Cryo-EM Structure of Actin Filaments from Zea …...2019/10/18 · BREAKTHROUGH REPORT Cryo-EM...
Transcript of Cryo-EM Structure of Actin Filaments from Zea …...2019/10/18 · BREAKTHROUGH REPORT Cryo-EM...
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BREAKTHROUGH REPORT
Cryo-EM Structure of Actin Filaments from Zea mays Pollen Zhanhong Ren1,#, Yan Zhang2,#, Yi Zhang1, Yunqiu He1, Pingzhou Du1, Zhanxin Wang1, Fei Sun2,3,4, * & Haiyun Ren1,*
1Key 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, China. 2National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. 3University of Chinese Academy of Sciences, Beijing, China. 4Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. #These authors contributed equally to this work. *Address correspondence to [email protected] or [email protected].
Short title: Structure of plant actin filaments
One-sentence summary: 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.
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Fei Sun ([email protected]) and Haiyun Ren ([email protected]).
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 is 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 strands running parallel to each other) 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 (D-loop) bends further 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 for their roles as tracks for long-distance vesicle and organelle transportation.
Key Words: Cryo-electron microscopy; Structure of plant actin filaments; D-loop conformation; Single-molecule magnetic tweezers; F-actin stability.
Plant Cell Advance Publication. Published on October 18, 2019, doi:10.1105/tpc.18.00973
©2019 American Society of Plant Biologists. All Rights Reserved
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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 (Breuer et al., 2017; Takatsuka et al., 2018;
Szymanski and Staiger, 2018; Fu, 2015; Li et al., 2018; Romarowski et al., 2018; Uraji et al., 2018). Actin exists
as 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 RSMA in different nucleotide states with resolution ranging from 3.3
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 (Lowe et al., 2016), the 3.4 Å resolution structure of
AlfA filaments from Bacillus subtilis (Szewczak-Harris and Lowe, 2018) and the 3.8 Å resolution structure of
Pyrobaculum calidifontis crenactin filaments (Izore 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 (Jing et al., 2003; Ren et al., 1997; 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 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.
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RESULTS AND DISCUSSION
Overall Structure
To determine the structure of plant actin filaments, we obtained highly purified proteins of Zea 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-proline column (Supplemental Figure 1A; Reference 7). Protein mass
spectrometry analysis revealed that the ZMPA samples contained five actin isoforms with approximately 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 Files 1 and 2). ZMPA filaments existed as a two-stranded structure composed of staggered actin
subunits, with a refined helical symmetry of –166.77° rotation and 27.5 Å rise per subunit, resembling those of
RSMA and jasplakinolide-stabilized RSMA (JASP-RSMA) filaments (Figures 1A and 1B; References 1,2,4).
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). The
current resolution enabled us to build a pseudo-atomic model of the ZMPA filament with accuracy in the
backbone level. A few charged residues (e.g., K115, E197, D246, E272, and D290) lack a clear side chain
density, which might be due to potential radiation damage or to the intrinsic 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 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–G). 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).
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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 actin filament covers similar areas to those in mammalian actin and JASP-PfAct1 filaments,
according to analysis using the PISA (Proteins, Interfaces, Surfaces and Assemblies) server
(http://www.ebi.ac.uk/pdbe/pisa/) (Krissinel and Henrick, 2007) (Supplemental Figure 4). 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–C). 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 result of Mical-mediated oxidation modification showed that
the intrastrand interactions are important for actin polymerization and stability (Murakami et al., 2010; Wertman
et al., 1992; 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.
In addition to the intrastrand contacts, the interstrand interactions are also important for actin polymerization
and stability (Pospich et al., 2017; Wertman et al., 1992). As in RSMA filaments, there is also a hydrophobic
contact site between residues 196–203 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
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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 of 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 filaments in vitro.
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;
Galkin et al., 2015; von der Ecken et al., 2015; Pospich et al., 2017; Grintsevich et al., 2017; Oztug Durer et al.,
2010). Compared to the D-loop (residues 39–53) of RSMA, there are two residue substitutions (glutamine to
threonine 43 and serine to alanine 54) in that of ZMPA (Supplemental Figure 1C). In contrast to that of
jasplakinolide-stabilized 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–4C).
Interestingly, the conformation of the D-loop in ZMPA resembles that in JASP-RSMA and that in RSMA
bound to ADP-BeFx (Beryllium fluoride) (Figures 4C–4E; Figure 5; Supplemental Movie 2). Previous studies
showed that JASP-RSMA and RSMA bound to ADP-BeFx are more structurally stable than that in the ADP
state (Visegrady et al., 2004; Kardos et al., 2007; Isambert et al., 1995), 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 reported closed
conformations of the actin filament with AMPPNP or ADP-Pi (Chou and Pollard, 2019). This may indicate that
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.
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To test the mechanical stability of actin filaments, we stretched a single F-actin using single-molecule
magnetic tweezers (Figure 6A), which is a powerful tool to assess the mechanical characteristics of single
biomolecules in vitro (Hinterdorfer et al., 2009; Yu et al., 2017; Chen et al., 2018; Strick et al., 1996). When the
stretching force increased, the F-actin was elongated until it was broken (Figure 6B). Since 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 pN (pico-Newton)) applied to F-actin (Supplemental Movies 3 and 4),
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 approximately 37.8 ± 2.2 pN,
which is significantly stronger than that of the RSMA filament (26.5 ± 1.8 pN, mean ± SE) (Supplemental
Movies 5 and 6; Figure 6C). The 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 (Kishino and Yanagida, 1988; Tsuda et al., 1996; Liu and Pollack, 2002). It may
result from the fact that the stretched actin filaments in previous studies were stablized with phalloidin but those
in our study were not. A previous study shows that phalloidin binds to the intersubunit interfaces of the actin
filament and couples neighboring actin subunits (Mentes et al., 2018). The stoichiometry of binding is one
phalloidin for one actin subunit and phalloidin can enhance F-actin stability (Visegrady et al., 2004; Mentes et
al., 2018). Besides, our results of stretching experiments of F-actin seeds (Supplemental Movie 3 and 4) show
that the interaction strength between the neighboring actin subunits stabilized by phalloidin is greater than 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 (Kishino and Yanagida, 1988) is about 1/4 of that by Tsuda (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 three-dimensional
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 to the sample. At each step, a constant force is exerted on the
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filament and the system achieves equilibrium. Therefore, in contrast to the stepwise pulling used in the
magnetic tweezers method, the continuous pulling in the optical tweezers method makes the system deviate
from equilibrium and the greater rupture force may be measured (Dhakal et al., 2013; You et al., 2014).
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-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
(Merino et al., 2018; von der Ecken et al., 2016). The previously reported complex structure of mammalian
actomyosin shows that the helix-loop-helix (HLH) motif of myosin enters the hydrophobic groove on the
adjacent actin subunit and contacts the D-loop (von der Ecken et al., 2016; Mentes et al., 2018; Fujii and
Namba, 2017), 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 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, the present structural and biochemical data suggest that ZMPA filaments are more stable than
RSMA filaments, which provides a possible explanation for their functional differences in cells.
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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℃. The cultivar of maize (Zea mays) plants was Longping No. 206.
Maize pollen actin was prepared as previously described (Ren et al., 1997). Purified pollen actin in buffer G (5
mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.01% NaN3, 0.2 mM ATP, and 0.5 mM DTT) was divided into small
aliquots (approximately 100 μL), flash-frozen in liquid nitrogen and stored at -80℃. 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 KCl, 10 mM MgCl2, 10 mM EGTA, and 100 mM imidazole, pH
7.0) (Neidt et al., 2008) at room temperature (25℃) for 4 h. To minimize the effect of nonpolymerizable actin
on the electron microscopy studies and mass spectrometry analysis, the polymerized plant actin filaments were
spun down (100,000 ×g at 4℃) 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.
Mass spectrometry analysis of ZMPA
Mass spectrometry analysis was performed by the Beijing Huada Protein R&D Center Co., Ltd. (Beijing, P.R.
China). 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 (Zea mays) database using 15 ppm peptide mass tolerance and 20 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 approximately 100 K using a semiautomatic plunge device (Thermo
Fisher Scientific Vitrobot Mark Ⅳ) with a blotting time of 5.5 s and blotting force of level 2 at 100% humidity,
25℃. 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 (Gatan K2 camera) in a 300-kV
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Titan Krios electron microscope (ThermoFisher 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 approximately 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 4 sessions of microscopy data collection hours.
Motion corrections and defocus estimations for all these micrographs were performed using 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 e2helixboxer.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 RELION 2.0 (Reference 9). 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 (Shaikh et al.,
2008) script 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 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 by 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 by 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 in Coot (Emsley et al., 2010). Further refinement was conducted with Phenix
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(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 in previous reports (Hinterdorfer et al., 2009;
Zlatanova and Leuba, 2003). The experimental setup of our single-molecule magnetic tweezers was constructed
in house, as described in the previous study (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 (DynabeadTM 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 (Olympus 60×1.4, oil immersion) with an AVT
Giga-Ethernet charge-coupled device (CCD) camera 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 carried
out. 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 LabView software according to the magnet’s position. In the stretching
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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 LabView software.
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% ethanol (pH 2.0) at 70℃ overnight. The reference
beads (2 µm polystyrene beads; QDSphere) were pipetted onto the coverslip and placed on a hot plate at 150℃
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 BSA, 1 mM EDTA, 10 mg/mL Pluronic® F-127 surfactant, 3 mM
NaN3, 10 mM phosphate buffer, pH 7.4) overnight at 37℃ to limit the nonspecific interaction between the actin
sample and the coverslip. Following streptavidin treatment, the coverslip was washed with 1× KMEI buffer (50
mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole, 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 G-actin in buffer G (5 mM Tris-HCl pH 7.0, 0.2
mM CaCl2, 0.01% NaN3, 0.2 mM ATP, and 0.5 mM DTT) and 1× KMEI buffer, the instant mixed G-actin
solution of 0.75 μM 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 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 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 (Santa Cruz; Lot #
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D1514) was coated on the superparamagnetic beads used for the stretching measurement of the single RSMA
filament and anti-plant actin antibody (Sigma-Aldrich; Lot # 492635537) for that of the single ZMPA filament
by referring to the protocol of DynabeadsTM 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 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 present study. All measurements
were carried out at 25ºC.
Figure Preparation
Multiple sequence alignments of the actin isoforms were performed using Clustal Omega (Sievers et al., 2011).
Figures were prepared using ResMap (Kucukelbir et al., 2014), Chimera (Pettersen et al., 2004), PyMOL
Molecular Graphics System, Version 2.0 Schrödinger, LLC (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 UniProt with accession codes of α-actin (P68135), PfAct1
(P86287), and five Zea mays pollen actin isoforms (B6TQ08, B4FRH8, C0HDZ6, B6SI11 and B4F989).
Cryo-EM maps of ZMPA filaments have been deposited into Electron Microscopy Data Bank (accession code,
EMD-9734) and the corresponding atomic coordinates have been deposited into Protein Data Bank (accession
code 6IUG).
13 / 18
Supplemental Data
Supplemental Figure 1. SDS-PAGE and LC-MS/MS (QE) results of ZMPA.
Supplemental Figure 2. Cryo-EM micrographs and analysis of ZMPA filaments.
Supplemental Figure 3. Comparison of the nucleotide-binding sites from various actin subunits.
Supplemental Figure 4. Longitudinal and lateral contacts of actin filaments.
Supplemental Figure 5. The hydrophobic groove in ZMPA filaments.
Supplemental Table 1. The residues predicted to be involved in intersubunit interactions.
Supplemental Table 2. Data collection and refinement statistics.
Supplemental Table 3. Statistical Analyses Results.
The following Supplemental Data Movies and were submitted to the Data Dryad 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-loop conformation.
Supplemental Movie 3. The binding strength between the anti-plant actin antibody and ZMPA.
Supplemental Movie 4. The binding strength between the anti-rabbit α-actin antibody and RSMA.
Supplemental Movie 5. The stretching measurement of a single ZMPA filament.
Supplemental Movie 6. The stretching measurement of a single RSMA filament.
Supplemental Movie Legends.
Supplemental File 1. Map of ZMPA filament.
Supplemental File 2. Validation report from wwPDB.
ACKNOWLEDGMENTS
We thank Prof. Edward H. Egelman from University of Virginia for his generous help with the data analysis for
the project. We thank Prof. 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 (CBI, http://cbi.ibp.ac.cn), Core Facilities for Protein Science,
at the Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS). We thank Dr. Xiaojun Huang, Wei
14 / 18
Ding, Boling Zhu, Tongxin Niu, and other staff members at the Center for Biological Imaging (CBI, CAS) for
their support during data collection and image processing. We thank Dr. Wei Li from Institute of Physics in
Chinese Academy of Sciences (CAS) for his advice on the single-molecule magnetic tweezers analysis. This
work was supported by grants from the National Natural Science Foundation of China (91854206 and
31770206 to H.Y.R.; 31770794 to Yan Z.) and from the Ministry of Science and Technology of China
(2013CB126902 to H.Y.R. and 2017YFA0504700 to F.S.).
AUTHOR CONTRIBUTIONS
H.Y.R. and Z.H.R. conceived and coordinated the project. The isolation and purification of ZMPA and structural
analysis was performed in Haiyun Ren’s lab and the work for image processing, model building and refinement
was performed in Fei Sun’s and Edward H. Egelman’s labs. 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 paper. Z.X.W. helped with the structural analysis and provided suggestions for
the revision of figures. The manuscript was written and revised by Z.H.R., Yan Z., Yi Z., H.Y.R. and F.S.
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Figure 1. Cryo-EM structure of ZMPA filaments.
(A) Stereo view of the three-dimensional (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 actin subunit A (SU-A) with its density. The four subdomains (SD1–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 subdomain 3 (SD3) and α helix in subdomain 4 (SD4) of SU-A.
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. Two important longitudinal interfaces will be formed by SU-B and subdomain 2 (SD2)
of SU-D (A–D) and by SU-B and subdomain 4 (SD4) of SU-D (E–F). (E–F) is a 180° right-handed rotation of (A–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) is an zoom-in display of
the interface formed by SU-B and D-loop of SU-D. (B) is a 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 tyrosine 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) is an enlarged display of
the same area as (A). (E) is an zoom-in display of 40° left-handed rotation of the interface formed by SU-D and
subdomain 3 (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) are the 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.
Figure 3. The interstrand contacts in ZMPA filaments.
(A–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. (A) is an zoom-in display of 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 196–203 in SD4 and SD1 of the neighboring
interstrand subunit C (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.
Figure 4. The ZMPA filament shows an open D-loop conformation.
(A–E) Visualization of actin subunit B (SU-B), subunit D (SU-D) and their corresponding densities in longitudinal
interfaces of the jasplakinolide-stabilized P. falciparum actin 1 (JASP-PfAct1; PDB ID code: 5OGW) (A), rabbit
skeletal muscle actin (RSMA; PDB ID code: 5ONV) (B), Zea mays pollen actin (ZMPA) (C), jasplakinolide-stabilized
rabbit skeletal muscle actin (JASP-RSMA; PDB ID code: 5OOC) (D), and rabbit skeletal muscle actin (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) 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). 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. 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.
Figure 6. 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 rabbit skeletal muscle actin (RSMA) (blue) and Zea mays pollen actin (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.
DOI 10.1105/tpc.18.00973; originally published online October 18, 2019;Plant Cell
Zhanhong Ren, Yan Zhang, Yi Zhang, Yunqiu He, Pingzhou Du, Zhanxin Wang, Fei Sun and Haiyun RenCryo-EM Structure of Actin Filaments from Zea mays Pollen
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