteins to the plasma membrane of the parasite and promote host invasion. While T. cruzi Rab11 (GFP-TcRab11) localized to the CVC, a dominant negative (DN) mutant tagged with GFP (GFP-TcRab11DN) localized to the cytosol, and epimastigotes expressing this mutant were less responsive to hyposmotic and hyperosmotic stress. Mutant parasites were still able to differentiate into metacyclic forms and infect host cells. GPI-anchored trans-sialidase (TcTS), mucins of the 60–200 KDa family, and trypomastigote small surface antigen (TcTSSA II) co-localized with GFP-TcRab11 to the CVC during transformation of intracellular amastigotes into trypomastigotes. Mucins of the gp35/50 family also co-localized with the CVC during metacyclogenesis. Parasites expressing GFP-TcRab11DN prevented TcTS, but not other membrane proteins, from reaching the plasma membrane, and were less infective as compared to wild type cells. Incubation of these mutants in the presence of exogenous recombinant active, but not inactive, TcTS, and a sialic acid donor, before infecting host cells, partially rescued infectivity of trypomastigotes. Taking together these results reveal roles of TcRab11 in osmoregulation and trafficking of trans-sialidase to the plasma membrane

  • tcpho91 is a Contractile Vacuole phosphate sodium symporter that regulates phosphate and polyphosphate metabolism in trypanosoma cruzi
    Molecular Microbiology, 2015
    Co-Authors: Veronica Jimenez, Roberto Docampo
    Abstract:

    Summary We have identified a phosphate transporter (TcPho91) localized to the bladder of the Contractile Vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease. TcPho91 has 12 transmembrane domains, an N-terminal regulatory SPX (named after SYG1, Pho81 and XPR1) domain and an anion permease domain. Functional expression in Xenopus laevis oocytes followed by two-electrode voltage clamp showed that TcPho91 is a low-affinity transporter with a Km for Pi in the millimolar range, and sodium-dependency. Epimastigotes overexpressing TcPho91-green fluorescent protein have significantly higher levels of pyrophosphate (PPi) and short-chain polyphosphate (polyP), suggesting accumulation of Pi in these cells. Moreover, when overexpressing parasites were maintained in a medium with low Pi, they grew at higher rates than control parasites. Only one allele of TcPho91 in the CL strain encodes for the complete open reading frame, while the other one is truncated encoding for only the N-terminal domain. Taking advantage of this characteristic, knockdown experiments were performed resulting in cells with reduced growth rate as well as a reduction in PPi and short-chain polyP levels. Our results indicate that TcPho91 is a phosphate sodium symporter involved in Pi homeostasis in T. cruzi.

  • Rab11 regulates trafficking of trans-sialidase to the plasma membrane through the Contractile Vacuole complex of Trypanosoma cruzi.
    Public Library of Science (PLoS), 2014
    Co-Authors: Sayantanee Niyogi, Juan Mucci, Oscar Campetella, Roberto Docampo
    Abstract:

    Trypanosoma cruzi is the etiologic agent of Chagas disease. Although this is not a free-living organism it has conserved a Contractile Vacuole complex (CVC) to regulate its osmolarity. This obligate intracellular pathogen is, in addition, dependent on surface proteins to invade its hosts. Here we used a combination of genetic and biochemical approaches to delineate the contribution of the CVC to the traffic of glycosylphosphatidylinositol (GPI)-anchored proteins to the plasma membrane of the parasite and promote host invasion. While T. cruzi Rab11 (GFP-TcRab11) localized to the CVC, a dominant negative (DN) mutant tagged with GFP (GFP-TcRab11DN) localized to the cytosol, and epimastigotes expressing this mutant were less responsive to hyposmotic and hyperosmotic stress. Mutant parasites were still able to differentiate into metacyclic forms and infect host cells. GPI-anchored trans-sialidase (TcTS), mucins of the 60-200 KDa family, and trypomastigote small surface antigen (TcTSSA II) co-localized with GFP-TcRab11 to the CVC during transformation of intracellular amastigotes into trypomastigotes. Mucins of the gp35/50 family also co-localized with the CVC during metacyclogenesis. Parasites expressing GFP-TcRab11DN prevented TcTS, but not other membrane proteins, from reaching the plasma membrane, and were less infective as compared to wild type cells. Incubation of these mutants in the presence of exogenous recombinant active, but not inactive, TcTS, and a sialic acid donor, before infecting host cells, partially rescued infectivity of trypomastigotes. Taking together these results reveal roles of TcRab11 in osmoregulation and trafficking of trans-sialidase to the plasma membrane, the role of trans-sialidase in promoting infection, and a novel unconventional mechanism of GPI-anchored protein secretion

  • Fluorescence microscopy analysis of TcRab11 in different stages of T. cruzi.
    2014
    Co-Authors: Sayantanee Niyogi, Juan Mucci, Oscar Campetella, Roberto Docampo
    Abstract:

    (A–C) GFP fusion protein of TcRab11 was detected in the Contractile Vacuole bladder of epimastigotes (Epi, A), trypomastigotes (Trypo, B), and intracellular amastigotes (Ama, C) using antibodies against GFP. Upper panels show differential interference contrast microscopy (DIC) images merged with DAPI staining of DNA (in blue) and GFP-TcRab11 (in green). Lower panels show fluorescence images. (D) GFP-TcRab11 (green) co-localizes with antibodies against T. cruzi aquaporin 1 (α-AQP, red), a marker for the Contractile Vacuole, under hyposmotic conditions. (E) Antibodies against TbRab11 (α-Rab11, red) co-localize with GFP-TcRab11 (green). (F) Antibodies against TbRab11 (red) localize to a compartment that resembles the Contractile Vacuole in (E). DAPI staining is in blue. Arrowheads in D–F show co-localization between antibodies against TcAQP1 and GFP (D), TbRab11 antibody and GFP (E) and labeling with antibodies against TbRab11 (F), respectively. Bars in A–F = 10 µm. (G) Western blot analyses with TbRab11 antibody of lysates of epimastigotes overexpressing GFP-TcRab11 (E-OE), or wild-type epimastigotes (E), trypomastigotes (T) and amastigotes (A) showing bands (arrows) corresponding to the endogenous TcRab11 (24 kDa) and to GFP-TcRab11 (50 kDa). The blots were sequentially probed with αTbRab11 and anti-tubulin antibodies, used as loading control.

  • Defining the role of a FYVE domain in the localization and activity of a cAMP phosphodiesterase implicated in osmoregulation in Trypanosoma cruzi
    Molecular microbiology, 2010
    Co-Authors: Alejandra Cecilia Schoijet, Wanderley De Souza, Kildare Miranda, Lia Carolina Soares Medeiros, Mirtha M. Flawiá, Héctor N. Torres, Omar Pedro Pignataro, Roberto Docampo, Guillermo D. Alonso
    Abstract:

    Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Trypanosoma cruzi, the causative agent of Chagas disease, encodes four different PDE families. One of these PDEs, T. cruzi PDE C2 (TcrPDEC2) has been characterized as a FYVE domain containing protein. Here, we report a novel role for TcrPDEC2 in osmoregulation in T. cruzi and reveal the relevance of its FYVE domain. Our data show that treatment of epimastigotes with TcrPDEC2 inhibitors improves their regulatory volume decrease, whereas cells overexpressing this enzyme are unaffected by the same inhibitors. Consistent with these results, TcrPDEC2 localizes to the Contractile Vacuole complex, showing strong labelling in the region corresponding to the spongiome. Furthermore, transgenic parasites overexpressing a truncated version of TcrPDEC2 without the FYVE domain show a failure in its targeting to the Contractile Vacuole complex and a marked decrease in PDE activity, supporting the importance of this domain to the localization and activity of TcrPDEC2. Taking together, the results here presented are consistent with the importance of the cyclic AMP signalling pathway in regulatory volume decrease and implicate TcrPDEC2 as a specifically localized PDE involved in osmoregulation in T. cruzi.

Seiji Sonobe - One of the best experts on this subject based on the ideXlab platform.

  • presence of aquaporin and v atpase on the Contractile Vacuole of amoeba proteus
    Biology of the Cell, 2008
    Co-Authors: Eri Nishihara, Teruo Shimmen, Etsuo Yokota, Akira Tazaki, Hidefumi Orii, Maki Katsuhara, Kensuke Kataoka, Hisako Igarashi, Yoshinori Moriyama, Seiji Sonobe
    Abstract:

    Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (Contractile Vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85–90]. Results. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected. Conclusions. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.

  • new aspects of membrane dynamics of amoeba proteus Contractile Vacuole revealed by vital staining with fm 4 64
    Protoplasma, 2007
    Co-Authors: Eri Nishihara, Teruo Shimmen, Seiji Sonobe
    Abstract:

    The Contractile Vacuole (CV) cycle of Amoeba proteus has been studied by phase contrast and electron microscopy. However, the understanding of membrane dynamics in this cycle is still poor. In this study, we used live imaging by fluorescence microscopy to obtain new insights. We succeeded in staining the CV with a styryl dye, FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide), and obtained the following results. (1) The CV membrane was directly stained with the dye in the external medium when the CV pore opened upon contraction. This indicates that transfer of plasma membrane to the CV does not occur. (2) The membrane dynamics during the CV cycle were elucidated. In particular, the fluorescent CV membrane was maintained as an aggregate just after contraction and the Vacuole re-formed from the aggregate. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle.

  • functional characterization of Contractile Vacuole isolated from amoeba proteus
    Cell Structure and Function, 2004
    Co-Authors: Eri Nishihara, Teruo Shimmen, Seiji Sonobe
    Abstract:

    Contractile Vacuoles (CVs) released from cells of Amoeba proteus were used to analyze its function in vitro. When CV was transferred to a hypertonic medium, its volume decreased within 10 sec. When it was subsequently returned to its original medium, it quickly started swelling. However, it ruptured before recovering its initial volume. These results suggested that the CV membrane is semi-permeable and that the fluid is collected by the osmotic gradient in vivo. The water permeability of membrane of isolated CV was calculated from the rate of osmotic volume change to be 0.94 microm/sec . OsM. This high value suggested that CV membrane is equipped with water channel. CV contracted (or burst) quickly upon addition of 1 mM ATP. Contraction was induced by ATP, but not by other nucleotides, GTP, ITP, ADP, or the analogues of ATP, AMP-PNP and ATPgammaS. It was suggested that the contraction of isolated CV was caused by increase in the tension of its membrane by ATP.

Yutaka Naitoh - One of the best experts on this subject based on the ideXlab platform.

  • osmoregulation in paramecium in situ ion gradients permit water to cascade through the cytosol to the Contractile Vacuole
    Journal of Cell Science, 2002
    Co-Authors: Christian Stock, Richard D. Allen, Heidi K Gronlien, Yutaka Naitoh
    Abstract:

    In vivo K+, Na+, Ca2+ and Cl- activities in the cytosol and the Contractile Vacuole fluid of Paramecium multimicronucleatum were determined in cells adapted to a number of external osmolarities and ionic conditions by using ion-selective microelectrodes. It was found that: (1) under standardized saline conditions K+ and Cl- were the major osmolytes in both the cytosol and the Contractile Vacuole fluid; and (2) the osmolarity of the Contractile Vacuole fluid, determined from K+ and Cl- activities only, was always more than 1.5 times higher than that of the cytosol. These findings indicate that excess cytosolic water crosses the Contractile Vacuole complex membrane osmotically. Substitution of choline or Ca2+ for K+ in the external solution or the external application of furosemide caused concomitant decreases in the cytosolic K+ and Cl- activities that were accompanied by a decrease in the water segregation activity of the Contractile Vacuole complex. This implies that the cytosolic K+ and Cl- are actively coimported across the plasma membrane. Thus, the osmotic gradients across both the plasma membrane and the membrane of the Contractile Vacuole complex ensure a controlled cascade of water flow through the cell that can provide for osmoregulation as well as the possible extrusion of metabolic waste by the Contractile Vacuole complex.

  • How external osmolarity affects the activity of the Contractile Vacuole complex, the cytosolic osmolarity and the water permeability of the plasma membrane in Paramecium multimicronucleatum
    The Journal of Experimental Biology, 2001
    Co-Authors: Christian Stock, Richard D. Allen, Yutaka Naitoh
    Abstract:

    The rate of fluid expulsion, R(CVC), from the Contractile Vacuole complex (CVC) of Paramecium multimicronucleatum was estimated from the volume of the Contractile Vacuoles (CVs) immediately before the start of fluid discharge and from the time elapsing between discharges. The R(CVC) increased when the cell was exposed to a strongly hypotonic solution and decreased in a weakly hypotonic solution. When the cell was exposed to an isotonic or a hypertonic solution, R(CVC) fell to zero. The time constant, tau, used to describe the change in R(CVC) in response to a change in external osmolarity shortened after a short-term exposure to a strongly hypotonic solution and lengthened after a short-term exposure to a less hypotonic solution. A remarkable lengthening of tau occurred after a short-term exposure to isotonic or hypertonic solution. Under natural conditions, mechanisms for controlling R(CVC) are effective in maintaining the cytosolic osmolarity hypertonic within a narrow concentration range despite changes in the external osmolarity, which is normally hypotonic to the cytosol. Cells exposed to an isotonic or hypertonic solution resumed CV activity when left in the solution for 12 h. The cytosolic osmolarity was found to increase and to remain hypertonic to the external solution. This will permit cells to continue to acquire water. The increase in the cytosolic osmolarity occurred in a stepwise fashion, rather than linearly, as the external osmolarity increased. That is, the cytosolic osmolarity first remained more-or-less constant at an increased level until the external osmolarity exceeded this level. Thereupon, the cytosolic osmolarity increased to a new higher level in 12 h, so that the cytosol again became hypertonic to the external solution and the cells resumed CV activity. These results imply that the cell needs to maintain water segregation activity even after it has been exposed to an isotonic or hypertonic environment. This supports the idea that the CVC might be involved not only in the elimination of excess cytosolic water but also in the excretion of some metabolic waste substances.

  • electrophysiology of the in situ Contractile Vacuole complex of paramecium reveals its membrane dynamics and electrogenic site during osmoregulatory activity
    The Journal of Experimental Biology, 1998
    Co-Authors: Takashi Tominaga, Richard D. Allen, Yutaka Naitoh
    Abstract:

    In the freshwater protozoan Paramecium multomicronucleatum, excess cytosolic water, acquired osmotically, is segregated and expelled to the cell exterior through the activity of the Contractile Vacuole complex. This process keeps the cell volume constant. The electrophysiological parameters of the organelle were measured in situ using a fine-tipped microelectrode inserted into the Contractile Vacuole, the exocytotic vesicle of the organelle to which the segregated fluid is transported before being expelled to the exterior. The input capacitance decreased markedly immediately before fluid expulsion and regained its previous value when fluid filling resumed after fluid expulsion. This change in the capacitance proved that the Contractile Vacuole became disconnected from its radial arms, which project from the Vacuole, before fluid expulsion occurred and then reconnected with the arms after fluid expulsion. A positive electrical potential was recorded from the Contractile Vacuole only when it was connected to the radial arms. This implies that the electrogenic mechanism resides exclusively in the radial arms and supports the idea that the decorated spongiomes, V-type proton-pump-covered terminal tubules of the radial arms that end blindly in the cytosol, are electrogenic. The linear current­voltage relationship of the Contractile Vacuole membrane also implies that few voltage-activated ion channels are present in the membrane. To explain the movement of water into the Contractile Vacuole complex, we favour the hypothesis that the potential generated across the decorated spongiome membrane can be used to drive counter-anions from the cytosol into the lumen of the complex. The anions could then act as an osmolite to pull cytosolic water into the lumen of the organelle.

  • how does the Contractile Vacuole of paramecium multimicronucleatum expel fluid modelling the expulsion mechanism
    The Journal of Experimental Biology, 1997
    Co-Authors: Yutaka Naitoh, Takashi Tominaga, Masaki Ishida, Agnes K Fok, Marilynn S Aihara, Richard D. Allen
    Abstract:

    To examine the forces needed for discharge of the fluid contents from the Contractile Vacuole of Paramecium multimicronucleatum, the time course of the decrease in Vacuole diameter during systole (the fluid-discharging period) was compared with that of various Vacuole discharge models. The observed time course did not fit that predicted by a model in which contraction of an actin­myosin network surrounding the Vacuole caused discharge nor that predicted by a model in which the surface tension of the lipid bilayer of the Vacuole caused discharge. Rather, it fitted that predicted by a model in which the cell's cytosolic pressure was responsible for discharge. Cytochalasin B, an effective inhibitor of actin polymerization, had no effect on the in vivo time course of systole. An injection of a monoclonal antibody raised against the proton pumps of the decorated spongiomes (now known to be the locus of fluid segregation in P. multimicronucleatum) disrupted the decorated spongiomes and reduced the rate of fluid segregation, whereas it did not alter the time course of systole. We conclude that in P. multimicronucleatum the internal pressure of the Contractile Vacuole is caused predominantly by the cytosolic pressure and that the fluid-segregation mechanism does not directly affect the fluid-discharge mechanism. Elimination of this cytosolic pressure by rupturing the cell revealed the presence of a novel fluid-discharge mechanism, apparently centered in the Vacuole membrane. The involvement of tubulation of the Vacuole membrane as the force-generating mechanism for fluid discharge in disrupted cells is discussed.

Matthew Turner - One of the best experts on this subject based on the ideXlab platform.

  • Hydro-osmotic Instabilities in Active Membrane Tubes
    Physical Review Letters, 2018
    Co-Authors: Sami Al-izzi, George Rowlands, Pierre Sens, Matthew Turner
    Abstract:

    We study a membrane tube with unidirectional ion pumps driving an osmotic pressure difference. A pressure-driven peristaltic instability is identified, qualitatively distinct from similar tension-driven Rayleigh-type instabilities on membrane tubes. We discuss how this instability could be related to the function and biogenesis of membrane bound organelles, in particular, the Contractile Vacuole complex. The unusually long natural wavelength of this instability is in agreement with that observed in cells.

Gong Jun - One of the best experts on this subject based on the ideXlab platform.

  • FIGURE 2 in Description of a new marine cyrtophorid ciliate, Brooklynella sinensis n. sp. from the China Sea with a new definition of the genus Brooklynella (Protozoa, Ciliophora, Cyrtophorida)
    2018
    Co-Authors: Gong Jun, Song Weibo
    Abstract:

    FIGURE 2. Photomicrographs of Brooklynella sinensis n. sp. from live cells (A – C), after protargol (D – J, M – P) and Chatton­Lwoff impregnation (K – L). (A) Ventral view of a typical individual, arrow indicates the podite. (B) Dorsal view. (C) Ventral view, showing the two Contractile Vacuoles (arrows). (D, M) Infraciliature, arrows mark posterior ends of right kineties. (E) Showing the equatorial fragment (arrow) and postoral kineties (arrowheads). (F) Right­ventral side view, arrows mark the three kineties extending apically. (G) Dorsal views, arrow refers to the terminal fragment. (H) An individual in early morphogenetic stage, to note the three thickened primordium (arrow). (I) Showing the two Contractile Vacuole pores (arrows). (J) To note the cytostome (arrow) encircled by kinetosome­like dots. (K, L) Silverline system. (N) Dorsal view, showing the terminal fragment (arrow) and cyrtos (arrowhead). (O) Showing the two adjacent Contractile Vacuole pores (arrow) and kinetosome­like dots (arrowhead) at the base of podite. (P) Focus on the cytostome (arrow) and kinetosome­like dots (arrowhead) near the posterior Contractile Vacuole pore. Cy = cyrtos; Ma = macronucleus. Scale bars = 20 m

  • FIGURES 11–18 in Resdescription of two synhymeniid ciliates, Chilodontopsis simplex Ozaki & Yagiu, 1941 and Zosterodasys transverses (Kahl, 1928) Foissner et al., 1994 (Alveolata, Ciliophora, Phyllopharyngea)
    2012
    Co-Authors: Wang Lijuan, Zhao Yuanjun, Gong Jun
    Abstract:

    FIGURES 11–18. Photomicrographs of Chilodontopsis simplex from live cells (11, 12) and after protargol impregnation (13– 18). (11, 12) Ventral views of typical individuals, arrow indicates the caudal Contractile Vacuole. (13–16) Ventral (13, 15, 16) and dorsal (14) views of the infraciliature, arrows indicate the synhymenium. (17, 18) Ingested diatoms. D = diatom; Ma = macronucleus. Scale bars = 30 µm

  • Resdescription of two synhymeniid ciliates, Chilodontopsis simplex Ozaki & Yagiu, 1941 and Zosterodasys transverses (Kahl, 1928) Foissner et al., 1994 (Alveolata, Ciliophora, Phyllopharyngea)
    2012
    Co-Authors: Wang Lijuan, Zhao Yuanjun, Gong Jun
    Abstract:

    The morphology and infraciliature of two synhymeniid ciliates, Chilodontopsis simplex Ozaki & Yagiu, 1941 and Zosterodasys transverses (Kahl, 1928) Foissner et al., 1994, collected from coasts of Qingdao, China, and Incheon, South Korea, respectively, have been investigated using live observation and protargol impregnation method. Chilodontopsis. simplex is characterized by: cell size 90-160 x 45-95 mu m in vivo, oval to long elliptical in outline; 59-78 somatic kineties, 11-13 nematodesmal rods; synhymenium constricted to ventral side, composed of 45-90 dikinetids, running across cell width; single Contractile Vacuole located in posterior cell end. The marine population of Zosterodasys transverses is briefly described, linked to the previously reported 18S ribosomal RNA gene (Genbank accession number EU286812), and compared to the freshwater population.The morphology and infraciliature of two synhymeniid ciliates, Chilodontopsis simplex Ozaki & Yagiu, 1941 and Zosterodasys transverses (Kahl, 1928) Foissner et al., 1994, collected from coasts of Qingdao, China, and Incheon, South Korea, respectively, have been investigated using live observation and protargol impregnation method. Chilodontopsis. simplex is characterized by: cell size 90-160 x 45-95 mu m in vivo, oval to long elliptical in outline; 59-78 somatic kineties, 11-13 nematodesmal rods; synhymenium constricted to ventral side, composed of 45-90 dikinetids, running across cell width; single Contractile Vacuole located in posterior cell end. The marine population of Zosterodasys transverses is briefly described, linked to the previously reported 18S ribosomal RNA gene (Genbank accession number EU286812), and compared to the freshwater population