The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input.

Biotechnology Biomedicine Centre of the Academy of Sciences České Budějovice 370 05 Czech Republic

Department of Molecular Genetics and Cell Biology University of Chicago Chicago IL 60637 USA

Department of Pharmacology University of Minnesota Minneapolis MN 55455 USA

Institute of Molecular Biology Academia Sinica Taipei 115 Taiwan

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bioRxiv. 2023 Nov 08:2023.11.07.566071. doi: 10.1101/2023.11.07.566071. PubMed

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Allen, R. D. (2000). The contractile vacuole and its membrane dynamics. PubMed DOI

Allen, R. D. and Naitoh, Y. (2002). Osmoregulation and contractile vacuoles of protozoa. PubMed DOI

Ashworth, J. M. and Watts, D. J. (1970). Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture. PubMed DOI PMC

Balderhaar, H. J. and Ungermann, C. (2013). CORVET and HOPS tethering complexes - coordinators of endosome and lysosome fusion. PubMed DOI

Becker, M., Matzner, M. and Gerisch, G. (1999). Drainin required for membrane fusion of the contractile vacuole in Dictyostelium is the prototype of a protein family also represented in man. PubMed DOI PMC

Betz, W. J. and Bewick, G. S. (1992). Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. PubMed DOI

Betz, W. J., Mao, F. and Smith, C. B. (1996). Imaging exocytosis and endocytosis. PubMed DOI

Bolte, S. and Cordelières, F. P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. PubMed DOI

Bowman, G. R. and Turkewitz, A. P. (2001). Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila. PubMed DOI PMC

Bright, L. J., Kambesis, N., Nelson, S. B., Jeong, B. and Turkewitz, A. P. (2010). Comprehensive analysis reveals dynamic and evolutionary plasticity of Rab GTPases and membrane traffic in Tetrahymena thermophila. PubMed DOI PMC

Briguglio, J. S., Kumar, S. and Turkewitz, A. P. (2013). Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena. PubMed DOI PMC

Bush, J., Nolta, K., Rodriguez-Paris, J., Kaufmann, N., O'halloran, T., Ruscetti, T., Temesvari, L., Steck, T. and Cardelli, J. (1994). A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H(+)-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes. PubMed DOI

Cassidy-Hanley, D., Bowen, J., Lee, J. H., Cole, E., Verplank, L. A., Gaertig, J., Gorovsky, M. A. and Bruns, P. J. (1997). Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. PubMed DOI PMC

Cheng, C. Y., Young, J. M., Lin, C. G., Chao, J. L., Malik, H. S. and 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. PubMed DOI PMC

Cole, E. and Gaertig, J. (2022). Anterior-posterior pattern formation in ciliates. PubMed DOI PMC

Collins, M. P. and Forgac, M. (2020). Regulation and function of V-ATPases in physiology and disease. PubMed DOI PMC

Cox, J. and Mann, M. (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. PubMed DOI

Cox, J., Hein, M. Y., Luber, C. A., Paron, I., Nagaraj, N. and Mann, M. (2014). Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. PubMed DOI PMC

Damstra, H. G. J., Mohar, B., Eddison, M., Akhmanova, A., Kapitein, L. C. and Tillberg, P. W. (2022). Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). PubMed DOI PMC

Docampo, R., Jimenez, V., Lander, N., Li, Z. H. and Niyogi, S. (2013). New insights into roles of acidocalcisomes and contractile vacuole complex in osmoregulation in protists. PubMed DOI PMC

Du, F., Edwards, K., Shen, Z., Sun, B., De Lozanne, A., Briggs, S. and Firtel, R. A. (2008). Regulation of contractile vacuole formation and activity in Dictyostelium. PubMed DOI PMC

Eisen, J. A., Coyne, R. S., Wu, M., Wu, D., Thiagarajan, M., Wortman, J. R., Badger, J. H., Ren, Q., Amedeo, P., Jones, K. M.et al. (2006). Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PubMed DOI PMC

Elde, N. C., Morgan, G., Winey, M., Sperling, L. and Turkewitz, A. P. (2005). Elucidation of clathrin-mediated endocytosis in tetrahymena reveals an evolutionarily convergent recruitment of dynamin. PubMed DOI PMC

Elliott, A. M. and Bak, I. J. (1964). Contractile vacuole+related structures in tetrahymena pyriformis. PubMed DOI

Essid, M., Gopaldass, N., Yoshida, K., Merrifield, C. and Soldati, T. (2012). Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole. PubMed DOI PMC

Fok, A. K., Clarke, M., Ma, L. and Allen, R. D. (1993). Vacuolar H(+)-ATPase of Dictyostelium discoideum. A monoclonal antibody study. PubMed DOI

Fok, A. K., Aihara, M. S., Ishida, M., Nolta, K. V., Steck, T. L. and Allen, R. D. (1995). The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps. PubMed DOI

Frankel, J. (1992). Positional information in cells and organisms. PubMed DOI

Frankel, J. (2000). Cell biology of Tetrahymena thermophila. PubMed DOI

Gabriel, D., Hacker, U., Kohler, J., Muller-Taubenberger, A., Schwartz, J. M., Westphal, M. and Gerisch, G. (1999). The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein. PubMed DOI

Gaertig, J., Cruz, M. A., Bowen, J., Gu, L., Pennock, D. G. and Gorovsky, M. A. (1995). Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila. PubMed DOI PMC

Gerald, N. J., Siano, M. and De Lozanne, A. (2002). The Dictyostelium LvsA protein is localized on the contractile vacuole and is required for osmoregulation. PubMed DOI

Gerisch, G., Heuser, J. and Clarke, M. (2002). Tubular-vesicular transformation in the contractile vacuole system of Dictyostelium. PubMed DOI

Girard-Dias, W., Alcantara, C. L., Cunha-E-Silva, N., De Souza, W. and Miranda, K. (2012). On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography. PubMed DOI

Gorovsky, M. A., Yao, M. C., Keevert, J. B. and Pleger, G. L. (1975). Isolation of micro- and macronuclei of Tetrahymena pyriformis. PubMed DOI

Gronlien, H. K., Stock, C., Aihara, M. S., Allen, R. D. and Naitoh, Y. (2002). Relationship between the membrane potential of the contractile vacuole complex and its osmoregulatory activity in Paramecium multimicronucleatum. PubMed DOI

Hankins, H. M., Baldridge, R. D., Xu, P. and Graham, T. R. (2015). Role of flippases, scramblases and transfer proteins in phosphatidylserine subcellular distribution. PubMed DOI PMC

Harris, E., Yoshida, K., Cardelli, J. and Bush, J. (2001). Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in dictyostelium. PubMed DOI

Heuser, J. (2006). Evidence for recycling of contractile vacuole membrane during osmoregulation in Dictyostelium amoebae–a tribute to Gunther Gerisch. PubMed DOI

Heuser, J., Zhu, Q. and Clarke, M. (1993). Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. PubMed DOI PMC

Ishida, M., Fok, A. K., Aihara, M. S. and Allen, R. D. (1996). Hyperosmotic stress leads to reversible dissociation of the proton pump-bearing tubules from the contractile vacuole complex in Paramecium. PubMed DOI

Ishida, M., Hori, M., Ooba, Y., Kinoshita, M., Matsutani, T., Naito, M., Hagimoto, T., Miyazaki, K., Ueda, S., Miura, K.et al. (2021). A functional Aqp1 gene product localizes on the contractile vacuole complex in paramecium multimicronucleatum. PubMed DOI

Iwamoto, M., Mori, C., Hiraoka, Y. and Haraguchi, T. (2014). Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena. PubMed DOI

Jiang, Y. Y., Maier, W., Baumeister, R., Minevich, G., Joachimiak, E., Ruan, Z., Kannan, N., Clarke, D., Frankel, J. and Gaertig, J. (2017). The Hippo pathway maintains the equatorial division plane in the ciliate tetrahymena. PubMed DOI PMC

Jiang, Y. Y., Maier, W., Baumeister, R., Joachimiak, E., Ruan, Z., Kannan, N., Clarke, D., Louka, P., Guha, M., Frankel, J.et al. (2019a). Two antagonistic hippo signaling circuits set the division plane at the medial position in the ciliate tetrahymena. PubMed DOI PMC

Jiang, Y. Y., Maier, W., Baumeister, R., Minevich, G., Joachimiak, E., Wloga, D., Ruan, Z., Kannan, N., Bocarro, S., Bahraini, A.et al. (2019b). LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena. PubMed DOI PMC

Jiang, Y. Y., Maier, W., Chukka, U. N., Choromanski, M., Lee, C., Joachimiak, E., Wloga, D., Yeung, W., Kannan, N., Frankel, J.et al. (2020). Mutual antagonism between Hippo signaling and cyclin E drives intracellular pattern formation. PubMed DOI PMC

Jimenez, V. and Docampo, R. (2015). TcPho91 is a contractile vacuole phosphate sodium symporter that regulates phosphate and polyphosphate metabolism in Trypanosoma cruzi. PubMed DOI PMC

Jimenez, V., Miranda, K. and Augusto, I. (2022). The old and the new about the contractile vacuole of Trypanosoma cruzi. PubMed DOI PMC

Kitching, J. (1939). The physiology of contractile vacuoles: IV. A note on the sources of the water evacuated, and on the function of contractile vacuoles in marine protozoa. DOI

Linder, J. C. and Staehelin, L. A. (1979). A novel model for fluid secretion by the trypanosomatid contractile vacuole apparatus. PubMed DOI PMC

Mahajan, D., Tie, H. C., Chen, B. and Lu, L. (2019). Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking. PubMed DOI PMC

Manna, P. T., Barlow, L. D., Ramirez-Macias, I., Herman, E. K. and Dacks, J. B. (2023). Endosomal vesicle fusion machinery is involved with the contractile vacuole in Dictyostelium discoideum. PubMed DOI

Marchesini, N., Ruiz, F. A., Vieira, M. and Docampo, R. (2002). Acidocalcisomes are functionally linked to the contractile vacuole of Dictyostelium discoideum. PubMed DOI

Marshansky, V. and Futai, M. (2008). The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function. PubMed DOI PMC

Mckanna, J. A. (1976). Fine structure of fluid segregation organelles of Paramecium contractile vacuoles. PubMed DOI

Mochizuki, K. (2008). High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene. PubMed DOI

Moliere, A., Beer, K. B. and Wehman, A. M. (2022). Dopey proteins are essential but overlooked regulators of membrane trafficking. PubMed DOI

Montalvetti, A., Rohloff, P. and Docampo, R. (2004). A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi. PubMed DOI

Naitoh, Y., Tominaga, T., Ishida, M., Fok, A., Aihara, M. and Allen, R. (1997). How does the contractile vacuole of Paramecium multimicronucleatum expel fluid? Modelling the expulsion mechanism. PubMed DOI

Ng, S. F. (1977). Analysis of contractile vacuole pore morphogenesis in Tetrahymena pyriformis by 180 degree rotation of ciliary meridians. PubMed DOI

Ng, S. F. and Frankel, J. (1977). 180 degrees rotation of ciliary rows and its morphogenetic implications in Tetrahymena pyriformis. PubMed DOI PMC

Nishihara, E., Shimmen, T. and Sonobe, S. (2007). New aspects of membrane dynamics of Amoeba proteus contractile vacuole revealed by vital staining with FM 4-64. PubMed DOI

Nolta, K. V., Padh, H. and Steck, T. L. (1993). An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum. PubMed DOI

Obado, S. O., Field, M. C., Chait, B. T. and Rout, M. P. (2016). High-efficiency isolation of nuclear envelope protein complexes from trypanosomes. PubMed DOI

Patterson, D. J. (1980). Contractile vacuoles and associated structures: their organization and function. DOI

Perez-Riverol, Y., Csordas, A., Bai, J., Bernal-Llinares, M., Hewapathirana, S., Kundu, D. J., Inuganti, A., Griss, J., Mayer, G., Eisenacher, M.et al. (2019). The PRIDE database and related tools and resources in 2019: improving support for quantification data. PubMed DOI PMC

Plattner, H. (2010). Membrane trafficking in protozoa SNARE proteins, H+-ATPase, actin, and other key players in ciliates. PubMed DOI

Plattner, H. (2013). Contractile vacuole complex--its expanding protein inventory. PubMed DOI

Plattner, H. (2015). The contractile vacuole complex of protists--new cues to function and biogenesis. PubMed DOI

Ritter, M., Bresgen, N. and Kerschbaum, H. H. (2021). From pinocytosis to methuosis-fluid consumption as a risk factor for cell death. PubMed DOI PMC

Rohloff, P., Montalvetti, A. and Docampo, R. (2004). Acidocalcisomes and the contractile vacuole complex are involved in osmoregulation in Trypanosoma cruzi. PubMed DOI

Ruehle, M. D., Orias, E. and Pearson, C. G. (2016). Tetrahymena as a unicellular model eukaryote: genetic and genomic tools. PubMed DOI PMC

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B.et al. (2012). Fiji: an open-source platform for biological-image analysis. PubMed DOI PMC

Schonemann, B., Bledowski, A., Sehring, I. M. and 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. PubMed DOI

Spallanzani, L. (1776).

Sparvoli, D., Richardson, E., Osakada, H., Lan, X., Iwamoto, M., Bowman, G. R., Kontur, C., Bourland, W. A., Lynn, D. H., Pritchard, J. K.et al. (2018). Remodeling the specificity of an endosomal CORVET tether underlies formation of regulated secretory vesicles in the ciliate Tetrahymena thermophila. PubMed DOI PMC

Sparvoli, D., Zoltner, M., Cheng, C. Y., Field, M. C. and Turkewitz, A. P. (2020). Diversification of CORVET tethers facilitates transport complexity in Tetrahymena thermophila. PubMed DOI PMC

Spector, D. L., Goldman, R. D. and Leinwand, L. A. (1998).

Stock, C., Allen, R. D. and Naitoh, Y. (2001). 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. PubMed DOI

Stock, C., Gronlien, H. K. and Allen, R. D. (2002a). The ionic composition of the contractile vacuole fluid of Paramecium mirrors ion transport across the plasma membrane. PubMed DOI

Stock, C., Gronlien, H. K., Allen, R. D. and Naitoh, Y. (2002b). Osmoregulation in Paramecium: in situ ion gradients permit water to cascade through the cytosol to the contractile vacuole. PubMed DOI

Stover, N. A., Krieger, C. J., Binkley, G., Dong, Q., Fisk, D. G., Nash, R., Sethuraman, A., Weng, S. and Cherry, J. M. (2006). Tetrahymena Genome Database (TGD): a new genomic resource for Tetrahymena thermophila research. PubMed DOI PMC

Tani, T., Allen, R. D. and Naitoh, Y. (2000). Periodic tension development in the membrane of the in vitro contractile vacuole of Paramecium multimicronucleatum: modification by bisection, fusion and suction. PubMed DOI

Tani, T., Allen, R. D. and Naitoh, Y. (2001). Cellular membranes that undergo cyclic changes in tension: direct measurement of force generation by an in vitro contractile vacuole of Paramecium multimicronucleatum. PubMed DOI

Tani, T., Tominaga, T., Allen, R. D. and Naitoh, Y. (2002). Development of periodic tension in the contractile vacuole complex membrane of paramecium governs its membrane dynamics. PubMed DOI

Temesvari, L. A., Rodriguez-Paris, J. M., Bush, J. M., Zhang, L. and Cardelli, J. A. (1996). Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum. PubMed DOI

Tominaga, T., Allen, R. D. and Naitoh, Y. (1998a). Cyclic changes in the tension of the contractile vacuole complex membrane control its exocytotic cycle. PubMed DOI

Tominaga, T., Allen, R. D. and Naitoh, Y. (1998b). Electrophysiology of the in situ contractile vacuole complex of Paramecium reveals its membrane dynamics and electrogenic site during osmoregulatory activity. PubMed DOI

Tominaga, T., Naitoh, Y. and Allen, R. D. (1999). A key function of non-planar membranes and their associated microtubular ribbons in contractile vacuole membrane dynamics is revealed by electrophysiologically controlled fixation of Paramecium. PubMed DOI

Turkewitz, A. P. and Bright, L. J. (2011). A Rab-based view of membrane traffic in the ciliate Tetrahymena thermophila. PubMed DOI PMC

Ulrich, P. N., Jimenez, V., Park, M., Martins, V. P., Atwood, J., 3rd, Moles, K., Collins, D., Rohloff, P., Tarleton, R., Moreno, S. N.et al. (2011). Identification of contractile vacuole proteins in Trypanosoma cruzi. PubMed DOI PMC

Van Der Beek, J., Jonker, C., Van Der Welle, R., Liv, N. and Klumperman, J. (2019). CORVET, CHEVI and HOPS - multisubunit tethers of the endo-lysosomal system in health and disease. PubMed DOI

Velle, K. B., Garner, R. M., Beckford, T. K., Weeda, M., Liu, C., Kennard, A. S., Edwards, M. and Fritz-Laylin, L. K. (2023). A conserved pressure-driven mechanism for regulating cytosolic osmolarity. PubMed DOI PMC

Wang, J., Chitsaz, F., Derbyshire, M. K., Gonzales, N. R., Gwadz, M., Lu, S., Marchler, G. H., Song, J. S., Thanki, N., Yamashita, R. A.et al. (2023). The conserved domain database in 2023. PubMed DOI PMC

Warren, A., Patterson, D. J., Dunthorn, M., Clamp, J. C., Achilles-Day, U. E. M., Aescht, E., Al-Farraj, S. A., Al-Quraishy, S., Al-Rasheid, K., Carr, M.et al. (2017). Beyond the “code”: a guide to the description and documentation of biodiversity in ciliated protists (Alveolata, Ciliophora). PubMed DOI PMC