a b c Plasma mem. Cytoplasm Total

Transcription

1 DOI: /nc1886 c Plsm mem. Cytoplsm Totl 0 µm -0.3 to -1.8 µm 0 to -1.8 µm Rel. Signl Density Time (h) Control Isoxen Figure S1 Isoxen cuses loss of YFP::CESA6 signl from the plsm memrne. Seedlings expressing YFP::CESA6 were treted with 0.01% DMSO (control) or 100 nm isoxen in 0.01% DMSO. Z-series of 10 opticl plnes, spced 0.3 µm prt, were cquired. Propidium iodide ws used to stin the cell wll, providing fiducil mrker for the strt of the z-series. Bsed on exmintion of mny imges, we determined tht the outer three plnes (z = +0.3 to +0.9 µm) represented the extrcellulr spce nd the fourth (z = 0 µm) represented the plsm memrne. The next six plnes (z = -0.3 to -1.8 µm) smpled the cellulr spce interior to the plsm memrne. Reltive YFP::CESA6 signl densities were mesured in ckground-sutrcted imges representing the plsm memrne (), cytoplsm () nd totl (c). Ech dt point represents signl density (reltive intensity per unit volume) from t lest 3 cells from 3 unique plnts. Isoxen significntly decreses YFP::CESA6 signl t the plsm memrne () compred to control (P = 0.027, F-test for equlity of slopes of regression lines). Totl integrted signl does not chnge significntly (c), suggesting there is no net loss of CESA6. Since most of the totl CESA6 protein resides in cytoplsmic orgnelles, nd mesured vlues for cytoplsmic signl hve high vrince (), it is not possile to determine if the protein lost t the plsm memrne could e ccounted for in cytoplsmic pools. Mesurements in were corrected for out-offocus signl cused y Golgi odies nd smll comprtments. First, the plnes representing the cytoplsm were summed nd thresholded y eye. Structures lrger thn 12 squre pixels were identified using the Anlyze prticles tool in ImgeJ. Regions of the plsm memrne tht were directly ove these structures were eliminted from nlysis. Thus the reported intensities in primrily represent signl from CESA puncte in the plsm memrne. Error rs represent s.e.m. 1

2 Control Isoxen Mnnitol BFA AE F Time verge Still imge Time verge Still imge Figure S2 Effects of isoxen, mnnitol or BFA tretment on CESA6 re reversile. Seedlings expressing YFP::CESA6 were treted with 0.05% DMSO (control), 100 nm isoxen in 0.01% DMSO, 200 mm mnnitol, 50 µm BFA in 0.05% DMSO or 10 nm AE F in 0.01% DMSO for 3 h (). In ll non-control tretments, few puncte re visile in the plsm memrne, nd mny SmCCs re oserved in the cell cortex. The plnts were then incuted in wter for 5 h. After wshing, CESA6 complexes ccumulte in the plsm memrne nd re motile in ll tretments (). Oservtions were performed on three plnts per tretment. Representtive imges re shown. Wshout ws not performed for AE F treted plnts. Time-verged projections were mde from 61 imges collected over 5 min. Scle r, 5 µm. 2

3 BRI1::GFP Plsm mem µm Plsm mem µm YFP::NPSN12 BFA Mnnitol Isoxen Control Figure S3 Isoxen does not generlly ffect the locliztion of plsm memrne proteins. Seedlings expressing plsm memrne mrkers BRI1::GFP () or YFP::NPSN12 () were treted with 0.05% DMSO (control), 100 nm isoxen in 0.01% DMSO, 200 mm mnnitol or 50 µm BFA in 0.05% DMSO. Focl plnes t the plsm memrne nd lower cell cortex re shown. Isoxen does not noticely disrupt locliztion of either leled protein.mnnitol cuses some detectle internliztion of BRI1 signl ut does not induce plsmolysis t the concentrtion used. BFA cuses intrcellulr ccumultion of leled BRI1 nd NPSN12. Scle r, 5 µm. 3

4 YFP::CESA6 FM4-64 Merge 10 min 30 min Figure S4 FM4-64 dye is internlized into SmCCs slowly. YFP::CESA6 seedlings were light-grown for 3 dys nd incuted in 200 mm mnnitol nd 20 µm FM4-64 for 10 min () or 30 min (). Epiderml hypocotyl cells were imged. () At 10 min, the dye lels endosomes 1 (red rrowheds) tht re not coincident with corticlly tethered SmCCs (green rrowheds), which were identified sed on their dynmics. () After 30 min, the dye lels Golgi odies (circles) nd some SmCCs (yellow rrowheds), indicting tht it is possile to eventully lod enough dye into SmCC memrnes for positive detection. The fct tht dye is not detected in SmCCs t erlier time points, lthough the plsm memrne is well leled, suggests tht most oserved SmCCs re not directly formed y endocytosed plsm memrne. Scle r, 5 µm. 4

5 supplementry informtion YFP::CESA6 RFP::TUA5 Merge BFA Mnnitol CFP::CESA6 c 1.5 Isoxen Isx + Txol 5 min Velocity (µm/min) Isoxen Isx + Tx Figure S5 () SmCCs coloclize with corticl microtuules in cells treted with BFA or mnnitol. Seedlings expressing YFP::CESA6 nd mcherry::tua5 were treted with 200 mm mnnitol or 50 µm BFA for 3 h. SmCCs (rrowheds) show colocliztion with microtuules. Scle r, 5 µm. (, c) Stiliztion of corticl microtuules y txol lowers SmCC velocity under isoxen tretment. Seedlings expressing CFP::CESA6 nd YFP::TUA5 were treted with 100 nm isoxen in 0.11% DMSO or 100 nm isoxen + 20 µm txol in 0.11% DMSO for 2 h. Visuliztion of leled microtuules confirmed tht txol slowed down depolymeriztion of microtuules. () Representtive kymogrphs of SmCCs. (c) Velocities of SmCCs in the cell cortex. For ech tretment, n = 24 SmCCs from 4 cells (from 4 seedlings). Appliction of isoxen nd txol together reduces SmCC velocity more thn isoxen does lone (P < 0.001, two-level nested ANOVA). Error rs represent s.e.m. Scle r, 1 µm. 5

6 0 s c stedy movement s RFP::TUA5 Merge 0 60 s d 0 s e RFP::TUA5 Merge s Figure S6 Two dditionl exmples of CESA tip trcking under norml conditions. Seedlings expressing nd mcherry::tua5 were imged in the sence of drugs. (-c) First exmple. () Still imges t t = 0 showing (rrowhed) nd n ssocited microtuule. () Kymogrph long cyn trce in. A CESA prticle ppers in the focl plne of the plsm memrne nd ecomes sttionry. This sttic ehvior is interrupted y depolymerizing end. trcks with the end for severl frmes () nd then ecomes sttic gin. (c) Kymogrph long mgent trce in. At 78 s, the CESA prticle moves stedily. (d, e) Second exmple. (d) Still imges t t = 0 showing (rrowhed) nd n ssocited microtuule. (e) Kymogrph long cyn trce in d. As in, trcks on depolymerizing end (). Scle r, 1 µm. 6

7 s CESA3 Kymogrph RFP::TUA s s RFP::TUA5 CESA3 Kymogrph s Figure S7 Two dditionl exmples of CESA delivery events tht re ssocited with SmCCs. Seedlings were treted with 200 mm mnnitol for 3 h nd then wshed with wter for 0.5 h. Kymogrphs were tken long the cyn trce. Yellow rrows mrk ifurction on the kymogrphs. () First exmple. A SmCC (open rrowhed) trcks on depolymerizing end efore splitting into two structures of unequl intensity t 104 s. The righter prticle (open rrowhed) moves in mnner consistent with SmCC motility. The dimmer prticle (closed rrowhed) moves t slow nd stedy rte, consistent with CESA complex motility in the plsm memrne. () Second exmple. A similr event occurs in different cell. The splitting event occurs t round 146 s. Scle r, 1 µm. 7

8 Control Isoxen Mnnitol YFP::CESA6 [ [ YFP::CESA s YFP::CESA6 CFP::TUA1 Figure S8 Golgi odies ssocite with SmCCs. () YFP::CESA6 seedlings were treted with 0.01% DMSO (control), 100 nm isoxen in 0.01% DMSO or 200 mm mnnitol for 3 h. In the control, physicl ssocition etween Golgi odies (rcs) nd SmCCS (rrowheds) is frequently oserved. With isoxen or mnnitol tretment, this ssocition is more conspicuous: the two structures re sometimes oserved to e connected y detectle strnd of YFP::CESA6 signl (rcket), which my represent memrne ridge. () Assocition etween Golgi odies nd corticlly loclized SmCCs persists for up to severl minutes in the presence of cytoplsmic streming. Seedlings expressing YFP::CESA6 nd CFP::TUA1 were treted with 100 nm isoxen for 2 h. A short distnce (< 1.3 µm) is mintined etween Golgi ody nd moving SmCC for 120 s, suggesting the two re physiclly tethered. These oservtions suggest tht some SmCCs cn form tight ssocitions with Golgi odies, which might e the cse if these SmCCs re derived from them or interct closely with them to exchnge crgo. Scle r, 1 µm. See Movie

9 YFP::CESA6 RFP::SYP42 Merge c Mnnitol (Time Averge) Mnnitol Control Figure S9 SYP42 lels suset of SmCCs. Seedlings expressing YFP::CESA6 nd mcherry::syp42 were incuted in wter () or 200 mm mnnitol (, c) for 3 h nd imged t the cell cortex. () Some smll comprtments re leled y YFP only (green rrowheds) or oth YFP nd mcherry (yellow rrowheds). (, c) SYP42 fils to lel corticlly tethered SmCCs (green rrowheds) fter mnnitol tretment. (, ) Still imges. (c) Averges of 61 frmes representing 1 min. Scle r, 5 µm. 9

10 Mrker Puttive locliztion GFP::KOR1 KOR1-contining comprtments 2 YFP::RA4 TGN-like comprtments 3 YFP::RF2 Erly endocytic comprtments 3 YFP::RG3c Prevcuolr comprtment/vcuole 3 YFP::RF2 Endosome 4 YFP::RC1 Endosome 5 YFP::RA1g Endosome 5 YFP::RA5d TGN 5 YFP::RG3f Lte endosome/vcuole 5 Supplementry Tle 1 Visulized endomemrne mrkers. Aridopsis seedlings expressing different fluorescent fusion proteins were drk-grown for 3 dys, treted with 100 nm isoxen for 2 h, nd visulized. The lines were ssyed for the leling of smll comprtments tht ecome tethered to the cell cortex in response to isoxen. None of the lines were oserved to disply such ehvior; however, it is possile tht one or more of these mrkers lel suset of SmCCs. 10

11 Supplementry Movie Legends All movies were cquired in epiderml cells of the upper hypocotyl of Aridopsis seedlings tht were drk-grown for 3 dys (see Methods). Movie 1 Photoleching used to visulize delivery of GFP-leled CESA complexes to the plsm memrne. A cell expressing nd mcherry::tua5 ws imged in the plne of the plsm memrne. At t = 0 s, GFP is leched. During the recovery, severl CESA prticles (rrowheds) re delivered to plsm memrne. The mjority of delivery sites re coincident with corticl microtuules. A 2-frme running verge ws pplied to the time series. Scle r, 5 µm. Movie 2 Delivery of single CESA prticle to the plsm memrne. A cell expressing nd mcherry::map4-mbd ws imged in the plne of the plsm memrne. The CESA prticle (rrowhed) undergoes three stges of ehvior: errtic motility (t = -26 to 0 s), sttic locliztion (t = 0 to 44 s), nd stedy movement (t = 44 to 490 s). At the strt of the sttic phse, the prticle coloclizes with microtuule signl. A 2-frme running verge ws pplied to the time series. Scle r, 1 µm. Movie 3 Delivery of multiple CESA prticles t the sme time nd position. A cell expressing nd mcherry::map4-mbd ws imged in the plne of the plsm memrne. A CESA prticle (lrge rrowhed) rrives t the plsm memrne nd splits into two puncte (smll rrowheds) t t = 56 s. A 2-frme running verge ws pplied to the time series. Scle r, 1 µm. Movie 4 Dynmics of CESA6-contining orgnelles re ffected y isoxen, osmotic stress or BFA. Seedlings expressing YFP::CESA6 were treted with 0.05% DMSO ( nd e), 100 nm isoxen in 0.01% DMSO (), 50 µm BFA in 0.05% DMSO (c) or 200 mm mnnitol (d). Time series were cquired t the plsm memrne or lower cell cortex, s indicted. () In the controls, SmCCs (open rrowheds) nd Golgi odies (crosses) move rpidly through the cell cortex. These two structures re often in close proximity nd move together in coordinted fshion (red rrowhed nd cross). (-d) With the other tretments, orgnelles show reduced velocity nd ltered dynmics t the cortex. SmCCs, in prticulr, exhiit slttory movement long liner pths. (e) These structures re distinguished from ctive CESA complexes (closed rrowhed) in the plsm memrne, which move t slow nd stedy rtes long liner trjectories. A 3-frme running verge ws pplied to -e. Scle r, 5 µm. Movies 5-8 SmCCs re coincident with corticl microtuules. Seedlings expressing YFP::CESA6 nd mcherry::tua5 were treted with 0.05% DMSO (5), 100 nm isoxen in 0.01% DMSO (6), 200 mm mnnitol (7) or 50 µm BFA in 0.05% DMSO (8) for 3 h. (5) In the control, CESA complexes (closed rrowheds) in the plsm memrne move slowly long pths coincident with microtuules. (6-8) With the other tretments, CESA complexes re predominntly clered from the plsm memrne. The SmCCs (open rrowheds) ccumulte t the cortex, displying colocliztion with microtuules. The motility of these SmCCs is often oserved to e coincident with depolymerizing microtuule ends (dshes). A 3-frme running verge ws pplied to 5. Scle r, 5 µm. Movies 9-11 Corticlly tethered SmCCs move with depolymerizing ends of microtuules. Seedlings expressing mrkers for CESA (green) nd microtuules (red) were treted with 100 nm isoxen or 200 mm mnnitol. Microtuule-ound SmCCs (rrowheds) move in concert with depolymerizing ends (dshes). (9) A SmCC is initilly sttionry nd then moves long the microtuule when encountering depolymerizing end. (10) SmCCs trck with the depolymerizing plus nd minus ends of the sme microtuule. Two SmCCS on the left re collected y the depolymerizing end. The increse in CESA signl t tht end suggests oth SmCCs remin ssocited with the microtuule. (11) A SmCC moves with shrinking end in microtuule undle. Scle r, 1 µm. Movie 12 CESA exhiits tip-trcking ehvior under norml conditions. A cell expressing nd mcherry::tua5 ws imged in the plne of the plsm memrne. During delivery, prticle (rrowhed) moves with the depolymerizing end of microtuule (dsh). A 3-frme running verge ws pplied to the time series. Scle r, 1 µm. Movie 13 SmCCs re ssocited with CESA delivery to the plsm memrne. Seedlings were treted with 200 mm mnnitol for 3 h nd then wshed with wter for 0.5 h. A SmCC (open rrowhed) trcks depolymerizing end (dsh) for the first 136 s nd then jumps to n djcent microtuule. At pproximtely t = 308 s, the SmCC egins to split into two structures of unequl intensity. The righter structure (open rrowhed) leves the focl plne (t 348 s) in mnner consistent with SmCC motility. The dimmer prticle (closed rrowhed) moves t slow nd stedy rte, consistent with CESA complex motility in the plsm memrne. A 3-frme running verge ws pplied to the time series. Scle r, 1 µm. Movie 14 Successive rekup of one SmCC into multiple comprtments. Seedlings treted with 200 mm mnnitol for 3 h nd then wshed with wter for 0.5 h. A SmCC (rrowhed) splits into two comprtments, clerly visile t 116 s. The righter dughter SmCC then proceeds to split into 3 comprtments, clerly visile t 198 s. All structures exhiit motility chrcteristic of SmCCs. Scle r, 1 µm. Movie 15 Golgi ody tethering to SmCC under isoxen tretment. Seedlings expressing YFP::CESA6 nd CFP::TUA1 were treted with 100 nm isoxen for 2 h. A Golgi ody (cross) shows pprent tethering to moving SmCC (rrowhed). A short distnce is mintined etween these structures for 120 s. Scle r, 1 µm. References 1. Bolte, S. et l. FM-dyes s experimentl proes for dissecting vesicle trfficking in living plnt cells. J Microsc 214, (2004). 2. Roert, S. et l. An Aridopsis endo-1,4-et-d-glucnse involved in cellulose synthesis undergoes regulted intrcellulr cycling. Plnt Cell 17, (2005). 3. Preuss, M.L., Sern, J., Flel, T.G., Bednrek, S.Y. & Nielsen, E. The Aridopsis R GTPse RA4 loclizes to the tips of growing root hir cells. Plnt Cell 16, (2004). 4. Ued, T., Uemur, T., Sto, M.H. & Nkno, A. Functionl differentition of endosomes in Aridopsis cells. Plnt J. 40, (2004). 5. Rutherford, S. & Moore, I. The Aridopsis R GTPse fmily: nother enigm vrition. Curr. Opin. Plnt Biol. 5, (2002). 11