Research Article 2885 The SEC6 protein is required for contractile vacuole function in Chlamydomonas reinhardtii Karin Komsic-Buchmann, Lisa Marie Stephan and Burkhard Becker* Botany, Cologne Biocenter, University of Cologne, 50674 Cologne, Germany *Author for correspondence ([email protected]) Accepted 14 February 2012 Journal of Cell Science 125, 2885–2895 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.099184 Summary Contractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion, rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and systole. These results indicate that CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and water expulsion during systole. In addition, CreSEC6 is not only necessary for CV function, but possibly influences the CV cycle in an indirect manner and flagellar length in Chlamydomonas. Key words: Contractile vacuole, Flagella, Osmoregulation, SEC6, Exocyst, Chlamydomonas 2 Journal of Cell Science Introduction system) has ever been observed. Therefore, HCO3 has been Contractile vacuoles (CVs) are osmoregulatory organelles found postulated to be the anion species continuously eliminated from in many unicellular freshwater protists without cell walls and the cell through the CV (Robinson et al., 1998; Tominaga et al., some sponges (Allen and Naitoh, 2002). CVs are membrane- 1998). This would be similar to the situation for water transport bound cell compartments that periodically accumulate (diastole) in animal epithelia (Hoffmann, 1986; Zeuthen, 1992), but 2 and expel (systole) water out of the cell, allowing cells to survive experimental evidence for a role of HCO3 in CVs has never under hypotonic conditions. Based on structure and behavior been presented. By contrast, experimental evidence points to the about six basic types of CV have been described (Patterson, involvement of phosphate in CV function in Trypanosoma and 1980). Despite this structural diversity the basic functions seem Chlamydomonas (Rohloff et al., 2004; Ruiz et al., 2001) and K+ to be conserved between different eukaryotes because the same and Cl2 have been identified as the major osmolytes in the proteins and cellular processes have been found in Amoeba, cytosol and CV in Paramecium (Stock et al., 2002). Dictyostelium, Paramecium, Trypanosoma and green algae [e.g. The structure and function of the CV in Chlamydomonas have V-ATPase (Becker and Hickisch, 2005; Fok et al., 2002; Heuser been investigated in some detail (Luykx et al., 1997a; Luykx et al., 1993; Montalvetti et al., 2004; Nishihara et al., 2008; et al., 1997b; Robinson et al., 1998). At the end of diastole the Robinson et al., 1998; Wassmer et al., 2005), aquaporin contractile vacuole of Chlamydomonas is spherical, expels the (Montalvetti et al., 2004; Nishihara et al., 2008), vesicular liquid into the medium and the CV fragments into smaller transport (Becker and Hickisch, 2005; Buchmann and Becker, vacuoles (systolic phase; Fig. 1C). During diastole these smaller 2009; Bush et al., 1994; Harris et al., 2001; Kissmehl et al., 2007; vacuoles swell and fuse with each other to form again the Schilde et al., 2006; Stavrou and O’Halloran, 2006); see Komsic- spherical vacuole at the end of a cycle (Luykx et al., 1997b) Buchmann and Becker for a summary of identified proteins and (Fig. 1A). Several questions remain regarding the situation in cellular processes (Komsic-Buchmann and Becker, 2012)]. Chlamydomonas and more generally. (1) Exocytotic pore-like There are many accounts of the osmoregulatory role of CVs structures were identified in ciliates (McKanna, 1973) but have (Allen, 2000; Allen and Naitoh, 2002), and it has been proposed been very difficult to demonstrate in many green algae that water enters the CV by osmosis. V-ATPase and/or V-PPase (Buchmann and Becker, 2009; Luykx et al., 1997b). (2) How drive secondary active transport systems, allowing water to the liquid leaves the cell in these systems is not clear, but follow passively through aquaporins. However, no acidification conspicuous intra-membrane particle arrays (up to 180 nm in of the CV (as expected for a proton-pump-mediated uptake diameter) have been observed in the plasma membrane overlying 2886 Journal of Cell Science 125 (12) Journal of Cell Science Fig. 1. The contractile vacuole of Chlamydomonas reinhardtii CC3395. (A–C) The ultrastructure of the CVs in CC3395. The two CVs are located close to the basal body (A). At the end of diastole (B) the CV membrane forms a contact zone with the plasma membrane marked by cytosolic electron dense material between the membranes (arrows). In the systolic phase the CV fragments into smaller vesicles (C). The contact zone persists apparently until the end of systole (arrow). The ‘bracelet’, a specialized plasma membrane region at the basis of the flagellum (F), is marked by an ellipse in A. M, mitochondrion; N, nucleus. (D) Frames from a light microscope time-lapse recording. Numbers indicate the time passed since the end of last diastole. The white arrow marks the CV, scale bar: 5 mm. (E) The growth of CC3395 in four different media (TAP/2, TAP, TAP-S and TAP-SS). The strain can grow in every medium tested. (F) The relationship between the CV period, the CV volume and the efflux of each CV to the cell surface (n545). The bigger the cell surface is, the longer the CV period, the higher the CV efflux and the larger the CV volume. (G) The mean values and the standard deviation of the data set in F, given as non-normalized and normalized to the cell surface. The CV period shows higher variation in the normalized data set, whereas the normalized data set for the CV volume and the water efflux from a cell shows less variation than the non-normalized data set. Numbers above the bars indicate the coefficient of variation for the different data sets. the CV region (Weiss et al., 1977). These arrays apparently form adapted to the need of the cell is at best fragmentary. Calcium, only during systole and are often matched by a similar array in protein kinases and cAMP have been implicated (Rohloff and the CV membrane opposing the plasma membrane array (Weiss Docampo, 2008), but in no system is the CV really understood. et al., 1977). Both array are connected by cytosolic electron Chlamydomonas is a well-established protist model system dense material (Weiss et al., 1977) (Fig. 1A) and similar (Grossman et al., 2003). The genome of Chlamydomonas has cytosolic electron dense material has also been detected in recently been sequenced (Merchant et al., 2007). Chlamydomonas another green alga Mesostigma viride (Buchmann and Becker, can be transformed using several methods (Coll, 2006; Grossman 2009). (3) A role for cytoskeletal elements during the CV cycle et al., 2003). Silencing of genes using RNA interference (RNAi) could only be demonstrated in Dictyostelium (Taft et al., 2008), has been successfully introduced in Chlamydomonas and is indicating that force generation during systole by cytoskeletal continuously improving (Schroda, 2006), and several proteins elements does not play any role in most systems. Changes in have been expressed as GFP-tagged constructs (Fuhrmann et al., membrane structure have been implicated in water expulsion 1999; Huang et al., 2007; Ruiz-Binder et al., 2002; Schoppmeier during systole in Paramecium (Allen and Naitoh, 2002), but et al., 2005), making it possible to observe the in vivo dynamics whether this is a general mechanism remains to be seen. In of subcellular structures and/or proteins. For this reason we addition, our knowledge of how the CV cycle is controlled and have started a forward genetic approach to analyze CV function Contractile vacuole of Chlamydomonas 2887 in Chlamydomonas. Osmoregulatory mutants isolated after normalized data set (Fig. 1G). These results indicate that cells use insertional mutagenesis showed defects in CV structure and mainly variation of the size of the CV to adapt to the increasing function. We have analyzed a mutant in which membrane fusion water influx during cell growth, whereas the contraction period is events related to CV function are apparently impaired. We show apparently regulated by a different factor. that the deletion of the single Chlamydomonas SEC6 protein accounts for the observed phenotype, indicating a role for Mutant screen SEC6, and probably the exocyst complex, in CV function in To isolate osmoregulatory insertional mutants we used the Chlamydomonas. mutant screen designed by Luykx et al. in combination with insertional mutagenesis using the hygromycin B resistance Results marker developed by Berthold et al. (see Materials and Characterization of the contractile vacuole of Methods for details) (Luykx et al., 1997a; Berthold et al., Chlamydomonas reinhardtii CC3395 2002).