BREAKTHROUGH REPORT

Cryo-EM Structure of Actin Filaments from Zeamays Pollen[OPEN]

Zhanhong Ren,a,1 Yan Zhang,b,1 Yi Zhang,a Yunqiu He,a Pingzhou Du,a Zhanxin Wang,a Fei Sun,b,c,d,2 andHaiyun Rena,2

a Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, Center for Biological Science and Technology,College of Life Sciences, Beijing Normal University, Zhuhai 519087, ChinabNational Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, ChineseAcademy of Sciences, Beijing 100101, ChinacUniversity of Chinese Academy of Sciences, Beijing 100049, ChinadCenter for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

ORCID IDs: 0000-0002-3582-008X (Z.H.R.); 0000-0001-5575-6882 (Yan Z.); 0000-0003-4801-800X (Yi Z.); 0000-0001-6892-1998(Y.Q.H.); 0000-0002-3562-8098 (P.Z.D.); 0000-0001-9956-9376 (Z.X.W.); 0000-0002-0351-5144 (F.S.); 0000-0002-2164-0678(H.Y.R.).

Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures thatperform vital roles in key cellular processes. Although large amounts of data on the biochemical activities, dynamic behaviors,and important cellular functions of plant actin filaments have accumulated, their structural basis remains elusive. Here, wereport a 3.9 Å structure of the plant actin filament from Zea mays pollen (ZMPA) using cryo-electron microscopy. Thestructure shows a right-handed, double-stranded (two parallel strands) and staggered architecture that is stabilized by intra-and interstrand interactions. While the overall structure resembles that of other actin filaments, its DNase I binding loopbends farther outward, adopting an open conformation similar to that of the jasplakinolide- or beryllium fluoride (BeFx)-stabilized rabbit skeletal muscle actin (RSMA) filament. Single-molecule magnetic tweezers analysis revealed that the ZMPAfilament can resist a greater stretching force than the RSMA filament. Overall, these data provide evidence that plant actinfilaments have greater stability than animal actin filaments, which might be important to their role as tracks for long-distancevesicle and organelle transportation.

INTRODUCTION

The actin cytoskeleton plays vital roles in many fundamentalprocesses including vesicle and organelle transportation, endo-and exocytosis, and cell division and growth (Fu, 2015; Breueret al., 2017;Li et al., 2018;Romarowski et al., 2018;Szymanski andStaiger, 2018;Takatsukaetal., 2018;Uraji et al., 2018).Actinexistsin two states in vivo: globular actin (G-actin) and filamentous actin(F-actin), which are subject to a dynamic equilibrium of poly-merization and depolymerization. Inmost instances, F-actin is thefunctional form of actin proteins. Thus, studying the structure ofF-actin is of particular importance for understanding its functionalmechanism. Recently, the evolution of cryo-electron microscopy(cryo-EM) technology has enabled the determination of filamen-tous structures of rabbit skeletal muscle actin (RSMA) in differentnucleotide states with resolution ranging from 3.3 Å to 4.7 Åand the structure of jasplakinolide-stabilized malaria parasite

Plasmodium falciparum actin 1 (JASP-PfAct1) at 3.8 Å resolution(Galkin et al., 2015; von der Ecken et al., 2015; Pospich et al.,2017; Merino et al., 2018). In addition to those of eukaryoticF-actins, high-resolution cryo-EM structures of bacterial andarchaeal actin-like filaments have also been revealed in the lastfew years, including the 3.6 Å resolution structure of MamKfilaments frommagnetotactic bacteria (Löwe et al., 2016), the 3.4Å resolution structure of AlfA filaments from Bacillus subtilis(Szewczak-Harris and Löwe, 2018), and the 3.8 Å resolutionstructure of Pyrobaculum calidifontis crenactin filaments (Izoréet al., 2016).Despite the high protein sequence identity between plant and

animal actins (Kandasamyet al., 2012), their biochemical activitiesand cellular functions are different (Ren et al., 1997; Jing et al.,2003; Kandasamy et al., 2012; Rula et al., 2018). However, thestructural basis accounting for these differences remains poorlyunderstood, largely because none of the plant F-actin structureshave been resolved.Here, we report a 3.9 Å resolution structure of Zea mays pollen

actin (ZMPA) filaments determined by cryo-EM and the ruptureforces of actin filaments measured by single-molecule magnetictweezers. Our structural data show that the ZMPA filament re-sembles jasplakinolide- or beryllium fluoride (BeFx)-stabilizedmammalian actin filament, implying that plant actin filaments haveenhanced stability. Furthermore, the recorded rupture events of

1 These authors contributed equally to this work.2 Address correspondence to feisun@ibp.ac.cn and hren@bnu.edu.cn.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Fei Sun (feisun@ibp.ac.cn) and Haiyun Ren (hren@bnu.edu.cn).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.18.00973

The Plant Cell, Vol. 31: 2855–2867, December 2019, www.plantcell.org ã 2019 ASPB.

actin filaments confirm that the ZMPA filament has greater me-chanical stability than RSMA.

RESULTS AND DISCUSSION

Overall Structure

To determine the structure of plant actin filaments, we obtainedhighly purified proteins of Z. mays (maize) pollen actin by takingadvantage of the high binding affinity between actin and profilinand the ability of the actin-profilin complex to bind a poly-L-Procolumn (Ren et al. 1997; Supplemental Figure 1A) . Protein massspectrometry analysis revealed that theZMPAsamples containedfive actin isoforms with ;98% protein sequence identity(Supplemental Figures 1B and 1C). The ZMPA samples weresubsequently polymerized into long and straight filaments in vitroand applied to structural studies by cryo-EM.

ZMPA filaments were highly contrasted to show the double-helical natureof thefilaments (Supplemental Figures2Aand2B).Acryo-EM dataset was collected, and the structure of the ZMPAfilament was reconstructed using a real-space helical re-construction approach (Figure 1A; Supplemental Movie 1;SupplementalMovieLegends;Supplemental Files1and2).ZMPAfilaments existed as a two-stranded structure composed ofstaggered actin subunits, with a refined helical symmetry with–166.77° rotation and 27.5 Å rise per subunit, resembling thestructures of RSMA and jasplakinolide-stabilized RSMA (JASP-RSMA) filaments (Figures 1A and 1B; Galkin et al., 2015; Merinoetal., 2018;ChouandPollard, 2019). Thefinal3D reconstructionofZMPA filaments had an overall resolution of 3.9 Å, using Fouriershell correlation (FSC)5 0.143 gold-standard criterion (Rosenthal

andHenderson, 2003;Figures1Cand1D).This resolutionenabledus to build a pseudo-atomic model of the ZMPA filament withaccuracy at the backbone level. A few charged residues (e.g.K115, E197, D246, E272, and D290) lacked clear side chaindensity,whichmightbedue topotential radiationdamageor to theintrinsic flexibilities of those residues.The structure of the ZMPA subunit comprises four subdomains

(SDs), SD1–SD4, and the D-loop in SD2 and hydrophobic plug inSD4 (Figure1B).TheADPbound in thecatalyticpocket, theb-sheetin SD3, and the a-helix in SD4 of the ZMPA subunit are clearlyresolved, and side-chain densities for the bulkier residues arevisible (Figures 1E to 1G). When amino acid differences wereoverlaid onto the structure of the ZMPA subunit, the sequencedivergences among the five ZMPA isoformsweremainly locatedin SD1 and SD4 but not in the regions important for nucleotidebinding or for intersubunit interactions that involve SD2 andthe hydrophobic groove (Supplemental Figure 2C). In addition,the nucleotide binding pockets are highly conserved among thestructures of ZMPA,PfAct1, andRSMA filaments (SupplementalFigure 3).

Intersubunit Interactions

Similar to other actin filaments, the ZMPA filament is stabilized byinteractions between actin subunits of the same strand (intra-strand) and the opposing strand (interstrand). Intra- and in-terstrand interaction sites are involved in the longitudinal andlateral interfaces, respectively (SupplementalTable1).The internalinterface in the ZMPA filament covers similar areas to those inmammalian actin and JASP-PfAct1 filaments, according toanalysis using the Proteins, Interfaces, Surfaces, and Assembliesserver (http://www.ebi.ac.uk/pdbe/pisa/; Supplemental Figure 4;

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Figure 1. Cryo-EM Structure of ZMPA Filaments.

(A)Stereo viewof the 3D reconstruction. The five central fitted actin subunits are shown (SU-A: coral; SU-B: green; SU-C: turquoise; SU-D: blue; SU-E: red).(B) Front and back view of SU-A with its density. SD1 to SD4 of SU-A are shown in gray, tan, salmon, and coral, respectively. The D-loop (purple) andhydrophobic plug (yellow) are located at SD2 and SD4, respectively.(C) The cryo-EM density used to build the model of ZMPA filaments, colored according to the local resolution of the map.(D) FSC curve for final ZMPA reconstruction indicates the two correlations (black and red dashed lines). One correlation is between the half-datasets (blackline), and the other is between the model and the map (red line).

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Krissinel and Henrick, 2007). The longitudinal interface is formedmainly between SD3 of one actin subunit and SD2 and SD4 of thenext actin subunit of the same strand (Figure 2). Similar to thesituations in RSMA and JASP-PfAct1 filaments (von der Eckenet al., 2015; Pospich et al., 2017), there are two similar lock-and-key hydrophobic interaction sites in ZMPA filaments. At the firsthydrophobic contact site, the major hydrophobic patch in theD-loop of ZMPA filaments interacts with the hydrophobic grooveon the adjacent actin subunit and encloses Y171 in SD3 of theneighboring actin subunit (Figures 2A to 2C). The second contactsite consists of V289 in SD3 and the hydrophobic pocket in SD4 ofthe next subunit on the same strand (Figure 2E). Two potential saltbridgesbetweenD290ofSD3andR64ofSD2andbetweenR292ofSD3 andD246 of SD4 form an intrastrand connection between twoneighboring subunits (Figures 2D and 2F). These two electrostaticinteraction sites were also reported to be highly conserved in thestructures of RSMA (von der Ecken et al., 2015) and JASP-PfAct1filaments (PDB ID code: 5OGW). Previous amino acid mutationalanalyses and the results of Mical-mediated oxidation modificationshowed that the intrastrand interactions are important for actinpolymerization and stability (Wertman et al., 1992; Murakami et al.,2010; Hung et al., 2011; Grintsevich et al., 2017). Thus, theabovementionedpotential saltbridges in the intrastrand interfaceofF-actin, together with the hydrophobic interactions, may providestructural support for ZMPA polymerization.

In addition to the intrastrand contacts, the interstrand inter-actions are also important for actin polymerization and stability(Wertman et al., 1992; Pospich et al., 2017). As inRSMA filaments,there is ahydrophobic contact site between residues196 to203ofSD4 and SD1 of the adjacent subunit in the opposite strand of theZMPA filament (Figure 3A). In addition, two potential electrostaticcontacts are present in the lateral interfaces of ZMPA filaments,which were also reported in RSMA and JASP-PfAct1 filaments(von der Ecken et al., 2015; Pospich et al., 2017). The first contactsite is formed by the interstrand interaction of the positivelycharged residue R41 of the D-loop with the negatively chargedresidue E272 of the hydrophobic plug of the opposing subunit(Figure 3B). The second interaction site involves two residues(E197 and K115) that form a potential salt bridge (Figure 3C).Previous studies indicated that mutations at these sites woulddestabilize the hydrophobic and electrostatic interstrand inter-actions of the PfAct1 filament and that PfAct1 mutants couldpolymerize into longfilaments invitroonly in thepresenceof JASP,an actin-filament-stabilizing agent (Pospich et al., 2017). Ourobservation of the interstrand interactions is therefore consistentwith the effective polymerization of ZMPA monomers into longactin filaments in vitro.

The D-Loop/Hydrophobic Groove Interface

It has been well documented that the D-loop is one of the majorregions involved in the intrastrand contacts of F-actins and plays

a critical role in actin polymerization and stability (Holmes et al.,1990; Fujii et al., 2010; OztugDurer et al., 2010; Galkin et al., 2015;von der Ecken et al., 2015; Grintsevich et al., 2017; Pospich et al.,2017). Compared with the D-loop (residues 39 to 53) of RSMA,there are two residue substitutions (Gln to Thr-43 and Ser to Ala-54) in thatofZMPA(Supplemental Figure1C).Bycontrast to thatofJASP-PfAct1 (Pospich et al., 2017) and of RSMA filaments in theADP state (Merino et al., 2018), the D-loop of ZMPA in the ADPstate shifts outwardwith respect to the filament axis (Figures 4A to4C). Interestingly, the conformation of the D-loop in ZMPA re-sembles that in JASP-RSMAand that in RSMAbound to berylliumfluoride (ADP-BeFx; Figures 4C to 4E and 5; Supplemental Movie2; Supplemental Movie Legends). Previous studies showed thatJASP-RSMAandRSMAbound toADP-BeFx aremore structurallystable than the form bound to ADP alone (Isambert et al., 1995;Visegrády et al., 2004; Kardos et al., 2007), while the prominentstructural difference is that the former structures have an openD-loop and the latter has a closed D-loop (Merino et al., 2018),implying apossible association between theD-loop conformationand filament stability. Therefore, our data suggest that the ZMPAfilament in the ADP statemay bemore stable and rigid thanRSMAand JASP-PfAct1 filaments in the same nucleotide state. Wenoted that Chou and Pollard (2019) reported closed con-formationsof theactin filamentwithAMPPNPorADP-Pi. Thismayindicate that a small difference between ATP analogs can greatlychange the D-loop conformation. Although the ADP-Pi state isvery stable (Fujiwara et al., 2007), the ratio of the open confor-mation is smaller in the case of ADP-Pi than that of ADP-BeFx

(Merino et al., 2018). Therefore, besides the open conformationbeing the main factor of the stability, other factors would con-tribute, which merits further studies.To test the mechanical stability of actin filaments, we stretched

a single F-actin using single-molecule magnetic tweezers (Fig-ure6A), apowerful tool toassess themechanical characteristicsofsingle biomolecules in vitro (Strick et al., 1996; Hinterdorfer et al.,2009; Yu et al., 2017; Chen et al., 2018).When the stretching forceincreased, the F-actin was elongated until it was broken(Figure 6B). Because the interaction between biotin and strep-tavidin (Tsai et al., 2016) and that betweenactin antibodyandactinare much stronger than the maximum stretching force (90 pico-Newton [pN]) applied to F-actin (Supplemental Movies 3 and 4;Supplemental Movie Legends), the breakage of a single tetherwithin the measurement range of our study reflects the disruptionof a single F-actin under tension. Thedisruption force of theZMPAfilament is centered at ;37.8 6 2.2 pN, which is significantlygreater than that of the RSMA filament (26.56 1.8 pN, mean6 SE;Figure 6C; Supplemental Movies 5 and 6; Supplemental MovieLegends). This result indicates that the ZMPA filament is morestable than theRSMA filament. The rupture forceof a singleRSMAfilament measured in our study is relatively small compared withthe values previously reported by Kishino and Yanagida (1988),Tsuda et al. (1996), and Liu and Pollack (2002). It may result from

Figure 1. (continued).

(E) A close-up view of ADP in the nucleotide binding cleft of actin shown with respective densities.(F) and (G) A close-up view of the b-sheet in SD3 and a-helix in SD4 of SU-A.

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Figure 2. The Intrastrand Interactions in ZMPA Filaments.

Surfaces are colored from high (yellow) to low (white) hydrophobicity. Subunit B (SU-B) and Subunit D (SU-D) are shown in their respective color. Twoimportant longitudinal interfaces will be formed by SU-B and SD2 of SU-D (A) to (D) and by SU-B and SD4 of SU-D (E) to (F). (E) to (F) Shows 180° right-handed rotation of (A) to (D) using the longitudinal axis of rotation.(A)and (B)SU-BandSU-Daredisplayedassurfaceand ribbon representations, respectively. Protein residuesare instick representationcoloredbyelementin gray for carbon and yellow for sulfur atoms. (A) Zoom-in display of the interface formed by SU-B and the D-loop of SU-D. (B) Shows 120° left-handedrotation of (A) using the longitudinal axis of rotation. Side view (A) and front view (B) show the interaction of the D-loop with the hydrophobic groove of theneighboring F-actin subunit in ZMPA filaments. The D-loop encloses Tyr-171 of the adjacent subunit.(C)and (E)SU-BandSU-Daredisplayedas ribbonandsurface representations, respectively.Protein residuesare instick representationcoloredbyelementin gray for carbon and red for oxygen atoms. (C) Enlarged display of the same area as (A). (E) Zoom-in display of a 40° left-handed rotation of the interfaceformed by SU-D and SD3 of SU-B using the lateral axis of rotation. Side view (C) shows the interaction of the D-loop with SD3 of the neighboring F-actinsubunit in ZMPA filaments. The Y171 in SD3 inserts into the hydrophobic D-loop (C), and V289 in SD3 inserts into the hydrophobic groove of SD4 of theneighboring subunit (E), resembling two lock-and-key hydrophobic interaction sites.(D) and (F) Enlarged display of the interface formed by SD3 of SU-B and SU-D. Two potential electrostatic contacts are formed in the intrastrand interface.The backbones of the charged residues involved in the interactions are shown as red (negative charge) and deep sky blue (positive charge) spheres.

Structure of Plant Actin Filaments 2859

the fact that the stretched actin filaments in previous studies werestabilized with phalloidin but those in our study were not. Aprevious study showed that phalloidin binds to the intersubunitinterfaces of the actin filament and couples neighboring actinsubunits together (Mentes et al., 2018). The stoichiometry ofbinding is one phalloidin per actin subunit, and phalloidin canenhance F-actin stability (Visegrády et al., 2004; Mentes et al.,2018). Besides, the results of our stretching experiments onF-actin seeds (SupplementalMovies3and4;SupplementalMovieLegends) show that the interaction strength between the neigh-boring actin subunits stabilized by phalloidin is >90 pN, which isused as the maximum stretching force in our study. In addition,stretchingsystemsmayalsocontribute to thedifferenceof ruptureforce measured in the previous and our studies. The rupture forcereported by Kishino and Yanagida (1988) is approximately onefourth of that by Tsuda et al. (1996) for the actin filament using thesame microneedle method. It was thought that the former did notconsider the randomizing effect of the rotational Brownianmotionand had system errors in the calibration of needles (Tsuda et al.,1996). The actin filament is pulled continuously in themicroneedlemethod, similar to that in optical tweezers. In optical tweezers,a laser is focused on dielectric beads. The beads experience a 3Drestoring force directed toward the focus (Neuman and Nagy,2008). During the stretching experiment, optical tweezers move

the trap continually to pull the sample and the system does notachieve equilibrium. For magnetic tweezers, the tension exertedon the samples is dependent on thepositionof themagnets. Inourmeasurements, the magnets move step by step toward thesample. At each step, a constant force is exerted on the filamentand the system achieves equilibrium. Therefore, in contrast withthe stepwise pulling used in the magnetic tweezers method, thecontinuous pulling in the optical tweezers method causes thesystem todeviate fromequilibriumandagreater rupture forcemaybe measured (Dhakal et al., 2013; You et al., 2014).The conformation of the D-loop implies that the interaction

between theD-loop and SD1 of the adjacent actin subunit may bestrengthened in theZMPA filament. Indeed, there is a clear densityconnecting the D-loop and the C-terminal tail of the neighboringintrastrand subunit of ZMPA, while the corresponding density isabsent in the JASP-PfAct1 and RSMA filaments in the ADP state(Figures 4A to 4C). In addition, the D-loop/hydrophobic grooveinterface is an important recognition site for the binding of manyactinbindingproteins toF-actin (vonderEckenetal., 2016;Merinoet al., 2018). The previously reported complex structure ofmammalian actomyosin shows that the helix–loop–helix motif ofmyosin enters the hydrophobic groove on the adjacent actinsubunit and contacts the D-loop (von der Ecken et al., 2016; Fujiiand Namba, 2017; Mentes et al., 2018), illustrating that myosin

Figure 3. The Interstrand Contacts in ZMPA Filaments.

(A) to (C) Actin subunit B (SU-B), subunit C (SU-C), and subunit D (SU-D) are shown in ribbons in the color of the respective subunit.(A)Surfacesarecolored fromhigh (yellow) to low (white) hydrophobicity. Zoom-indisplayof the90° left-handed rotationof the interface formedbySU-CandSU-Dusing the longitudinal axis of rotation. Protein residues are in stick representation coloredby element in gray for carbon and red for oxygen atoms. Theinterstrand hydrophobic contact in ZMPA filaments is mediated by residues 196 to 203 in SD4 and SD1 of the neighboring interstrand SU-C.(B)and (C)Thepotential electrostatic interactionsare formed in the interstrand interface. Thebackbonesof thecharged residues involved in the interactionsare shown as red (negative charge) and deep sky blue (positive charge) spheres.

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Figure 4. The ZMPA Filament Shows an Open D-Loop Conformation.

(A) to (E)Visualization of actin subunitB (SU-B), subunit D (SU-D), and their corresponding densities in longitudinal interfacesof JASP-PfAct1, PDB IDcode:5OGW (A); RSMA,PDB IDcode: 5ONV (B); ZMPA (C); JASP-RSMA,PDB IDcode: 5OOC (D); andRSMA filaments bound toADP-BeFx, PDB IDcode: 5OOF(E). The jasplakinolide and its corresponding density are not shown in (A) and (D). SU-B andSU-Dof JASP-PfAct1 are shown in orange andmagenta, thoseof RSMA in cyan and dark gray, those of ZMPA in green and blue, those of JASP-RSMA in orchid and turquoise, and those of RSMAbound to ADP-BeFx insalmon and brown, respectively. The C-terminal tail (the last amino acid in the C terminus) of SU-B is labeled with a black star. The D-loop is bent fartheroutward with respect to the filament axis in ZMPA (C) than in JASP-PfAct1 (A) and in RSMA (B).(C to E) There is a clear additional density between theD-loop and theC-terminal tail of the neighboring actin subunit in the ZMPA filament (indicated by theredellipse;C). Thestateof theD-loop in theZMPA filament is similar to that in theJASP-RSMAfilament (D)and in theRSMAfilamentbound toADP-BeFx (E).

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Figure 5. Two Distinct D-Loop States of Actin Subunits.

When actin subunit Ds (SU-Ds) from five kinds of F-actin are aligned, a magnified view of the boxed D-loops shows that the ZMPA subunit adopts an openD-loop state similar to that of JASP-RSMA and RSMA bound to ADP-BeFx, while JASP-PfAct1 and RSMA show a closed D-loop state.

Figure 6. The ZMPA Filament Resists a Greater Stretching Force than RSMA.

(A)Schematic setupof themagnetic tweezersused in thestudy (not toscale). Theanti-actin-coatedmagneticbeadbinds to theactinfilament that is tetheredto a coverslip through interactions between the preformed biotin-labeled F-actin seed (stabilized with phalloidin) and streptavidin, which forms a singletether. The stretching force imposed on a magnetic bead will decrease or increase when the permanent magnets are moved up or down.(B) The stretching curves of RSMA (blue) and ZMPA (red) filaments. The time-force (upper), time-extension (middle), and force-extension (lower) curves ofa single actin filament are shown. The rupture force of a single ZMPA filament is 35 pN, which is 9.5 pN greater than that of a single RSMA filament.(C)Statistical analysis of thedisruption forceofRSMAandZMPAfilaments. Theaverageddisruption forceof theRSMAfilament is 26.561.8pN (mean6 SE)and that of the ZMPA filament is 37.86 2.2 pN (mean6 SE). ***P value < 0.001, as determined by a two-tailed Student’s t test. n5 75 for RSMAand n5 50 forZMPA.

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could directly sense the changes occurring in the D-loop/hy-drophobic groove interface. Our model shows that the D-loop/hydrophobicgroove interfaceofZMPA in theADPstate isdifferentfrom that of RSMA in the same state (Figures 4B and 4C;Supplemental Figures5Aand5C). This differencemayaccount forthe different interaction modes with myosin and other actinbinding proteins that bind to the D-loop/hydrophobic groove in-terface. This result is consistent with a recent report that themeasurements of enzymatic and motile activities of Arabidopsis(Arabidopsis thaliana) myosins using Arabidopsis actins weredistinct from thosemeasuredusinganimal skeletalmusclea-actin(Rula et al., 2018). These abovementioned characteristics areimportant for plant actin filaments, as they need to serve as tracksfor long-distance vesicle and organelle transportation processesthat generate extensive tensions, which is different from themicrotubule-centric cargo transportation systems in mammaliancells (Hammer andSellers, 2011;Brandizzi andWasteneys, 2013).

In summary, this structural and single molecule force-spectroscopy experimental data suggest that ZMPA filamentsare more stable than RSMA filaments, which provides a possibleexplanation for their functional differences in cells.

METHODS

Purification and Polymerization of ZMPA

Plant actin was purified frompollen collected frommaize (Zeamays) plantsduring July and August 2016 and July and August 2017 and then stored at280°C. The cultivar of maize plants was Longping No. 206. Maize pollenactin was prepared as described in Ren et al. (1997). Purified pollen actin inbuffer G (5 mM of Tris-HCl at pH 8.0, 0.2 mM of CaCl2, 0.01% [w/v] NaN3,0.2 mM of ATP, and 0.5 mM of DTT) was divided into small aliquots (;100mL), flash-frozen in liquid nitrogen, and stored at 280°C. The polymeri-zation of freshly frozen plant actin was tested. The plant actin was dialyzedagainst bufferGwith pH7.0 overnight to change fromalkaline pH to neutral(Sampath and Pollard, 1991) and was polymerized at a final concentrationof 9.5 mM by the addition of 103 KMEI (500 mM of KCl, 10 mM of MgCl2,10 mM of EGTA, and 100 mM of imidazole at pH 7.0; Neidt et al., 2008) atroom temperature (25°C) for 4 h. To minimize the effect of non-polymerizableactinon theEMstudiesandmassspectrometryanalysis, thepolymerized plant actin filaments were spun down (100,000g at 4°C) andgently suspended in buffer G (pH 7.0) with 13 KMEI. The protein con-centration was determined with Bradford reagent (Bio-Rad), using BSA asa standard.

MS Analysis of ZMPA

MS analysis was performed by the Beijing Huada Protein R&DCenter. Theidentities of the plant actin filaments were revealed by liquidchromatography-tandemmass spectrometry (LC-MS/MS) analysis. TheQExactivemassspectrometrydata (ThermoFisherScientific)were searchedagainst the UniProt maize database using 15mL/L peptidemass toleranceand 20 millimass units (mmu) fragment mass tolerance.

Grid Preparation and Image Acquisition for Cryo-EM

ZMPA filaments (3 mL) were applied to glow-discharged GIG holey carbongrids (R1.2/1.3, 400 meshes). The grid was flash-frozen in liquid ethane at;100 K using a Vitrobot Mark IV Semiautomatic Plunge Device (ThermoFisher Scientific) with a blotting time of 5.5 s and blotting force of level 2 at100% humidity, 25°C. Screening for sample and blotting conditions was

performed on a Talos F200C 200-kV electron microscope equipped witha 4K34K Ceta camera (Thermo Fisher Scientific). Finally, micrographs ofplant actin filaments for structure determination were collected on a directelectron device (model no. K2 camera; Gatan) in a 300-kV Titan KriosElectron Microscope (Thermo Fisher Scientific) using the automated ac-quisition software SerialEM (Mastronarde, 2005). A total of 3,605 micro-graphs were recorded at a calibrated pixel size of 1.063 Å with a total doseof ;39 e2/Å2 and a defocus range from 1.2 to 2.0 mm.

Image Processing of the Cryo-EM Data Set

In total, 3,605 micrograph movie stacks were collected during four ses-sionsofmicroscopydatacollectionhours.Motioncorrectionsanddefocusestimations for all these micrographs were performed using the softwaresMotionCorr2 (Zheng et al., 2017) and GCTF (Zhang, 2016), respectively.Micrographswith ice contamination, poor Thon rings, largedefocus valuesand other defects, were excluded before filament boxing. A total of 1,540micrographs were finally selected. These selected micrographs weremultiplied by their theoretical contrast transfer function (CTF) for initialcorrection ofCTF (Galkin et al., 2015). A total of 8,609ZMPA filamentswereboxed using e2 helixboxer.py in the package of EMAN2 (Tang et al., 2007)with a box width of 168 and 77% box overlap. A total of 40,943 segmentsweregeneratedwith abox sizeof 384, and2Dclassificationwascalculatedto check the diameter distribution anddata quality by the programRELION2.0 (Scheres, 2012). For the 2D class averages without any downsize orbinning, the secondary structures could be observed from some of the 2Dclass averages (Supplemental Figure 2B). Helical parameters were cal-culated by indexing the layer lines of the Fourier transform of the ZMPAfilament. An initial helical riseof 27.5Åand twist of2166.77°wereobtainedand used as the initial helical parameters for helical reconstruction. Weused the real-space helical reconstruction algorithm IHRSR (Egelman,2000, 2007), which was integrated into the SPIDER script (Shaikh et al.,2008) to perform 3D reconstruction, and 35,684 particles were finally in-cluded to obtain a 3.9 Å map (Figure 1). The helical parameters convergedto 27.5 Å for the helical rise and2166.77° for the helical twist. We dividedCTF2 to correct CTF aswehad alreadymultipliedCTF at the beginning.Weused the software SPIDER (Shaikh et al., 2008) to perform postprocessing(B-sharping). The gold-standard FSC curve was calculated by dividing theparticles into halves at the beginning. The local resolution was estimatedusing the program ResMap (Kucukelbir et al., 2014).

Model Building and Refinement of ZMPA Filaments

Weused themodel of actin fromOryctolaguscuniculus (Merinoet al., 2018;PDB ID code: 5OOC) as a startingmodel, and first fitted it into themapwiththeprogramChimera asa rigid body (Pettersenet al., 2004). Then, residueswere mutated to those in the ZMPA sequence (UniProt ID code: B6TQ08),and thesepoorly fitted regionswere adjustedmanually and refinedwith thesoftwareCoot (Emsleyet al., 2010). Further refinementwasconductedwiththe program Phenix (Adams et al., 2011). The geometry parameters for thefinal model are presented in Supplemental Table 2.

Single-Molecule Magnetic Tweezers Analysis

The basic principle of magnetic tweezers was described previously byHinterdorfer etal. (2009)andZlatanovaandLeuba (2003). Theexperimentalsetup of our single-molecule magnetic tweezers was constructed in-house, as described previously by Chen et al. (2018). In our study, twoends of a single F-actin were bound to a streptavidin-coated coverslip andan anti-actin-coated superparamagnetic bead (Dynabead M-270 Epoxy;Invitrogen) via the biochemical reactions between biotin and streptavidinand the bonds between actin and anti-actin antibody, respectively, asshown in Figure 6A. Magnetic tweezers were used to stretch the actin

Structure of Plant Actin Filaments 2863

filament vertically by the application of a horizontal magnetic field gen-erated by a pair of magnets (small NdFeB magnets with a distance of 0.5mm) to a superparamagnetic bead to which the actin filament was at-tached. The superparamagnetic bead was illuminated by a parallel lightplacedabove themagnets. The interferenceof the illuminating lightwith thelight scattered by the superparamagnetic bead produced concentric dif-fraction rings in the focal plane of the objective placed below the flow cell.The image of the diffraction pattern was recorded through a microscopeobjective (oil immersion type, 6031.4; Olympus) with a Giga-Ethernetcharge-coupled device camera (AVT) at 200 Hz. The real-time position(x, y, z) of the bead at various forces was recorded by comparing thediffraction pattern of the bead with calibration images at various distancesfrom the focal point of the objective. Themotion of the superparamagneticbead tethered to the coverslip with a linear bio-molecule can be describedas an inverse pendulumwith a tension in the z direction. The fluctuations ofthebead in the x-yplaneare related to theapplied forcebyF5 kBTl/⟨dx2⟩according to the equipartition theorem, where l is the average end-to-endextension of the molecule, kB is the Boltzmann constant, T is the absolutetemperature of the environment, and dx is fluctuation of the bead in the xdirection (Yan et al., 2004). The force exerted on the sample increases ina mono-exponential relationship with the magnet’s position in the z di-rection. Before the real measurements for actin filaments were made, theforce calibration was performed. Using a well-studied DNA tether (lambdaDNA), thepositionsof thebead in thex-yplaneateachmagnetpositionwasrecorded and the tensions were calculated according to F5 kBTl/⟨dx2⟩.The relationship between tension and magnet position was obtained byfitting the tension and magnet position with the mono-exponential re-lationship. In themeasurement of actin filaments, the tension was read outdirectly based on the fitted mono-exponential relationship in the softwareLabView (http://www.ni.com/zh-cn/shop/labview/labview-details.html)according to the magnet’s position. In the stretching measurements ofactin filament, the measurement was initiated when the magnets were3mmaway from the surface of the flow cell and quite low tension (<0.5 pN)was exerted on the sample. Then, the magnets were lowered to 0.1 mmfrom the surface of the flow cell finally with a step size of 0.02 mm, stayingfor 2 s at each step, and the extension of the sample was traced. Thecorresponding stretching force on the single tether was thereby increasedfrom 0 to;90 pN exponentially. The tension was read out by the softwareLabView.

Stretching Measurement of a Single F-Actin

To anchor the F-actin, the surface of the coverslip needs to be function-alized. First, the coverslip was cleaned by sonicating multiple times (1 h indetergent, 1 h in ethanol, 2 h with piranha solution, and 10min in deionizedwater). The coverslip was then incubated with 2-mg/mL methoxy-PEG-silane (Mw 5,000; Laysan Bio) and 2-mg/mL Biotin-PEG-silane (Mw 3,400;Laysan Bio) in 95% (v/v) ethanol (pH 2.0) at 70°C overnight. The referencebeads (2-mm polystyrene beads; QDSphere) were pipetted onto thecoverslip and placed on a hot plate at 150°C for 10 min to melt onto thecoverslip. Second, the coverslip was made to be part of the flow cell andthen incubated in passivation buffer (10 mg/mL of BSA, 1 mM of EDTA,10 mg/mL of Pluronic F-127 surfactant [BASF], 3 mM of NaN3, and 10mMof phosphate buffer at pH 7.4) overnight at 37°C to limit the nonspecificinteraction between the actin sample and the coverslip. After streptavidintreatment, the coverslip was washed with 13 KMEI buffer (50 mM of KCl,1mMofMgCl2, 1mMofEGTA,and10mMof imidazoleatpH7.0) to removefree streptavidin, and then incubated with the preformed biotin-labeledF-actin seeds (stabilizedwithphalloidin) for 5min toallowseeds tobind to itvia the interactions between biotin and streptavidin. After the removal offree seeds bywashing the coverslipwith the instantmixedG-actin solutionof 0.15 mM of G-actin in buffer G (5 mM of Tris-HCl at pH 7.0, 0.2 mM ofCaCl2, 0.01% [w/v] of NaN3, 0.2 mM of ATP, and 0.5 mM of DTT) and 13

KMEI buffer, the instant mixed G-actin solution of 0.75 mM of G-actin inbuffer G and 13 KMEI buffer was flowed into the channel for actin poly-merization and elongation from the barbed ends of immobilized seeds.After actin polymerization for 6 min, the instant mixed solution of anti-actin–coatedsuperparamagneticbeads,0.15mMofG-actin inbufferGand13 KMEI buffer were gently injected into the flow cell, and incubated for10min to allow the beads to bind to the F-actin. Finally, the free beadswereremoved bywashing the coverslip gently with the instantmixed solution of0.15 mM of G-actin in buffer G and 13 KMEI buffer. To prevent the de-polymerization of F-actin, the solution used to wash the channel shouldcontain a low concentration of G-actin (slightly higher than the criticalconcentration of actin polymerization). The anti-rabbit a-actin mousemonoclonal antibody (Lot no. D1514; Santa Cruz Biotechnology) wascoated on the superparamagnetic beads used for the stretching mea-surement of the singleRSMA filament and anti-plant actin antibody (Lot no.492,635,537; Sigma-Aldrich) for that of the single ZMPA filament by re-ferring to the protocol of Dynabeads Antibody Coupling Kit (Invitrogen).

Thebreakageof asingle tether resulted in the target superparamagneticbead moving suddenly out of the viewing area. To ensure that the singleF-actin is tethered between beads and the coverslip, the magnets wererotated 50 turns before the measurements were taken. Those beads witha single filament rotated freely and were chosen for measurements, whilethose with more than one filament did not rotate freely and were notconsidered. If multiple actin filaments are anchored between the coverslipand the superparamagnetic bead, the actin filaments will be tangled whenwe rotate themagnetsand thebeadcannot rotateaccordingly,whichhelpsus to discriminate the bead with a single actin filament anchored. Finally,the breaking force of a single tether was measured and defined as thedisruption force of a single F-actin in our study. All measurements wereperformed at 25°C.

Figure Preparation

Multiple sequence alignments of the actin isoforms were performed usingthe software Clustal Omega (Sievers et al., 2011). Figures were preparedusing the softwares ResMap (Kucukelbir et al., 2014) and Chimera(Pettersen et al., 2004), the PyMOL Molecular Graphics System, Version2.0 (Schrödinger; http://www.pymol.org/), and Adobe Illustrator CC.

Statistical Analysis

Statistical significance was analyzed by unpaired Student’s t test for two-groupdata. TheP value is shown inSupplemental Table 3. The sample sizeand the significance level of theP value aredescribed in the figure legendofFigure 6C.

Accession Numbers

Sequence data from this article can be found in the UniProt database withaccession codes of a-actin (P68135), PfAct1 (P86287), and five ZMPAisoforms (B6TQ08, B4FRH8, C0HDZ6, B6SI11, and B4F989). Cryo-EMmaps of ZMPA filaments have been deposited into the Electron Micros-copy Data Bank (accession code EMD-9734) and the correspondingatomic coordinates have been deposited into the Protein DataBank (ac-cession code 6IUG).

Supplemental Data

Supplemental Figure 1. SDS-PAGE and liquid chromatography-tandem MS (Q exactive) results of ZMPA.

Supplemental Figure 2. Cryo-EM micrographs and analysis of ZMPAfilaments.

2864 The Plant Cell

Supplemental Figure 3. Comparison of the nucleotide binding sitesfrom various actin subunits.

Supplemental Figure 4. Longitudinal and lateral contacts of actinfilaments.

Supplemental Figure 5. The hydrophobic groove in ZMPA filaments.

Supplemental Table 1. The residues predicted to be involved inintersubunit interactions.

Supplemental Table 2. Data collection and refinement statistics.

Supplemental Table 3. Statistical analyses results.

The following Supplemental Data Movies were submitted to the DataDryad Repository and are available at https://doi.org/10.5061/dryad.k0p2ngf42.

Supplemental Movie 1. Cryo-EM reconstruction of ZMPA filament.

Supplemental Movie 2. The ZMPA filament adopts an open-D-loopconformation.

Supplemental Movie 3. The binding strength between the anti-plantactin antibody and ZMPA.

Supplemental Movie 4. The binding strength between the anti-rabbita-actin antibody and RSMA.

Supplemental Movie 5. The stretching measurement of a singleZMPA filament.

Supplemental Movie 6. The stretching measurement of a singleRSMA filament.

Supplemental Movie Legends. Legends for Supplemental Movies1 to 6.

Supplemental File 1. Map of ZMPA filament.

Supplemental File 2. Validation report from wwPDB.

ACKNOWLEDGMENTS

We thank Edward H. Egelman from the University of Virginia for hisgenerous help with the data analysis for the project. The isolation andpurification of ZMPA and structural analysis was performed in H.Y.R.’slab and the work for image processing, model building, and refinementwas performed in F.S.’s and Edward H. Egelman’s labs. We thank YunXiang, Prof. Li Zhu, Bin Yuan, and Dr. Xia Deng from Lanzhou Universityfor their support and advice for this research program. Cryo-EM samplepreparation and data collection were carried out at the Center for Bi-ological Imaging (http://cbi.ibp.ac.cn), Core Facilities for Protein Sci-ence, at the Institute of Biophysics, Chinese Academy of Sciences. Wethank Xiaojun Huang, Wei Ding, Boling Zhu, Tongxin Niu, and other staffmembers at the Center for Biological Imaging for their support duringdata collection and image processing. We thankWei Li from the Instituteof Physics at the Chinese Academy of Sciences for his advice on thesingle-molecule magnetic tweezers analysis. This work was supportedby the National Natural Science Foundation of China (grants 91854206and 31770206 to H.Y.R., and 31770794 to Yan Z.) and the Ministry ofScience and Technology of China (grant 2013CB126902 to H.Y.R. and2017YFA0504700 to F.S.).

AUTHOR CONTRIBUTIONS

H.Y.R. and Z.H.R. conceived and coordinated the project; Z.H.R., Y.Q.H.,P.Z.D., and H.Y.R. purified the ZMPA frommaize pollen; Z.H.R. performed

cryo-EM sample preparation, data collection, and preliminary data pro-cessing; Yan Z. performed image processing, model building, and re-finement; Z.H.R., F.S., andH.Y.R. analyzed the structure; Z.H.R. performedthe single-molecule magnetic tweezers analysis; Z.H.R. prepared thefigures, tables, and video of this article; Z.X.W. helped with the structuralanalysis and provided suggestions for the revision of figures; article waswritten and revised by Z.H.R., Yan Z., Yi Z., H.Y.R., and F.S.

Received January 2, 2019; revised September 17, 2019; acceptedOctober 16, 2019; published October 18, 2019.

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Structure of Plant Actin Filaments 2867

DOI 10.1105/tpc.18.00973; originally published online October 18, 2019; 2019;31;2855-2867Plant Cell

RenZhanhong Ren, Yan Zhang, Yi Zhang, Yunqiu He, Pingzhou Du, Zhanxin Wang, Fei Sun and Haiyun

PollenZea maysCryo-EM Structure of Actin Filaments from

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