Biology subjects

Fujiwara, I.

Publications and source records attributed to Fujiwara, I..

9 recordsLinked to original sources

bioRxiv2026

Coordinated Membrane Deformation Driven by a Minimal Set of Spiroplasma MreB Isoforms

Spiroplasma are wall-less helical bacteria that swim by switching the handedness of their helices. In most bacteria, MreB, a bacterial actin, forms relatively static filaments beneath the membrane to organize cell wall synthesis. In contrast, Spiroplasma eriocheiris encodes five MreB isoforms (SpeMreBs), and swimming requires a pair of isoforms, SpeMreB5 together with SpeMreB4 (or the related isoform SpeMreB1) (1) Yet how these MreBs generate force and membrane deformation remains unclear. To examine the membrane-deforming activities of SpeMreBs, we demonstrated a simple reconstitution system using the non-motile synthetic bacterium JCVI-syn3B and purified SpeMreBs expressed in Escherichia coli. Lysates of syn3B expressing SpeMreB5 deformed liposomes in a concentration-dependent manner. In contrast, lysates co-expressing both SpeMreB5 and SpeMreB4 showed a plateau in the frequency of deformation, suggesting that SpeMreB4 suppresses membrane deformation driven by SpeMreB5. Deformed liposomes exhibited either fluctuating or stable behaviors. ATP depletion changed both the frequency and behavior of deformation, indicating that membrane remodeling depends on the nucleotide state of SpeMreBs. Reconstitution with purified SpeMreBs from E. coli confirmed that SpeMreB5 alone deforms membranes, whereas SpeMreB1, a member of the same class as SpeMreB4, suppresses deformation. These results suggest that membrane shape in Spiroplasma is dynamically regulated by antagonistic interactions among isoforms of SpeMreBs isoforms and their nucleotide-dependent assembly states.

synthetic biology↗

bioRxiv2025

Microscopic and structural observations of actin filament capping and severing by Cytochalasin D

Cytochalasin D (CytoD) is widely used to inhibit actin polymerization, but the underlying regulatory mechanism is unclear. We addressed this using Total Internal Reflection Fluorescence (TIRF) microscopy. Our time course depolymerization assay of individual actin filaments showed that CytoD tightly caps the barbed end, with an estimated Km value for inhibition of 4.1 nM and a duration time of [~]1 min. Consistently, in polymerization assays, CytoD at concentrations near the Km value completely suppressed barbed end elongation. Interestingly, at lower concentrations, CytoD acted as a leaky capper, allowing actin monomer addition by rapidly binding to and dissociating from barbed ends. We interpreted this contradictory behavior as arising from differences in binding modes: capping one strand (fast dissociation) or both strands (slow dissociation). CytoD severs actin filaments at micromolar levels, a concentration range commonly used in cell biological studies. Although the severing rate is slower than cofilin, the frequency is higher, resulting in the fragmentation of filaments into shorter pieces. Severing activity was suppressed by inorganic phosphate or cofilin. We determined the crystal structure of CytoD bound to filamentous conformation (F-form) actin and found that CytoD fits better in the hydrophobic cleft of F-form actin than in the monomeric conformation actin, explaining the preferential binding towards barbed end subunits. The structure further indicates that CytoD prevents barbed end depolymerization by stabilizing the terminal subunits in the F-form, which is supported by our MD simulations. Collectively, our results demonstrate how CytoD regulates actin dynamics at the molecular level.

biophysics↗

bioRxiv2024

A highly active bacterial actin actuates the polymerization of another isoform essential for swimming motility of Spiroplasma

Spiroplasma is a wall-less helical bacterium that is characterized by a unique swimming motility involving five isoforms of the bacterial actin MreBs (SMreB1-5). The functions of SMreBs are unique in the MreB family proteins, as their counterparts in walled-bacteria are involved in maintaining the cell shape by scaffolding the cell-wall synthesis complex through their static properties. In vitro analyses of individual SMreBs provide clues to understand the detailed molecular mechanism of Spiroplasma swimming. However, the purification difficulties have hampered in vitro analyses of one of the SMreBs, SMreB1, which drives the swimming. Here, we isolated soluble SMreB1 of Spiroplasma eriocheiris (SpeMreB1) and evaluated its activity. SpeMreB1 was expressed as a fusion with a solubilization-tag, ProteinS (PrS), which allowed us to purify it in the soluble fraction. SpeMreB1 exhibited the highest phosphate release rate and the fold changes of critical concentrations for polymerization across the nucleotide states among the MreB family proteins. SpeMreB1 interacted with polymerized SpeMreB5, another SMreB essential for Spiroplasma swimming. In the AMPPNP- or ADP-bound state, SpeMreB1 decreased the amount of SpeMreB5 filaments, possibly reflecting their disassembly. Regardless of the nucleotide state, SpeMreB1 bound to negatively charged lipids. These results suggest that SpeMreB1 utilizes its highest activity to manage SpeMreB5 filaments underneath the cell membrane to drive Spiroplasma swimming. ImportanceIn most bacterial species, MreB is involved in cell-shape maintenance by localizing the bacterial cell-wall synthesis complex with its static properties. In contrast, five isoforms of MreBs in a pathogenic wall-less helical bacterium Spiroplasma are involved in its unique motility system driven by a kink propagation along the helical cell. Our integrated biochemical assays show that one isoform of MreBs involved in the swimming of a crustacean pathogen S. eriocheiris (SpeMreB1) is exceptionally active in the MreB family proteins and manages the polymerization of another MreB essential for the swimming (SpeMreB5). This study sheds light on an evolutionary mystery how Spiroplasma has adapted static MreB proteins to a dynamic phenomenon like its swimming motility.

biophysics↗

bioRxiv2023

Assembly properties of Spiroplasma MreB involved in swimming motility

Bacterial actin MreB forms filaments in which the unit of the structure is an antiparallel double strand. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), a part of which is composed of an intra-cellular ribbon for driving its swimming motility. The interaction modes of each ribbon component are unclear, although these are clues for understanding Spiroplasma swimming. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), which forms sheets and is a component protein of the ribbon. Electron microscopy (EM) revealed that sheet formation was inhibited under acidic conditions and paracrystal structures were formed under acidic and neutral conditions with low ionic strength. Solution assays found four properties of paracrystals as follows: (I) their formation followed sheet formation, (II) electrostatic interactions were required for their formation, (III) the positively charged and unstructured C-terminal region contributed to the nucleation of their formation, and (IV) their formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic conditions. During these studies, we found two aggregation modes of SpeMreB5, with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.

biophysics↗

bioRxiv2022

The difference in FRET efficiency fluctuation at each end of the actin filament

Actin filaments are involved in various cell motility processes. Actin polymerisation is primarily governed by monomer association and dissociation occurring at the rapid-growing end called the barbed end, which generates the force to push the plasma membrane forward. Individual actin filaments bind to one nucleotide and its hydrolysis energy is used to maintain the filamentous form by changing the characteristics of the subunits. The asymmetry of the individual actin filaments is also important for detecting the asymmetry of the cell. However, asymmetry at the subunit level including conformational and temporal changes in actin filaments has not been visualized yet. Here, we used "Forster (or fluorescence) resonance energy transfer (FRET)-actin filament" by copolymerising an equal amount of donor and acceptor labelled actin. FRET efficiency change was measured along each actin filament under a light microscope. The FRET efficiency was lower near the end region than in the interior regions. Fluctuations in the FRET efficiency (fFRET) were used to monitor local flexibilities along each actin filament. The fFRET was larger near the end region than the interior region. Our quantified data showed that spatial change of fFRET along actin filaments was rapidly decayed from the barbed end from near the pointed end toward internal region, suggesting that the behaviour of actin subunits near ends is affected from each end. Our result revealed that actin filaments have different orientations locally. These orientations appear when actin forms filaments, which may contribute the cell motility.

biophysics↗

bioRxiv2021

Structure and polymerization dynamics of bacterial actin MreB3 and MreB5 involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. It polymerises into an antiparallel double-stranded filament that generally functions in cell shape determination by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five classes of MreB homologs (SpeMreB1-5) that are likely to be involved in swimming motility. Here, we investigated the structure, ATPase activity, and polymerisation dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerised into an antiparallel double-stranded filament, and SpeMreB5 formed sheets, including the antiparallel filament, upon the binding of a nucleotide. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our crystal structures of SpeMreB3 and analyses of the mutant variants showed that the amino acid motif most likely plays a role in eliminating the proton of the nucleophilic water for ATP hydrolysis. Our sedimentation assay suggests that SpeMreB3 has a lower polymerisation activity than SpeMreB5, while their polymerisation dynamics are qualitatively similar to those of other actin superfamily proteins, in which ATP hydrolysis stabilises the filament, and Pi release leads to depolymerisation.

biochemistry↗

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

Updated phylogeny and sequence features of MreB from the Wall-less bacteria Spiropaslma

Spiroplasma are wall-less bacteria which belong to the phylum Tenericutes that evolved from Firmicutes including Bacillus subtilis. Spiroplasma swim by a mechanism unrelated to widespread bacterial motilities, such as flagellar motility, and caused by helicity switching with kinks traveling along the helical cell body. The swimming force is likely generated by five classes of bacterial actin homolog MreBs (SMreBs 1-5) involved in the helical bone structure. We analyzed sequences of SMreBs to clarify their phylogeny and sequence features. The maximum likelihood method based on around 5,000 MreB sequences showed that the phylogenetic tree was divided into several radiations. SMreBs formed a clade adjacent to the radiation of MreBH, an MreB isoform of Firmicutes. Sequence comparisons of SMreBs and Bacillus MreBs were also performed to clarify the features of SMreB. Catalytic glutamic acid and threonine were substituted to aspartic acid and lysine, respectively, in SMreB3. In SMreBs 2 and 4, amino acids involved in inter- and intra-protofilament interactions were significantly different from those in Bacillus MreBs. A membrane-binding region was not identified in most SMreBs 1 and 4 unlike many walled-bacterial MreBs. SMreB5 had a significantly longer C-terminal region than the other MreBs, which possibly forms a protein-protein interaction. These features may support the functions responsible for the unique mechanism of Spiroplasma swimming.

bioinformatics↗

bioRxiv2020

Myosin 18Aα targets guanine nucleotide exchange factor β-Pix to dendritic spines of cerebellar Purkinje neurons to promote spine maturation

Dendritic spines are signaling microcompartments that serve as the primary site of synapse formation in neurons. Actin assembly and myosin 2 contractility play major roles in the maturation of spines from filopodial precursors, as well as in the subsequent, activity-dependent changes in spine morphology that underly learning and memory. Myosin 18A is a myosin 2-like protein conserved from flies to man that lacks motor activity, is sub-stochiometric to myosin 2, and co-assembles with myosin 2 to make mixed filaments. Myosin 18A is alternatively spliced to create multiple isoforms that contain unique N- and C-terminal extensions harboring both recognizable and uncharacterized protein: protein interaction domains. These observations suggest that myosin 18A serves to recruit proteins to mixed filaments of myosin 2 and myosin 18A. One protein known to bind to myosin 18A is {beta}-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42. Notably, {beta}-Pix has been shown to promote spine maturation by activating both Arp2/3 complex-dependent branched actin filament assembly and myosin 2 contractility within spines. Here we show that myosin 18A is expressed in cerebellar Purkinje neurons and concentrates in spines along with myosin 2 and F-actin. Myosin 18A is targeted to spines by co-assembling with myosin 2 and by an actin binding site present in its N-terminal extension. miRNA-mediated knockdown of myosin 18A results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A. Importantly, {beta}-Pix co-localizes with myosin 18A in spines, and its spine localization is lost upon myosin 18A knockdown or when its myosin 18A binding site is deleted. Finally, we show that the spines of myosin 18A knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data demonstrate that mixed filaments of myosin 2 and myosin 18A form a complex with {beta}-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of {beta}-Pixs GEF activity.

neuroscience↗