Multiflagellate Swimming Controlled by Hydrodynamic Interactions.

Many eukaryotic microorganisms propelled by multiple flagella can swim very rapidly with distinct gaits. Here, we model a three-dimensional mutiflagellate swimmer, resembling the microalgae. When the flagella are actuated synchronously, the swimming efficiency can be enhanced or reduced by interflagella hydrodynamic interactions (HIs), determined by the intrinsic tilting angle of the flagella. The asynchronous gait with a phase difference between neighboring flagella can reduce oscillatory motion via the basal mechanical coupling. In the presence of a spherical body, simulations taking into account the flagella-body interactions reveal the advantage of anterior configuration compared with posterior configuration, where in the latter case an optimal flagella number arises. Apart from understanding the role of HIs in the multiflagellate microorganisms, this work could also guide laboratory fabrications of novel microswimmers.

Intraflagellar transport speed is sensitive to genetic and mechanical perturbations to flagellar beating.

Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.

The younger flagellum sets the beat for Chlamydomonas reinhardtii.

Eukaryotes swim with coordinated flagellar (ciliary) beating and steer by fine-tuning the coordination. The model organism for studying flagellate motility, Chlamydomonas reinhardtii, employs synchronous, breaststroke-like flagellar beating to swim, and it modulates the beating amplitudes differentially to steer. This strategy hinges on both inherent flagellar asymmetries (e.g. different response to chemical messengers) and such asymmetries being effectively coordinated in the synchronous beating. In C. reinhardtii, the synchrony of beating is known to be supported by a mechanical connection between flagella; however, how flagellar asymmetries persist in the synchrony remains elusive. For example, it has been speculated for decades that one flagellum leads the beating, as its dynamic properties (i.e. frequency, waveform, etc.) appear to be copied by the other one. In this study, we combine experiments, computations, and modeling efforts to elucidate the roles played by each flagellum in synchronous beating. With a non-invasive technique to selectively load each flagellum, we show that the coordinated beating essentially only responds to load exerted on the cis flagellum; and that such asymmetry in response derives from a unilateral coupling between the two flagella. Our results highlight a distinct role for each flagellum in coordination and have implication for biflagellates' tactic behaviors.

Many single-cell organisms use tiny hair-like structures called flagella to move around. To direct this movement, the flagella must work together and beat in a synchronous manner. In some organisms, coordination is achieved by each flagellum reacting to the flow generated by neighbouring flagella. In others, flagella are joined together by fiber connections between their bases, which allow movement to be coordinated through mechanical signals sent between flagella. One such organism is Chlamydomonas reinhardtii, a type of algae frequently used to study flagellar coordination. Its two flagella ­ named trans and cis because of their positions relative to the cell's eyespot ­ propel the cell through water using breaststroke-like movements. To steer, C. reinhardtii adjusts the strength of the strokes made by each flagellum. Despite this asymmetry, the flagella must continue to beat in synchrony to move efficiently. To understand how the cell manages these differences, Wei et al. exposed each flagellum to carefully generated oscillations in water so that each was exposed to different forces and their separate responses could be measured. A combination of experiments, modelling and computer simulations were then used to work out how the two flagella coordinate to steer the cell. Wei et al. found that only the cis flagellum coordinates the beating, with the trans flagellum simply copying the motion of the cis. A direct consequence of such one-way coupling is that only forces on the cis flagellum influence the coordinated beating dynamics of both flagella. These findings shed light on the unique roles of each flagellum in the coordinated movement in C. reinhardtii and have implications for how other organisms with mechanically-connected flagella navigate their environments.

The lack of Tex44 causes severe subfertility with flagellar abnormalities in male mice.

By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1, that is associated with male hypofertility and ultrastructural sperm abnormalities. Within this locus, we identified a new candidate gene that could be implicated in a reproductive phenotype: Tex44 (Testis-expressed protein 44). We thus performed a CRISPR/Cas9-mediated complete deletion of this gene in mice in order to study its function. Tex44-KO males were severely hypofertile in vivo and in vitro due to a drastic reduction of sperm motility which itself resulted from important morphological sperm abnormalities. Namely, Tex44-KO sperm showed a disorganized junction between the midpiece and the principal piece of the flagellum, leading to a 180° flagellar bending in this region. In addition, the loss of some axonemal microtubule doublets and outer dense fibers in the flagellum's principal piece has been observed. Our results suggest that, in mice, TEX44 is implicated in the correct set-up of the sperm flagellum during spermiogenesis and its absence leads to flagellar abnormalities and consequently to severe male hypofertility.

Effect of fluid elasticity on the emergence of oscillations in an active elastic filament.

Many microorganisms propel themselves through complex media by deforming their flagella. The beat is thought to emerge from interactions between forces of the surrounding fluid, the passive elastic response from deformations of the flagellum and active forces from internal molecular motors. The beat varies in response to changes in the fluid rheology, including elasticity, but there are limited data on how systematic changes in elasticity alter the beat. This work analyses a related problem with fixed-strength driving force: the emergence of beating of an elastic planar filament driven by a follower force at the tip of a viscoelastic fluid. This analysis examines how the onset of oscillations depends on the strength of the force and viscoelastic parameters. Compared to a Newtonian fluid, it takes more force to induce the instability in viscoelastic fluids, and the frequency of the oscillation is higher. The linear analysis predicts that the frequency increases with the fluid relaxation time. Using numerical simulations, the model predictions are compared with experimental data on frequency changes in the bi-flagellated alga Chlamydomonas reinhardtii. The model shows the same trends in response to changes in both fluid viscosity and Deborah number and thus provides a possible mechanistic understanding of the experimental observations.

Spatiotemporal dynamics of the proton motive force on single bacterial cells.

Electrochemical gradients across biological membranes are vital for cellular bioenergetics. In bacteria, the proton motive force (PMF) drives essential processes like adenosine triphosphate production and motility. Traditionally viewed as temporally and spatially stable, recent research reveals a dynamic PMF behavior at both single-cell and community levels. Moreover, the observed lateral segregation of respiratory complexes could suggest a spatial heterogeneity of the PMF. Using a light-activated proton pump and detecting the activity of the bacterial flagellar motor, we perturb and probe the PMF of single cells. Spatially homogeneous PMF perturbations reveal millisecond-scale temporal dynamics and an asymmetrical capacitive response. Localized perturbations show a rapid lateral PMF homogenization, faster than proton diffusion, akin to the electrotonic potential spread observed in passive neurons, explained by cable theory. These observations imply a global coupling between PMF sources and consumers along the membrane, precluding sustained PMF spatial heterogeneity but allowing for rapid temporal changes.

Escherichia coli self-organizes developmental rosettes.

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.

Characterization of tryptanthrin as an antibacterial reagent inhibiting Vibrio splendidus.

Isolates of Vibrio splendidus are ubiquitously presented in various marine environments, and they can infect diverse marine culture animals, leading to high mortality and economic loss. Therefore, a control strategy of the infection caused by V. splendidus is urgently recommended. Tryptanthrin is a naturally extracted bioactive chemical with antimicrobial activity to other bacteria. In this study, the effects of tryptanthrin on the bacterial growth and virulence-related factors of one pathogenic strain V. splendidus AJ01 were determined. Tryptanthrin (10 µg/mL) could completely inhibit the growth of V. splendidus AJ01. The virulence-related factors of V. splendidus AJ01 were affected in the presence of tryptanthrin. Tryptanthrin resulted an increase in biofilm formation, but lead to reduction in the motility and hemolytic activity of V. splendidus cells. In the cells treated with tryptanthrin, two distinctly differentially expressed extracellular proteins, proteases and flagellum, were identified using SDS-PAGE combined with LC-MS. Real-time reverse transcriptase PCR confirmed that the genes involved in the flagellar formation and hemolysin decreased, whereas specific extracellular proteases and the genes involved in the biofilm formation were upregulated. Two previously annotated luxOVs genes were cloned, and their expression levels were analyzed at different cell densities. Molecular docking was performed to predict the interaction between LuxOVs and ATP/tryptanthrin. The two sigma-54-dependent transcriptional regulators showed similar ATP or tryptanthrin binding capacity but with different sites, and the direct competitive binding between ATP and tryptanthrin was present only in their binding to LuxO1. These results indicated that tryptanthrin can be used as a bactericide of V. splendidus by inhibiting the growth, bacterial flagella, and extracellular proteases, but increasing the biofilm. Sigma-54-dependent transcriptional regulator, especially the quorum sensing regulatory protein LuxO1, was determined to be the potential target of tryptanthrin. KEY POINTS: • Tryptanthrin inhibited the growth of V. splendidus in a dose-dependent manner. • The effect of tryptanthrin on the virulence factors of V. splendidus was characterized. • LuxO was the potential target for tryptanthrin based on molecular docking.

Foraging mechanisms in excavate flagellates shed light on the functional ecology of early eukaryotes.

The phagotrophic flagellates described as "typical excavates" have been hypothesized to be morphologically similar to the Last Eukaryotic Common Ancestor and understanding the functional ecology of excavates may therefore help shed light on the ecology of these early eukaryotes. Typical excavates are characterized by a posterior flagellum equipped with a vane that beats in a ventral groove. Here, we combined flow visualization and observations of prey capture in representatives of the three clades of excavates with computational fluid dynamic modeling, to understand the functional significance of this cell architecture. We record substantial differences amongst species in the orientation of the vane and the beat plane of the posterior flagellum. Clearance rate magnitudes estimated from flow visualization and modeling are both like that of other similarly sized flagellates. The interaction between a vaned flagellum beating in a confinement is modeled to produce a very efficient feeding current at low energy costs, irrespective of the beat plane and vane orientation and of all other morphological variations. Given this predicted uniformity of function, we suggest that the foraging systems of typical excavates studied here may be good proxies to understand those potentially used by our distant ancestors more than 1 billion years ago.

Trapping and scattering of a multiflagellated bacterium by a hard surface.

Thiovulum majus, which is one of the fastest known bacteria, swims using hundreds of flagella. Unlike typical pusher cells, which swim in circular paths over hard surfaces, T. majus localize near hard boundaries by turning their flagella to exert a net force normal to the surface. To probe the torques that stabilize this hydrodynamically bound state, the trajectories of several thousand collisions between a T. majus cell and a wall of a quasi-two-dimensional microfluidic chamber are analyzed. Measuring the fraction of cells escaping the wall either to the left or to the right of the point of contact-and how this probability varies with incident angle and time spent in contact with the surface-maps the scattering dynamics onto a first passage problem. These measurements are compared to the prediction of a Fokker-Planck equation to fit the angular velocity of a cell in contact with a hard surface. This analysis reveals a bound state with a narrow basin of attraction in which cells orient their flagella normal to the surface. The escape angle predicted by matching these near field dynamics with the far-field hydrodynamics is consistent with observation. We discuss the significance of these results for the ecology of T. majus and their self-organization into active chiral crystals.

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner.

The flagellar motor proteins, MotA and MotB, form a complex that rotates the flagella by utilizing the proton motive force (PMF) at the bacterial cell membrane. Although PMF affects the susceptibility to aminoglycosides, the effect of flagellar motor proteins on the susceptibility to aminoglycosides has not been investigated. Here, we found that MotB overexpression increased susceptibility to aminoglycosides, such as kanamycin and gentamicin, in Bacillus subtilis without affecting swimming motility. MotB overexpression did not affect susceptibility to ribosome-targeting antibiotics other than aminoglycosides, cell wall-targeting antibiotics, DNA synthesis-inhibiting antibiotics, or antibiotics inhibiting RNA synthesis. Meanwhile, MotB overexpression increased the susceptibility to aminoglycosides even in the motA-deletion mutant, which lacks swimming motility. Overexpression of the MotB mutant protein carrying an amino acid substitution at the proton-binding site (D24A) resulted in the loss of the enhanced aminoglycoside-sensitive phenotype. These results suggested that MotB overexpression sensitizes B. subtilis to aminoglycosides in a motility-independent manner. Notably, the aminoglycoside-sensitive phenotype induced by MotB requires the proton-binding site but not the MotA/MotB complex formation.

Ecological relevance of flagellar motility in soil bacterial communities.

Flagellar motility is a key bacterial trait as it allows bacteria to navigate their immediate surroundings. Not all bacteria are capable of flagellar motility, and the distribution of this trait, its ecological associations, and the life history strategies of flagellated taxa remain poorly characterized. We developed and validated a genome-based approach to infer the potential for flagellar motility across 12 bacterial phyla (26 192 unique genomes). The capacity for flagellar motility was associated with a higher prevalence of genes for carbohydrate metabolism and higher maximum potential growth rates, suggesting that flagellar motility is more prevalent in environments with higher carbon availability. To test this hypothesis, we applied a method to infer the prevalence of flagellar motility in whole bacterial communities from metagenomic data and quantified the prevalence of flagellar motility across four independent field studies that each captured putative gradients in soil carbon availability (148 metagenomes). We observed a positive relationship between the prevalence of bacterial flagellar motility and soil carbon availability in all datasets. Since soil carbon availability is often correlated with other factors that could influence the prevalence of flagellar motility, we validated these observations using metagenomic data from a soil incubation experiment where carbon availability was directly manipulated with glucose amendments. This confirmed that the prevalence of bacterial flagellar motility is consistently associated with soil carbon availability over other potential confounding factors. This work highlights the value of combining predictive genomic and metagenomic approaches to expand our understanding of microbial phenotypic traits and reveal their general environmental associations.

Motility-activating mutations upstream of flhDC reduce acid shock survival of Escherichia coli.

Many neutralophilic bacterial species try to evade acid stress with an escape strategy, which is reflected in the increased expression of genes coding for flagellar components. Extremely acid-tolerant bacteria, such as Escherichia coli, survive the strong acid stress, e.g., in the stomach of vertebrates. Recently, we were able to show that the induction of motility genes in E. coli is strictly dependent on the degree of acid stress, i.e., they are induced under mild acid stress but not under severe acid stress. However, it was not known to what extent fine-tuned expression of motility genes is related to fitness and the ability to survive periods of acid shock. In this study, we demonstrate that the expression of FlhDC, the master regulator of flagellation, is inversely correlated with the acid shock survival of E. coli. We encountered this phenomenon when analyzing mutants from the Keio collection, in which the expression of flhDC was altered by an insertion sequence element. These results suggest a fitness trade-off between acid tolerance and motility.IMPORTANCEEscherichia coli is extremely acid-resistant, which is crucial for survival in the gastrointestinal tract of vertebrates. Recently, we systematically studied the response of E. coli to mild and severe acidic conditions using Ribo-Seq and RNA-Seq. We found that motility genes are induced at pH 5.8 but not at pH 4.4, indicating stress-dependent synthesis of flagellar components. In this study, we demonstrate that motility-activating mutations upstream of flhDC, encoding the master regulator of flagella genes, reduce the ability of E. coli to survive periods of acid shock. Furthermore, we show an inverse correlation between motility and acid survival using a chromosomal isopropyl ß-D-thio-galactopyranoside (IPTG)-inducible flhDC promoter and by sampling differentially motile subpopulations from swim agar plates. These results reveal a previously undiscovered trade-off between motility and acid tolerance and suggest a differentiation of E. coli into motile and acid-tolerant subpopulations, driven by the integration of insertion sequence elements.

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans.

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

CryoEM structures reveal how the bacterial flagellum rotates and switches direction.

Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or 'switch', limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0 Å) and isolated C-ring in a clockwise pose alone (4.6 Å) and bound to a regulator (5.9 Å). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation.

The MBO2/FAP58 heterodimer stabilizes assembly of inner arm dynein b and reveals axoneme asymmetries involved in ciliary waveform.

Cilia generate three-dimensional waveforms required for cell motility and transport of fluid, mucus, and particles over the cell surface. This movement is driven by multiple dynein motors attached to nine outer doublet microtubules that form the axoneme. The outer and inner arm dyneins are organized into 96-nm repeats tandemly arrayed along the length of the doublets. Motility is regulated in part by projections from the two central pair microtubules that contact radial spokes located near the base of the inner dynein arms in each repeat. Although much is known about the structures and protein complexes within the axoneme, many questions remain about the regulatory mechanisms that allow the cilia to modify their waveforms in response to internal or external stimuli. Here, we used Chlamydomonas mbo (move backwards only) mutants with altered waveforms to identify at least two conserved proteins, MBO2/CCDC146 and FAP58/CCDC147, that form part of a L-shaped structure that varies between doublet microtubules. Comparative proteomics identified additional missing proteins that are altered in other motility mutants, revealing overlapping protein defects. Cryo-electron tomography and epitope tagging revealed that the L-shaped, MBO2/FAP58 structure interconnects inner dynein arms with multiple regulatory complexes, consistent with its function in modifying the ciliary waveform.

Structure-guided discovery of protein and glycan components in native mastigonemes.

Mastigonemes, the hair-like lateral appendages lining cilia or flagella, participate in mechanosensation and cellular motion, but their constituents and structure have remained unclear. Here, we report the cryo-EM structure of native mastigonemes isolated from Chlamydomonas at 3.0 Å resolution. The long stem assembles as a super spiral, with each helical turn comprising four pairs of anti-parallel mastigoneme-like protein 1 (Mst1). A large array of arabinoglycans, which represents a common class of glycosylation in plants and algae, is resolved surrounding the type II poly-hydroxyproline (Hyp) helix in Mst1. The EM map unveils a mastigoneme axial protein (Mstax) that is rich in heavily glycosylated Hyp and contains a PKD2-like transmembrane domain (TMD). Mstax, with nearly 8,000 residues spanning from the intracellular region to the distal end of the mastigoneme, provides the framework for Mst1 assembly. Our study provides insights into the complexity of protein and glycan interactions in native bio-architectures.

Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport.

Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.