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.

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.

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.

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.

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.

Flagellotropic phages: common yet diverse host interaction strategies.

Many bacteriophages (phages) interact with flagella and rely on bacterial motility for successful infection of their hosts. Yet, limited information is available on how phages have evolved to recognize and bind both flagella and subsequent surface receptors for phage DNA injection. Here, we present an update on the current knowledge of flagellotropic phages using a few well-studied phages as examples to unravel the molecular details of bacterial host recognition. We discuss the recent advances in the role of globular exposed flagellin domains and flagella glycosylation in phage binding to the flagella. In addition, we present diverse types of surface receptors and phage components responsible for the interaction with the host. Finally, we point to questions remaining to be answered and new approaches to study this unique group of phages.

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.

Rotation of the Fla2 flagella of Cereibacter sphaeroides requires the periplasmic proteins MotK and MotE that interact with the flagellar stator protein MotB2.

The bacterial flagellum is a complex structure formed by more than 25 different proteins, this appendage comprises three conserved structures: the basal body, the hook and filament. The basal body, embedded in the cell envelope, is the most complex structure and houses the export apparatus and the motor. In situ images of the flagellar motor in different species have revealed a huge diversity of structures that surround the well-conserved periplasmic components of the basal body. The identity of the proteins that form these novel structures in many cases has been elucidated genetically and biochemically, but in others they remain to be identified or characterized. In this work, we report that in the alpha proteobacteria Cereibacter sphaeroides the novel protein MotK along with MotE are essential for flagellar rotation. We show evidence that these periplasmic proteins interact with each other and with MotB2. Moreover, these proteins localize to the flagellated pole and MotK localization is dependent on MotB2 and MotA2. These results together suggest that the role of MotK and MotE is to activate or recruit the flagellar stators to the flagellar structure.

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite.

The honey bee trypanosomatid parasite, Lotmaria passim, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and entire body membrane of L. passim through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in Leishmania, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the FCaBP gene are present in several Trypanosoma species and Leptomonas pyrrhocoris, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of L. passim FCaBP1 is in close proximity to the BBSome complex, while that of Trypanosoma brucei FCaBP does not direct GFP to the flagellum in L. passim. Deletion of the two FCaBP genes in L. passim affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, FCaBP represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.

The swimming defect caused by the absence of the transcriptional regulator LdtR in Sinorhizobium meliloti is restored by mutations in the motility genes motA and motS.

The flagellar motor is a powerful macromolecular machine used to propel bacteria through various environments. We determined that flagellar motility of the alpha-proteobacterium Sinorhizobium meliloti is nearly abolished in the absence of the transcriptional regulator LdtR, known to influence peptidoglycan remodeling and stress response. LdtR does not regulate motility gene transcription. Remarkably, the motility defects of the ΔldtR mutant can be restored by secondary mutations in the motility gene motA or a previously uncharacterized gene in the flagellar regulon, which we named motS. MotS is not essential for S. meliloti motility and may serve an accessory role in flagellar motor function. Structural modeling predicts that MotS comprised an N-terminal transmembrane segment, a long-disordered region, and a conserved ß-sandwich domain. The C terminus of MotS is localized in the periplasm. Genetics based substitution of MotA with MotAG12S also restored the ΔldtR motility defect. The MotAG12S variant protein features a local polarity shift at the periphery of the MotAB stator units. We propose that MotS may be required for optimal alignment of stators in wild-type flagellar motors but becomes detrimental in cells with altered peptidoglycan. Similarly, the polarity shift in stator units composed of MotB/MotAG12S might stabilize its interaction with altered peptidoglycan.

Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Roles for CEP170 in cilia function and dynein-2 assembly.

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.

The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery.

Type III secretion systems (T3SSs) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS contain at least two export signals in their N-terminal D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and cytoplasmic ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of type III secretion systems: targeting of subunits to and their sequential interactions with key components of the export machinery. IMPORTANCE: Many bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface "nanomachines." Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway.

Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor.

The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.

The bacterial division protein MinDE has an independent function in flagellation.

Before preparing for division, bacteria stop their motility. During the exponential growth phase in Escherichia coli, when the rate of bacterial division is highest, the expression of flagellar genes is repressed and bacterial adhesion is enhanced. Hence, it is evident that cell division and motility in bacteria are linked; however, the specific molecular mechanism by which these two processes are linked is not known. While observing E. coli, we found that compared to the WT, the E. coli (Δmin) cells show higher motility and flagellation. We demonstrated that the higher motility was due to the absence of the Min system and can be restored to normal in the presence of Min proteins, where Min system negatively regulates flagella formation. The Min system in E. coli is widely studied for its role in the inhibition of polar Z-ring formation through its pole-to-pole oscillation. However, its role in bacterial motility is not explored. MinD homologs, FlhG and FleN, are known to control flagellar expression through their interaction with FlrA and FleQ, respectively. AtoC, a part of the two-component system AtoSC complex, is homologous to FlrA/FleQ, and the complex is involved in E. coli flagellation via its interaction with the fliA promoter. We have shown that MinD interacts directly with the AtoS of AtoSC complex and controls the fliA expression. Our findings suggest that the Min system acts as a link between cell division and motility in E. coli.

Regulation of ciliary homeostasis by intraflagellar transport-independent kinesins.

Cilia are highly conserved eukaryotic organelles that protrude from the cell surface and are involved in sensory perception, motility, and signaling. Their proper assembly and function rely on the bidirectional intraflagellar transport (IFT) system, which involves motor proteins, including antegrade kinesins and retrograde dynein. Although the role of IFT-mediated transport in cilia has been extensively studied, recent research has highlighted the contribution of IFT-independent kinesins in ciliary processes. The coordinated activities and interplay between IFT kinesins and IFT-independent kinesins are crucial for maintaining ciliary homeostasis. In this comprehensive review, we aim to delve into the specific contributions and mechanisms of action of the IFT-independent kinesins in cilia. By shedding light on their involvement, we hope to gain a more holistic perspective on ciliogenesis and ciliopathies.

STYXL1 regulates CCT complex assembly and flagellar tubulin folding in sperm formation.

Tubulin-based microtubule is a core component of flagella axoneme and essential for sperm motility and male fertility. Structural components of the axoneme have been well explored. However, how tubulin folding is regulated in sperm flagella formation is still largely unknown. Here, we report a germ cell-specific co-factor of CCT complex, STYXL1. Deletion of Styxl1 results in male infertility and microtubule defects of sperm flagella. Proteomic analysis of Styxl1-/- sperm reveals abnormal downregulation of flagella-related proteins including tubulins. The N-terminal rhodanese-like domain of STYXL1 is important for its interactions with CCT complex subunits, CCT1, CCT6 and CCT7. Styxl1 deletion leads to defects in CCT complex assembly and tubulin polymerization. Collectively, our findings reveal the vital roles of germ cell-specific STYXL1 in CCT-facilitated tubulin folding and sperm flagella development.

The emerging functions of intraflagellar transport 52 in ciliary transport and ciliopathies.

Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction.

Wave-like oscillations of clamped microtubules driven by collective dynein transport.

Microtubules (MTs) are observed to move and buckle driven by ATP-dependent molecular motors in both mitotic and interphasic eukaryotic cells as well as in specialized structures such as flagella and cilia with a stereotypical geometry. In previous work, clamped MTs driven by a few kinesin motors were seen to buckle and occasionally flap in what was referred to as flagella-like motion. Theoretical models of active-filament dynamics and a following force have predicted that, with sufficient force and binding-unbinding, such clamped filaments should spontaneously undergo periodic buckling oscillations. However, a systematic experimental test of the theory and reconciliation to a model was lacking. Here, we have engineered a minimal system of MTs clamped at their plus ends and transported by a sheet of dynein motors that demonstrate the emergence of spontaneous traveling-wave oscillations along single filaments. The frequencies of tip oscillations are in the millihertz range and are statistically indistinguishable in the onset and recovery phases. We develop a 2D computational model of clamped MTs binding and unbinding stochastically to motors in a "gliding-assay" geometry. The simulated MTs oscillate with a frequency comparable to experiment. The model predicts the effect of MT length and motor density on qualitative transitions between distinct phases of flapping, regular oscillations, and looping. We develop an effective "order parameter" based on the relative deflection along the filament and orthogonal to it. The transitions predicted in simulations are validated by experimental data. These results demonstrate a role for geometry, MT buckling, and collective molecular motor activity in the emergence of oscillatory dynamics.