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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
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
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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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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
This information is current as of December 22, 2020
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