ivniSeao uin en n eeiaBrandizzi* Federica and Renna Luciana Stefano, Giovanni expansion streaming cell organelle during over control exerts reticulum endoplasmic The REPORT SHORT ß eevd5Ags 03 cetd9Dcme 2013 December 9 Accepted 2013; August 5 Received ([email protected]) correspondence for *Author MI Lansing, East University, State USA. Michigan 48824, Lab, Research Plant MSU-DOE organelles cellular other the bearing a of has positioning movement and ER dynamics the pattern the that streaming Proving on the similar membrane 2010). a and al., on large ER et based (Ueda the suggested, a of been streaming a has with Indeed cytosol the These cytosol. organelles between the relationship in that additional embedded plant causative 2010). organelles possible in other that stir movement al., is of extension underscore organelle motors It to et primary also cells. contribute as 2009; forces Ueda but myosins al., unidentified for streaming 2008; et not role cytoplasmic (Avisar a but al., define streaming reduction observations et organelle single substantial of of Prokhnevsky cause loss loss have endoplasmic as myosins, well complete mitochondria the as multiple and inhibitors, of and myosin peroxisomes streaming that Golgi, demonstrated the (ER), of reticulum analyses microscopy Nebenfu 2010; Goldstein, Mathur and and Brandizzi Verchot-Lubicz 2008; Vick 2009b; 2002a; al., al., Nebenfu et Sparkes al., et 2004; 2002; et Esseling-Ozdoba al., actin In (Brandizzi 2013; et on established. depend motors Wasteneys, well to as and believed is myosins is proteins movement with motor organelle as cells with eukaryotic motility plant kinesin organelle of and least microtubule-based environment yet cells dynein and crowded as animal crucial In the to most cells. of referred the feature of commonly one understood organelles, is of streaming, cytoplasmic movement rapid The INTRODUCTION streaming, Cytoplasmic ER, WORDS: KEY ER the by cell cytoskeleton. co-exerted the forces during motor and on that depends changes it that demonstrate profound and expansion we undergoes streaming, streaming and live-cell cytoplasmic structure quantitative ER the Through compromised cytoplasmic of wild-type for expansion. role in force a cell analyses propelling tested during as we streaming (ER) Here reticulum movement. endoplasmic yet-unknown organelle and and of chemical for existence the to homeostasis motors In supports due myosins unknown. streaming of cell largely ablation cytoplasmic genetic are of for forces loss driving partial crucial plants, precise the is but expansion streaming Cytoplasmic ABSTRACT 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,9793doi:10.1242/jcs.139907 947–953 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. h,21) oehls,qatttv confocal quantitative Nonetheless, 2012). ¨hr, rbdpi thaliana Arabidopsis h ta. 99 hme n Yokota, and Shimmen 1999; al., et ¨hr rbdpi thaliana Arabidopsis el n uat with mutants and cells hte h oeetpteno ln raelsi uniform is 2008). organelles unknown al., yet plant et is (Prokhnevsky 2010; of it growth al., pattern cell 2010), during al., movement et et been the (Peremyslov Ueda have whether 2008; cells al., plants expanded et in Prokhnevsky al., fully et analyses on However, (Peremyslov 2013). streaming conducted al., growth et cytoplasmic Tominaga cell 2010; because al., plant et for Peremyslov in 2008; crucial defects is expansion streaming Cell cells. thaliana plant in plants. patterns in motility are intracellular cytoskeleton, unique actin–myosin the of the nature for to the addition responsible of in puzzle that, the solve motors to the piece missing a identify would obndfre xre ytectseeo n h ER. the and cytoskeleton the by exerted the forces on depends combined streaming organelle expansion cell plant support during experimental that offering by ER streaming cytoplasmic existing for other the aberrant model into the perspectives of new that provide motility analyses the Our show organelles. affect negatively we streaming and ER structure RHD3, conserved in the defective dynamin-like in mutation cells streaming. a ER-shaping of to organelle because studies streaming a and and our architecture highlight expansion extending thus by cell and Furthermore, the between expand, of streaming parallel cells of the as as new forces well increase as propelling organelles, ER, the other as of dynamics and function that shape demonstrate the ER, We expansion. cell the large during streaming that cytoplasmic as was streaming hypothesis such organelle underlying organelles, Our efficient expansion. maintain cell to during cytoskeleton the with Rmrhlg nege hne uighpctlcell model hypocotyl broad a support during data these supplementary 1999), changes al., the 1A; et undergoes (Fig. that (Ridge observations expansion DAG morphology earlier 12 with Combined ER at 1). of strands was Movie presence material predominant and morphology a tubules ER with radically ER expansion, of organization the cell Such sheets during DAG 1A). changed perforated Fig. 3 (Col-0/ER; al., enlarged cisternae et at large by Zhang after that 2002; predominantly al., days found characterized et [3 (Qiu We DAG) early 2011). fully (12 and phases at DAG), 9 cell and cotyledons expanded (6 expanding of rapidly the (DAG)], in region germination cells marker epidermal lumen one-third analyzed ER and apical 2007), inert al., ER, the et the expressing (Nelson visualize ER-YK stably To 2011). seedlings al., used et phases we (Zhang expansion defined well which we been for are have so, cells do streaming pavement on To epidermal analyses and expansion. cotyledon microscopy cell confocal architecture live-cell during if quantitative ER performed the establish of to features constant aimed first expand We morphology cells in as changes dynamics substantial and undergoes architecture ER The DISCUSSION AND RESULTS eakbyltl skonaotctpamcstreaming cytoplasmic about known is little Remarkably nti ok eamdt dniytefre htcooperate that forces the identify to aimed we work, this In ysnmtnsspottehptei htcytoplasmic that hypothesis the support mutants myosin Arabidopsis 947

Journal of Cell Science HR REPORT SHORT rgesvl iil sclsudretrpdexpansion comparable rapid was 948 streaming ER underwent that verified cells only also were We as defects 1C). ER (Fig. the visible that found confocal progressively we an Interestingly, [ of live-cell of 1C]. occurrence ER-YK cells Fig. 2011), in the with tubules ER established ER transformed al., ER in unbranched and the et protein long of this (Chen analyses of microscopy reports with involvement Consistent expansion. the published cell during test streaming 2012) and al., to architecture et Stefano in us 2011; (RHD3) al., prompted et 3 (Chen DEFECTIVE organization network HAIR ER ROOT protein dynamin-like architecture ( expand. to significant cells addition as undergoes in streaming ER that Movie the evidence S1; expanded Fig. provide changes, fully material data in supplementary These and 1B; 1). DAG) Fig. 9 DAG; low and (12 (6 S1; a transition cells cells the by Fig. expanding through increased rapidly characterized material progressively to it was supplementary that found streaming but We 1) 1B; ER Movie Fig. 2010). employed the al., (Col-0/ER; DAG et velocity successfully 3 Ueda ER 2012; at the approaches al., that of et imaging velocity (Stefano maximal previously live-cell the measured using we expansion, cell changes profound undergo can network growth. ER cell the during of shape manner. a the RHD3-dependent and that A) an (Col-0/ER; in wild-type expansion in cell expansion to cell correlated of phases are different ER at 5 plant cells the bars: epidermal of cotyledon dynamics of ER and cortical architecture The 1. Fig. h eetyetbihdrl fteER-membrane-associated the of role established recently The during variations streaming ER of occurrence the for assay To m .()Mxmlsraigvlct fE nwl-ye(o-)and (Col-0) wild-type in ER of velocity streaming Maximal (B) m. rhd3-7 E Seaoe l,2012); al., et (Stefano /ER P , .0)icessin increases 0.001) rhd3 ulallele null rhd3-7 Rascae rtisc-xr oto fE temn scells as and streaming cytoskeletal ER that of expand. suggest control cell also co-exert during proteins streaming data and ER-associated ER our 2010) prevents 1B), al., RHD3 (Fig. et of (Ueda expansion loss streaming that ER for results in ER evidence XI-K our tubular published myosin to the of cisternal role of the that from light findings transition in the Furthermore, the support architecture. in data required These is 1). RHD3 Movie S1; Fig. material ( significantly was maximal streaming in is ER streaming type, ER wild where in cells expanded fully in however, and type wild between uatlreypritd(i.2,) hs eut sals that establish results RHD3 the These of 2A,B). defects (Fig. ER the persisted that largely depolymerization found mutant and actin ER 1998) al., through et the wild-type (Boevink cytoskeleton in a actin–myosin treated actin the of we (25 of this restoration B latrunculin test depletion in To then result phenotype. case, test would If cytoskeleton. the to background actin–myosin the aimed is in in defects organization we to this linked network 2003), is 1C) ER al., (Fig. compromised et the RHD3 (Hu an whether in seen mutant been have the loss-of-function organization from actin independent in is defects that Because integrity network cytoskeleton ER in actin role a has RHD3 oyeosepesn h Rlmnmre ER-YK. marker lumen ER the expressing cotyledons rhd3-7 ora fCl cec 21)17 4–5 doi:10.1242/jcs.139907 947–953 127, (2014) Science Cell of Journal oprdwt idtp el Fg B supplementary 1B; (Fig. cells wild-type with compared m o or,wihdsut h nert of integrity the disrupts which hour), 1 for M rhd3 rhd3-7 ncotmtn ( mutant knockout nerypae fcl expansion; cell of phases early in rhd3-7 AC ofcliae of images Confocal (A,C) rhd3-7 E-Kclswith cells /ER-YK P , E;C el.Scale cells. C) /ER; .0)reduced 0.001) rhd3 rhd3-7 null

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ER to actin due cortical the integrity of network absence ER the in Defects 2. Fig. of In loss 1B). the that (Fig. for evidence expansion responsible cell the components during of sole streaming light ER the that not us in be to do variations suggested not 1B) myosins may (Fig. ER other expand that myosins cells and with 2010), as al., changes combination et streaming (Ueda in streaming ER or abolish completely alone XI-K of rhd3-7 E-Kbcgon.Saebr 5 bar: Scale background. /ER-YK rhd3-7 ncotmtn oyeo epidermal cotyledon mutant knockout AB ofcliae fthe of images Confocal (A,B) rhd3 eue Rstreaming ER reduces m m. eue Rmmrn liiylne otels fRHD3 of thus and loss cytoskeleton. move, the to the from the ER independently the to streaming of that ER compromises linked ability overall speculate fluidity the affects We membrane negatively cytoskeleton. 2013). ER the cells reduced al., expanded the fully from in in diffusion independently et membrane flow ER membrane optimal (Lee maintain reduced on co- RTNLB13 effect rhd3 when the reticulon only of morphology RHD3 Independently with the ER that affects observation expressed 2 the the as RHD3-Like by for such supported isoform required properties, is balance ER-shaping This the other reticulons. compromises of RHD3 possible homeostasis also of functional is could loss It absence a phenotype. its flow that therefore membrane the bilayer; observed influences RHD3 the lipid region cause of this ER that role the possible of ER-shaping is organization It the 2012). for al., et necessary (Stefano is the domain that shown membrane-insertedhairpin a been encompasses has that It RHD3 of 2013). region al., C-terminal et morphology (Lee ER movement disrupt Golgi not and does overexpression mutants GTPase that 2 and RHD3-Like 2011) of al., et (Chen phenotypes 2 unlike vegetative that, RHD3-Like evidence the and by rhd3 supported RL1) is other This (At1g72960; RL2). the (At5g45160; 1 with RHD3-Like compared 2013) function al., isoforms et dominant Zhang 2012; an a al., is et with Stefano RHD3 2013; al., al., et membrane. et (Chen McNew ER ability 2011; fusogenic the membrane with of protein flow ER-shaping the affects specifically 2.Tedfeecsi ebaefo n h bec of absence between the content and lumen ER flow and of flow membrane wild-type the same in in the differences Fig. at differences significant material mutant supplementary The 3D,E; the (Figs S2). and expansion type cell of wild stages the between differences iia ntetobcgons(i.1) oee,i fully in than However, is type wild 1B). the streaming in the (Fig. higher ER is in backgrounds streaming ER the two where cells when expanded the S2), in Fig. similar material supplementary 0.56 is expanded B fully latrunculin in ER with the of velocity the streaming and type wild the between when rhd3 velocities 2C), streaming (Fig. DAG ER 12 in at type differences wild the of that with compared that ue akrGPHE Badzie l,20)i fully in expansion of 2003) and stages wild-type early al., the in both cells et in with compared (Brandizzi cells GFP–HDEL expanded a marker verified analyses lumen ( FRAP significant Conversely, but 3A,B). slight (Fig. 2012) the al., in et reduced greatly was CNX–GFP ebaemre ossigo F uint the to was fusion mobility protein GFP membrane (Irons that lectin between a established ER similar an 2003), of of al., ER tail et cytosolic an consisting and (CNX–GFP), domain transmembrane calnexin–GFP marker of membrane Analyses treatment. of B and lumen wild-type and photobleaching in after experiments membrane recovery (FRAP) of fluorescence performed flow we the ER, the ensure to role independent streaming. ER in RHD3 to role data a sole attribute the These and be motors streaming. streaming not ER may ER myosins that on hypothesis underlying motors the support acto-myosin and RHD3 of (0.85 (1.55 cells wild-type untreated in than ots hte H3cudas aeacytoskeleton- a have also could RHD3 whether test To elto fteeioom ln osntcueobvious cause not does alone isoforms these of depletion , rhd3 uat u eut niaeta H3i eesr to necessary is RHD3 that indicate results our mutant, uataemrel ifrn Fg B.Temaximal The 1B). (Fig. different markedly are mutant RHD3 ora fCl cec 21)17 4–5 doi:10.1242/jcs.139907 947–953 127, (2014) Science Cell of Journal osrsle nardcino Rsraigvelocity streaming ER of reduction a in resulted loss m /eod Fg B,cnitn iha diierole additive an with consistent 1B), (Fig. m/second) uat(i.1) efudta h oiiyof mobility the that found we 1B), (Fig. mutant rhd3 rhd3-7 uatclsidct htls fRHD3 of loss that indicate cells mutant P , n idtp el t3DG(i.3A; (Fig. DAG 3 at cells wild-type and rhd3 .0)ices ntefo fteER the of flow the in increase 0.001) m /eod(i.2) hc slower is which 2C), (Fig. m/second ncotcls u osignificant no but cells, knockout m /eod n untreated and m/second) rhd3 RHD3 el pnlatrunculin upon cells ncot(Stefano knockout rhd3 el treated cells Arabidopsis rhd3 949

Journal of Cell Science HR REPORT SHORT 950 ER The organelles. these al., propel et to place Ueda in are 2008; 2002a; forces al., cytoskeletal Nebenfu al., et and et Prokhnevsky Vick actin–myosin Brandizzi 2008; 2010; 2009; al., peroxisomes al., et the Golgi, et Peremyslov as (Avisar such of completely mitochondria organelles not and of do movement disruption myosins the of abolish ablation organelles chemical other genetic of and streaming cytoskeleton that the on influence 5 crucial bar: Evidence a Scale photobleaching. has spot ER of The sites indicate circles White DAG. 3 at D) (GFP-HDEL; marker membrane. lumen ER ER the in the availability of RHD3 A) by influenced (CNX–GFP; is calnexin–GFP fluidity membrane ER 3. Fig. BCEF nlsso RPeprmnssoigtehl ieadmbl rcino N–F BC n F–DL(,)i idtp and wild-type in (E,F) GFP–HDEL and (B,C) CNX–GFP of fraction mobile and time half DAG. the 12 showing and experiments FRAP of Analysis (B,C,E,F) h,21)idctsta diinlnon- additional that indicates 2012) ¨hr, uigcl xaso.W xetdta fteE temn is streaming ER the first if we that a expected so, We has do mitochondria expansion. and To network peroxisomes cell organelles. ER Golgi, during other of the movement of whether the motility analyzed test cytoskeleton the the to on organelles on us bearing depend other prompted entirely the 2C), not membrane (Fig. to does above, streaming largest proximity described ER evidence the close The that S3). with in Fig. is material organelles (supplementary it the and of extension one arguably is AD RPeprmn nclsepesn h Rmmrn marker membrane ER the expressing cells in experiment FRAP (A,D) ora fCl cec 21)17 4–5 doi:10.1242/jcs.139907 947–953 127, (2014) Science Cell of Journal rhd3-7 m el t3 at cells m.

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Journal of Cell Science oetbihteifuneo Ro rael temn,we streaming, organelle on ER by of abolished influence during completely the not S4). increased establish Fig. was material To (supplementary peroxisomes but cytoskeleton actin 1B). 4) of and (Fig. disruption (Fig. expansion expansion mitochondria streaming cell the cell Golgi, that verified during we of hypothesis, observed our with accordance streaming In increase ER of the streaming parallel in then should mitochondria organelles, and other peroxisomes of Golgi, movement the in involved REPORT SHORT uattemvmn ftettesta onc h Rt the to ER the 952 connect that tethers the the in of that possible movement also the is mutant it the that of speculate membrane independently we lipid RHD3 ER cytoskeleton, of of or availability fluidity the the proteins on depends that between proteins evidence the contacts on Based on bilayers. membrane depend heterotypic contact that the through to organelles connections contributes can other ER the the ER that of possible movement is the (Mehrshahi it Therefore, domains that 2013). al., membrane et hypothesized hemifused yet been is through organelles chloroplasts has other proteins with the it ER of the unknown, al., of nature connections et the the Hawes Although enable 2007; 2009a). that al., al., et et that (Andersson Sparkes stacks tweezers 2010; Golgi the been or by chloroplasts have pulled follow are stacks tubules ER Golgi tweezers that optical and showing laser on chloroplasts Furthermore, based experiments to through 1997). demonstrated ER (Staehelin, in of observed analyses attachment other been microscopy have non- the plant sites electron In other either 2013). attachment of Voeltz, and ER–mitochondria and are (English ER motility cells, exist the that to the known between are interactions Connections organelles on The structured. through or ER be regulated organelles. the may non-ER by cytosol; organelles with the interfere exerted of would in influence ER organelles streaming the other of actin–myosin-driven the movement propel reduced components conversely, ER to membrane the ER contribute of as movement may effective well model as this cisternae In and fluidity. tubules and ER cytoskeleton of actin–myosin the movement on based forces depends cooperative streaming membrane on cytoplasmic ER that and therefore streaming propose We ER found and fluidity. compromises RHD3, also RHD3 of of absence We the loss peak. in that altered ER movement is the streaming protein cytoplasmic the when membrane that of ER cells streaming the and expanded and and ER appearance fully reticulated streaming in typical a of highest non- assumes of levels is streaming low organelles the by ER Conversely movement. characterized and of protein is enlarged membrane is ER We membrane component an ER the at cisternae. the cells when a when in and expansion lowest of is tubules include phase organelles early non-ER ER of also streaming the that found analyses protein of ER movement the feature translational work although this in Mathur flow streaming 1998; ER al., of Nebenfu measurements et 2002; (Boevink al., on et cytoskeleton depends largely actin–myosin of mitochondria streaming the and the plants, peroxisomes land stacks, In Golgi organelles. other the ER. of the streaming streaming of ensure streaming to cells the necessary besides vegetative is organelles of RHD3 other of growth of presence the the during that constant and organelle in general not role that stages major is support organelle a data later streaming These has at 1B). 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(SALK_106309) al., et (Stefano aitosi Rmmrn ifso ol ntr affect turn in would Therefore, diffusion compromised. organelles. membrane other the is of ER motility organelles in other variations of membranes h og ebaemre TGP hc sbsdo F uinto signal fusion HDEL; GFP the a tetrapeptide on to the based fused by is which ER YFP ER-YK ST–GFP, the of marker marker in membrane composed lumenal Golgi retained the is and ER AtWAK2 which the of 2007), were: peptide study al., EYFP. and this et mCherry in (Nelson 1997), al., used et markers (Haseloff proteins GFP5 The were fluorescent work The this analyses. (http://www. in confocal used 1000 for META used FluoView were LSM510 Olympus olympus.com) Zeiss an microscope and confocal (http://www.zeiss.com/) scanning laser inverted An microscopy scanning laser Confocal agar. w/v 0.8% and B5 antibiotics appropriate Gamborg’s Gom8 the with sucrose, the supplemented w/v on or 1% medium selection vitamins, (MS) subsequent crosses Skoog and floral 1998), and Bent, either Murashige and through (Clough method obtained floral-dip were ER-YK expressing transgenic conditions Homozygous growth and materials Plant METHODS AND MATERIALS el a mgd oyeo el t3ad1 A eeicbtdin incubated were DAG 12 and pavement 3 epidermal at cotyledon cells abaxial Cotyledon of imaged. region was cortical cells the in ER The analyses FRAP a at frames Time-lapse recorded. were (3.0–5.0 region cells cortical Golgi 512 the the pavement of in 90 velocity sequence with cotyledon the sequences each analyze time-lapse for To 30 frames 2007). mitochondria, and al., peroxisomes et Faso stacks, Hanton 2002b; or 2009; al., fluorochromes et al., single (Brandizzi et for previously Ser, settings described as Imaging tetrapeptide were YFP. the combinations to signal, fused 1 is Leu, and type is Lys which which peroxisome-targeting YFP–PTS1, a marker MT-RB, cytochrome peroxisome of the of composed marker and acids mCherry; mitochondria amino to transferase fused 29 yeast the sialyl first rat the 1998); a of composed al., of tail et cytosolic (Boevink and domain transmembrane the conditions. ae oe ie 0 fa ro 8 mlsrln)t avoid to line) laser nm 488 argon an of 10% 3 and (i.e. photobleaching, power laser e eunei h otclE (3.0–5.0 ER cortical the in each sequence of per values maximal sample. averaging each by for sequence estimated the Maximal time-lapse sequences. as were time-lapse 512 calculated 50 values 90 the were in velocity the values estimated in by velocities then the stacks sequence; of calculated mean Golgi time-lapse were the each all in values frames of Velocity velocity Cybernetics). the averaging (Media Image using 6 post-acquisition calculated Pro-Plus was Velocity objective. 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Journal of Cell Science atn .L,Car,L,Rna . ahsn .A n rniz,F. Brandizzi, and A. L. Matheson, L., Renna, L., Chatre, L., S. Hanton, ao . hn .N,Tmr,K,Hl,M,Zmls . at,L,Saravanan, L., Marti, S., Zemelis, M., Held, K., Tamura, N., Y. Chen, C., Faso, seigOdb,A,Humn . a amrn .A,Esr .and E. Eiser, A., A. Lammeren, Van D., Houtman, A., Esseling-Ozdoba, F. A. Bent, and J. S. Clough, H. Zheng, and F. Brandizzi, G., Stefano, J., Chen, K., Oparka, D., Evans, P., Boevink, L., L. DaSilva, S., Hanton, F., Brandizzi, Hawes, and J. Lippincott-Schwartz, G., A. Roberts, L., E. Snapp, F., Brandizzi, C. Hawes, and M. Fricker, F., Brandizzi, O. Wasteneys, G. and F. Brandizzi, Hawes, and A. Betteridge, B., Martin, S., Cruz, Santa K., Oparka, P., Boevink, Sparkes, and C. Hawes, E., Sadot, E., Belausov, M., Abu-Abied, D., Avisar, nls,A .adVet,G K. G. Voeltz, and R. A. English, Gokso X., M. Andersson, arnui (25 B latrunculin REPORT SHORT aeof . imrn,K . rse,D .adHde S. Hodge, and C. D. Prasher, R., K. Siemering, J., Haseloff, P., Shinn, H., Chen, J., C. Kim, J., T. Leisse, N., A. Stepanova, M., J. Alonso, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139907/-/DC1 online available material Supplementary material Supplementary and F.B.]. (Molecular to Foundation 1243792 Science number National [grant the Biosciences) by Cellular supported was study This Funding the wrote and experiments the designed manuscript. conceived, F.B. and L.R. G.S., contributions Author interests. competing no declare authors The interests Competing A)tenme fbecigeet a 6 2 5ad63, and 75 software 62, 5 2010). 46, Prism al., et was using (Stefano previously plotted events described as were bleaching Software), data (GraphPad of Normalized and number respectively. DAG) the (12 CNX–GFP DAG), DAG) Col-0 (3 DAG), CNX–GFP DAG), (3 Col-0 (3 and GFP For GFP–HDEL DAG) respectively. 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