Biology subjects

Morimoto, Y. V.

Publications and source records attributed to Morimoto, Y. V..

8 recordsLinked to original sources

bioRxiv2026

Thermal fractuations assist high-speed rotation of the bacterial flagellar motor at low load

The bacterial flagellar motor is a proton-driven rotary nanomachine that converts ion flow into mechanical motion. Proton-coupled rotation of the MotA5-MotB2 stator complex generates torque, yet how this process drives high-speed rotation remains unclear. Here we use single-molecule nanophotometry to resolve stepwise rotation under low-load conditions. Lowering intracellular pH selectively prolongs dwell times without affecting step durations, indicating that proton dissociation triggers torque generation. Structural analysis further suggests that the rotor disengages from the stator before completing the elementary step angle (~11 degree), implying that the power stroke alone is insufficient. Notably, the rotational diffusion coefficient of stator-less motors closely matches that inferred from wild-type motors at low load. These findings support a model in which intrinsic thermal fluctuations compensate for the limited reach of the power stroke, enabling rapid rotation. Our results reveal a hybrid mechanism in which ion-driven conformational changes bias stochastic motion to achieve efficient energy transduction.

biophysics↗

bioRxiv2025

Front-rear polarity of intracellular signaling uncovered via giant Dictyostelium cells

Intracellular signaling dynamics are often obscured by the spatial and temporal limitations of cell size. Here, we developed a method to enlarge Dictyostelium discoideum cells by partial cytokinesis inhibition, generating multinucleated yet functional giant cells. These cells retained chemotactic signaling, polarity, and motility, enabling high-resolution live-cell imaging. Using fluorescent probes for cAMP and Ca2+, we uncovered a directional, front-to-rear propagation of cAMP signaling and a biphasic Ca2+ response coordinated with actin wave dynamics. Grid-based mapping revealed asymmetric cAMP synthesis and decay kinetics, and vesicle localization suggested spatially regulated cAMP secretion. Combining giant cells with super-resolution or electron microscopy allowed detailed visualization of intracellular local structures at high resolution. Our findings demonstrate that intracellular signaling involves self-organized, spatially structured propagation events aligned with cellular polarity. The giant cell platform offers a powerful and generalizable strategy for dissecting the spatiotemporal logic of single-cell signaling.

cell biology↗

bioRxiv2025

Live imaging of bacterial actin MreBs causing helicity switching of a minimal synthetic cell

Spiroplasma swim by switching their helical body into right- and left-handed. Helicity formation and switching can be reconstituted in an immotile minimal synthetic bacterium, JCVI-syn3B by introducing the pair of bacterial actin, MreB4 and MreB5. Here, we analyzed MreB behaviors optically, to investigate this unknown mechanism. We tried MreB4 fluorescence labeling by protein fusion. The labeling was not achieved, because the fusion of fluorescent protein or peptide to 16 positions resulted in immotile constructs. These results may suggest that MreB4 has many interaction interfaces with other proteins. Induced expression of MreB4 to cells with constitutive MreB5 expression resulted in earlier and higher frequency of motile cells, distinct from the results of constitutive MreB4 and inducible MreB5. Next, the behavior of labeled MreB5 was analyzed. Photobleaching and photoactivation suggested static behavior of MreB5 during cell movements. Cell treatment by A22, a MreB polymerization inhibitor caused helix deformation, movement stall, and diffusion of MreB5 fluorescence, suggesting that A22 sensitive MreB5 interaction should be involved in helix formation and motility. SignificanceSpiroplasma MreB is a unique bacterial actin that causes motility with a different mechanism from other actins. Here, we investigated the helicity switching involved in the motility. Fluorescently traced MreB behaviors suggested that the motility mechanism is not coupled with its replacement in the internal structure.

biophysics↗

bioRxiv2021

Calcium signaling mediates mechanotransduction at the multicellular stage of Dictyostelium discoideum

Calcium acts as a second messenger and regulates cellular functions, including cell motility. In Dictyostelium discoideum, the cytosolic calcium level oscillates synchronously, and calcium signal waves propagate in the cell population during the early stages of development, including aggregation. At the unicellular phase, the calcium response through Piezo channels also functions in mechanosensing. However, calcium signaling dynamics during multicellular morphogenesis is still unclear. Here, live-imaging of cytosolic calcium levels revealed that calcium wave propagation, depending on cAMP relay, temporarily disappeared at the onset of multicellular body formation. Alternatively, the occasional burst of calcium signals and their propagation were observed in both anterior and posterior regions of migrating multicellular bodies. Calcium signaling in multicellular bodies occurred in response to mechanical stimulation. Both pathways, calcium release from the endoplasmic reticulum via IP3 receptor and calcium influx from outside the cell, were involved in calcium waves induced by mechanical stimuli. These show that calcium signaling works on mechanosensing in both the unicellular and multicellular phases of Dictyostelium using different molecular mechanisms during development. Summary statementFluorescence imaging revealed that calcium signaling via both endoplasmic reticulum and extracellular pathways plays an important role in mechanosensing during the multicellular stage of Dictyostelium.

developmental biology↗

bioRxiv2021

Light-dependent synthesis of a nucleotide second messenger controls bacterial motility

Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and function, such as virulence, biofilm formation, and others mainly via gene regulation. Here we show that the swimming motility of a soil bacterium is rapidly modulated by cyclic adenosine monophosphate (cAMP) synthesized upon light exposure. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis determined the novel sensory gene, and its expression in Escherichia coli through codon optimization revealed the light-dependent synthesis of cAMP. GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. The present findings highlight the new role of cAMP that rapidly controls the flagella-dependent bacterial motility and the global distribution of the discovered photoactivated cyclase among diverse microbial species.

microbiology↗

bioRxiv2020

Membrane voltage-dependent activation of the flagellar protein export engine

Ion motive force (IMF) consists of the electric potential difference ({Delta}{Psi}) and the ion concentration difference ({Delta}pI) across the cytoplasmic membrane. The flagellar protein export machinery is an ion/protein antiporter utilizing IMF to drive ion-coupled protein export, but it remains unknown how. Here, we report a {Delta}{Psi}-dependent activation mechanism of the transmembrane export gate complex. Depletions of both H+ and Na+ gradients nearly diminished flagellar protein export in the absence of the cytoplasmic ATPase complex, but an increase in {Delta}{Psi} by an upward shift of external pH from 7.5 to 8.5 dramatically recovered it. An increase in the cytoplasmic level of export substrates and gain-of-function mutations in FlhA enhanced protein export at external pH 7.5 in the absence of Na+ in a similar manner to {Delta}{Psi} increase. We propose that the export gate complex has a voltage-gated mechanism to activate the ion/protein antiporter of the flagellar protein export engine.

microbiology↗

bioRxiv2020

The FlgN chaperone activates the Na+-driven engine of the flagellar protein export apparatus

The bacterial flagellar protein export machinery promotes H+-coupled translocation of flagellar proteins to the cell exterior. When the cytoplasmic ATPase complex does not function, the transmembrane export gate complex opens its Na+ channel and continues protein transport. However, it remains unknown how. Here we report that the FlgN chaperone acts as a switch to activate a backup export mechanism for the ATPase complex by activating the Na+-driven engine. Impaired interaction of FlhA with the FliJ subunit of the ATPase complex increased Na+-dependence of flagellar protein export. Deletion of FlgN inhibited protein export in the absence of the ATPase complex but not in its presence. Gain-of-function mutations in FlhA restored not only the FlgN defect but also the FliJ defect. We propose that the interaction of FlgN with FlhA opens the Na+ channel in the export engine, thereby maintaining the protein export activity in the absence of the active ATPase complex.

microbiology↗

bioRxiv2020

Distinct chemotactic behavior in the original Escherichia coli K-12 depending on forward-and-backward swimming, not on run-tumble movements

Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of rotatory direction which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150{degrees}. The flagellar helicity remained right-handed with a 1.3 m pitch and 0.14 m helix radius, which is assumed to be a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not form rings on a semi-solid surface. Based on these findings, we propose a flagellar polymorphism-dependent migration mechanism in structured environments.

microbiology↗