Vps8Dp concentrates at junctions between adjacent vacuoles
A: Live imaging of numerous large Dop1p-labeled vacuoles in cells expressing Dop1p-mNeon, following hypo -osmotic shock. The cell shown was imaged to capture a cross-sectional view, in a time-lapse video with 4 sec frame intervals (Movie 6). Four successive images extracted from the video are shown. Growing cell cultures were hypo-osmotically challenged with 10 mM Tris-HCl buffer, pH 7.4 and immediately immobilized for imaging using CyGEL, as described in Materials and Methods. Videos were captured using a Zeiss Axio Observer 7 system.
B: Live imaging of the numerous Dop1p-labeled vacuoles followinghyper -osmotic shock in cells expressing Dop1p-mNeon. The cell shown was imaged to capture a cross-sectional view, in a time-lapse video with 0.15 sec frame intervals (Movie 7). Four successive images extracted from the video are shown. Growing cell cultures were hyper-osmotically challenged with 10 mM sorbitol and immediately immobilized for imaging using CyGEL, as described in Materials and Methods. Videos were captured using a Marianas spinning disc confocal microscope.
C: Live imaging of numerous large Vps8Dp-labeled vacuoles followinghypo -osmotic shock in cells expressing Vps8Dp-mNeon. The cell shown was imaged to capture a cross-sectional view, in a time-lapse video with 0.5 sec frame intervals (Movie 8). Four successive images extracted from the video are shown. Cells were treated as described in Fig. 5A. Videos were captured using a Marianas spinning disc confocal microscope.
D-E: Imaging of extracellular Vps8Dp-labeled vacuoles that leaked from ruptured cells expressing Vps8Dp-mNeon.
D. Vps8Dp localizes densely at contact site between adjacent vacuoles, which in some cases are sites of subsequent membrane fusion (arrowhead). Four successive images extracted from Movie 9 are shown.
E: Vps8Dp re-distributes at the time that two vacuoles come into contact. The two large vacuoles shown were separated by ~1µ at the beginning of the video (t=0) (Movie 10). When they moved into contact starting at t=10s, Vps8Dp accumulated at the junction (boxed). A second example of this phenomenon is shown in Movie 11.
F. Plot of fluorescence intensity change with time for the boxed area shown in Fig. 5E, in Movie 10. The data were plotted using GraphPad Software Prism. The three red arrows correspond to the images shown in panel E.
Reference
ALLEN, R. D. 2000. The contractile vacuole and its membrane dynamics.Bioessays, 22, 1035-42.
ALLEN, R. D. & NAITOH, Y. 2002. Osmoregulation and contractile vacuoles of protozoa. Int Rev Cytol, 215, 351-94.
ASENSIO, C. S., SIRKIS, D. W., MAAS, J. W., JR., EGAMI, K., TO, T. L., BRODSKY, F. M., SHU, X., CHENG, Y. & EDWARDS, R. H. 2013. Self-assembly of VPS41 promotes sorting required for biogenesis of the regulated secretory pathway. Dev Cell, 27, 425-37.
BAKER, R. W., JEFFREY, P. D., ZICK, M., PHILLIPS, B. P., WICKNER, W. T. & HUGHSON, F. M. 2015. A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly. Science, 349,1111-4.
BOWMAN, G. R. & TURKEWITZ, A. P. 2001. Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila. Genetics, 159, 1605-16.
BRIGHT, L. J., KAMBESIS, N., NELSON, S. B., JEONG, B. & TURKEWITZ, A. P. 2010. Comprehensive analysis reveals dynamic and evolutionary plasticity of Rab GTPases and membrane traffic in Tetrahymena thermophila. PLoS Genet, 6, e1001155.
BRIGUGLIO, J. S., KUMAR, S. & TURKEWITZ, A. P. 2013. Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena.J Cell Biol, 203, 537-50.
CASSIDY-HANLEY, D., BOWEN, J., LEE, J. H., COLE, E., VERPLANK, L. A., GAERTIG, J., GOROVSKY, M. A. & BRUNS, P. J. 1997. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics, 146, 135-47.
CHENG, C. Y., ROMERO, D. P., ZOLTNER, M., YAO, M. C. & TURKEWITZ, A. P. 2023. Structure and dynamics of the contractile vacuole complex in Tetrahymena thermophila. J Cell Sci .
CHENG, C. Y., YOUNG, J. M., LIN, C. Y. G., CHAO, J. L., MALIK, H. S. & YAO, M. C. 2016. The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila. Genes & Development, 30, 2724-2736.
DAVID L. SPECTOR, R. D. G., LESLIE A. LEINWAND 1998. Cells: A Laboratory Manual, Volume 1, Chapter 18, Culture and Manipulation of Tetrahymena .
DU, F., EDWARDS, K., SHEN, Z., SUN, B., DE LOZANNE, A., BRIGGS, S. & FIRTEL, R. A. 2008. Regulation of contractile vacuole formation and activity in Dictyostelium. EMBO J, 27, 2064-76.
ELLIOTT, A. M. & BAK, I. J. 1964. The Contractile Vacuole and Related Structures in Tetrahymena Pyriformis. J Protozool, 11,250-61.
ESSID, M., GOPALDASS, N., YOSHIDA, K., MERRIFIELD, C. & SOLDATI, T. 2012. Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole. Mol Biol Cell, 23,1267-82.
FRATTI, R. A., JUN, Y., MERZ, A. J., MARGOLIS, N. & WICKNER, W. 2004. Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles. J Cell Biol, 167, 1087-98.
GABRIEL, D., HACKER, U., KOHLER, J., MULLER-TAUBENBERGER, A., SCHWARTZ, J. M., WESTPHAL, M. & GERISCH, G. 1999. The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein (vol 112, pg 3995, 1999). Journal of Cell Science, 112, U3-U3.
GERALD, N. J., SIANO, M. & DE LOZANNE, A. 2002. The Dictyostelium LvsA protein is localized on the contractile vacuole and is required for osmoregulation. Traffic, 3, 50-60.
GOROVSKY, M. A., YAO, M. C., KEEVERT, J. B. & PLEGER, G. L. 1975. Isolation of micro- and macronuclei of Tetrahymena pyriformis.Methods Cell Biol, 9, 311-27.
HARRIS, E., YOSHIDA, K., CARDELLI, J. & BUSH, J. 2001. Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in dictyostelium. J Cell Sci,114, 3035-45.
HOWARD-TILL, R. A. & YAO, M. C. 2006. Induction of gene silencing by hairpin RNA expression in Tetrahymena thermophila reveals a second small RNA pathway. Mol Cell Biol, 26, 8731-42.
JIMENEZ, V., MIRANDA, K. & AUGUSTO, I. 2022. The old and the new about the contractile vacuole of Trypanosoma cruzi. J Eukaryot Microbiol, 69, e12939.
KISSMEHL, R., FROISSARD, M., PLATTNER, H., MOMAYEZI, M. & COHEN, J. 2002. NSF regulates membrane traffic along multiple pathways in Paramecium. J Cell Sci, 115, 3935-46.
KLAUKE, N. & PLATTNER, H. 2000. ”Frustrated Exocytosis”–a novel phenomenon: membrane fusion without contents release, followed by detachment and reattachment of dense core vesicles in Paramecium cells.J Membr Biol, 176, 237-48.
KLINGER, C. M., KLUTE, M. J. & DACKS, J. B. 2013. Comparative genomic analysis of multi-subunit tethering complexes demonstrates an ancient pan-eukaryotic complement and sculpting in Apicomplexa. PLoS One,8, e76278.
LADENBURGER, E. M., KORN, I., KASIELKE, N., WASSMER, T. & PLATTNER, H. 2006. An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system. J Cell Sci, 119, 3705-17.
LADENBURGER, E. M., SEHRING, I. M., KORN, I. & PLATTNER, H. 2009. Novel types of Ca2+ release channels participate in the secretory cycle of Paramecium cells. Mol Cell Biol, 29, 3605-22.
LINKNER, J., WITTE, G., ZHAO, H., JUNEMANN, A., NORDHOLZ, B., RUNGE-WOLLMANN, P., LAPPALAINEN, P. & FAIX, J. 2014. The inverse BAR domain protein IBARa drives membrane remodeling to control osmoregulation, phagocytosis and cytokinesis. J Cell Sci,127, 1279-92.
LORINCZ, P., LAKATOS, Z., VARGA, A., MARUZS, T., SIMON-VECSEI, Z., DARULA, Z., BENKO, P., CSORDAS, G., LIPPAI, M., ANDO, I., HEGEDUS, K., MEDZIHRADSZKY, K. F., TAKATS, S. & JUHASZ, G. 2016. MiniCORVET is a Vps8-containing early endosomal tether in Drosophila. Elife, 5.
MACRO, L., JAISWAL, J. K. & SIMON, S. M. 2012. Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium. J Cell Sci, 125, 5721-32.
MALCHOW, D., LUSCHE, D. F., DE LOZANNE, A. & SCHLATTERER, C. 2008. A fast Ca2+-induced Ca2+-release mechanism in Dictyostelium discoideum.Cell Calcium, 43, 521-30.
MANNA, P. T., BARLOW, L. D., RAMIREZ-MACIAS, I., HERMAN, E. K. & DACKS, J. B. 2023. Endosomal vesicle fusion machinery is involved with the contractile vacuole in Dictyostelium discoideum. J Cell Sci, 136.
MORLON-GUYOT, J., EL HAJJ, H., MARTIN, K., FOIS, A., CARRILLO, A., BERRY, L., BURCHMORE, R., MEISSNER, M., LEBRUN, M. & DAHER, W. 2018. A proteomic analysis unravels novel CORVET and HOPS proteins involved in Toxoplasma gondii secretory organelles biogenesis. Cell Microbiol, 20, e12870.
NAITOH, Y., TOMINAGA, T. & ALLEN, R. 1997. The contractile vacuole fluid discharge rate is determined by the vacuole size immediately before the start of discharge in Paramecium multimicronucleatum. J Exp Biol, 200, 1737-44.
PARKINSON, K., BAINES, A. E., KELLER, T., GRUENHEIT, N., BRAGG, L., NORTH, R. A. & THOMPSON, C. R. 2014. Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel.Nat Cell Biol, 16, 87-98.
PATEL, S. & DOCAMPO, R. 2010. Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling. Trends Cell Biol, 20, 277-86.
PEPLOWSKA, K., MARKGRAF, D. F., OSTROWICZ, C. W., BANGE, G. & UNGERMANN, C. 2007. The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis.Dev Cell, 12, 739-50.
PLATTNER, H. 2013. Contractile vacuole complex–its expanding protein inventory. Int Rev Cell Mol Biol, 306, 371-416.
REUTER, A. T., STUERMER, C. A. & PLATTNER, H. 2013. Identification, localization, and functional implications of the microdomain-forming stomatin family in the ciliated protozoan Paramecium tetraurelia.Eukaryot Cell, 12, 529-44.
SCHINDELIN, J., ARGANDA-CARRERAS, I., FRISE, E., KAYNIG, V., LONGAIR, M., PIETZSCH, T., PREIBISCH, S., RUEDEN, C., SAALFELD, S., SCHMID, B., TINEVEZ, J. Y., WHITE, D. J., HARTENSTEIN, V., ELICEIRI, K., TOMANCAK, P. & CARDONA, A. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods, 9, 676-82.
SCHONEMANN, B., BLEDOWSKI, A., SEHRING, I. M. & PLATTNER, H. 2013. A set of SNARE proteins in the contractile vacuole complex of Paramecium regulates cellular calcium tolerance and also contributes to organelle biogenesis. Cell Calcium, 53, 204-16.
SHANG, Y., SONG, X., BOWEN, J., CORSTANJE, R., GAO, Y., GAERTIG, J. & GOROVSKY, M. A. 2002. A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila.Proc Natl Acad Sci U S A, 99, 3734-9.
SIVARAMAKRISHNAN, V. & FOUNTAIN, S. J. 2012. A mechanism of intracellular P2X receptor activation. J Biol Chem, 287,28315-26.
SPANG, A. 2016. Membrane Tethering Complexes in the Endosomal System.Front Cell Dev Biol, 4, 35.
SPARVOLI, D., RICHARDSON, E., OSAKADA, H., LAN, X., IWAMOTO, M., BOWMAN, G. R., KONTUR, C., BOURLAND, W. A., LYNN, D. H., PRITCHARD, J. K., HARAGUCHI, T., DACKS, J. B. & TURKEWITZ, A. P. 2018. Remodeling the Specificity of an Endosomal CORVET Tether Underlies Formation of Regulated Secretory Vesicles in the Ciliate Tetrahymena thermophila.Curr Biol, 28, 697-710 e13.
SPARVOLI, D., ZOLTNER, M., CHENG, C. Y., FIELD, M. C. & TURKEWITZ, A. P. 2020. Diversification of CORVET tethers facilitates transport complexity in Tetrahymena thermophila. J Cell Sci, 133.
STAVROU, I. & O’HALLORAN, T. J. 2006. The monomeric clathrin assembly protein, AP180, regulates contractile vacuole size in Dictyostelium discoideum. Mol Biol Cell, 17, 5381-9.
STEVENS, T. H. & FORGAC, M. 1997. Structure, function and regulation of the vacuolar (H+)-ATPase. Annu Rev Cell Dev Biol, 13,779-808.
TANI, T., TOMINAGA, T., ALLEN, R. D. & NAITOH, Y. 2002. Development of periodic tension in the contractile vacuole complex membrane of paramecium governs its membrane dynamics. Cell Biol Int,26, 853-60.
TOMINAGA, T., ALLEN, R. D. & NAITOH, Y. 1998a. Cyclic changes in the tension of the contractile vacuole complex membrane control its exocytotic cycle. J Exp Biol, 201 (Pt 18), 2647-58.
TOMINAGA, T., ALLEN, R. D. & NAITOH, Y. 1998b. Electrophysiology of the in situ contractile vacuole complex of Paramecium reveals its membrane dynamics and electrogenic site during osmoregulatory activity. J Exp Biol, 201, 451-60.
ULRICH, P. N., JIMENEZ, V., PARK, M., MARTINS, V. P., ATWOOD, J., 3RD, MOLES, K., COLLINS, D., ROHLOFF, P., TARLETON, R., MORENO, S. N., ORLANDO, R. & DOCAMPO, R. 2011. Identification of contractile vacuole proteins in Trypanosoma cruzi. PLoS One, 6, e18013.
VAN DER BEEK, J., JONKER, C., VAN DER WELLE, R., LIV, N. & KLUMPERMAN, J. 2019. CORVET, CHEVI and HOPS - multisubunit tethers of the endo-lysosomal system in health and disease. J Cell Sci, 132.
WANG, L., MERZ, A. J., COLLINS, K. M. & WICKNER, W. 2003. Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion. J Cell Biol, 160, 365-74.
WEN, Y., STAVROU, I., BERSUKER, K., BRADY, R. J., DE LOZANNE, A. & O’HALLORAN, T. J. 2009. AP180-mediated trafficking of Vamp7B limits homotypic fusion of Dictyostelium contractile vacuoles. Mol Biol Cell, 20, 4278-88.
YAO, M. C. & YAO, C. H. 1991. Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement. Proc Natl Acad Sci U S A, 88, 9493-7.