SUPPLEMENTARY INFORMATION

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1 DOI: /nc2885 kd M ΔNZipA ZipA x His NiNTA 27.0 c 1.,, 2. evnescent field supported memrne Supplementry Figure 1 Experimentl ssy. () Illustrtion of protein interctions (dpted from ref. ): nd ZipA ind to sme highly conserved Ctil of (shown in green), which is connected to the rest of the protein y flexile linker. inds to the memrne surfce using n mphipthix helix, locted t the end of Cterminl flexile linker. ZipA is trnsmemrne protein. The Nterminl trnsmemrnedomin hs een sustituted y Histg otining His Δ22ZipA, which ws then permnently ttched to the memrne using Ni 2+ chelting. () Commssiestined SDSPge gel of purified proteins used for this study. (c) Schemtic drwing of the experimentl ssy. A plstic ring ws glued to glss cover slip to crete rection chmer nd supported lipid ilyer ws formed on the glss surfce., nd were dded to the uffer efore polymeriztion of ws initited y dding. 1

2 + [] = 1mM, R286W, M167I, V277M 1.0 Intensity (.u.) time (min) Supplementry Figure 2 Remodeling of cytoskeletl structures depends on the presence of, ut not on polymeriztion. () Representtive intensity curve corresponding to Supplementry Video 4, showing the role of for cossemly: The initil trnsient inding of to the memrne is likely due to residul mounts of eing copurified with. After detchment of nd, the filment network ressemled s soon s fresh is dded (t ornge rrowhed nd dshed line). Similr intensity curves were otined in 5 experiments. () polymeriztion is not importnt for the selforgniztion of nd in our in vitro system. When we used selfinterction mutnts of ( R286W (left), M167I (middle), V277M (right)), formed the sme cytoskeletl pttern s with the wildtype protein. Similr microgrphs were otined in n=10 experiments. 2

3 0 s +1.2 s 0 s +1.6 s MIP 0 s +0.8 s 0 s +0.8 s 0 s + 2 s 0 s + 4 s x t Kymogrph trvel time (34.04 s ± s) 2. trvel length (1.18 µm ± 0.30 µm) 3. polymeriztion rte (1.89 ± 0.48 µm/min) 4. depolymeriztion rte (3.97 ±1.07 µm/min) 4. 0 s +1.2 s 0 s +1.2 s 0 s + 2 s 0 s + 2 s Supplementry Figure 3 Dynmics of single filments recruited to the memrne y. Snpshots, mximum intensity projections (MIP) nd kymogrphs of single filments from movies cquired t frmerte of 2s or 400 ms ( = 0.45 µm with 30 mol% Alex488, = 0.2 µm). Cyn rrowheds in MIPs indicte the first inding of to the memrne. Yellow rrows in kymogrph show the polymeriztion direction. Top left, with grey ckground. Scle rs correspond to or. Vlues in grey ox (top left) represent verge vlues of n=38 nlyzed filments from 5 independent experiments. 3

4 8% Ni 2+ NTA 2 µm 2 µm 0 min 5 min 10 min 40 min 90 min 4% Ni 2+ NTA 0 min 5 min 10 min min 100 min 2% Ni 2+ NTA 0 min 5 min 15 min 30 min 60 min 1% Ni 2+ NTA 0 min 3 min 10 min 25 min 45 min c [] = 1 µm; [] = 0.15 µm 0.7 µm 1.5 [] (µm) t = 0 min [] = 1 µm; [] = 3 µm + 2 min 2 µm t = 0 min, 2 min [] (µm) µm t = 0 min + 2 min 2 µm t = 0 min, 2 min Supplementry Figure 4 Influence of different concentrtions of memrne nchors on filment ptterns. () Representtive snpshots of filments recruited to the memrne y ZipA t indicted time points fter ddition of. The percentge of Ni 2+ chelting (18:1 DGSNTANi 2+ ) defines the mount of ZipA immoilized nd the mount of recruited to the memrne. With 8% Ni 2+ chelting, undles densely covered the memrne. At lower concentrtions (1%, 2% nd 4%), undles of filments strt to pper fter out min. On memrnes with 1% Ni 2+ chelting, individul filments could riefly e resolved (see 3 min fter ddition of ), efore the filment density ecme too high. Similr microgrphs were otined in 5 experiments. () Simplified phse digrm of filment networks on the memrne. Filled green dots represent experiments with protein concentrtions llowing for the formtion of dynmic filment network (t protein concentrtions of [] > 0.7 µm nd µm < [] <1.7 µm). Empty dots represent experiments where filments were either too short for continuous polymer network (with < 0.7 µm) or where they did not show rpid rerrngements (with < µm). Light green re represents the concentrtion rtio for nd found in vivo 26. Yellow filled circle represents the concentrtion rtio used for single filment experiments. (c) Representtive snpshots nd overlys of two individul frmes seprted y 2 min of timelpse movie. At low concentrtions (top), forms sttic, long filments, wheres t high concentrtions (ottom), filments were short nd dynmic, ut did not form continuous filment network. Similr microgrphs were otined in 5 experiments. 4

5 Frequency SUPPLEMENTARY INFORMATION Monomer lifetime distriution lifetime (s) Normlized se rte // /ZipA/ / / YFPMTS/ YFPMTS/ Supplementry Figure 5 The memrne nchor does not influence the se rte nd lifetime of monomers. () With ZipA, the verge lifetime of ws slightly longer thn with. Linerlog plot of lifetime distriution with ZipA s memrne nchor (filled red circles) out 30 min fter ddition of. Blck line represents the verged liner fit to individul lifetime distriutions with <t> = s ± 2.33 s (s.d. n = 24 videos with 1500 prticles otined from 5 independent experiments), which is slightly longer thn for / (p=0.0174). () se ctivity ws not ffected y the memrne nchor. Br grph shows se ctivity of (5 µm) or YFPMTS (5 µm) nd the influence of memrne nchors (HisZipA or, 3 µm) nd phospho (1 mg/ml). The corresponding rtes were normlized to the ctivity of lone (0.083/s). We found tht the se ctivity of YFPMTS to e out 30% lower thn of wildtype. se ctivities were determined using the EnzChek Phosphte Assy Kit (Moleculr Proes). Error rs correspond to s.d, from n=3 independent experiments. 5

6 1.0 Intensity (.u.) time (min) disssemly time (s) 40 0 (, ZipA) 50 s 100 s 300 s (, ZipA) Supplementry Figure 6 destilizes the filment network lso in presence of ZipA. Left, men intensity trces for depolymeriztion upon rpid dilution for filments recruited to the memrne y ZipA nd. Right: men disssemly times otined from douleexponentil fits, error rs nd thin lines correspond to s.e.m, (n=3 independent experiments). Bottom: Representtive snpshots showing disssemly of filments fter dilution (time point of dilution is indicted y ornge rrowhed nd dshed line). In the presence of oth nchors, ZipA nd, no thick undles of persist on the memrne. Fluorescence intensity of ech frme ws normlized. 6

7 GMPCPP ADP M ZipA Supplementry Figure 7 Uncropped Commssiestined SDSPge gels corresponding to Figs. 3c () nd 4d (). P = Pellet, SN = Superntnt; 1 nd 1 in indicte replictes using identicl experimentl conditions. 7