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. 2019 Oct 18;31(12):2855–2867. doi: 10.1105/tpc.18.00973

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

Zhanhong Ren a,1,✉, Yan Zhang b,1, Yi Zhang a, Yunqiu He a, Pingzhou Du a,1,✉, Zhanxin Wang a, Fei Sun b,c,d,2, Haiyun Ren a,2
PMCID: PMC6925000  PMID: 31628168

Our cryo-EM structural data, together with the single-molecule magnetic tweezers analysis, reveal that the plant actin filament from Zea mays pollen is more structurally stable than the rabbit skeletal muscle actin filament.

Abstract

Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures that perform 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, we report a 3.9 Å structure of the plant actin filament from Zea mays pollen (ZMPA) using cryo-electron microscopy. The structure 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 loop bends 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 ZMPA filament can resist a greater stretching force than the RSMA filament. Overall, these data provide evidence that plant actin filaments have greater stability than animal actin filaments, which might be important to their role as tracks for long-distance vesicle and organelle transportation.

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INTRODUCTION

The actin cytoskeleton plays vital roles in many fundamental processes including vesicle and organelle transportation, endo- and exocytosis, and cell division and growth (Fu, 2015; Breuer et al., 2017; Li et al., 2018; Romarowski et al., 2018; Szymanski and Staiger, 2018; Takatsuka et al., 2018; Uraji et al., 2018). Actin exists in two states in vivo: globular actin (G-actin) and filamentous actin (F-actin), which are subject to a dynamic equilibrium of polymerization and depolymerization. In most instances, F-actin is the functional form of actin proteins. Thus, studying the structure of F-actin is of particular importance for understanding its functional mechanism. Recently, the evolution of cryo-electron microscopy (cryo-EM) technology has enabled the determination of filamentous structures of rabbit skeletal muscle actin (RSMA) in different nucleotide states with resolution ranging from 3.3 Å to 4.7 Å and 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 eukaryotic F-actins, high-resolution cryo-EM structures of bacterial and archaeal actin-like filaments have also been revealed in the last few years, including the 3.6 Å resolution structure of MamK filaments from magnetotactic bacteria (Löwe et al., 2016), the 3.4 Å resolution structure of AlfA filaments from Bacillus subtilis (Szewczak-Harris and Löwe, 2018), and the 3.8 Å resolution structure of Pyrobaculum calidifontis crenactin filaments (Izoré et al., 2016).

Despite the high protein sequence identity between plant and animal actins (Kandasamy et al., 2012), their biochemical activities and cellular functions are different (Ren et al., 1997; Jing et al., 2003; Kandasamy et al., 2012; Rula et al., 2018). However, the structural basis accounting for these differences remains poorly understood, largely because none of the plant F-actin structures have been resolved.

Here, we report a 3.9 Å resolution structure of Zea mays pollen actin (ZMPA) filaments determined by cryo-EM and the rupture forces of actin filaments measured by single-molecule magnetic tweezers. Our structural data show that the ZMPA filament resembles jasplakinolide- or beryllium fluoride (BeFx)-stabilized mammalian actin filament, implying that plant actin filaments have enhanced stability. Furthermore, the recorded rupture events of actin filaments confirm that the ZMPA filament has greater mechanical stability than RSMA.

RESULTS AND DISCUSSION

Overall Structure

To determine the structure of plant actin filaments, we obtained highly purified proteins of Z. mays (maize) pollen actin by taking advantage of the high binding affinity between actin and profilin and the ability of the actin-profilin complex to bind a poly-L-Pro column (Ren et al. 1997; Supplemental Figure 1A) . Protein mass spectrometry analysis revealed that the ZMPA samples contained five actin isoforms with ∼98% protein sequence identity (Supplemental Figures 1B and 1C). The ZMPA samples were subsequently polymerized into long and straight filaments in vitro and applied to structural studies by cryo-EM.

ZMPA filaments were highly contrasted to show the double-helical nature of the filaments (Supplemental Figures 2A and 2B). A cryo-EM dataset was collected, and the structure of the ZMPA filament was reconstructed using a real-space helical reconstruction approach (Figure 1A; Supplemental Movie 1; Supplemental Movie Legends; Supplemental Files 1 and 2). ZMPA filaments existed as a two-stranded structure composed of staggered actin subunits, with a refined helical symmetry with –166.77° rotation and 27.5 Å rise per subunit, resembling the structures of RSMA and jasplakinolide-stabilized RSMA (JASP-RSMA) filaments (Figures 1A and 1B; Galkin et al., 2015; Merino et al., 2018; Chou and Pollard, 2019). The final 3D reconstruction of ZMPA filaments had an overall resolution of 3.9 Å, using Fourier shell correlation (FSC) = 0.143 gold-standard criterion (Rosenthal and Henderson, 2003; Figures 1C and 1D). This resolution enabled us to build a pseudo-atomic model of the ZMPA filament with accuracy at the backbone level. A few charged residues (e.g. K115, E197, D246, E272, and D290) lacked clear side chain density, which might be due to potential radiation damage or to the intrinsic flexibilities of those residues.

Figure 1.

Figure 1.

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Cryo-EM Structure of ZMPA Filaments.

(A) Stereo view of 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) and hydrophobic 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 (black line), and the other is between the model and the map (red line).

(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 β-sheet in SD3 and α-helix in SD4 of SU-A.

The structure of the ZMPA subunit comprises four subdomains (SDs), SD1–SD4, and the D-loop in SD2 and hydrophobic plug in SD4 (Figure 1B). The ADP bound in the catalytic pocket, the β-sheet in SD3, and the α-helix in SD4 of the ZMPA subunit are clearly resolved, and side-chain densities for the bulkier residues are visible (Figures 1E to 1G). When amino acid differences were overlaid onto the structure of the ZMPA subunit, the sequence divergences among the five ZMPA isoforms were mainly located in SD1 and SD4 but not in the regions important for nucleotide binding or for intersubunit interactions that involve SD2 and the hydrophobic groove (Supplemental Figure 2C). In addition, the nucleotide binding pockets are highly conserved among the structures of ZMPA, PfAct1, and RSMA filaments (Supplemental Figure 3).

Intersubunit Interactions

Similar to other actin filaments, the ZMPA filament is stabilized by interactions between actin subunits of the same strand (intrastrand) and the opposing strand (interstrand). Intra- and interstrand interaction sites are involved in the longitudinal and lateral interfaces, respectively (Supplemental Table 1). The internal interface in the ZMPA filament covers similar areas to those in mammalian actin and JASP-PfAct1 filaments, according to analysis using the Proteins, Interfaces, Surfaces, and Assemblies server (http://www.ebi.ac.uk/pdbe/pisa/; Supplemental Figure 4; Krissinel and Henrick, 2007). The longitudinal interface is formed mainly between SD3 of one actin subunit and SD2 and SD4 of the next actin subunit of the same strand (Figure 2). Similar to the situations in RSMA and JASP-PfAct1 filaments (von der Ecken et al., 2015; Pospich et al., 2017), there are two similar lock-and-key hydrophobic interaction sites in ZMPA filaments. At the first hydrophobic contact site, the major hydrophobic patch in the D-loop of ZMPA filaments interacts with the hydrophobic groove on the adjacent actin subunit and encloses Y171 in SD3 of the neighboring actin subunit (Figures 2A to 2C). The second contact site consists of V289 in SD3 and the hydrophobic pocket in SD4 of the next subunit on the same strand (Figure 2E). Two potential salt bridges between D290 of SD3 and R64 of SD2 and between R292 of SD3 and D246 of SD4 form an intrastrand connection between two neighboring subunits (Figures 2D and 2F). These two electrostatic interaction sites were also reported to be highly conserved in the structures of RSMA (von der Ecken et al., 2015) and JASP-PfAct1 filaments (PDB ID code: 5OGW). Previous amino acid mutational analyses and the results of Mical-mediated oxidation modification showed that the intrastrand interactions are important for actin polymerization and stability (Wertman et al., 1992; Murakami et al., 2010; Hung et al., 2011; Grintsevich et al., 2017). Thus, the abovementioned potential salt bridges in the intrastrand interface of F-actin, together with the hydrophobic interactions, may provide structural support for ZMPA polymerization.

Figure 2.

Figure 2.

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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. Two important 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-B and SU-D are displayed as surface and ribbon representations, respectively. Protein residues are in stick representation colored by element in 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-handed rotation 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 the neighboring F-actin subunit in ZMPA filaments. The D-loop encloses Tyr-171 of the adjacent subunit.

(C) and (E) SU-B and SU-D are displayed as ribbon and surface representations, respectively. Protein residues are in stick representation colored by element in 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 interface formed 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-actin subunit 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 the neighboring 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.

In addition to the intrastrand contacts, the interstrand interactions are also important for actin polymerization and stability (Wertman et al., 1992; Pospich et al., 2017). As in RSMA filaments, there is a hydrophobic contact site between residues 196 to 203 of SD4 and SD1 of the adjacent subunit in the opposite strand of the ZMPA filament (Figure 3A). In addition, two potential electrostatic contacts 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 contact site is formed by the interstrand interaction of the positively charged residue R41 of the D-loop with the negatively charged residue 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 would destabilize the hydrophobic and electrostatic interstrand interactions of the PfAct1 filament and that PfAct1 mutants could polymerize into long filaments in vitro only in the presence of JASP, an actin-filament-stabilizing agent (Pospich et al., 2017). Our observation of the interstrand interactions is therefore consistent with the effective polymerization of ZMPA monomers into long actin filamentsin vitro.

Figure 3.

Figure 3.

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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) Surfaces are colored from high (yellow) to low (white) hydrophobicity. Zoom-in display of the 90° left-handed rotation of the interface formed by SU-C and SU-D using the longitudinal axis of rotation. Protein residues are in stick representation colored by element in gray for carbon and red for oxygen atoms. The interstrand 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) The potential electrostatic interactions are formed in the interstrand interface. The backbones of the charged residues involved in the interactions are shown as red (negative charge) and deep sky blue (positive charge) spheres.

The D-Loop/Hydrophobic Groove Interface

It has been well documented that the D-loop is one of the major regions 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; Oztug Durer 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 that of ZMPA (Supplemental Figure 1C). By contrast to that of JASP-PfAct1 (Pospich et al., 2017) and of RSMA filaments in the ADP state (Merino et al., 2018), the D-loop of ZMPA in the ADP state shifts outward with respect to the filament axis (Figures 4A to 4C). Interestingly, the conformation of the D-loop in ZMPA resembles that in JASP-RSMA and that in RSMA bound to beryllium fluoride (ADP-BeFx; Figures 4C to 4E and 5; Supplemental Movie 2; Supplemental Movie Legends). Previous studies showed that JASP-RSMA and RSMA bound to ADP-BeFx are more structurally stable than the form bound to ADP alone (Isambert et al., 1995; Visegrády et al., 2004; Kardos et al., 2007), while the prominent structural difference is that the former structures have an open D-loop and the latter has a closed D-loop (Merino et al., 2018), implying a possible association between the D-loop conformation and filament stability. Therefore, our data suggest that the ZMPA filament in the ADP state may be more stable and rigid than RSMA and JASP-PfAct1 filaments in the same nucleotide state. We noted that Chou and Pollard (2019) reported closed conformations of the actin filament with AMPPNP or ADP-Pi. This may indicate that a small difference between ATP analogs can greatly change the D-loop conformation. Although the ADP-Pi state is very stable (Fujiwara et al., 2007), the ratio of the open conformation is smaller in the case of ADP-Pi than that of ADP-BeFx (Merino et al., 2018). Therefore, besides the open conformation being the main factor of the stability, other factors would contribute, which merits further studies.

Figure 4.

Figure 4.

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The ZMPA Filament Shows an Open D-Loop Conformation.

(A) to (E) Visualization of actin subunit B (SU-B), subunit D (SU-D), and their corresponding densities in longitudinal interfaces of JASP-PfAct1, PDB ID code: 5OGW (A); RSMA, PDB ID code: 5ONV (B); ZMPA (C); JASP-RSMA, PDB ID code: 5OOC (D); and RSMA filaments bound to ADP-BeFx, PDB ID code: 5OOF (E). The jasplakinolide and its corresponding density are not shown in (A) and (D). SU-B and SU-D of JASP-PfAct1 are shown in orange and magenta, those of RSMA in cyan and dark gray, those of ZMPA in green and blue, those of JASP-RSMA in orchid and turquoise, and those of RSMA bound to ADP-BeFx in salmon 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 farther outward 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 the D-loop and the C-terminal tail of the neighboring actin subunit in the ZMPA filament (indicated by the red ellipse; C). The state of the D-loop in the ZMPA filament is similar to that in the JASP-RSMA filament (D) and in the RSMA filament bound to ADP-BeFx (E).

Figure 5.

Figure 5.

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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 open D-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.

To test the mechanical stability of actin filaments, we stretched a single F-actin using single-molecule magnetic tweezers (Figure 6A), a powerful tool to assess the mechanical characteristics of single biomolecules in vitro (Strick et al., 1996; Hinterdorfer et al., 2009; Yu et al., 2017; Chen et al., 2018). When the stretching force increased, the F-actin was elongated until it was broken (Figure 6B). Because the interaction between biotin and streptavidin (Tsai et al., 2016) and that between actin antibody and actin are much stronger than the maximum stretching force (90 picoNewton [pN]) applied to F-actin (Supplemental Movies 3 and 4; Supplemental Movie Legends), the breakage of a single tether within the measurement range of our study reflects the disruption of a single F-actin under tension. The disruption force of the ZMPA filament is centered at ∼37.8 ± 2.2 pN, which is significantly greater than that of the RSMA filament (26.5 ± 1.8 pN, mean ± se; Figure 6C; Supplemental Movies 5 and 6; Supplemental Movie Legends). This result indicates that the ZMPA filament is more stable than the RSMA filament. The rupture force of a single RSMA filament measured in our study is relatively small compared with the values previously reported by Kishino and Yanagida (1988), Tsuda et al. (1996), and Liu and Pollack (2002). It may result from the fact that the stretched actin filaments in previous studies were stabilized with phalloidin but those in our study were not. A previous study showed that phalloidin binds to the intersubunit interfaces of the actin filament and couples neighboring actin subunits together (Mentes et al., 2018). The stoichiometry of binding is one phalloidin per actin subunit, and phalloidin can enhance F-actin stability (Visegrády et al., 2004; Mentes et al., 2018). Besides, the results of our stretching experiments on F-actin seeds (Supplemental Movies 3 and 4; Supplemental Movie Legends) show that the interaction strength between the neighboring actin subunits stabilized by phalloidin is >90 pN, which is used as the maximum stretching force in our study. In addition, stretching systems may also contribute to the difference of rupture force measured in the previous and our studies. The rupture force reported by Kishino and Yanagida (1988) is approximately one fourth of that by Tsuda et al. (1996) for the actin filament using the same microneedle method. It was thought that the former did not consider the randomizing effect of the rotational Brownian motion and had system errors in the calibration of needles (Tsuda et al., 1996). The actin filament is pulled continuously in the microneedle method, similar to that in optical tweezers. In optical tweezers, a laser is focused on dielectric beads. The beads experience a 3D restoring 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 not achieve equilibrium. For magnetic tweezers, the tension exerted on the samples is dependent on the position of the magnets. In our measurements, the magnets move step by step toward the sample. At each step, a constant force is exerted on the filament and the system achieves equilibrium. Therefore, in contrast with the stepwise pulling used in the magnetic tweezers method, the continuous pulling in the optical tweezers method causes the system to deviate from equilibrium and a greater rupture force may be measured (Dhakal et al., 2013; You et al., 2014).

Figure 6.

Figure 6.

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The ZMPA Filament Resists a Greater Stretching Force than RSMA.

(A) Schematic setup of the magnetic tweezers used in the study (not to scale). The anti-actin-coated magnetic bead binds to the actin filament that is tethered to a coverslip through interactions between the preformed biotin-labeled F-actin seed (stabilized with phalloidin) and streptavidin, which forms a single tether. 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 of a 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 the disruption force of RSMA and ZMPA filaments. The averaged disruption force of the RSMA filament is 26.5 ± 1.8 pN (mean ± se) and that of the ZMPA filament is 37.8 ± 2.2 pN (mean ± se). ***P value < 0.001, as determined by a two-tailed Student’s t test. n = 75 for RSMA and n = 50 for ZMPA.

The conformation of the D-loop implies that the interaction between the D-loop and SD1 of the adjacent actin subunit may be strengthened in the ZMPA filament. Indeed, there is a clear density connecting the D-loop and the C-terminal tail of the neighboring intrastrand subunit of ZMPA, while the corresponding density is absent in the JASP-PfAct1 and RSMA filaments in the ADP state (Figures 4A to 4C). In addition, the D-loop/hydrophobic groove interface is an important recognition site for the binding of many actin binding proteins to F-actin (von der Ecken et al., 2016; Merino et al., 2018). The previously reported complex structure of mammalian actomyosin shows that the helix–loop–helix motif of myosin enters the hydrophobic groove on the adjacent actin subunit and contacts the D-loop (von der Ecken et al., 2016; Fujii and Namba, 2017; Mentes et al., 2018), illustrating that myosin could directly sense the changes occurring in the D-loop/hydrophobic groove interface. Our model shows that the D-loop/hydrophobic groove interface of ZMPA in the ADP state is different from that of RSMA in the same state (Figures 4B and 4C; Supplemental Figures 5A and 5C). This difference may account for the different interaction modes with myosin and other actin binding proteins that bind to the D-loop/hydrophobic groove interface. This result is consistent with a recent report that the measurements of enzymatic and motile activities of Arabidopsis (Arabidopsis thaliana) myosins using Arabidopsis actins were distinct from those measured using animal skeletal muscle α-actin (Rula et al., 2018). These abovementioned characteristics are important for plant actin filaments, as they need to serve as tracks for long-distance vesicle and organelle transportation processes that generate extensive tensions, which is different from the microtubule-centric cargo transportation systems in mammalian cells (Hammer and Sellers, 2011; Brandizzi and Wasteneys, 2013).

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

METHODS

Purification and Polymerization of ZMPA

Plant actin was purified from pollen collected from maize (Zea mays) plants during July and August 2016 and July and August 2017 and then stored at −80°C. The cultivar of maize plants was Longping No. 206. Maize pollen actin was prepared as described in Ren et al. (1997). Purified pollen actin in buffer 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 (∼100 μL), flash-frozen in liquid nitrogen, and stored at −80°C. The polymerization of freshly frozen plant actin was tested. The plant actin was dialyzed against buffer G with pH 7.0 overnight to change from alkaline pH to neutral (Sampath and Pollard, 1991) and was polymerized at a final concentration of 9.5 μM by the addition of 10× 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) at room temperature (25°C) for 4 h. To minimize the effect of nonpolymerizable actin on the EM studies and mass spectrometry analysis, the polymerized plant actin filaments were spun down (100,000g at 4°C) and gently suspended in buffer G (pH 7.0) with 1× KMEI. The protein concentration was determined with Bradford reagent (Bio-Rad), using BSA as a standard.

MS Analysis of ZMPA

MS analysis was performed by the Beijing Huada Protein R&D Center. The identities of the plant actin filaments were revealed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The Q Exactive mass spectrometry data (Thermo Fisher Scientific) were searched against the UniProt maize database using 15 μL/L peptide mass tolerance and 20 millimass units (mmu) fragment mass tolerance.

Grid Preparation and Image Acquisition for Cryo-EM

ZMPA filaments (3 μL) were applied to glow-discharged GIG holey carbon grids (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 (Thermo Fisher Scientific) with a blotting time of 5.5 s and blotting force of level 2 at 100% humidity, 25°C. Screening for sample and blotting conditions was performed on a Talos F200C 200-kV electron microscope equipped with a 4K×4K Ceta camera (Thermo Fisher Scientific). Finally, micrographs of plant actin filaments for structure determination were collected on a direct electron device (model no. K2 camera; Gatan) in a 300-kV Titan Krios Electron Microscope (Thermo Fisher Scientific) using the automated acquisition software SerialEM (Mastronarde, 2005). A total of 3,605 micrographs were recorded at a calibrated pixel size of 1.063 Å with a total dose of ∼39 e−/Å2 and a defocus range from 1.2 to 2.0 μm.

Image Processing of the Cryo-EM Data Set

In total, 3,605 micrograph movie stacks were collected during four sessions of microscopy data collection hours. Motion corrections and defocus estimations for all these micrographs were performed using the softwares MotionCorr2 (Zheng et al., 2017) and GCTF (Zhang, 2016), respectively. Micrographs with ice contamination, poor Thon rings, large defocus values and other defects, were excluded before filament boxing. A total of 1,540 micrographs were finally selected. These selected micrographs were multiplied by their theoretical contrast transfer function (CTF) for initial correction of CTF (Galkin et al., 2015). A total of 8,609 ZMPA filaments were boxed 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 segments were generated with a box size of 384, and 2D classification was calculated to check the diameter distribution and data quality by the program RELION 2.0 (Scheres, 2012). For the 2D class averages without any downsize or binning, the secondary structures could be observed from some of the 2D class averages (Supplemental Figure 2B). Helical parameters were calculated by indexing the layer lines of the Fourier transform of the ZMPA filament. An initial helical rise of 27.5 Å and twist of −166.77° were obtained and used as the initial helical parameters for helical reconstruction. We used 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 included to obtain a 3.9 Å map (Figure 1). The helical parameters converged to 27.5 Å for the helical rise and −166.77° for the helical twist. We divided CTF2 to correct CTF as we had already multiplied CTF at the beginning. We used the software SPIDER (Shaikh et al., 2008) to perform postprocessing (B-sharping). The gold-standard FSC curve was calculated by dividing the particles into halves at the beginning. The local resolution was estimated using the program ResMap (Kucukelbir et al., 2014).

Model Building and Refinement of ZMPA Filaments

We used the model of actin from Oryctolagus cuniculus (Merino et al., 2018; PDB ID code: 5OOC) as a starting model, and first fitted it into the map with the program Chimera as a rigid body (Pettersen et al., 2004). Then, residues were mutated to those in the ZMPA sequence (UniProt ID code: B6TQ08), and these poorly fitted regions were adjusted manually and refined with the software Coot (Emsley et al., 2010). Further refinement was conducted with the program Phenix (Adams et al., 2011). The geometry parameters for the final model are presented in Supplemental Table 2.

Single-Molecule Magnetic Tweezers Analysis

The basic principle of magnetic tweezers was described previously by Hinterdorfer et al. (2009) and Zlatanova and Leuba (2003). The experimental setup of our single-molecule magnetic tweezers was constructed in-house, as described previously by Chen et al. (2018). In our study, two ends of a single F-actin were bound to a streptavidin-coated coverslip and an anti-actin-coated superparamagnetic bead (Dynabead M-270 Epoxy; Invitrogen) via the biochemical reactions between biotin and streptavidin and the bonds between actin and anti-actin antibody, respectively, as shown in Figure 6A. Magnetic tweezers were used to stretch the actin filament vertically by the application of a horizontal magnetic field generated by a pair of magnets (small NdFeB magnets with a distance of 0.5 mm) to a superparamagnetic bead to which the actin filament was attached. The superparamagnetic bead was illuminated by a parallel light placed above the magnets. The interference of the illuminating light with the light scattered by the superparamagnetic bead produced concentric diffraction rings in the focal plane of the objective placed below the flow cell. The image of the diffraction pattern was recorded through a microscope objective (oil immersion type, 60×1.4; Olympus) with a Giga-Ethernet charge-coupled device camera (AVT) at 200 Hz. The real-time position (x, y, z) of the bead at various forces was recorded by comparing the diffraction pattern of the bead with calibration images at various distances from the focal point of the objective. The motion of the superparamagnetic bead tethered to the coverslip with a linear bio-molecule can be described as an inverse pendulum with a tension in the z direction. The fluctuations of the bead in the x-y plane are related to the applied force by F = kBTl/⟨δx2⟩ according to the equipartition theorem, where l is the average end-to-end extension of the molecule, kB is the Boltzmann constant, T is the absolute temperature of the environment, and δx is fluctuation of the bead in the x direction (Yan et al., 2004). The force exerted on the sample increases in a mono-exponential relationship with the magnet’s position in the z direction. Before the real measurements for actin filaments were made, the force calibration was performed. Using a well-studied DNA tether (lambda DNA), the positions of the bead in the x-y plane at each magnet position was recorded and the tensions were calculated according to F = kBTl/⟨δx2⟩. The relationship between tension and magnet position was obtained by fitting the tension and magnet position with the mono-exponential relationship. In the measurement of actin filaments, the tension was read out directly based on the fitted mono-exponential relationship in the software LabView (http://www.ni.com/zh-cn/shop/labview/labview-details.html) according to the magnet’s position. In the stretching measurements of actin filament, the measurement was initiated when the magnets were 3 mm away 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 mm from the surface of the flow cell finally with a step size of 0.02 mm, staying for 2 s at each step, and the extension of the sample was traced. The corresponding stretching force on the single tether was thereby increased from 0 to ∼90 pN exponentially. The tension was read out by the software LabView.

Stretching Measurement of a Single F-Actin

To anchor the F-actin, the surface of the coverslip needs to be functionalized. First, the coverslip was cleaned by sonicating multiple times (1 h in detergent, 1 h in ethanol, 2 h with piranha solution, and 10 min in deionized water). The coverslip was then incubated with 2-mg/mL methoxy-PEG-silane (Mw 5,000; Laysan Bio) and 2-μg/mL Biotin-PEG-silane (Mw 3,400; Laysan Bio) in 95% (v/v) ethanol (pH 2.0) at 70°C overnight. The reference beads (2-µm polystyrene beads; QDSphere) were pipetted onto the coverslip and placed on a hot plate at 150°C for 10 min to melt onto the coverslip. Second, the coverslip was made to be part of the flow cell and then 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 10 mM of phosphate buffer at pH 7.4) overnight at 37°C to limit the nonspecific interaction between the actin sample and the coverslip. After streptavidin treatment, the coverslip was washed with 1× KMEI buffer (50 mM of KCl, 1 mM of MgCl2, 1 mM of EGTA, and 10 mM of imidazole at pH 7.0) to remove free streptavidin, and then incubated with the preformed biotin-labeled F-actin seeds (stabilized with phalloidin) for 5 min to allow seeds to bind to it via the interactions between biotin and streptavidin. After the removal of free seeds by washing the coverslip with the instant mixed G-actin solution of 0.15 μM of G-actin in buffer G (5 mM of Tris-HCl at pH 7.0, 0.2 mM of CaCl2, 0.01% [w/v] of NaN3, 0.2 mM of ATP, and 0.5 mM of DTT) and 1× KMEI buffer, the instant mixed G-actin solution of 0.75 μM of G-actin in buffer G and 1× KMEI buffer was flowed into the channel for actin polymerization and elongation from the barbed ends of immobilized seeds. After actin polymerization for 6 min, the instant mixed solution of anti-actin–coated superparamagnetic beads, 0.15 μM of G-actin in buffer G and 1× KMEI buffer were gently injected into the flow cell, and incubated for 10 min to allow the beads to bind to the F-actin. Finally, the free beads were removed by washing the coverslip gently with the instant mixed solution of 0.15 μM of G-actin in buffer G and 1× KMEI buffer. To prevent the depolymerization of F-actin, the solution used to wash the channel should contain a low concentration of G-actin (slightly higher than the critical concentration of actin polymerization). The anti-rabbit α-actin mouse monoclonal antibody (Lot no. D1514; Santa Cruz Biotechnology) was coated on the superparamagnetic beads used for the stretching measurement of the single RSMA filament and anti-plant actin antibody (Lot no. 492,635,537; Sigma-Aldrich) for that of the single ZMPA filament by referring to the protocol of Dynabeads Antibody Coupling Kit (Invitrogen).

The breakage of a single tether resulted in the target superparamagnetic bead moving suddenly out of the viewing area. To ensure that the single F-actin is tethered between beads and the coverslip, the magnets were rotated 50 turns before the measurements were taken. Those beads with a single filament rotated freely and were chosen for measurements, while those with more than one filament did not rotate freely and were not considered. If multiple actin filaments are anchored between the coverslip and the superparamagnetic bead, the actin filaments will be tangled when we rotate the magnets and the bead cannot rotate accordingly, which helps us to discriminate the bead with a single actin filament anchored. Finally, the breaking force of a single tether was measured and defined as the disruption force of a single F-actin in our study. All measurements were performed at 25°C.

Figure Preparation

Multiple sequence alignments of the actin isoforms were performed using the software Clustal Omega (Sievers et al., 2011). Figures were prepared using the softwares ResMap (Kucukelbir et al., 2014) and Chimera (Pettersen et al., 2004), the PyMOL Molecular Graphics System, Version 2.0 (Schrödinger; http://www.pymol.org/), and Adobe Illustrator CC.

Statistical Analysis

Statistical significance was analyzed by unpaired Student’s t test for two-group data. The P value is shown in Supplemental Table 3. The sample size and the significance level of the P value are described in the figure legend of Figure 6C.

Accession Numbers

Sequence data from this article can be found in the UniProt database with accession codes of α-actin (P68135), PfAct1 (P86287), and five ZMPA isoforms (B6TQ08, B4FRH8, C0HDZ6, B6SI11, and B4F989). Cryo-EM maps of ZMPA filaments have been deposited into the Electron Microscopy Data Bank (accession code EMD-9734) and the corresponding atomic coordinates have been deposited into the Protein DataBank (accession code 6IUG).

Supplemental Data

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

Supplemental Movie Legends. Legends for Supplemental Movies 1 to 6.

DIVE Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

We thank Edward H. Egelman from the University of Virginia for his generous help with the data analysis for the project. The isolation and purification of ZMPA and structural analysis was performed in H.Y.R.’s lab and the work for image processing, model building, and refinement was performed in F.S.’s and Edward H. Egelman’s labs. We thank Yun Xiang, Prof. Li Zhu, Bin Yuan, and Dr. Xia Deng from Lanzhou University for their support and advice for this research program. Cryo-EM sample preparation and data collection were carried out at the Center for Biological Imaging (http://cbi.ibp.ac.cn), Core Facilities for Protein Science, at the Institute of Biophysics, Chinese Academy of Sciences. We thank Xiaojun Huang, Wei Ding, Boling Zhu, Tongxin Niu, and other staff members at the Center for Biological Imaging for their support during data collection and image processing. We thank Wei Li from the Institute of Physics at the Chinese Academy of Sciences for his advice on the single-molecule magnetic tweezers analysis. This work was supported by the National Natural Science Foundation of China (grants 91854206 and 31770206 to H.Y.R., and 31770794 to Yan Z.) and the Ministry of Science and Technology of China (grant 2013CB126902 to H.Y.R. and 2017YFA0504700 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 from maize pollen; Z.H.R. performed cryo-EM sample preparation, data collection, and preliminary data processing; Yan Z. performed image processing, model building, and refinement; Z.H.R., F.S., and H.Y.R. analyzed the structure; Z.H.R. performed the single-molecule magnetic tweezers analysis; Z.H.R. prepared the figures, tables, and video of this article; Z.X.W. helped with the structural analysis and provided suggestions for the revision of figures; article was written and revised by Z.H.R., Yan Z., Yi Z., H.Y.R., and F.S.

Footnotes

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