Journal of Cell Science Dictyostelium oay oon icne,Uiest fClge 07 oon,Germany Cologne, 50674 Cologne, Becker* of Burkhard University and Biocenter, Cologne Stephan Botany, Marie Lisa Komsic-Buchmann, Karin vacuole contractile for in required function is protein SEC6 The Article Research fteC a xetdfrapoo-upmdae uptake proton-pump-mediated a for expected to (as water CV acidification allowing no the However, systems, of aquaporins. through transport V-PPase passively and/or active follow V-ATPase proposed osmosis. secondary been by has CV drive it the and enters 2002), water Naitoh, that and Allen 2000; (Allen, and 2012)]. proteins Becker, identified and of (Komsic-Buchmann summary processes a Komsic- cellular for see 2006); 2007; Becker al., O’Halloran, vesicular and aquaporin et and Buchmann Kissmehl Stavrou 2008), Becker, 2001; 2006; 2005), al., al., and et al., et Harris Schilde Buchmann al., 1994; et al., 2005; et 2008; et Bush Hickisch, Nishihara 2009; al., and 2004; Wassmer et (Becker al., Nishihara transport 1998; 2004; et al., al., (Montalvetti et et Heuser Montalvetti 2002; Robinson al., 1993; et al., Fok in 2005; et found Hickisch, and been (Becker V-ATPase have same processes the (Patterson, cellular because eukaryotes described and seem different functions been proteins between basic have conserved the be diversity CV behavior to structural of and this types Despite structure 1980). basic on survive six Based to cells about conditions. allowing cell, hypotonic the membrane- of under out are (diastole) water CVs (systole) accumulate and expel periodically 2002). and that walls Naitoh, compartments cell cell and without bound (Allen protists sponges freshwater some unicellular found many organelles osmoregulatory in are (CVs) vacuoles Contractile Introduction an in cycle CV the influences and possibly diastole but and during words: function, diastole fusion Key CV late vesicle for during homotypic in necessary observed for only length the membrane required not flagellar CV that and is and the function showed CreSEC6 and manner CV addition, cells membrane for indirect In Osmo75 plasma essential systole. of the is during analysis between CreSEC6 expulsion zones that water microscopy contact indicate Electron close results the rates. the These of efflux form systole. Transformation water to exocytosis. able increased efficient is to for mutant leading required unchanged, is remained that interval complex in exocyst homologue exocytotic SEC6 the and only visible of overexpressed vacuolar the no a component is homotypic or with genes in a CVs The affected in impaired mutant enlarged is the detail. is Osmo75 two of protein function mutant in or One the SEC6 one analyzed flagella. CV that addition, The was long suggest In had of (Osmo75) (CreSEC6). findings CVs. mutants These line small the mechanism microscopy. cell multiple Furthermore light had fusion. mutant the by membrane cells One observed most investigate conditions. were and all, hypotonic cells, To at strong mutant CVs in under protists. variable grow was many to morphology unable in CV lines osmoregulation cell the for rendering essential are Chlamydomonas (CVs) vacuoles Contractile Summary 10.1242/jcs.099184 doi: 2885–2895 125, ß Science Cell of Journal 2012 February 14 Accepted ( correspondence for *Author 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. hr r ayacut fteomrgltr oeo CVs of role osmoregulatory the of accounts many are There otatl aul,Fael,Omrglto,SC,Exocyst, SEC6, Osmoregulation, Flagella, vacuole, Contractile , CreSEC6 Paramecium eioae oe soeuaoymtns oro h sltdmtn ellnscridtesm 361bs deletion, base 33,641 same the carried lines cell mutant isolated the of Four mutants. osmoregulatory novel isolated we , [email protected] CreSEC6-GFP a F-agdpoen n ipae oiidC ciiy V eelre,weesteC contraction CV the whereas larger, were CVs activity. CV modified a displayed and protein) GFP-tagged a (as , hayooa reinhardtii Chlamydomonas Trypanosoma ) osrc ece h uatcmltl omrglto n lgla egh.Rsudstrains Rescued length). flagellar and (osmoregulation completely mutant the rescued construct Chlamydomonas n re la [e.g. algae green and Amoeba . , ytm a vrbe bevd hrfr,HCO Therefore, observed. been ever has system) novmn fpopaei Vfnto in function CV the in Chlamydomonas to phosphate points of evidence experimental involvement contrast, By presented. been n Cl and h iudlae h eli hs ytm sntcer but clear, not in nm overlying is membrane 180 algae plasma systems to the in these green (up observed How been in arrays have (2) many diameter) cell particle 1997b). the intra-membrane in al., conspicuous leaves et demonstrate Luykx liquid 2009; to the Becker, have and difficult but (Buchmann 1973) very (McKanna, 1997b) ciliates in been the in al., situation identified et again the were (Luykx structures form regarding cycle remain to a questions Chlamydomonas other of Several smaller end each 1A). the into (Fig. with at fragments fuse vacuole smaller CV spherical and these diastole the swell During 1C). and vacuoles Fig. the phase; medium diastole (systolic of the vacuoles end Luykx into the 1997a; At liquid 1998). al., of al., et vacuole et (Luykx contractile Robinson 1997b; detail al., some et in investigated been in CV and cytosol naia ptei Hfmn,18;Zuhn 92,but 1992), HCO Zeuthen, of role 1986; a transport (Hoffmann, for water evidence for epithelia experimental al., situation et the animal Tominaga to 1998; similar in al., be et would from (Robinson This CV eliminated 1998). the continuously through species cell anion the the be to postulated Chlamydomonas h tutr n ucino h Vin CV the of function and structure The 2 aebe dniida h ao soye nthe in osmolytes major the as identified been have Rhofe l,20;Ri ta. 01 n K and 2001) al., et Ruiz 2004; al., et (Rohloff n oegnrly 1 xcttcpore-like Exocytotic (1) generally. more and Paramecium Chlamydomonas Soke l,2002). al., et (Stock sshrcl xesthe expels spherical, is Chlamydomonas 3 2 nCshsnever has CVs in Trypanosoma Chlamydomonas 3 2 a been has 2885 have and + Journal of Cell Science h Vpro hw ihrvraini h omlzddt e,weestenraie aastfrteC oueadtewtrefu rmacl hw l shows cell a from sets. efflux data water different the the and for volume variation CV of the coefficient for the set indicate data bars normalized the the above whereas Numbers set, set. data data normalized non-normalized the the in than variation variation higher shows period ( surface CV cell ( the The volume. to CV CV the each larger of the efflux and the efflux and volume CV the period, CV the between arw.Te‘rclt,aseilzdpam ebaergo ttebsso h lglu F,i akdb nelpei .M iohnro;N nucl N, mitochondrion; M, A. in ellipse o an end by the material marked until dense is apparently electron (F), persists cytosolic flagellum zone by the contact marked of The membrane basis 5 (C). plasma the vesicles the at smaller with region into zone membrane fragments contact plasma ( CV a specialized the forms a phase membrane ‘bracelet’, systolic CV The the (arrow). the In (B) (arrows). diastole membranes of the end between the At (A). body basal hte hsi eea ehns ean ob en In and seen. controlled be is cycle to CV remains the mechanism how of general knowledge a our expulsion addition, is in water Changes this in systems. whether in implicated most systole been in during role have cytoskeletal any structure by play membrane systole not during does generation elements cycle force CV that the in in similar during indicating demonstrated elements detected be and cytoskeletal only for been could 1A) role also (Fig. A (3) has 2009). 1977) electron alga material cytosolic al., green by dense another et connected electron (Weiss are cytosolic array material in Both (Weiss array dense array 1977). similar membrane al., a plasma the by et opposing matched membrane often form CV are apparently the and arrays These systole 1977). during al., only et (Weiss region CV the of vacuole contractile The 1. Fig. 2886 D m rmsfo ih irsoetm-as eodn.Nmesidct h iepse ic h n fls isoe h ht ro ak h V sc CV, the marks arrow white The diastole. last of end the since passed time the indicate Numbers recording. time-lapse microscope light a from Frames ) .( m. E h rwho C35i ordfeetmda(A/,TP A- n A-S.Tesri a rwi vr eimtse.( tested. medium every in grow can strain The TAP-SS). and TAP-S TAP, (TAP/2, media different four in CC3395 of growth The ) ora fCl cec 2 (12) 125 Science Cell of Journal Paramecium eotgaviride Mesostigma hayooa reinhardtii Chlamydomonas G Dictyostelium h envle n h tnaddvaino h aasti ,gvna o-omlzdadnraie otecl surface. cell the to normalized and non-normalized as given F, in set data the of deviation standard the and values mean The ) AlnadNio,20) but 2002), Naitoh, and (Allen Bcmn n Becker, and (Buchmann Tf ta. 2008), al., et (Taft CC3395. ( A–C h lrsrcueo h V nC39.TetoCsaelctdcoet the to close located are CVs two The CC3395. in CVs the of ultrastructure The ) n 5 fsbellrsrcue n/rpoen.Frti esnwe function reason CV analyze this to approach For genetic dynamics proteins. forward vivo a and/or in started the have structures observe to subcellular possible Schoppmeier it 2002; of making al., 2005), et Ruiz-Binder al., al., 2007; et et al., proteins (Fuhrmann et constructs Huang several GFP-tagged 1999; as and expressed been 2006), in have (Schroda, introduced improving (RNAi) interference successfully continuously RNA using been genes of has Grossman Silencing 2006; 2003). (Coll, al., methods et several using transformed of be 2007). genome can al., et The (Merchant sequenced 2003). been recently al., understood. really et CV and (Grossman the is (Rohloff system Calcium, implicated no fragmentary. been in best but have at 2008), is cAMP Docampo, cell and the kinases of protein need the to adapted 5.Tebge h elsraei,telne h Vpro,tehge h CV the higher the period, CV the longer the is, surface cell the bigger The 45). Chlamydomonas sawl-salse rts oe system model protist well-established a is Chlamydomonas Chlamydomonas F Chlamydomonas h relationship The ) systole f eus. n is and l bar: ale has ess Journal of Cell Science ) rmteerslsi a ecluae hti A eim(64 medium 11.9 TAP in that a calculated be mosM) can it results these From 1). otato nevlo h V hncmae ihtenon- the with compared when CV, the standard for the the bigger contrast, was of set By interval data 1G). normalized contraction Fig. the for in obtained variation bars deviation less of coefficients the the showed (compare above cell and set set indicated volume a CV from data data efflux the standard for water normalized set the non-normalized and data the non-normalized values the and expected, than variation mean normalized As the 1G). the (Fig. calculated for then deviations We characterize surface our to CVs. used cell videos in the the the using synchronized from cell data not each CV for the determined were areas normalize cells to tried the we Because cultures, cycle. cell the area)]. C ouecl ufc area), surface volume–cell (CV E6 n rbbyteeoytcmlx nC ucinin for function CV role in a complex, indicating exocyst the phenotype, probably observed and single the SEC6, the for show of We accounts deletion impaired. apparently and the fusion are membrane function structure that which CV in CV to mutant related a in events analyzed have defects We function. showed mutagenesis insertional in n h ytl 1.3 systole the the and of end 1.78 the at 20.6 was vacuole round large diastole the average the of medium and diameter TAP Materials In maximum (see culturing). analysis cell our on in details used for were Methods 1E) Fig. own phase, (unpublished (end varies log subculturing cells of after days osmolarity the 4–6 cells of cytosolic only status therefore observations), osmolarity and the conditions cytosolic that growth with indicate the data that Preliminary indicates result of this therefore, used We of reinhardtii vacuole Chlamydomonas contractile the of Characterization Results Chlamydomonas i.1,altrefcossoe odcreaint h cell the to correlation good directly a from evident showed is is [ factors As area cell. surface three cell a and all of period area 1F, a surface a Fig. CV cell performed the volume, therefore and in CV efflux we water between area; uptake analysis surface correlation cell Water linear the minute). to proportional per volume iil ihalgtmcocp.Csaeol iil ihtelight the were with that visible CVs only in exhibited are microscope cells CVs microscope. the light of a 5% with visible than mosM). less 204 medium sucrose, to this medium mM the In 120 of strength (containing osmotic TAP-SS total end the anterior except increasing the tested at media CVs two all had mineralin Cells the media. of other half the only contains of TAP/2 nutrients that note 8A); Fig. 1C). diastole also see (Fig. of systole end during other the persist at apparently in membrane which close 1B), plasma forms (Fig. the As and with 1A) zones (Fig. 1A–D). vacuoles contact (Fig. small from easily develops microscopy using is diastole strain and electron this wall of Chlamydomonas cell and CV a the have characterized light first not We does transformed. which screen, mutant C35gosi ei fdfeetomtcsrnts(i.1E, (Fig. strengths osmotic different of media in grows CC3395 Chlamydomonas Chlamydomonas 6 6 8.75 . eod ( seconds 5.3 Chlamydomonas hayooa reinhardtii Chlamydomonas Chlamydomonas m m r 2 3 Chlamydomonas 5 mnt apoiaey2 ftettlcell total the of 2% (approximately /minute . .58(Vpro–elsraearea), surface period–cell (CV 0.6568 tan,telrerudC iil tlate at visible CV round large the strains, 6 n 6 soeuaoymtnsioae after isolated mutants Osmoregulatory . 5 . eod splmnaymtra Movie material (supplementary seconds 0.5 0.43 C35i prxmtl 0 mosM. 200 approximately is CC3395 5.Tedatl atd19.4 lasted diastole The 45). el osdrbyices nsz during size in increase considerably cells m C35cl xesapproximately expels cell CC3395 n h otato interval contraction the and m CC3395 hni si yooi medium; hypotonic in is it when Chlamydomonas r 2 5 .82(flxcl surface (efflux–cell 0.7872 tanC39 o the for CC3395 strain E6protein SEC6 6 . seconds 5.0 r 2 5 0.7907 l ute okwscridotwt so5a representative 75. so a and as known, 67 Osmo75 not 65, with is Osmo64, out 32 of carried and two was 28 work Osmo12, these further in At all Osmo32, size event. insertion deletion and same the the present from Osmo12 originated also for probably clones identical are deletions sequences large strains 2C). these in (Fig. ( also 32 that and indicated Osmo12 walking after 2B), Primer (Fig. time Osmo28 the phenotype: the recovery determine strong to after the able division during only deletion were 3 gene, cell we contrast, marker by base By transformation). the (possibly from 33,641 of event originated have insertion insertion same might same clones the the the that isolated indicating exactly four 2A), All (Fig. 75. contained and 67 66, strains Osmo64, for sequences flanking anyvraino h ieo h Vt dp oteincreasing factor. the is different period to a contraction adapt by the regulated to whereas apparently CV growth, cell the during of influx use size water cells the that of indicate results variation These mainly 1G). (Fig. set data normalized hntp n rwhi A eim rwhi TAP-S strong a in showing growth 75) medium, 67, TAP 66, in cell 64, seven growth as 32, on (no 75 to 28, work phenotype of referred our (Osmo12, total a concentrated (hereafter lines obtained We mutants to we Osmo1–Osmo75). able Altogether osmoregulatory still In medium. were in potential but medium. TAP phenotype grown sucrose, in TAP growth cells M than 0.06 grow in different etc.) containing all a color, medium different TAP at showed rate, grow lines growth Seven (different to cell medium. 68 failed TAP in addition, lines al., grow to cell and et failure 144 mutant and for Berthold Materials (TAP-S, obtained, screened sucrose were were 1997a; (see M these clones 0.06 al., hygromycin-B-resistant containing al. 2858 et plates mosM) et TAP resistance (Luykx On B details) Berthold 2002). with for hygromycin combination by in the Methods the developed al. used using et marker we Luykx mutagenesis by mutants insertional designed insertional screen osmoregulatory mutant isolate To screen Mutant hte h bevdgot eeti eae oCV to related is CV the defect indeed that growth confirmed microscopy observed Video malfunction. the whether hypotonic strong under die, conditions even or hypotonic ( grow, mild conditions to strain, under fail lines parental grow but mosM) the to in cell (144 of able grow mutant that are to to the cells osmolarity able Osmo75 that cytosolic different not Assuming similar was the a osmolarity. mutant have in low The Osmo75 of 8A). for Fig. media 3). curves see (Fig. growth (also microscopy the electron media and shows video media 3A characterized by Fig. same and lines strain the cell parental mutant in the the Osmo75 of characterization for for curves used growth determined We Osmo75 of Characterization 5 the obtained for We marker 75. resistance and 67 B 66, hygromycin 64, the 32, 28, of Osmo12, insertion of locus (RESDA- amplification-PCR (Gonza PCR) site-directed enzyme the of Restriction insertion same marker the B carry hygromycin 75 67, 66, Osmo64, medium). . 9 ie n lcrnmcocp a sdt investigate to used was microscopy electron and Video netfakn eune o h te he uat hwn a showing mutants three other the for sequences flanking insert b a curd(i.2,) eas h 5 the Because 2B,C). (Fig. occurred had kb) 9 otatl aul of vacuole Contractile # ´ e-alse ta. 05 a sdt eemn the determine to used was 2005) al., et lez-Ballester 4mosM). 64 Chlamydomonas 9 netflanking insert 9 n 3 and 2887 9 Journal of Cell Science bevtos(i.3,) ecudas eettetypical the detect also could We 3I,J). (Fig. observations mixed a smaller cells multiple 3H). and examined (Fig. CV the CVs enlarged of one 3.3% detected: the was in morphotype Movie of Finally, material in visible. supplementary and 8.0% were 3C; visible, 2) (Fig. was In CVs 3G) enlarged 4). (Fig. microscopy two CV 3.7% light Movie enlarged using one material visible cells investigated were supplementary that 3D,E; CVs 3F; (Fig. (Fig. any bodies cells have given basal the not of the a 23.7% did to Surprisingly 3). close in Movie multiple region material variable had supplementary the (61.3%) in quite 3B–L). cells CVs the (Fig. was of smaller medium Many the phenotype population. TAP-S and cell diastole in CV Osmo75 size CV CV) the the the showed a CVs, of However, cells of of end Osmo75 number interval the all the contraction at contrast, in the (changes By vacuole of dysfunctions 3B). round cells Fig. CV large 1D, all alternating (Fig. typical a tested, a with following media material CVs cycle hypotonic two (supplementary have all cells strain parental In Osmo75 2–4). in Movies aberrant 5 the was a amplify site to insertion cycle failed identified the that indicate primers arrows Vertical of detected. positions be the could kb indicate 9 arrows grey of Horizontal deletion the minimal cassette. by ( a marker genome. indicated walking the the primer is in By mutants, cassette determined. marker these be the could for cassette of Methods) insertion and the to Material owing (see deletions. Cre20.g760000.t1.2) RESDA-PCR huge by caused determined casette deletion, ( marker The B shown. hygromycin are the (http://www.phytozome.net) of Insertion 2. Fig. 2888 A nOm7 orgnsaecmltl eee Ce0g580t.–r2.795.12 n w ee r rnae Ce0g570t. and (Cre20.g759750.t1.2 truncated are genes two and (Cre20.g759800.t1.2–Cre20.g759950.t1.2) deleted completely are genes four Osmo75 In ) lcrnmcocp ofre h ih microscopy light the confirmed microscopy Electron ora fCl cec 2 (12) 125 Science Cell of Journal B h orsodn ra ftegnm of genome the of areas corresponding The reua nrae n erae fteC imtro appear or diameter CV the show of cells decreases Osmo75 light and completely of always increases CVs the contrast, CC3395 irregular 4), By of (Fig. in shown). CV empty (not visible the TAP-S, completely disappears medium always in is TAP not that in do CV noteworthy whereas cells is no CC3395 it of generally However, CVs 4). systole CV (Fig. a of at microscope systole; of during end diameter decreases in rapidly the The and CVs pattern. diastole also. during oscillating recorded increases was an strain of showed size parental the CC3395 the comparison, For in every 4). CVs CVs one (Fig. the individual or medium of TAP-S two size in with the seconds cells recorded 5 and of 3C,G) videos (Fig. also selected CVs we enlarged see detail more 3K,L; in Fig. strain with 1B Fig. cystosolic (compare dense 8H). than Fig. cells membrane electron CV CC3395 and less membrane in in plasma contain zones the contact between to material Often plasma 4). appeared the Fig. clearly below; with Osmo75 (see was membrane Osmo75 expulsion in water CV impaired although the systole, during by membrane formed zones contact , C nOm2 n so2ad3 nyoefakn euneo h marker the of sequence flanking one only 32 and Osmo12 and Osmo28 In ) oivsiaetebhvo fidvda V nteOsmo75 the in CVs individual of behavior the investigate To 9 fakn eino h akrcassette. marker the of region -flanking hayooa reinhardtii Chlamydomonas h 3 the t 9 n of end bar. Journal of Cell Science (6.97 eghpeoye lglao so5clswr uhlonger much were cells Osmo75 of (9.63 Flagella terminate phenotype. to length failure by expulsion. caused water achieve possibly be and are systole might diastole. CVs during vacuoles enlarged fusion the small vacuolar The during multiple homotypic inefficient events The by impaired. fusion caused are membrane rarely cycle Osmo75 CV discharges in that total indicates but 4), (Fig. constant time occurred. some for Osmo75. mutant osmoregulatory the of vacuoles contractile The 3. Fig. ebae(akdb rosadaesonelre nKL.()Temxdpeoyeo so5 n nagdC n utpesalrCVs. smaller multiple and CV enlarged one Osmo75, of phenotype mixed The (J) K,L). in 5 bar: enlarged Scale shown CV. are a displaying and normally region arrows cytoplasmic by the marks (marked (C–H) triplicate). F membrane in analyzed in arrowhead were type The ( asterisk. each TAP/2. white of cells and ( by (100 indicated morphologies TAP CV are in different CVs died, the phenotypes. with even CV cells parental or different and grow, mutant to various the failed of but proportion TAP-SS, the and TAP-S in grew strain I–L ial entdta so5clshdadsic flagellar distinct a had cells Osmo75 that noted we Finally phenotype osmoregulatory observed the together Taken lcrnmcorpso h V fOm7.()Fu V r iil noecl mlil V e el.Too hmso otc oe ihteplasma the with zones contact show them of Two cell). per CVs (multiple cell one in visible are CVs Four (I) Osmo75. of CVs the of micrographs Electron ) 6 6 1.05 1.55 m m ;Fg 5). Fig. m; )ta hs fteprna C35strain CC3395 parental the of those than m) ( A h rwho so5i ordfeetmda(A/,TP A- n A-S.The TAP-SS). and TAP-S TAP, (TAP/2, media different four in Osmo75 of growth The ) akrt netgt hte rti agtn oteC is CV the useful to not a targeting (data be protein cells might whether vegetative CreMIP1–GFP investigate in to that expressed marker reasoned is We CreMIP2 shown). CreMIP1 that not www.phytozome.net) showed of but RT-PCR (Cre12.g549300; (Cre17.g711250). CreMIP1 genome CreMIP2 and 2011) The al., 2004; 2008). et al., et al., (Montalvetti in reinhardtii in et organisms CV impaired for other Nishihara system is several marker in CV GFP function the a develop to to Chlamydomonas targeting tried protein Osmo75 we in Osmo75 whether impaired not test is CV To the to targeting Protein B–H otatl aul of vacuole Contractile el fOm7 hwvral Vmrhlge.Tegah()shows (B) graph The morphologies. CV variable show Osmo75 of Cells ) noe nytopttv qaois(Anderberg aquaporins putative two only encodes qaoishv enipiae nCV in implicated been have Aquaporins . Chlamydomonas Chlamydomonas xmlsfrthe for Examples m m. 2889 Journal of Cell Science epciey ett right. to left respectively, so5aeidctdb ahs( hashes by indicated are Osmo75 *** infcn ifrne rmC39 r niae yatrss(* asterisks by indicated are CC3395 (Osmo75-SEC6GFP). from strains differences rescued Significant three and Osmo75 of mutant length Flagellar 5. Fig. complex exocyst the of part be in to protein shown been SEC6 have putative proteins only encodes on Au9.Cre20.g759900.t1 information model the Gene available models. the gene summarizes six the proteins 1 putative Table additional truncated. two Four are and models. deleted gene are six proteins affects Osmo75 putative in (www.phytozome.net), deletion base sequence 33,641 genome the available the on Based Osmo75 of Rescue 6C). (Fig. CV cycle CV the the intermingle during that not other did each observed apparently with membrane always plasma we and membrane using background UVM4 that addition, genetic indicating the In UVM4 Osmo75. strains, in in 6D,E) impaired CV (Fig. the of the Osmo75 targeting protein and to 6B–E, Fig. 6B,C) from localized (Fig. evident is clearly as the However, CreMIP1–GFP in 6A). fluorescence (Fig. background channel resistance). Non- GFP some experiment. this for showed of (paromomycin cells results screened the transformed shows protein and linearized 6 Fig. selected expression. APHVIII a GFP were with the clones Paromomycin-resistant transformed and were GFP 2009) al., Sca pJR38 in et expression was vector (which GFP systems, (Neupert strain expression for UVM4 of the developed GFP other and cDNA specifically Osmo75 the full-length 2009). in al., into The et (Neupert CV. of cloned as the expression was to that, addition, CreMIP1 localized In confirm are line. aquaporins cell might Osmo75 CreMIP1–GFP the in impaired 2890 P I– # Xba .0) infcn ifrne ewe h ece tan and strains rescued the between differences Significant 0.001). rgeto J3-I1GP oigfrCreMIP1– for coding pJR38-MIP1-GFP, of fragment I ora fCl cec 2 (12) 125 Science Cell of Journal CreMIP1-GFP hayooa reinhardtii Chlamydomonas ### P # 0.001); osrc oteC snot is CV the to construct Chlamydomonas n 5 CreMIP1-GFP 7 7 9 3ad32, and 33 29, 37, 37, Chlamydomonas C35 the CC3395, P SEC6 . # nthe in 0.05, ) tan(i.7 ae –)udraltse soi conditions, osmotic tested all under 1–4) lanes 7, (Fig. strain s3%ietcl(7 iia)t the to similar) (47% Cre identical that showed 31% sequence is cDNA full-length the published This of the exon. analysis in the one present the also misses with that is Phytozome exon cDNA indicates in Phytozome sequence full-length protein in the both predicted sequence of excluding protein bases and Comparison long 2439 transcript was aa) long. cDNA (672 aa) gene full-length bases (812 isolated Augustus 2019 the the However, be UTRs. on to full- Based cDNA the the expected 5). length we from (www.phytozome.net) lane fragment Phytozome PCR in 7, model same (Fig. the strain amplify Osmo75 to failed we whereas h bevdpeoyeo so5 o hsrao we reason this CreSEC6. For as to referred Osmo75. protein, this of on possibly cycle. work phenotype SEC6, our CV concentrated for to cause a observed molecular similar probable during protein the a systole seems a function, of exocyst of affecting many beginning deletion the in the the during secretion Therefore occur at polarized events and fusion in diastole Membrane involved systems. is eukaryotic and membrane events efficient for fusion required factors tethering multi-subunit of (Bro ai fC:elsraeae n ae flxcl ufc area surface efflux:cell water and the area strains; contraction rescue surface the ten CV:cell all Whereas of in altered ratio strain. significantly revealed not parental strains was cycle Osmo75 interval the rescued CV and ten the the investigated of between hypotonic differences analyses strong interesting microscope on Cre light well the Detailed insertion as different of of grow because not sites possibly media, did isotonic strains and D10) some media tested, and conditions A5 osmotic all (e.g. at grew are strains lines rescue cell endogenous rescue the Cre than all the level higher that a at showed and analysis RT-PCR F9, H7). GFP F6, and E3, D10, H5 C11, lines G6, A9, (Osmo75-A5, cell selected randomly rescue were selected randomly three a rescued 5). completely in (Fig. indicated cells, nearly living analyzed was already of phenotype CV when microscopy flagellar this in light long defects by the so no and observed to medium, investigated be clones could resistant TAP all function For clones in cells rescue. Osmo75 grow 312 successful obtained. not plates were the paromomycin do as TAP and pJR38 mutant B On using the hygromycin again rescue vector. protein to fusion expression tried CreSEC6–GFP we a Osmo75 with of phenotype observed thaliana TPRrvae htCre that revealed RT-PCR ocaatrz h ece tan nmr eal e strains ten detail, more in strains rescued the characterize To the for responsible indeed is CreSEC6 that confirm To ce ta. 00.Teeoytcmlxblnst h group the to belongs complex exocyst The 2010). al., et ¨cker osrc sepesdi l e tan Fg ,lns6–15) lanes 7, (Fig. strains ten all in expressed is construct . el hwacmltl reua behavior. Osmo75 irregular of completely CVs a the pattern, show in reiterating cells CVs a Whereas show CV. strain Osmo75. different parental and a of CC3395 represents area strain line surface parental Each the the calculate in to CVs used individual and seconds cells 5 the every from liquid expel to efficiently. fails Osmo75 4. Fig. SEC6-GFP h imtro niiulCswsdetermined was CVs individual of diameter The SEC6 SEC6 osrc ntevrosstrains. various the in construct vrxrsos lhuhalten all Although overexpressors. sepesdi h parental the in expressed is Arabidopsis SEC6 SEC6 from nC39.Thus CC3395. in eune Blast sequence. 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(Fig. that area, surface efflux:cell confirmed water strains; rescue ten P area, the of surface seven volume:cell for (CV significantly increased # .0 o l e ecesris.Eeto microscopy Electron strains). rescue ten all for 0.001 Chlamydomonas al .(uaie rtisof proteins (Putative) 1. Table samdlsse and system model a as P # 0.001 Chlamydomonas Adrege l,21) oee,R-C niae that indicated RT-PCR isoform However, second a 2011). least al., at Cre of et presence the (Anderberg indicated MIPs algal of genome a the of localization cellular aquaporin. the investigated ta. 00,sgetn htptnilmmrn uinevents fusion membrane mechanism. potential kiss-and-run that Zanchi the 1981; suggesting follow (Patterson, 2010), cycle al., CV the et membrane during plasma intermingle the and in not membrane as do CV addition, In the 2008). systems al., other et many Nishihara the 2004; systems, al., et other (Montalvetti in the membrane. 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Osmo75, the in of CV sensitivity do the (exocytosis) in during efficiently systole events operate and not fusion fusion) membrane vacuolar that (homotypic diastole indicated phenotype the of the in encoded protein, respect. the this probably in important most more fragment, is DNA used method linear large transformation method small obtained we a transformation marker using as the However, deletions of 2011). type and al., the in plasmid) et on (Gonzalez-Ballester full uncommon depend to size, not proposed (large been the are have during and deletions gene 2011) marker large the Such Chlamydomonas of time). insertion the by recovery (possibly after event insertion division same cell clones the the from and that originated indicating 67 have deletion, 65, might base 33,641 (Osmo64, same mutants the show obtained 75) the of Four dysfunction. than CC3395 in higher 137c. slightly is in cells the of osmolarity cytosolic in uptake 2892 aulrfso.Scn,teSC eeinmtn in mutant deletion SEC6 the homotypic Second, complex-mediated HOPS in fusion. that in speculate involved vacuolar to also the tempting is in is SEC6 found it phenotype observed Osmo75 been the Given in not 2007). al., far et (Bro (Koumandou genome so subunits have different subunits six of yeast consists the in the (Bro by Work complex mediated HOPS is efficiently. fusion vacuolar place homotypic many that take indicates is to system not (leading Osmo75 does fusion CVs) vacuolar in smaller homotypic phenotype and to (leading CVs) observed inefficient enlarged is exocytosis the efficient First, aspects. respect for two in surprising required this be In to 2010). (Bro systems The shown various 2007). been in al., exocytosis has et (Koumandou event complex factor fusion and exocyst tethering membrane events, own every its fusion that requires membrane indicates of knowledge facilitators current ancient are (Bro MTCs to (MTCs) belongs factors which tethering complex, multi-subunit exocyst the the of part Osmo75. is in protein length for SEC6 flagellar The responsible and function is CV CreSEC6 in defect of observed the deletion the indeed that confirmed h 361bs eeini so5icue h nySEC6 only the includes Osmo75 in deletion base 33,641 The CV for screened and generated were mutants Insertional Chlamydomonas ora fCl cec 2 (12) 125 Science Cell of Journal fe rnfrain(ozlzBletre al., et (Gonzalez-Ballester transformation after ce ta. 00.TeHP ope nyeast in complex HOPS The 2010). al., et ¨cker Chlamydomonas oee,orrslsidct htthe that indicate results our However, . ce ta. 00 hn tal., et Zhang 2010; al., et ¨cker ce ta. 00;two 2010); al., et ¨cker eoe Characterization genome. SEC6-GFP ce ta. 2010). al., et ¨cker Chlamydomonas Chlamydomonas construct lsammrn n Vmmrn n oa erto in secretion polar and membrane CV the between and zones contact membrane close in the plasma of exocyst new formation these the that for and plausible required systems seems other in it the complex Given findings, exocyst 2012). the al., thus of et SEC9, (Morgera function from proceed SEC1 to exocytosis releases complex allowing SNARE SEC6 the exocystosis. t- of for the formation required to the inhibiting binds SEC9, proteins)] SNARE sec1/Munc18-like membrane (plasma olngriainadgot,idctn ao oei polar in role major a indicating growth, homozygous and produce germination pollen to fail expression (Ha progenies SEC6 disrupt and that 1992), al., lines et (Potenza yeast in Chlamydomonas ta. 02.SC teyatpam ebaeS protein SM membrane plasma yeast [the (Morgera SEC1 SEC1 2012). with interaction al., processes by et exocytosis cellular regulates SEC6 different two exocytosis). and of fusion vacuolar dysfunction (homotypic Osmo75 of a phenotype the that indicates idea the the supporting phenotype, of CV role a Dictyostelium to the in to to pointing function shown contrast CV complex, been in exocyst complex have exocyst and the exocytosis with during in interact involved docking are mutant vesicle which proteins), in (SM secA SecA proteins 2010). Munc18 al., a mammalian et (Zanchi CV the that the function. from is derived reported be CV to shown in al. was complex) discoideum et exocyst Dictyostelium Zanchi the probably Recently, two (and these SEC6 between behavior in clear. difference not currently the is systems for length. flagellar reason the change The not did of SEC6–GFP deletion SEC10 of contrast, overexpression By of cilia. in deletion primary elongated SEC6 and overexpression to whereas led 2004) 2009), SEC10 MDCK al., of al., in et cilia (Zuo et ciliogenesis primary the abolishes (Rogers of of base al., cells et the requirement Zuo epithelial to 2011; the localizes Guo to and Exocyst (Das contrast 2009). animals in striking ciliogenesis in in exocyst longer is form to This able are cells flagella. the as impaired, completely not in seems However, plants. in secretion neetnl,i eetsuyMreae l hwdthat showed al. et Morgera study recent a in Interestingly, of involvement an of report first the is this knowledge our To Dictyostelium Chlamydomonas ´ ae l,20) h atri asdb eet in defects by caused is latter The 2008). al., et la ed nyt nelre Vadntt multiple a to not and CV enlarged an to only leads SEC6 GFP xlie ntergt h eti eewsue as used was length: gene Expected centrin control. are The loading and right. used the templates on different explained Lane the top. to the at refer indicated genes numbers are The used primers panels). the lower by (two targeted 10 Osmo75 and of panel) strains different (upper rescue of Osmo75 media and different strengths, four osmotic in strain parental the Chlamydomonas of Expression 7. Fig. sval,weesSC sesnilfrgrowth for essential is SEC6 whereas viable, is 2 p eti,9 bp. 94 centrin, bp; 222 , eeini so5 the Osmo75, in deletion ooou fteyatSC n the and SEC1 yeast the of homologue eeoe ag aul,which vacuole, large a developed asdeogtdfael,whereas flagella, elongated caused strains. Chlamydomonas Arabidopsis SEC6 Dictyostelium TPRwspromdusing performed was RT-PCR nvarious in Chlamydomonas SEC6 SecA -N insertion T-DNA oa secretion polar 2 bp; 126 , oee,in However, . uainin mutation SEC6- is Journal of Cell Science elsoni ,telf Va i isoeadtergtC nerydatl.Tecnatznsse opritutlteedo h ytlcpae( arro (G phase systolic the of end the until persist the to in seem visible zones are contact CVs The Two diastole. H). early in in enlarged CV and right arrows, the (E, and membrane diastole plasma mid I). the at with in CV zones left enlarged contact the forms F, CV in the shown diastole cell of end the At Osmo75-SEC6GFP-G6. ( ece tan so5SCGPA oH ( H7 (* to significantly A5 Osmo75-SEC6GFP strains rescued infcnl ifrn Vprosfo hto C35(* CC3395 of that from periods CV different significantly h olwn tan eeue nti study: this in mt1 used cwd were strains following The cultures Cell Methods and Materials yeast. in block observed similar block a releasing SEC1 by the cycle CV as expulsion the water in for efficiently required proceed to be might SEC6 biogenesis. flagellar TAP mosM, (204 isotonic to TAP/2) mosM, (32 hypotonic strong from ranging strengths osmotic different with plates agar on ( listed) as Osmo75-SEC6GFP. H7, strains to rescued (A5 the SEC6GFP of Characterization 8. Fig. n B 5 oprsno h Vpro fC39 ( CC3395 of period CV the of Comparison ) 0.Teefu falrsudsri ssgiiatyhge hnteefu fC39 (*** CC3395 of efflux the than higher significantly is strain rescued all of efflux The 20). Sioaaae l,19)adUM (cwd UVM4 and 1998) al., et (Shimogawara ) P # .5 *** 0.05, P # .0) ( 0.001). D h Vefu eaiet h Vsraeae fC39 ( CC3395 of area surface CV the to relative efflux CV The ) Chlamydomonas n n 5 5 5 ihteC eid ftetnrsudsrisOm7-E6F 5t 7( H7 to A5 Osmo75-SEC6GFP strains rescued ten the of periods CV the with 45) 0.Ol w ece tan,C1adD0 aesmlrC oue oC39,teohr l differ all others the CC3395, to volumes CV similar have D10, and C11 strains, rescued two Only 20). mt+ r7 Nuete al., et (Neupert arg7) P # C39 ( 3395 CC .5.( 0.05). C oprsno h Vvlm eaiet h elsraeae fC39 ( CC3395 of area surface cell the to relative volume CV the of Comparison ) arg7-8 ( A h rwho C35 so5adtnrnol eetdrsudsrisOsmo75- strains rescued selected randomly ten and Osmo75 CC3395, of growth The ) l rnfrat eeawy etudrslcinpesr yadto of weeks. addition 6 every by least at pressure media new selection a into under using transferred and kept determined medium the always was to Germany). antibiotics were and media Berlin, TAP-S Gonotec, transformants all for 010, added (Osmomat All of was osmometer sucrose depression osmolarity point mM either freezing The 120 or was respectively. mM medium 60 Niederelbert, TAP-SS, with GmbH, or the the LED TAP/2), supplemented Electron 1965), osmolarities (for Thermo were X-CAD, Germany) Levine, lines different (TKA dest. cell and aqua achieve with mutant (Gorman diluted To derived medium arginine. all TAP and additional in CC3395 for cultured medium were Cells 2009). otatl aul of vacuole Contractile P # .0) ( 0.001). n 5 5 n h ece tan so5SCGPA oH7 to A5 Osmo75-SEC6GFP strains rescued the and 45) E–I lcrnmcorpso n ece strain, rescued one of micrographs Electron ) Chlamydomonas n 5 0.Ol 3adF have F6 and E3 Only 20). n 5 5 n the and 45) s and ws, 2893 -SS). Journal of Cell Science Nuete l,20)rsligi Cre in resulting the 2009) into ligated al., and et PCR, (Neupert by sequences coding dark the in TAP-S in hours 10 16 containing by plates followed TAP-S TAP in on hours plated 2 and for recover to allowed the with transformed ih irsoywspromda ecie yBcmn n ekr except Becker, and Buchmann by 7 described used as we that performed was microscopy Neubauer Light a microscopy Light using counted was concentration culture a to a medium TAP in with cells diluted 0.37 then of of were Cells number hematocytometer. The assay. light reduced hematocytometer. under Neubauer cultured a where using curves growth of (20–30 determination the for Cells media different in Growth Erlangen, (Peqlab, cDNA Strand kit 1 First using RNA Aid Revert (Fermentas) Plant the kit with peqGOLD Synthesis performed the was synthesis using cDNA isolated Germany). was RNA with Total transformed sucrose. additional were RT-PCR without medium cells TAP solid Osmo75 on cells phenotype, the plating before mutant medium the of Cre rescue For (supplementary rescue Mutant presented) are S3). S2, maps S1, Figs vector S1; and Table material sequences (primer, constructs Subsequently, GC-rich WI). for Madison, Cre (Promega, enhancer of cDNA an Cre complete with The and 2006). combination al., in et reactions (Ralser PCR used templates all First was In Aid Canada). (Fermentas) Revert Burlington, the DreamTaq (Fermentas, with following Kit Synthesis OH) synthesized cDNA Cincinnati, was Strand (MRC, cDNA REAGENT instructions. TRI manufacturer’s using the isolated was RNA Total constructs fusion GFP of protocol RESDA-PCR the using determined Gonza were regions flanking insertion site The insertion the screening of the Determination and selected were triplicate. TAP-S in with performed a showed compared was that TAP process lines Cell in in medium. growth TAP containing, or different TAP-S plates either 96-well containing plates into microtiter 200 transferred and well, picked each were clones Individual mutants osmoregulatory for (see Screening microscope fluorescence a Transformed using onto g/ml). fluorescence 10 plated GFP and MO; for media below). Louis, screened appropriate St. were the (Sigma, Cre cells in paromomycin the recovered with containing were Osmo75 plates cells and construct UVM4 of fusion transformation After Germany). (Kindle, mutagenesis method insertional bead glass the Kindle’s For using performed 1990). were transformations All of Transformation 21 at cultured were Cells 2894 ltswt ifrn soi tegh(ihu niitc) ntilct.Clswere Cells days. triplicate. in 21 antibiotics), for (without grown strength osmotic different with plates otaeIc,L ol,C) loecnemcocp a efre sn a using performed was microscopy Fluorescence CA). (GraphPad Jolla, software 5 5% La Prism (Student’s with GraphPad Inc., the tests fixed using Software significance were done were cells and correction) length, analysis Welch period flagella with regression and the size Linear the as measure iodine. using calculated To calculated Lugol’s were investigated. was cells cell cell and per the CVs rate of the efflux were of The cycles volume spheroid. three and prolate least a surface At The seconds. CV. 0.5 per every analyzed taken were images microscopy video are S1). (http://www. (primers Table templates quantprime cDNA material using GC-rich for supplementary designed in for specific were listed enhancer were primers an and The with 2006). biomedcentral.com/1471-2105/9/465) al., combination et in (Ralser used was (Fermentas) (Gonza al. et Matsuo by developed orlgtdr yl.I l xeiet -a-l utrs( cultures 5-day-old experiments all In cycle. light:dark hour ihacl est f10 of density cell a with rwho ei fdfeetomtcsrntswsas nlzduigaplate a using analyzed also was strengths osmotic different of media on Growth SEC6-GFP. ´ e-alse ta.adseii rmr o the for primers specific and al. et les-Ballester 6 m MIP1 10 Mol/m 6 ora fCl cec 2 (12) 125 Science Cell of Journal el/l h iue elsseso (3 suspension cell diluted The cells/ml. m Ce2g430 a mlfe n lndit pGEM-T-easy into cloned and amplified was (Cre12.g549300) 2 m elsseso nec ld Bcmn n ekr 09.For 2009). Becker, and (Buchmann slide each on suspension cell l scn) w ilgclrpiae eecutdfu ie each times four counted were replicates biological Two /second). fe rnfrainclswr loe orcvri TAP in recover to allowed were cells transformation After A-.Atr23wesaiut eetaserdit new into transferred were aliquots weeks 2–3 After TAP-S. l Chlamydomonas Hin 6 IIcset fpy3(etode l,20) el were Cells 2002). al., et (Berthold pHyg3 of cassette dIII ˚ –10 ihapoo lxo 70 of flux photon a with C 7 cells/ml. ´ m e-alse ta. 05 asoe l,2008). al., et Matsuo 2005; al., et les-Ballester oa N.I h C ecin DreamTaq reactions PCR the In RNA. total g cells Nde SEC6-GFP m Chlamydomonas /lhgoyi Rt,Karlsruhe, (Roth, B hygromycin g/ml etito ie eeaddt the to added were sites restriction I Nde m Hin n Cre and m etito ieo pJR38 of site restriction I mol/m )wsdopdot agar onto dropped was l) IIfamn fpHyg3 of fragment dIII SEC6 C35clswere cells CC3395 2 6 MIP1-GFP n 4hour:10 14 a and s a)wr used were day) 1 (Cre20.g759900) MIP1-GFP fusion t -test nebr,H . ailo,J A J. Danielson, I., H. Anderberg, UieslIaig op,BdodHls NY). Hills, 6.3r4 version Bedford The software, Corp., imaging MI). Imaging, Metamorph Heights, a with (Universal Sterling analyzed and Instruments, were 535/50) videos (Diagnostic 505; and camera (480/40; images digital set filter CCD GFP RT a Spot NY), Rochester, Du Associates, (Osram, (Vincent Du lamp short GmbH, mercury a (Nikon with 800 Eclipse Nikon el eecnetae ycnrfgn t500 at centrifuging by concentrated were Cells microscopy Electron Bro A. Hickisch, and B. Becker, Y. Naitoh, and D. R. Allen, D. R. Allen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.099184/-/DC1 online available material Supplementary g18013.t1; new gene ID: new (DUF789), annotation, gene No g18011.t1. function Cre20.g760000.t1 ID: and new unknown g18012.t1, ID: of Sec6, gene Protein component Cre20.g759900.t1 Cre20.g759950.t1 g18014.t1; complex ID: gene Cre20.g759850.t1Exocyst new g1801t.t1; related, SpoU ID: protein gene Cre20.g759800.t1 CGI-12 new g18016.t1; family, methylase ID: rRNA gene Phytoene new Cre20.g759750.t1 follow: therefore as dehydrogenase; and changed 17 been the chromosome have release IDs to gene new mapped all the been In has the 2012). 20 of scaffold June former release 8 (v5.3, new available a became paper genome this of acceptance After proof in added Note Forschungsgemeinschaft B.B.]. Deutsche to the Be1779/12-1 by number [grant supported was work This Funding mutant. Osmo75 the Alteko characterize A.-K. to students: Benstein, following helped K.-F. M. the R. thank Germany), USA) Langenbach, we MA, (Golm, addition, D. (Worcester, In Schroda Brown discussions. J. helpful M. and for USA) and GA, pHyg3 Germany) (Athens, (Regensburg, Lechtreck plasmid Mages plasmid W. the providing and strain, for for UVM4 Germany) the (Golm, and Bock pJR38 R. thank authors The Acknowledgements n meddi pn82 lrti etos(0n)wr u ihaLeika a with 45 cut (diatome, were knife nm) diamante (60 a sections and Ultrathin UC7 812. EM Epon microtome in embedded and etod . cmt,R n ae,W. Mages, and R. Schmitt, P., Berthold, fteples h elpleswr nuae vrih na1 qeu uranyl aqueous 1% a in overnight removal incubated before were 4 solution ice pellets at on glutaraldehyde solution cell minutes with acetate The 30 overlaid pellets. for incubated the and were of minutes) Samples medium) 15 medium). (500 in in pelleted for (2.5% (30% Germany), temperature solution Marburg, BSA room (Plano, in capsules at BEEM resuspended the into were during transferred Cells handling linked and cross follows. the easier were as fixation allow centrifugation BSA After by To obtained with ice. medium. pellets on cell fresh the minutes with procedure, 30 dehydration once additional fixation washed the of were and minute cells first aqueous temperature The room and 1%). at and glutaraldehyde was 1.25% final concentration sucrose with mM (final of (3 simultaneously tetroxide HEPES amount osmium fixation additional appropriate and before strength, an osmotic with concentration) respective the (HSM) reach medium to salt added high in resuspended laatn A.Iae eeaaye ihDgtlMcorp n Adobe and Gatan, Micrograph 1; Digital SC200W 1963). with (Orius analyzed (Reynolds, camera were citrate CS4. digital Photoshop Images lead a CA). and and Phillips, Pleasanton, 10, Netherlands) (CM acetate microscope The electron uranyl transmission Eindhoven, a with aqueous taken were 2% Micrographs with stained iest n osre motifs. conserved and diversity 1035-1042. ehrn opee n hi oei ebaefusion. membrane in role their and complexes tethering reinhardtii. Chlamydomonas 7 aph hygroscopicus A. brefeldin protozoa. ce,C,Engelbrecht-Vandre C., ¨cker, n.Rv Cytol. Rev. Int. 20) h otatl aul n t ebaedynamics. membrane its and vacuole contractile The (2000). ln elPhysiol. Cell Plant ˚ .Smlswr ahdaddhdae na tao series ethanol an in dehydrated and washed were Samples C. 0 eemdae oiatrssac gis yrmcnBin B hygromycin against resistance dominant mediates gene 215 Protist 20) niiino otatl aul ucinby function vacuole contractile of Inhibition (2005). 351-394. , 20) soeuainadcnrcievcoe of vacuoles contractile and Osmoregulation (2002). M vl Biol. Evol. BMC 46 ,S n nemn,C. Ungermann, and S. ´, ˚ n oasn U. Johanson, and . 201-212. , 153 sedr,Gray,Uilt hte control shutter Uniblitz Germany), ¨sseldorf, sedr,Gray irsoeequipped microscope Germany) ¨sseldorf, 401-412. , 20) negnee Streptomyces engineered An (2002). 11 g 110. , t20 at se n .Kh,who Kehl, K. and ¨ster ur Biol. Curr. 21) la Is high MIPs, Algal (2011). ˚ ˚ o 5mntsand minutes 15 for C nl) etoswere Sections angle). 21) Multisubunit (2010). Chlamydomonas 20 R943-R952. , Bioessays 22 g , , Journal of Cell Science ide .L. K. Kindle, un,K,Dee,D . icel . aor .J,Wta,G .and B. G. Witman, J., G. Pazour, A., Mitchell, R., D. Diener, K., Huang, K. M. E. Hoffmann, Clarke, and Q. Zhu, J., Heuser, ars . ohd,K,Crel,J n uh J. Bush, and J. Cardelli, K., Yoshida, E., Harris, Ha D., Rokhsar, D., Martinez, A., P. Lefebvre, C., Hauser, E., E. Harris, R., A. Grossman, P. R. Levine, and S. D. Gorman, otlet,A,Rhof .adDcmo R. J., Docampo, and S. P. Karpowicz, Rohloff, H., A., E. Montalvetti, Harris, O., Vallon, E., S. Prochnik, S., S. Merchant, A. J. McKanna, ozlzBletr . otka,W,Ms . ag .Q,Ctlnti C., Catalanotti, Q., W. Yang, F., Mus, W., Pootakham, D., Gonzalez-Ballester, ogr,F,Sla,M . uue .L,Gnh,P,Bee,D . ar .M. C. Carr, N., D. Brewer, P., Gandhi, L., M. Dubuke, R., M. Sallah, F., Morgera, M. Ishiura, and K. Shimogawara, Y., Niwa, K., Onai, K., Okamoto, T., Matsuo, Gonza omno,V . ak,J . olo,R .R n il,M C. M. Field, and R. M. R. Coulson, B., J. Dacks, L., V. Koumandou, B. Becker, and K. Komsic-Buchmann, uk,P,Hpert,M n oisn .G. D. Robinson, and M. Hoppenrath, P., Luykx, uran . etl .adHgmn,P. Hegemann, and W. Oertel, M., Fuhrmann, uk,P,Hpert,M n oisn .G. D. Robinson, and M. Hoppenrath, P., Luykx, and K. Lutter, K., Nuehse, C., Danzer, T., Wassmer, C., Schilde, R., Kissmehl, o,A . aaci . siaa . iaa .S,Ihd,M n le,R D. R. Allen, and M. Ishida, S., M. Aihara, A., Ishihara, K., Yamauchi, K., A. Fok, uh . ot,K,RdiuzPrs . afan . ’alrn . ucti T., Ruscetti, T., O’Halloran, N., Kaufmann, J., Rodriguez-Paris, K., Nolta, J., Bush, B. Becker, and K. Buchmann, a,A n u,W. Guo, and A. Das, M. J. Coll, l,M,Cl,R,Snk . Drdova L., Synek, R., Cole, M., ´la, enadi flagella. reinhardtii L. J. 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Higuera, Reverse A., Montaigu, de L., Magneschi, h MpoenSec1. protein the SM and the M. acidocalcisomes Munson, to and pyrophosphatase cruzi. proton Trypanosoma of vacuolar complex vacuole the contractile with localizes the of components identified analysis Protozool. genetic forward system. circadian systematic Chlamydomonas A (2008). etito nyest-ietdapiiainPR olt dniyregions identify to tool a PCR: DNA. amplification marker a site-directed flanking enzyme Restriction otatl aulsin vacuoles contractile Gruyter. de Walter Berlin: In Katsaros). function. and structure re loecn rti GP savraierpre nClmdmnsreinhardtii. Chlamydomonas in reporter versatile J. a Plant is (GFP) protein fluorescent green htafc h ucino h otatl aul in vacuole contractile the tethering of of function Protoplasma evolution the affect eukaryote; that ancestral the in proteins. SM fusion and complexes membrane for systems Paramecium. in pathways trafficking different the to assignment H. 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