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Molecular Biology of the Cell May 2024Cilia generate three-dimensional waveforms required for cell motility and transport of fluid, mucus, and particles over the cell surface. This movement is driven by...
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 (ove ackwards nly) 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.
Topics: Axoneme; Dyneins; Microtubules; Cilia; Proteins; Flagella
PubMed: 38568782
DOI: 10.1091/mbc.E23-11-0439 -
Asian Journal of Andrology Jul 2024The second-to-fourth digit (2D:4D) ratio is thought to be associated with prenatal androgen exposure. However, the relationship between the 2D:4D ratio and hypospadias...
The second-to-fourth digit (2D:4D) ratio is thought to be associated with prenatal androgen exposure. However, the relationship between the 2D:4D ratio and hypospadias is poorly understood, and its molecular mechanism is not clear. In this study, by analyzing the hand digit length of 142 boys with hypospadias (23 distal, 68 middle, and 51 proximal) and 196 controls enrolled in Shanghai Children's Hospital (Shanghai, China) from December 2020 to December 2021, we found that the 2D:4D ratio was significantly increased in boys with hypospadias ( P < 0.001) and it was positively correlated with the severity of the hypospadias. This was further verified by the comparison of control mice and prenatal low testosterone mice model obtained by knocking out the risk gene (dynein axonemal heavy chain 8 [ DNAH8 ]) associated with hypospadias. Furthermore, the discrepancy was mainly caused by a shift in 4D. Proteomic characterization of a mouse model validated that low testosterone levels during pregnancy can impair the growth and development of 4D. Comprehensive mechanistic explorations revealed that during the androgen-sensitive window, the downregulation of the androgen receptor (AR) caused by low testosterone levels, as well as the suppressed expression of chondrocyte proliferation-related genes such as Wnt family member 5a ( Wnt5a ), Wnt5b , Smad family member 2 ( Smad2 ), and Smad3 ; mitochondrial function-related genes in cartilage such as AMP-activated protein kinase ( AMPK ) and nuclear respiratory factor 1 ( Nrf-1 ); and vascular development-related genes such as myosin light chain ( MLC ), notch receptor 3 ( Notch3 ), and sphingosine kinase 1 ( Sphk1 ), are responsible for the limitation of 4D growth, which results in a higher 2D:4D ratio in boys with hypospadias via decreased endochondral ossification. This study indicates that the ratio of 2D:4D is a risk marker of hypospadias and provides a potential molecular mechanism.
Topics: Hypospadias; Male; Animals; Humans; Fingers; Mice; Female; Testosterone; Receptors, Androgen; Pregnancy; Child, Preschool; Child; Case-Control Studies
PubMed: 38563741
DOI: 10.4103/aja202377 -
BioRxiv : the Preprint Server For... Mar 2024Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the...
Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in and cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.
PubMed: 38562900
DOI: 10.1101/2024.03.20.585965 -
Scientific Reports Apr 2024The phenylpyrazole insecticide fipronil has wide-ranging applications from agriculture to public health to control undesirable organisms. However, several studies have...
The phenylpyrazole insecticide fipronil has wide-ranging applications from agriculture to public health to control undesirable organisms. However, several studies have reported the residual environmental hazards of fipronil and demonstrated its harmful effects even in mammalian reproduction. Therefore, this study was conducted to demonstrate the mode of action of fipronil on mouse spermatozoa. We treated fipronil to spermatozoa and performed comprehensive function evaluations. Moreover, proteomic analyses were conducted to identify the alteration of protein expression levels in spermatozoa. Most of sperm motility and kinematic parameters and intracellular ATP levels were diminished, and the spontaneous acrosome reaction was promoted after treatment with fipronil. Proteomic analyses revealed altered expression levels of 14 proteins after treatment. These proteins have been reported to be associated with sperm-specific pathways, prominently the cytoskeleton of the sperm, "9 + 2" axoneme composition, metabolism, and fertility. Collectively, our results showed that fipronil alters sperm functional-related proteins and therefore influences male fertility. This study elucidates the possible reproductive toxic hazards associated with male infertility through aberrant suppression of sperm proteins.
Topics: Male; Mice; Animals; Proteomics; Semen; Sperm Motility; Spermatozoa; Proteins; Mammals; Pyrazoles
PubMed: 38561386
DOI: 10.1038/s41598-024-57876-4 -
Biomolecular NMR Assignments Jun 2024The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog,...
The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the H, N, and C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.
Topics: Humans; Calcium-Binding Proteins; Calponins; Microfilament Proteins; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Protein Domains; Solutions
PubMed: 38551798
DOI: 10.1007/s12104-024-10170-w -
Cytoskeleton (Hoboken, N.J.) Mar 2024The axoneme is an intricate nanomachine responsible for generating the propulsive oscillations of cilia and flagella in an astonishing variety of organisms. New imaging...
The axoneme is an intricate nanomachine responsible for generating the propulsive oscillations of cilia and flagella in an astonishing variety of organisms. New imaging techniques based on cryoelectron-tomography (cryo-ET) and subtomogram averaging have revealed the detailed structures of the axoneme and its components with sub-nm resolution, but the mechanical function of each component and how the assembly generates oscillations remains stubbornly unclear. Most explanations of oscillatory behavior rely on the dynamic regulation of dynein by some signal, but this may not be necessary if the system of dynein-driven slender filaments is dynamically unstable. Understanding the possibility of instability-driven oscillations requires a multifilament model of the axoneme that accounts for distortions of the axoneme as it bends. Active bending requires forces and bending moments that will tend to change the spacing and alignment of doublets. We hypothesize that components of the axoneme resist and respond to these loads in ways that are critical to beating. Specifically, we propose (i) that radial spokes provide torsional stiffness by resisting misalignment (as well as spacing) between the central pair and outer doublets, and (ii) that the kinematics of dynein arms affect the relationships between active forces and bending moments on deforming doublets. These proposed relationships enhance the ability of theoretical, multifilament models of axonemal beating to generate propulsive oscillatory waveforms via dynamic mechanical instability.
PubMed: 38546291
DOI: 10.1002/cm.21856 -
Nature Communications Mar 2024Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core...
Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.
Topics: Humans; Cilia; Microtubule Proteins; Axoneme; Microtubules; Centrioles
PubMed: 38538594
DOI: 10.1038/s41467-024-46737-3 -
Auris, Nasus, Larynx Jun 2024Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause... (Review)
Review
OBJECTIVE
Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients.
METHODS
An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology-Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology.
RESULTS
The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells.
CONCLUSION
This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan.
Topics: Humans; Kartagener Syndrome; Genetic Testing; Diagnosis, Differential; Cilia; Japan; Axonemal Dyneins; Proteins
PubMed: 38537559
DOI: 10.1016/j.anl.2024.02.001 -
The American Journal of Case Reports Mar 2024BACKGROUND Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease that can present at different ages with different phenotypes. Missed and delayed...
BACKGROUND Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease that can present at different ages with different phenotypes. Missed and delayed diagnoses are fairly common. Many variants in the DNAH5 gene have been described that confirm the diagnosis of PCD. Advances in medicine, especially in molecular genetics, have led to increasingly early discoveries of such cases, especially in those with nonclassical presentations. CASE REPORT This report describes a patient with bronchiectasis, lung cysts, finger clubbing, and failure to thrive who was misdiagnosed for several years as having asthma. Many differentials were suspected and worked up, including a suspicion of PCD. Genetic tests with whole-exome sequencing (WES) and whole-genome sequencing (WGS) detected a heterozygous, likely pathogenic, variant in the DNAH5 gene associated with PCD. CONCLUSIONS Despite a thorough workup done for this case, including a genetic workup, a PCD diagnosis was not established. We plan to reanalyze the WGS in the future, and with advent of technology and better coverage of genes, a genetic answer for this challenging case may resolve this diagnostic quandary in the future.
Topics: Humans; Axonemal Dyneins; Genetic Testing; Kartagener Syndrome; Lung; Mutation
PubMed: 38521969
DOI: 10.12659/AJCR.942444 -
PLoS Genetics Mar 2024Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans,...
Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.
Topics: Humans; Chlamydomonas reinhardtii; Cilia; Mutation; Dyneins; Proteins; Chlamydomonas; Gene Dosage; Axoneme
PubMed: 38498551
DOI: 10.1371/journal.pgen.1011038