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Nature Communications Apr 2024Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport...
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.
Topics: Caenorhabditis elegans; Animals; Cilia; Caenorhabditis elegans Proteins; Kinesins; Flagella; Tubulin; Axoneme; Dyneins; Biological Transport; Single Molecule Imaging; Protein Transport
PubMed: 38658528
DOI: 10.1038/s41467-024-47807-2 -
The Journal of Cell Biology Sep 2017As essential components of the eukaryotic cytoskeleton, microtubules fulfill a variety of functions that can be temporally and spatially controlled by tubulin...
As essential components of the eukaryotic cytoskeleton, microtubules fulfill a variety of functions that can be temporally and spatially controlled by tubulin posttranslational modifications. Tubulin glycylation has so far been mostly found on motile cilia and flagella, where it is involved in the stabilization of the axoneme. In contrast, barely anything is known about the role of glycylation in primary cilia because of limitations in detecting this modification in these organelles. We thus developed novel glycylation-specific antibodies with which we detected glycylation in many primary cilia. Glycylation accumulates in primary cilia in a length-dependent manner, and depletion or overexpression of glycylating enzymes modulates the length of primary cilia in cultured cells. This strongly suggests that glycylation is essential for the homeostasis of primary cilia, which has important implications for human disorders related to primary cilia dysfunctions, such as ciliopathies and certain types of cancer.
Topics: Animals; Animals, Newborn; Antibodies; Antibody Specificity; Axoneme; Cilia; Dogs; Flagella; Glycosylation; HEK293 Cells; HeLa Cells; Homeostasis; Humans; Madin Darby Canine Kidney Cells; Mice, Inbred C57BL; Movement; Peptide Synthases; Protein Processing, Post-Translational; Time Factors; Transfection; Tubulin
PubMed: 28687664
DOI: 10.1083/jcb.201612050 -
Journal of the Royal Society, Interface Jul 2022The structure of the axoneme in motile cilia and flagella is emerging with increasing detail from high-resolution imaging, but the mechanism by which the axoneme creates...
The structure of the axoneme in motile cilia and flagella is emerging with increasing detail from high-resolution imaging, but the mechanism by which the axoneme creates oscillatory, propulsive motion remains mysterious. It has recently been proposed that this motion may be caused by a dynamic 'flutter' instability that can occur under steady dynein loading, and not by switching or modulation of dynein motor activity (as commonly assumed). In the current work, we have built an improved multi-filament mathematical model of the axoneme and implemented it as a system of discrete equations using the finite-element method. The eigenvalues and eigenvectors of this model predict the emergence of oscillatory, wave-like solutions in the absence of dynein regulation and specify the associated frequencies and waveforms of beating. Time-domain simulations with this model illustrate the behaviour predicted by the system's eigenvalues. This model and analysis allow us to efficiently explore the potential effects of difficult to measure biophysical parameters, such as elasticity of radial spokes and inter-doublet links, on the ciliary waveform. These results support the idea that dynamic instability without dynamic dynein regulation is a plausible and robust mechanism for generating ciliary beating.
Topics: Axoneme; Cilia; Dyneins; Flagella; Models, Biological
PubMed: 35857924
DOI: 10.1098/rsif.2022.0264 -
The Journal of Cell Biology Jan 2020Cells use motile cilia to generate force in the extracellular space. The structure of a cilium can be classified into three subdomains: the intracellular basal body (BB)...
Cells use motile cilia to generate force in the extracellular space. The structure of a cilium can be classified into three subdomains: the intracellular basal body (BB) that templates cilium formation, the extracellular axoneme that generates force, and the transition zone (TZ) that bridges them. While the BB is composed of triplet microtubules (TMTs), the axoneme is composed of doublet microtubules (DMTs), meaning the cilium must convert between different microtubule geometries. Here, we performed electron cryotomography to define this conversion, and our reconstructions reveal identifying structural features of the BB, TZ, and axoneme. Each region is distinct in terms of microtubule number and geometry, microtubule inner proteins, and microtubule linkers. TMT to DMT conversion occurs within the BB, and microtubule geometry changes to axonemal by the end of the TZ, followed by the addition of axoneme-specific components essential for cilium motility. Our results provide the highest-resolution images of the motile cilium to date and reveal how BBs template axonemes.
Topics: Animals; Axoneme; Basal Bodies; Cattle; Cilia; Cryoelectron Microscopy; Electron Microscope Tomography; Microtubule Proteins; Microtubules; Trachea
PubMed: 31874113
DOI: 10.1083/jcb.201907060 -
Journal of Cellular and Molecular... Mar 2018Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces....
Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non-motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down-regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α-tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α-tubulin and protect the acetylation level of α-tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α-tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo-expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia.
Topics: Animals; Asthenozoospermia; Axoneme; HEK293 Cells; Heat-Shock Proteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; NIH 3T3 Cells; Rats, Sprague-Dawley; Sperm Motility; Sperm Tail; Spermatozoa
PubMed: 29168316
DOI: 10.1111/jcmm.13457 -
Philosophical Transactions of the Royal... Feb 2020Nearly all motile cilia and flagella (terms here used interchangeably) have a '9+2' axoneme containing nine outer doublet microtubules and two central microtubules. The...
Nearly all motile cilia and flagella (terms here used interchangeably) have a '9+2' axoneme containing nine outer doublet microtubules and two central microtubules. The central pair of microtubules plus associated projections, termed the central apparatus (CA), is involved in the control of flagellar motility and is essential for the normal movement of '9+2' cilia. Research using the green alga , an important model system for studying cilia, has provided most of our knowledge of the protein composition of the CA, and recent work using this organism has expanded the number of known and candidate CA proteins nearly threefold. Here we take advantage of this enhanced proteome to examine the genomes of a wide range of eukaryotic organisms, representing all of the major phylogenetic groups, to identify predicted orthologues of the CA proteins and explore how widely the proteins are conserved and whether there are patterns to this conservation. We also discuss in detail two contrasting groups of CA proteins-the ASH-domain proteins, which are broadly conserved, and the PAS proteins, which are restricted primarily to the volvocalean algae. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.
Topics: Axoneme; Cilia; Eukaryota; Flagella; Microtubules
PubMed: 31884923
DOI: 10.1098/rstb.2019.0164 -
International Journal For Parasitology Jul 2008African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit... (Review)
Review
African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit sustained economic development in sub-Saharan Africa. T. brucei is a highly motile protozoan parasite and coordinated motility is central to both disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Therefore, understanding unique aspects of the T. brucei flagellum may uncover novel targets for therapeutic intervention in African sleeping sickness. Moreover, studies of conserved features of the T. brucei flagellum are directly relevant to understanding fundamental aspects of flagellum and cilium function in other eukaryotes, making T. brucei an important model system. The T. brucei flagellum contains a canonical 9+2 axoneme, together with additional features that are unique to kinetoplastids and a few closely-related organisms. Until recently, much of our knowledge of the structure and function of the trypanosome flagellum was based on analogy and inference from other organisms. There has been an explosion in functional studies in T. brucei in recent years, revealing conserved as well as novel and unexpected structural and functional features of the flagellum. Most notably, the flagellum has been found to be an essential organelle, with critical roles in parasite motility, morphogenesis, cell division and immune evasion. This review highlights recent discoveries on the T. brucei flagellum.
Topics: Animals; Axoneme; Flagella; Humans; Life Cycle Stages; Protozoan Proteins; RNA Interference; Trypanosoma brucei brucei; Trypanosomiasis, African; Tsetse Flies
PubMed: 18472102
DOI: 10.1016/j.ijpara.2008.03.003 -
Frontiers in Endocrinology 2022Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of sperm flagella and defects in these proteins...
Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of sperm flagella and defects in these proteins generally impair the axoneme structure and affect sperm flagella function. In this study, we performed whole exome sequencing for a cohort of 126 infertile patients with asthenozoospermia and identified homozygous mutation in one patient from a consanguineous family. This identified homozygous mutation was verified by Sanger sequencing. analysis showed that this homozygous mutation is very rare, highly pathogenic, and very conserved. Sperm routine analysis confirmed that the motility of the spermatozoa from the patient significantly decreased. Further sperm morphology analysis showed that the spermatozoa from the patient exhibited multiple flagella morphological defects and a specific loss in the inner dynein arms. Fortunately, the patient was able to have his child intracytoplasmic sperm injection treatment. Our study is the first to demonstrate that homozygous mutation may impair the integration of axoneme structure, affect sperm motility and cause asthenoteratozoospermia in human beings.
Topics: Humans; Male; Asthenozoospermia; Axoneme; Dyneins; Mutation; Semen; Sperm Motility
PubMed: 36726469
DOI: 10.3389/fendo.2022.1058651 -
Proceedings of the National Academy of... May 2019We report a complete 3D structural model of typical epithelial primary cilia based on structural maps of full-length primary cilia obtained by serial section electron...
We report a complete 3D structural model of typical epithelial primary cilia based on structural maps of full-length primary cilia obtained by serial section electron tomography. Our data demonstrate the architecture of primary cilia differs extensively from the commonly acknowledged 9+0 paradigm. The axoneme structure is relatively stable but gradually evolves from base to tip with a decreasing number of microtubule complexes (MtCs) and a reducing diameter. The axonemal MtCs are cross-linked by previously unrecognized fibrous protein networks. Such an architecture explains why primary cilia can elastically withstand liquid flow for mechanosensing. The nine axonemal MtCs in a cilium are found to differ significantly in length indicating intraflagellar transport processes in primary cilia may be more complicated than that reported for motile cilia. The 3D maps of microtubule doublet-singlet transitions generally display longitudinal gaps at the inner junction between the A- and B-tubules, which indicates the inner junction protein is a major player in doublet-singlet transitions. In addition, vesicles releasing from kidney primary cilia were observed in the structural maps, supporting that ciliary vesicles budding may serve as ectosomes for cell-cell communication.
Topics: Animals; Axoneme; Cell Line; Cilia; Cytoplasmic Vesicles; Dogs; Electron Microscope Tomography; Epithelial Cells; Imaging, Three-Dimensional; Microtubules
PubMed: 31004057
DOI: 10.1073/pnas.1821064116 -
The Journal of Cell Biology Jun 2020Mutations in the channel protein PKD2 cause autosomal dominant polycystic kidney disease, but the function of PKD2 in cilia remains unclear. Here, we show that PKD2...
Mutations in the channel protein PKD2 cause autosomal dominant polycystic kidney disease, but the function of PKD2 in cilia remains unclear. Here, we show that PKD2 targets and anchors mastigonemes, filamentous polymers of the glycoprotein MST1, to the extracellular surface of Chlamydomonas cilia. PKD2-mastigoneme complexes physically connect to the axonemal doublets 4 and 8, positioning them perpendicular to the plane of ciliary beating. pkd2 mutant cilia lack mastigonemes, and mutant cells swim with reduced velocity, indicating a motility-related function of the PKD2-mastigoneme complex. Association with both the axoneme and extracellular structures supports a mechanosensory role of Chlamydomonas PKD2. We propose that PKD2-mastigoneme arrays, on opposing sides of the cilium, could perceive forces during ciliary beating and transfer these signals to locally regulate the response of the axoneme.
Topics: Axoneme; Cell Movement; Chlamydomonas; Cilia; Glycoproteins; Mutation; Polymers; Transient Receptor Potential Channels
PubMed: 32348466
DOI: 10.1083/jcb.202001122