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In several pathogenic bacteria, including Vibrio species, the filament of the bacterial flagellum is encased by a membranous sheath, an extension of the bacterial outer membrane. It has been proposed that having sheathed flagella permit bacteria to evade an immune response against flagellar components, suggesting a role in virulence. However, the molecular details of the interaction between sheath and filament, and how it impacts filament rotation, remain largely uncharacterized. Here, we combine single-particle cryo-electron microscopy, cryo-electron tomography, and genetic analyses to resolve the molecular architecture and biogenesis of the sheathed flagellum in Vibrio alginolyticus . We show that the flagellar filament forms a canonical 11-stranded supercoil made of the flagellin FlaD2 and enveloped by a bilayered sheath. We report that the filament surface is highly electronegative, suggesting that electrostatic repulsion between filament and sheath may reduce friction and supports high-speed flagellar rotation. We also show that the filament cap protein FliD possesses a unique domain in sheathed flagella, that may coordinate sheath assembly with filament elongation. Collectively, this structural insight into the structure of the Vibrio alginolyticus flagellum suggests a molecular mechanism for the rotation of sheathed flagella. Most bacteria use a flagellum, to swim and disseminate in their environment, and it is essential for virulence. In Vibrio , the flagellum is surrounded by a sheath, a membrane-like shield that protects the bacterium from the host’s immune system. Here, authors have obtained a view of the sheathed flagellum to atomic levels, revealing the molecular details of how the flagellum rotates within the sheath.

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 infections 1 . Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes 2 . 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. Detailed atomic models of axonemes from algal flagella and human respiratory cilia, which are hair-like protrusions from cells that enable motility and clear mucus from human airways, could provide insights into how they function.

The bacterial flagellar motor can rotate in counterclockwise (CCW) or clockwise (CW) senses, and transitions are controlled by the phosphorylated form of the response regulator CheY (CheY-P). To dissect the mechanism underlying flagellar rotational switching, we use Borrelia burgdorferi as a model system to determine high-resolution in situ motor structures in cheX and cheY3 mutants, in which motors are locked in either CCW or CW rotation. The structures showed that CheY3-P interacts directly with a switch protein, FliM, inducing a major remodeling of another switch protein, FliG2, and altering its interaction with the torque generator. Our findings lead to a model in which the torque generator rotates in response to an inward flow of H+ driven by the proton motive force, and conformational changes in FliG2 driven by CheY3-P allow the switch complex to interact with opposite sides of the rotating torque generator, facilitating rotational switching.In situ cryo-ET analyses of Borrelia burgdorferi flagellar motors locked in clockwise or counterclockwise rotation provide insights into rotational switching.

The Helicobacter pylori flagellar motor contains several accessory structures that are not found in the archetypal Escherichia coli and Salmonella enterica motors. H . pylori hp0838 encodes a previously uncharacterized lipoprotein and is in an operon with flgP , which encodes a motor accessory protein. Deletion analysis of hp0838 in H . pylori B128 showed that the gene is not required for motility in soft agar medium, but the mutant displayed a reduced growth rate and an increased sensitivity to bacitracin, which is an antibiotic that is normally excluded by the outer membrane. Introducing a plasmid-borne copy of hp0838 into the H . pylori Δ hp0838 mutant suppressed the fitness defect and antibiotic sensitivity of the strain. A variant of the Δ hp0838 mutant containing a frameshift mutation in pflA , which resulted in paralyzed flagella, displayed wild-type growth rate and resistance to bacitracin, suggesting the fitness defect and antibiotic sensitivity of the Δ hp0838 mutant are dependent on flagellar rotation. Comparative analysis of in-situ structures of the wild type and Δ hp0838 mutant motors revealed the Δ hp0838 mutant motor lacked a previously undescribed ring structure with 18-fold symmetry located near the outer membrane. Given its role in formation of the motor outer ring, HP0838 was designated FapH ( f lagellar a ccessory p rotein in H elicobacter pylori ) and the motor accessory formed the protein was named the FapH ring. Our data suggest that the FapH ring helps to preserve outer membrane barrier function during flagellar rotation. Given that FapH homologs are present in many members of the phylum Campylobacterota, they may have similar roles in protecting the outer membrane from damage due to flagellar rotation in these bacteria.

Living systems maintain or increase local order by working against the second law of thermodynamics. Thermodynamic consistency is restored as they consume free energy, thereby increasing the net entropy of their environment. Recently introduced estimators for the entropy production rate have provided major insights into the efficiency of important cellular processes. In experiments, however, many degrees of freedom typically remain hidden to the observer, and, in these cases, existing methods are not optimal. Here, by reformulating the problem within an optimization framework, we are able to infer improved bounds on the rate of entropy production from partial measurements of biological systems. Our approach yields provably optimal estimates given certain measurable transition statistics. In contrast to prevailing methods, the improved estimator reveals nonzero entropy production rates even when nonequilibrium processes appear time symmetric and therefore may pretend to obey detailed balance. We demonstrate the broad applicability of this framework by providing improved bounds on the energy consumption rates in a diverse range of biological systems including bacterial flagella motors, growing microtubules, and calcium oscillations within human embryonic kidney cells.

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas . These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes. Microtubules in cilia are sufficiently stable to withstand the beating motion, but how they are stabilized while serving as tracks for intraflagellar transport and axonemal dyneins remains unknown. Here authors identify two microtubule inner proteins, FAP45 and FAP52, which stabilize the ciliary axonemes in Chlamydomonas .

Microorganisms must distribute their limited resources among different physiological functions, including those that do not directly contribute to growth. In this study, we investigate the allocation of resources to flagellar swimming, the most prominent and biosynthetically costly of such cellular functions in bacteria. Although the growth-dependence of flagellar gene expression in peritrichously flagellated Escherichia coli is well known, the underlying physiological limitations and regulatory strategies are not fully understood. By characterizing the dependence of motile behavior on the activity of the flagellar regulon, we demonstrate that, beyond a critical number of filaments, the hydrodynamics of propulsion limits the ability of bacteria to increase their swimming by synthesizing additional flagella. In nutrient-rich conditions, E. coli apparently maximizes its motility until reaching this limit, while avoiding the excessive cost of flagella production. Conversely, during carbon-limited growth motility remains below maximal levels and inversely correlates with the growth rate. The physics of swimming may further explain the selection for bimodal resource allocation in motility at low average expression levels. Notwithstanding strain-specific variation, the expression of flagellar genes in all tested natural isolates of E. coli also falls within the same range defined by the physical limitations on swimming and its biosynthetic cost. Microbial strategies for allocating limited resources to different cellular functions remain to be fully understood. Lisevich et al. show how the interplay between general physical and context-dependent physiological limitations determines resource allocation into a major bacterial cellular function, motility.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

BackgroundMale infertility is a prevalent issue worldwide, mostly due to the impaired sperm motility. Multiple morphological abnormalities of the sperm flagella (MMAF) present aberrant spermatozoa with absent, short, coiled, bent and irregular-calibre flagella resulting in severely decreased motility. Previous studies reported several MMAF-associated genes accounting for approximately half of MMAF cases.Methods and resultWe conducted genetic analysis using whole-exome sequencing in 88 Han Chinese MMAF probands. CFAP65 homozygous mutations were identified in four unrelated consanguineous families, and CFAP65 compound heterozygous mutations were found in two unrelated cases with MMAF. All these CFAP65 mutations were null, including four frameshift mutations (c.1775delC [p.Pro592Leufs*8], c.3072_3079dup [p.Arg1027Profs*41], c.1946delC [p.Pro649Argfs*5] and c.1580delT [p.Leu527Argfs*31]) and three stop-gain mutations (c.4855C>T [p.Arg1619*], c.5270T>A [p.Leu1757*] and c.5341G>T [p.Glu1781*]). Additionally, two homozygous CFAP65 variants likely affecting splicing were identified in two MMAF-affected men of Tunisian and Iranian ancestries, respectively. These biallelic variants of CFAP65 were verified by Sanger sequencing and were absent or very rare in large data sets aggregating sequence information from various human populations. CFAP65, encoding the cilia and flagella associated protein 65, is highly and preferentially expressed in the testis. Here we also generated a frameshift mutation in mouse orthologue Cfap65 using CRISPR-Cas9 technology. Remarkably, the phenotypes of Cfap65-mutated male mice were consistent with human MMAF.ConclusionsOur experimental observations performed on both human subjects and on Cfap65-mutated mice demonstrate that the presence of biallelic mutations in CFAP65 causes the MMAF phenotype and impairs sperm motility.