Biology subjects

Kodera, N.

Publications and source records attributed to Kodera, N..

31 records · Page 2Linked to original sources

bioRxiv2023

S100A11 promotes focal adhesion disassembly via myosin II-driven contractility and Piezo1-mediated Ca2+ entry

S100A11 is a small Ca2+-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays Ca2+-dependent F-actin binding activity and localizes to actin stress fibers (SFs), but its precise role in regulating these structures remains unclear. Analyzing endogenous S100A11 localization in HeLa and U2OS osteosarcoma cells confirmed SF association but in addition revealed steady localization to stable focal adhesions (FAs), typically at the end of dorsal stress fibers. In contrast, S100A11 levels at FAs increased sharply, but transiently, at the onset of peripheral FA disassembly. Elevating intracellular Ca2+ levels using the Ca2+ ionophore ionomycin reliably stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NM II) inhibitor blebbistatin, or with an inhibitor to the stretch-activatable Ca2+ channel Piezo1 effectively suppressed S100A11 recruitment, implicating S100A11 in an actomyosin contractility-driven FA disassembly mechanism involving Piezo1-dependent Ca2+ influx. Applying external mechanical forces on peripheral FAs via a micropipette likewise recruited S100A11 to FAs, even when NM II was inhibited by blebbistatin or in NM IIA knockout cells, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function was still indispensable, indicating that NM II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. Moreover, S100A11 knockout cells feature enlarged FAs and display delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel mechano-sensitive function for S100A11 in promoting actomyosin contractility-driven FA disassembly.

cell biology↗

bioRxiv2023

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions

Atomic force microscopy (AFM) and high-speed AFM allow direct observation of biomolecular structures and their functional dynamics. Based on scanning the molecular surface of a sample deposited on a supporting substrate by a probing tip, topographic images of its dynamic shape are obtained. Critical to successful AFM observations is a balance between immobilization of the sample while avoiding too strong perturbations of its functional conformational dynamics. Since the sample placement on the supporting substrate cannot be directly controlled in experiments, the relative orientation is a priori unknown, and, due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis. We present a method to predict the macromolecular placement of samples based on electrostatic interactions with the AFM substrate and demonstrate applications to HS-AFM observations of the Cas9 endonuclease, an aptamer-protein complex, and the ClpB molecular chaperone. The model also allows predictions of imaging stability under tip-scanning. We implemented the developed method within the freely available BioAFMviewer software package. Therefore, predictions based on available structural data can be made even prior to an actual experiment.

biophysics↗

bioRxiv2023

Evolutionarily acquired activity-dependent transformation of the CaMKII holoenzyme

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has long been central in synaptic plasticity research. CaMKII is a dodecameric serine/threonine kinase that has been essentially conserved across metazoans for over a million years. While the mechanisms of CaMKII activation are well studied, its "behavior" at the molecular level has remained unobserved. Here, high-speed atomic force microscopy was used to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII in various states at nanometer resolution. Among the species, rat CaMKII underwent internal kinase domain aggregation in an activity-dependent manner and showed a higher tolerance to dephosphorylation by phosphatase. Our findings suggest that mammalian CaMKII has evolutionarily acquired a new structural form and a tolerance to phosphatase to maintain robust CaMKII activity for proper neuronal function. One-Sentence SummaryHigh-speed atomic force microscopy reveals the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII

neuroscience↗

bioRxiv2022

Structural dynamics of E6AP E3 ligase HECT domain and involvement of flexible hinge loop in ubiquitin chain synthesis mechanism

Ubiquitin (Ub) ligases E3 are an important factor in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe containing the E2-binding site and a C-lobe containing the catalytic Cys residue that forms a thioester bond with Ub. These two lobes are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of this type of E3 ligases remain unclear. Here, we provide the first direct demonstration of structural dynamics of the E6AP HECT domain using high-speed atomic force microscopy. We also investigated structural dynamics of hinge loop flexibility restricted HECT domain, and we found that flexibility of the E6AP hinge loop has a great impact not only on its structural dynamics but also on the formation of free Ub chains mediated by E3-E3 interactions.

biophysics↗

bioRxiv2021

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

molecular biology↗

bioRxiv2021

Chained structure of dimeric F1-like ATPase in Mycoplasma mobile gliding machinery

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called chains that are each comprised of 17 repeating protein units called particles. These proteins include homologs of the catalytic - and {beta}-subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOBs 1660 (-subunit homolog), 1670 ({beta}-subunit homolog), 1630, 1620, and 4530, and showed ATP hydrolyzing activity. The 2D structure, with dimensions of 35 and 26 nm, showed a hexameric ring dimer about 12 nm in diameter, resembling F1-ATPase catalytic ({beta})3. We isolated the F1-like ATPase unit, which is composed of MMOBs 1660, 1670, and 1630. Furthermore, we isolated the chain and analyzed the 3D structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31 nm intervals. An atomic model of F1-ATPase catalytic ({beta})3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCEF1Fo- ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria, and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

microbiology↗

bioRxiv2021

Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for corynespora leaf fall disease in rubber trees. Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more susceptible to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo- SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and disrupt specific membranes. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/438527v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@17ef3f4org.highwire.dtl.DTLVardef@1c24d41org.highwire.dtl.DTLVardef@15e071aorg.highwire.dtl.DTLVardef@24740e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICas1 and Cas2 directly damage and cause necrosis in the leaves of specific rubber clones. C_LIO_LICas1 and Cas2 can form biofilm-like structures on specific lipid membranes (negative phospholipids, glycerolipids, and sterols). The biofilm-like formation of Cas toxin plays an important role in selective disruption on lipid membranes. C_LIO_LIVulnerability of the specific cytoplasmic membranes to the selective Cas toxin that is the most remarkable feature of cytotoxicity of Cas toxin on plant cells. C_LI

biochemistry↗

bioRxiv2021

Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the so-called hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown, potentially due to its conformational plasticity. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. The conformational change of the hinge domain might be essential for the dsDNA binding regulation and play important roles in condensin-mediated DNA-loop extrusion.

biophysics↗

bioRxiv2021

Movements of Mycoplasma mobile gliding machinery detected by high-speed atomic force microscopy

Mycoplasma mobile, a parasitic bacterium, glides on solid surfaces, such as animal cells and glass by a special mechanism. This process is driven by the force generated through ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells are unclear. In this study, we detected the movements of the internal structure by scanning cells immobilized on a glass substrate using high-speed atomic force microscopy (HS-AFM). By scanning the surface of a cell, we succeeded in visualizing particles, 2 nm in hight and aligned mostly along the cell axis with a pitch of 31.5 nm, consistent with previously reported features based on electron microscopy. Movements of individual particles were then analyzed by HS-AFM. In the presence of sodium azide, the average speed of particle movements was reduced, suggesting that movement is linked to ATP hydrolysis. Partial inhibition of the reaction by sodium azide enabled us to analyze particle behavior in detail, showing that the particles move 9 nm right, relative to the gliding direction, and 2 nm into the cell interior in 330 ms, then return to their original position, based on ATP hydrolysis. IMPORTANCEThe Mycoplasma genus contains bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide by a special mechanism linked to their infection and survival. The special machinery for gliding can be divided into surface and internal structures that have evolved from rotary motors represented by ATP synthases. This study succeeded in visualizing the real-time movements of the internal structure by scanning from the outside of the cell using an innovative high-speed atomic force microscope, and then analyzing their behaviors.

microbiology↗

bioRxiv2020

Native cyclase-associated protein and actin from Xenopus laevis oocytes form a 4:4 complex with a tripartite structure

Cyclase-associated protein (CAP) is a conserved actin-binding protein that regulates multiple aspects of actin filament dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. Intriguingly, CAP has been isolated from different cells and tissues as an equimolar complex with actin, and previous studies have shown that a CAP-actin complex contains six molecules each of CAP and actin. Here, we successfully isolated a complex of Xenopus cyclase-associated protein 1 (XCAP1) and actin from oocyte extracts and demonstrated that the complex contained four molecules each of XCAP1 and actin. The XCAP1-actin complex remained stable as a single population of 340 kDa in hydrodynamic analysis using gel filtration or analytical ultracentrifugation. Examination of the XCAP1-actin complex by high-speed atomic force microscopy revealed a tripartite structure: a middle globular domain and two globular arms. The two arms were connected with the middle globular domain by a flexible linker and observed in two states with different heights, presumably representing the presence or absence of G-actin. We hypothesize that the middle globular domain corresponds to a tetramer of the N-terminal helical-folded domain of XCAP1, and that each arm in the high state corresponds to a hetero-tetramer containing a dimer of the C-terminal CARP domain of XCAP1 and two G-actin molecules. This novel configuration of a CAP-actin complex may represent a functionally important aspect of this complex.

cell biology↗

bioRxiv2020

Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother

High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement of the instruments, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie, compared to that mimics the raw AFM movie, and the Kalman filter result. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhAc and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.

biophysics↗

bioRxiv2020

Direct visualization of translational GTPase factor-pool formed around the archaeal ribosomal P-stalk by high-speed atomic force microscopy

The ribosomal stalk protein plays an essential role in the recruitment of translational GTPase factors EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal stalk (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit, where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Intriguingly, archaeal aEF1A and aEF2 molecules spontaneously assembled around the ribosomal P-stalk up to the maximum number of available binding sites. These results provide the first visual evidence for the factor-pooling mechanism and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Actin binding domain of Rng2 strongly inhibits actin movement on myosin II HMM through structural changes of actin filaments

Substoichiometric binding of certain actin-binding proteins induces conformational changes in a disproportionally large number of actin protomers in actin filaments. Here, we report a case in which such conformational changes in actin filaments have profound functional consequences. Rng2 is an IQGAP protein implicated in the assembly and contraction of contractile rings in Schizosaccharomyces pombe. We found that the calponin-homology actin-binding domain of Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD halved the sliding speed of actin filaments at a binding ratio of 1.3% (=1/77), and virtually stopped movement at a binding ratio of 11% (=1/9). Rng2CHD also inhibited actin movements on Dictyostelium myosin II, but in this case by inducing the detachment of actin filaments from myosin II-coated surfaces. Rng2CHD induced cooperative structural changes of actin filaments accompanied by shortening of the filament helical pitch, and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II in the presence of ADP. Intriguingly, actin-activated ATPase of S1 was hardly inhibited by Rng2CHD. We suggest that sparsely bound Rng2CHD induces global structural changes of actin filaments and interferes with the force generation by actin-myosin II.

biophysics↗