Faculty Profiles - KOJIMA, Seiji

Site-Directed Cross-Linking Between Bacterial Flagellar Motor Proteins In Vivo Reviewed

Hiroyuki Terashima, Michio Homma, Seiji Kojima

Methods in Molecular Biology Vol. 2646 page： 71 - 82 2023.2

Ring formation by Vibrio fusion protein composed of FliF and FliG, MS-ring and C-ring component of bacterial flagellar motor in membrane Open Access

Kanji Takahashi, Tatsuro Nishikino, Hiroki Kajino, Seiji Kojima, Takayuki Uchihashi, Michio Homma

BIOPHYSICS AND PHYSICOBIOLOGY Vol. 20 ( 2 ) page： e200028 2023

Language：English Publishing type：Research paper (scientific journal) Publisher：BIOPHYSICAL SOC JAPAN

The marine bacterium Vibrio alginolyticus has a single flagellum as a locomotory organ at the cell pole, which is rotated by the Na+-motive force to swim in a liquid. The base of the flagella has a motor composed of a stator and rotor, which serves as a power engine to generate torque through the rotor-stator interaction coupled to Na+ influx through the stator channel. The MS-ring, which is embedded in the membrane at the base of the flagella as part of the rotor, is the initial structure required for flagellum assembly. It comprises 34 molecules of the twotransmembrane protein FliF. FliG, FliM, and FliN form a C-ring just below the MS-ring. FliG is an important rotor protein that interacts with the stator PomA and directly contributes to force generation. We previously found that FliG promotes MS-ring formation in E. coli. In the present study, we constructed a fliF-fliG fusion gene, which encodes an approximately 100 kDa protein, and the successful production of this protein effectively formed the MS-ring in E. coli cells. We observed fuzzy structures around the ring using either electron microscopy or high-speed atomic force microscopy (HS-AFM), suggesting that FliM and FliN are necessary for the formation of a stable ring structure. The HS-AFM movies revealed flexible movements at the FliG region.

DOI： 10.2142/biophysico.bppb-v20.0028

Mutations in the stator protein PomA affect switching of rotational direction in bacterial flagellar motor Reviewed Open Access

Hiroyuki Terashima, Kiyoshiro Hori, Kunio Ihara, Michio Homma, Seiji Kojima

Scientific Reports Vol. 12 ( 1 ) page： 2979 2022.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Abstract

The flagellar motor rotates bi-directionally in counter-clockwise (CCW) and clockwise (CW) directions. The motor consists of a stator and a rotor. Recent structural studies have revealed that the stator is composed of a pentameric ring of A subunits and a dimer axis of B subunits. Highly conserved charged and neighboring residues of the A subunit interacts with the rotor, generating torque through a gear-like mechanism. The rotational direction is controlled by chemotaxis signaling transmitted to the rotor, with less evidence for the stator being involved. In this study, we report novel mutations that affect the switching of the rotational direction at the putative interaction site of the stator to generate rotational force. Our results highlight an aspect of flagellar motor function that appropriate switching of the interaction states between the stator and rotor is critical for controlling the rotational direction.

DOI： 10.1038/s41598-022-06947-5

Other Link： https://www.nature.com/articles/s41598-022-06947-5

Structure of MotA, a flagellar stator protein, from hyperthermophile Reviewed

Tatsuro Nishikino, Norihiro Takekawa, Duy Phuoc Tran, Jun-ichi Kishikawa, Mika Hirose, Sakura Onoe, Seiji Kojima, Michio Homma, Akio Kitao, Takayuki Kato, Katsumi Imada

Biochemical and Biophysical Research Communications Vol. 631 page： 78 - 85 2022.11

Function and Structure of FlaK, a Master Regulator of the Polar Flagellar Genes in Marine Vibrio. International journal Open Access

Michio Homma, Tomoya Kobayakawa, Yuxi Hao, Tatsuro Nishikino, Seiji Kojima

Journal of bacteriology Vol. 204 ( 11 ) page： e0032022 2022.10

Language：English Publishing type：Research paper (scientific journal)

Vibrio alginolyticus has a flagellum at the cell pole, and the fla genes, involved in its formation, are hierarchically regulated in several classes. FlaK (also called FlrA) is an ortholog of Pseudomonas aeruginosa FleQ, an AAA+ ATPase that functions as a master regulator for all later fla genes. In this study, we conducted mutational analysis of FlaK to examine its ATPase activity, ability to form a multimeric structure, and function in flagellation. We cloned flaK and confirmed that its deletion caused a nonflagellated phenotype. We substituted amino acids at the ATP binding/hydrolysis site and at the putative subunit interfaces in a multimeric structure. Mutations in these sites abolished both ATPase activity and the ability of FlaK to induce downstream flagellar gene expression. The L371E mutation, at the putative subunit interface, abolished flagellar gene expression but retained ATPase activity, suggesting that ATP hydrolysis is not sufficient for flagellar gene expression. We also found that FlhG, a negative flagellar biogenesis regulator, suppressed the ATPase activity of FlaK. The 20 FlhG C-terminal residues are critical for reducing FlaK ATPase activity. Chemical cross-linking and size exclusion chromatography revealed that FlaK mostly exists as a dimer in solution and can form multimers, independent of ATP. However, ATP induced the interaction between FlhG and FlaK to form a large complex. The in vivo effects of FlhG on FlaK, such as multimer formation and/or DNA binding, are important for gene regulation. IMPORTANCE FlaK is an NtrC-type activator of the AAA+ ATPase subfamily of σ54-dependent promoters of flagellar genes. FlhG, a MinD-like ATPase, negatively regulates the polar flagellar number by collaborating with FlhF, an FtsY-like GTPase. We found that FlaK and FlhG interact in the presence of ATP to form a large complex. Mutational analysis revealed the importance of FlaK ATPase activity in flagellar gene expression and provided a model of the Vibrio molecular mechanism that regulates the flagellar number.

DOI： 10.1128/jb.00320-22

Formation of multiple flagella caused by a mutation of the flagellar rotor protein FliM in Vibrio alginolyticus. International journal

Michio Homma, Norihiro Takekawa, Kazushi Fujiwara, Yuxi Hao, Yasuhiro Onoue, Seiji Kojima

Genes to cells : devoted to molecular & cellular mechanisms Vol. 27 ( 9 ) page： 568 - 578 2022.9

Language：English Publishing type：Research paper (scientific journal)

Marine bacterium Vibrio alginolyticus forms a single flagellum at a cell pole. In Vibrio, two proteins (GTPase FlhF and ATPase FlhG) regulate the number of flagella. We previously isolated the NMB155 mutant that forms multiple flagella despite the absence of mutations in flhF and flhG. Whole-genome sequencing of NMB155 identified an E9K mutation in FliM that is a component of C-ring in the flagellar rotor. Mutations in FliM result in defects in flagellar formation (fla) and flagellar rotation (che or mot); however, there are a few reports indicating that FliM mutations increase the number of flagella. Here, we determined that the E9K mutation confers the multi-flagellar phenotype and also the che phenotype. The co-expression of wild-type FliM and FliM-E9K indicated that they were competitive in regard to determining the flagellar number. The ATPase activity of FlhG has been correlated with the number of flagella. We observed that the ATPase activity of FlhG was increased by the addition of FliM but not by the addition of FliM-E9K in vitro. This indicates that FliM interacts with FlhG to increase its ATPase activity, and the E9K mutation may inhibit this interaction. FliM may control the ATPase activity of FlhG to properly regulate the number of the polar flagellum at the cell pole.

DOI： 10.1111/gtc.12975

Functional analysis of the N-terminal region of Vibrio FlhG, a MinD-type ATPase in flagellar number control. Reviewed

Homma M, Mizuno A, Hao Y, Kojima S

Vol. 172 ( 2 ) page： 99 - 107 2022.6

The Periplasmic Domain of the Ion-Conducting Stator of Bacterial Flagella Regulates Force Generation Reviewed Open Access

Homma M, Kojima S

2022.4

Hoop-like role of the cytosolic interface helix in Vibrio PomA, an ion-conducting membrane protein, in the bacterial flagellar motor. International journal Open Access

Tatsuro Nishikino, Yugo Sagara, Hiroyuki Terashima, Michio Homma, Seiji Kojima

Journal of biochemistry Vol. 171 ( 4 ) page： 443 - 450 2022.1

Language：English Publishing type：Research paper (scientific journal)

Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The three-dimensional structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66, and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

DOI： 10.1093/jb/mvac001

Achievements in bacterial flagellar research with focus on Vibrio species

Michio Homma, Tatsuro Nishikino, Seiji Kojima

Microbiology and Immunology Vol. 66 ( 2 ) page： 75 - 95 2022.1

ZomB is essential for chemotaxis of Vibrio alginolyticus by the rotational direction control of the polar flagellar motor. International journal Open Access

Norihiro Takekawa, Tatsuro Nishikino, Kiyoshiro Hori, Seiji Kojima, Katsumi Imada, Michio Homma

Genes to cells : devoted to molecular & cellular mechanisms Vol. 26 ( 11 ) page： 927 - 937 2021.9

Language：English Publishing type：Research paper (scientific journal)

Bacteria exhibit chemotaxis by controlling flagellar rotation to move toward preferred places or away from non-preferred places. The change in rotation is triggered by the binding of the chemotaxis signaling protein CheY-Phosphate (CheY-P) to the C-ring in the flagellar motor. Some specific bacteria, including Vibrio spp. and Shewanella spp. have a single transmembrane protein called ZomB. ZomB is essential for controlling the flagellar rotational direction in Shewanella putrefaciens and Vibrio parahaemolyticus. In this study, we confirmed that the zomB deletion results only in the counterclockwise (CCW) rotation of the motor in Vibrio alginolyticus as previously reported in other bacteria. We found that ZomB is not required for a clockwise-locked phenotype caused by mutations in fliG and fliM, and that ZomB is essential for CW rotation induced by overproduction of CheY-P. Purified ZomB proteins form multimers, suggesting that ZomB may function as a homo-oligomer. These observations imply that ZomB interacts with protein(s) involved in either flagellar motor rotation, chemotaxis or both. We provide the evidence that ZomB is a new player in chemotaxis and is required for the rotational control in addition to CheY in Vibrio alginolyticus.

DOI： 10.1111/gtc.12895

A slight bending of an α-helix in FliM creates a counterclockwise-locked structure of the flagellar motor in Vibrio. International journal

Norihiro Takekawa, Tatsuro Nishikino, Toshiki Yamashita, Kiyoshiro Hori, Yasuhiro Onoue, Kunio Ihara, Seiji Kojima, Michio Homma, Katsumi Imada

Journal of biochemistry Vol. 170 ( 4 ) page： 531 - 538 2021.6

Language：English Publishing type：Research paper (scientific journal)

Many bacteria swim by rotating flagella. The chemotaxis system controls the direction of flagellar rotation. Vibrio alginolyticus, which has a single polar flagellum, swims smoothly by rotating the flagellar motor counterclockwise (CCW) in response to attractants. In response to repellents, the motor frequently switches its rotational direction between CCW and clockwise (CW). We isolated a mutant strain that swims with a CW-locked rotation of the flagellum, which pulls rather than pushes the cell. This CW phenotype arises from a R49P substitution in FliM, which is the component in the C-ring of the motor that binds the chemotaxis signaling protein, phosphorylated CheY. However, this phenotype is independent of CheY, indicating that the mutation produces a CW conformation of the C-ring in the absence of CheY. The crystal structure of FliM with the R49P substitution showed a conformational change in the N-terminal α-helix of the middle domain of FliM (FliMM). This helix should mediates FliM-FliM interaction. The structural models of wild-type and mutant C-ring showed that the relatively small conformational change in FliMM induces a drastic rearrangement of the conformation of the FliMM domain that generates a CW conformation of the C-ring.

DOI： 10.1093/jb/mvab074

Putative spanner function of the Vibrio PomB plug region in the stator rotation model for flagellar motor Open Access

Michio Homma, Hiroyuki Terashima, Hiroaki Koiwa, Seiji Kojima

Journal of Bacteriology Vol. 203 ( 16 ) 2021.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extending from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-crosslinking and disulfide crosslinking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after crosslinking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner.

Importance

The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by crosslinking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

DOI： 10.1128/jb.00159-21

Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring. Invited Reviewed Open Access

mBio. 2021.3

Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring Reviewed Open Access

Norihiro Takekawa, Akihiro Kawamoto, Mayuko Sakuma, Takayuki Kato, Seiji Kojima, Miki Kinoshita, Tohru Minamino, Keiichi Namba, Michio Homma, Katsumi Imada

mBio Vol. 12 ( 2 ) 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

<title>ABSTRACT</title>
The bacterial flagellum is a protein nanomachine essential for bacterial motility. The flagellar basal body contains several ring structures. The MS-ring is embedded in the cytoplasmic membrane and is formed at the earliest stage of flagellar formation to serve as the base for flagellar assembly as well as a housing for the flagellar protein export gate complex. The MS-ring is formed by FliF, which has two transmembrane helices and a large periplasmic region. A recent electron cryomicroscopy (cryoEM) study of the MS-ring formed by overexpressed FliF revealed a symmetry mismatch between the S-ring and inner part of the M-ring. However, the actual symmetry relation in the native MS-ring and positions of missing domains remain obscure. Here, we show the structure of the M-ring by combining cryoEM and X-ray crystallography. The crystal structure of the N-terminal half of the periplasmic region of FliF showed that it consists of two domains (D1 and D2) resembling PrgK D1/PrgH D2 and PrgK D2/PrgH D3 of the injectisome. CryoEM analysis revealed that the inner part of the M-ring shows a gear wheel-like density with the inner ring of C23 symmetry surrounded by cogs with C11 symmetry, to which 34 copies of FliFD1–D2 fitted well. We propose that FliFD1–D2 adopts two distinct orientations in the M-ring relative to the rest of FliF, with 23 chains forming the wheel and 11 chains forming the cogs, and the 34 chains come together to form the S-ring with C34 symmetry for multiple functions of the MS-ring.

<bold>IMPORTANCE</bold> The bacterial flagellum is a motility organelle formed by tens of thousands of protein molecules. At the earliest stage of flagellar assembly, a transmembrane protein, FliF, forms the MS-ring in the cytoplasmic membrane as the base for flagellar assembly. Here, we solved the crystal structure of a FliF fragment. Electron cryomicroscopy (cryoEM) structural analysis of the MS-ring showed that the M-ring and S-ring have different rotational symmetries. By docking the crystal structure of the FliF fragment into the cryoEM density map of the entire MS-ring, we built a model of the whole periplasmic region of FliF and proposed that FliF adopts two distinct conformations to generate three distinct C11, C23, and C34 symmetries within the MS-ring for its multiple functions.

DOI： 10.1128/mbio.03199-20

Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body. International journal Open Access

Seiji Kojima, Hiroki Kajino, Keiichi Hirano, Yuna Inoue, Hiroyuki Terashima, Michio Homma

Journal of bacteriology Vol. 203 ( 9 ) 2021.2

Language：English Publishing type：Research paper (scientific journal)

The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane in Vibrio species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

DOI： 10.1128/JB.00009-21

Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar motor. International journal Open Access

Hiroyuki Terashima, Seiji Kojima, Michio Homma

Journal of bacteriology Vol. 203 ( 9 ) 2021.2

Language：English Publishing type：Research paper (scientific journal)

The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses, but have not been demonstrated biochemically. Here, we used site-directed photo- and disulfide-crosslinking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-L-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA formed a crosslink with FliG. PomA residue K89 gave the highest yield of crosslinks, suggesting that it is the PomA residue nearest to FliG. UV-induced crosslinking stopped motor rotation, and the isolated hook-basal body contained the crosslinked products. pBPA inserted to replace residues R281 or D288 in FliG formed crosslinks with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide crosslinks with cysteine inserted in place of FliG residues R281 and D288, and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA.ImportanceThe bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo- and disulfide-crosslinking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

DOI： 10.1128/JB.00016-21

Stator Dynamics Depending on Sodium Concentration in Sodium-Driven Bacterial Flagellar Motors. International journal Open Access

Tsai-Shun Lin, Seiji Kojima, Hajime Fukuoka, Akihiko Ishijima, Michio Homma, Chien-Jung Lo

Frontiers in microbiology Vol. 12 page： 765739 - 765739 2021

Language：English Publishing type：Research paper (scientific journal)

Bacterial flagellar motor (BFM) is a large membrane-spanning molecular rotary machine for swimming motility. Torque is generated by the interaction between the rotor and multiple stator units powered by ion-motive force (IMF). The number of bound stator units is dynamically changed in response to the external load and the IMF. However, the detailed dynamics of stator unit exchange process remains unclear. Here, we directly measured the speed changes of sodium-driven chimeric BFMs under fast perfusion of different sodium concentration conditions using computer-controlled, high-throughput microfluidic devices. We found the sodium-driven chimeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the number of functional stator units shows a rapidly excessive reduction and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions.

DOI： 10.3389/fmicb.2021.765739

The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching. Reviewed International journal Open Access

Brittany L Carroll, Tatsuro Nishikino, Wangbiao Guo, Shiwei Zhu, Seiji Kojima, Michio Homma, Jun Liu

eLife Vol. 9 2020.9

Language：English Publishing type：Research paper (scientific journal)

The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

DOI： 10.7554/eLife.61446