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.

Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions1. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains2-5. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis2,6. SLC9C1 activation is further regulated by cAMP2,7, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH-sAC-cAMP signalling pathway in metazoa8,9, required for sperm motility and fertilization4. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration-the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

Fine structure and molecular characterization of two new parabasalid species that naturally colonize laboratory mice, Tritrichomonas musculus and Tritrichomonas casperi.

Tritrichomonas muris is a common flagellated protist isolated from the cecum of wild rodents. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, referred to as Tritrichomonas musculis and Tritrichomonas rainier, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, Tritrichomonas musculus n. sp., and Tritrichomonas casperi n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the 18S rRNA and trans-ITS genetic loci supported their designation as distinct species, related to T. muris. To assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice bred at the National Institutes of Health (NIH) were screened using pan-parabasalid primers that amplify the trans-ITS region. Forty-four percent of mice were positive for parabasalids, encompassing a total of eight distinct sequence types. Tritrichomonas casperi and Trichomitus-like protists were dominant. T. musculus and T. rainier were also detected, but T. muris was not. Our work establishes a previously underappreciated diversity of commensal trichomonad flagellates that naturally colonize the enteric cavity of laboratory mice.

Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography.

Proteus mirabilis is a Gram-negative Gammaproteobacterium and a major causative agent of urinary tract infections in humans. It is characterized by its ability to switch between swimming motility in liquid media and swarming on solid surfaces. Here, we used cryo-electron tomography and subtomogram averaging to reveal the structure of the flagellar motor of P. mirabilis at nanometer resolution in intact cells. We found that P. mirabilis has a motor that is structurally similar to those of Escherichia coli and Salmonella enterica, lacking the periplasmic elaborations that characterize other more specialized gammaproteobacterial motors. In addition, no density corresponding to stators was present in the subtomogram average suggesting that the stators are dynamic. Finally, several assembly intermediates of the motor were seen that support the inside-out assembly pathway.

Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Diving into the complexity of the spirochetal endoflagellum.

Spirochaetes, a phylum that includes medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, are in many ways highly unique bacteria. Their cell morphology, subcellular organization, and metabolism reveal atypical features. Spirochetal motility is also singular, dependent on the presence of periplasmic flagella or endoflagella, inserted subterminally at cell poles and not penetrating the outer membrane and elongating outside the cell as in enterobacteria. In this review we present a comprehensive comparative genomics analysis of endoflagellar systems in spirochetes, highlighting recent findings on the flagellar basal body and filament. Continued progress in understanding the function and architecture of spirochetal flagella is uncovering paradigm-shifting mechanisms of bacterial motility.

Flagellum tapering and midpiece volume in songbird spermatozoa.

In contrast to numerous studies on spermatozoa length, relatively little work focuses on the width of spermatozoa, and particularly the width of the midpiece and flagellum. In flagellated spermatozoa, the flagellum provides forward thrust while energy may be provided via mitochondria in the midpiece and/or through glycolysis along the flagellum itself. Longer flagella may be able to provide greater thrust but may also require stronger structural features and more or larger mitochondria to supply sufficient energy. Here, we use scanning electron microscopy to investigate the ultrastructure of spermatozoa from 55 passerine species in 26 taxonomic families in the Passerides infraorder. Our data confirm the qualitative observation that the flagellum tapers along its length, and we show that longer flagella are wider at the neck. This pattern is similar to mammals, and likely reflects the need for longer cells to be stronger against shearing forces. We further estimate the volume of the mitochondrial helix and show that it correlates well with midpiece length, supporting the use of midpiece length as a proxy for mitochondrial volume, at least in between-species studies where midpiece length is highly variable. These results provide important context for understanding the evolutionary correlations among different sperm cell components and dimensions.

In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains.

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

3D cryo-EM imaging of bacterial flagella: Novel structural and mechanistic insights into cell motility.

Bacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from Escherichia coli weighs ∼20 MDa, without considering its filament portion, which is by itself a ∼1.6 GDa structure arranged as a multimer of ∼30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way.

Wrapped Up: The Motility of Polarly Flagellated Bacteria.

A huge number of bacterial species are motile by flagella, which allow them to actively move toward favorable environments and away from hazardous areas and to conquer new habitats. The general perception of flagellum-mediated movement and chemotaxis is dominated by the Escherichia coli paradigm, with its peritrichous flagellation and its famous run-and-tumble navigation pattern, which has shaped the view on how bacteria swim and navigate in chemical gradients. However, a significant amount-more likely the majority-of bacterial species exhibit a (bi)polar flagellar localization pattern instead of lateral flagella. Accordingly, these species have evolved very different mechanisms for navigation and chemotaxis. Here, we review the earlier and recent findings on the various modes of motility mediated by polar flagella.

FliL ring enhances the function of periplasmic flagella.

SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of Borrelia burgdorferi, revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.

Structural basis of torque generation in the bi-directional bacterial flagellar motor.

The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA5B2) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor.

Regulator of ribonuclease activity modulates the pathogenicity of Vibrio vulnificus.

RraA, a protein regulator of RNase E activity, plays a unique role in modulating the mRNA abundance in Escherichia coli. The marine pathogenic bacterium Vibrio vulnificus also possesses homologs of RNase E (VvRNase E) and RraA (VvRraA1 and VvRraA2). However, their physiological roles have not yet been investigated. In this study, we demonstrated that VvRraA1 expression levels affect the pathogenicity of V. vulnificus. Compared to the wild-type strain, the VvrraA1-deleted strain (ΔVvrraA1) showed decreased motility, invasiveness, biofilm formation ability as well as virulence in mice; these phenotypic changes of ΔVvrraA1 were restored by the exogenous expression of VvrraA1. Transcriptomic analysis indicated that VvRraA1 expression levels affect the abundance of a large number of mRNA species. Among them, the half-lives of mRNA species encoding virulence factors (e.g., smcR and htpG) that have been previously shown to affect VvrraA1 expression-dependent phenotypes were positively correlated with VvrraA1 expression levels. These findings suggest that VvRraA1 modulates the pathogenicity of V. vulnificus by regulating the abundance of a subset of mRNA species.

Characterization of the Flagellar Collar Reveals Structural Plasticity Essential for Spirochete Motility.

Spirochetes are a remarkable group of bacteria with distinct morphology and periplasmic flagella that enable motility in viscous environments, such as host connective tissues. The collar, a spirochete-specific complex of the periplasmic flagellum, is required for this unique spirochete motility, yet it has not been clear how the collar assembles and enables spirochetes to transit between complex host environments. Here, we characterize the collar complex in the Lyme disease spirochete Borrelia burgdorferi. We discover as well as delineate the distinct functions of two novel collar proteins, FlcB and FlcC, by combining subtractive bioinformatic, genetic, and cryo-electron tomography approaches. Our high-resolution in situ structures reveal that the multiprotein collar has a remarkable structural plasticity essential not only for assembly of flagellar motors in the highly curved membrane of spirochetes but also for generation of the high torque necessary for spirochete motility. IMPORTANCE Many spirochetes cause serious human diseases. They are well recognized by their distinct morphology and motility. Spirochete motility is driven by a periplasmic flagellum, which possesses a unique collar essential for flagellar assembly and spirochete motility. Here, we discover two novel collar proteins in the Lyme disease spirochete Borrelia burgdorferi. We demonstrate, for the first time, that the collar is a multiprotein complex with a remarkable plasticity that enables the motor to accommodate the highly curved membrane of spirochetes and generate the high torque necessary for spirochete motility.

Molecular Determinants of Filament Capping Proteins Required for the Formation of Functional Flagella in Gram-Negative Bacteria.

Bacterial flagella are cell surface protein appendages that are critical for motility and pathogenesis. Flagellar filaments are tubular structures constructed from thousands of copies of the protein flagellin, or FliC, arranged in helical fashion. Individual unfolded FliC subunits traverse the filament pore and are folded and sorted into place with the assistance of the flagellar capping protein complex, an oligomer of the FliD protein. The FliD filament cap is a stool-like structure, with its D2 and D3 domains forming a flat head region, and its D1 domain leg-like structures extending perpendicularly from the head towards the inner core of the filament. Here, using an approach combining bacterial genetics, motility assays, electron microscopy and molecular modeling, we define, in numerous Gram-negative bacteria, which regions of FliD are critical for interaction with FliC subunits and result in the formation of functional flagella. Our data indicate that the D1 domain of FliD is its sole functionally important domain, and that its flexible coiled coil region comprised of helices at its extreme N- and C-termini controls compatibility with the FliC filament. FliD sequences from different bacterial species in the head region are well tolerated. Additionally, head domains can be replaced by small peptides and larger head domains from different species and still produce functional flagella.

Mechanosensitive recruitment of stator units promotes binding of the response regulator CheY-P to the flagellar motor.

Reversible switching of the bacterial flagellar motor between clockwise (CW) and counterclockwise (CCW) rotation is necessary for chemotaxis, which enables cells to swim towards favorable chemical habitats. Increase in the viscous resistance to the rotation of the motor (mechanical load) inhibits switching. However, cells must maintain homeostasis in switching to navigate within environments of different viscosities. The mechanism by which the cell maintains optimal chemotactic function under varying loads is not understood. Here, we show that the flagellar motor allosterically controls the binding affinity of the chemotaxis response regulator, CheY-P, to the flagellar switch complex by modulating the mechanical forces acting on the rotor. Mechanosensitive CheY-P binding compensates for the load-induced loss of switching by precisely adapting the switch response to a mechanical stimulus. The interplay between mechanical forces and CheY-P binding tunes the chemotactic function to match the load. This adaptive response of the chemotaxis output to mechanical stimuli resembles the proprioceptive feedback in the neuromuscular systems of insects and vertebrates.

Gaia Pigino: Inside the cell.

Gaia Pigino studies the molecular mechanisms and principles of self-organization in cilia using 3D cryo-EM.

Structural Dynamics of the Functional Nonameric Type III Translocase Export Gate.

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of ∼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a ∼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.

A novel mutation in DNAH17 is present in a patient with multiple morphological abnormalities of the flagella.

RESEARCH QUESTION: Asthenoteratospermia is characterized by malformed spermatozoa with motility defects, which results in male infertility. Multiple morphological abnormalities of the sperm flagella (MMAF) is a hallmark of asthenoteratospermia. The genetic causes of MMAF, however, are unknown in about one-third of cases. Which other MMAF-associated genes are waiting to be discovered? DESIGN: Whole-exome sequencing was conducted to identify causative genes in a man with MMAF. Immunofluorescence staining and western blot were applied to assess the pathogenicity of the identified variant. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with MMAF. RESULT: Sanger sequencing of the family demonstrated that the infertile man carried a homozygous DNAH17 variant (c. 4810C>T [p.R1604C]). The obviously decreased DNAH17 expression was observed in HEK293T cells transfected with MUT-DNAH17 plasmid compared with cells with WT-DNAH17 plasmid. Immunofluorescence analysis showed that this mutation induced significant decrease in DNAH17 expression, which negatively affected the DNAH8 expression in the patient's spermatozoa. Moreover, the outcome of ICSI in the patient was unsuccessful. CONCLUSION: Our study revealed a novel homozygous missense mutation in DNAH17 involved in MMAF phenotype. The finding of the novel mutation in DNAH17 enriches the gene variant spectrum of MMAF, further contributing to diagnosis, genetic counselling and prognosis for male infertility.

Structure of the molecular bushing of the bacterial flagellar motor.

The basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface.