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Current Biology : CB Nov 2014In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at...
BACKGROUND
In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear.
RESULTS
Here we report that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. We show that Cep290 is essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity and find that MKS proteins also delimit a centriole-independent compartment in mouse spermatids.
CONCLUSIONS
Our findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly.
Topics: Animals; Axoneme; Cilia; Drosophila; Drosophila Proteins; Male; Mice; Microtubule-Associated Proteins; Spermatids
PubMed: 25447994
DOI: 10.1016/j.cub.2014.09.047 -
Journal of Cell Science Dec 2022The assembly and maintenance of most cilia and eukaryotic flagella depends on intraflagellar transport (IFT), the bidirectional movement of multi-megadalton IFT trains... (Review)
Review
The assembly and maintenance of most cilia and eukaryotic flagella depends on intraflagellar transport (IFT), the bidirectional movement of multi-megadalton IFT trains along the axonemal microtubules. These IFT trains function as carriers, moving ciliary proteins between the cell body and the organelle. Whereas tubulin, the principal protein of cilia, binds directly to IFT particle proteins, the transport of other ciliary proteins and complexes requires adapters that link them to the trains. Large axonemal substructures, such as radial spokes, outer dynein arms and inner dynein arms, assemble in the cell body before attaching to IFT trains, using the adapters ARMC2, ODA16 and IDA3, respectively. Ciliary import of several membrane proteins involves the putative adapter tubby-like protein 3 (TULP3), whereas membrane protein export involves the BBSome, an octameric complex that co-migrates with IFT particles. Thus, cells employ a variety of adapters, each of which is substoichiometric to the core IFT machinery, to expand the cargo range of the IFT trains. This Review summarizes the individual and shared features of the known cargo adapters and discusses their possible role in regulating the transport capacity of the IFT pathway.
Topics: Dyneins; Flagella; Protein Transport; Axoneme; Cilia; Biological Transport; Membrane Proteins
PubMed: 36533425
DOI: 10.1242/jcs.260408 -
Cellular Microbiology Mar 2020Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid...
Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal '9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.
Topics: Animals; Axoneme; Culicidae; Female; Gene Knockout Techniques; Genes, Protozoan; Kinesins; Life Cycle Stages; Malaria; Mice; Mitosis; Models, Animal; Mosquito Vectors; Organisms, Genetically Modified; Plasmodium berghei; Protozoan Proteins
PubMed: 31634979
DOI: 10.1111/cmi.13121 -
International Review of Cell and... 2008Insects do not possess ciliated epithelia, and cilia/flagella are present in the sperm tail and--as modified cilia--in mechano- and chemosensory neurons. The core... (Review)
Review
Insects do not possess ciliated epithelia, and cilia/flagella are present in the sperm tail and--as modified cilia--in mechano- and chemosensory neurons. The core cytoskeletal component of these organelles, the axoneme, is a microtubule-based structure that has been conserved throughout evolution. However, in insects the sperm axoneme exhibits distinctive structural features; moreover, several insect groups are characterized by an unusual sperm axoneme variability. Besides the abundance of morphological data on insect sperm flagella, most of the available molecular information on the insect axoneme comes from genetic studies on Drosophila spermatogenesis, and only recently other insect species have been proposed as useful models. Here, we review the current knowledge on the cell biology of insect axoneme, including contributions from both Drosophila and other model insects.
Topics: Animals; Axoneme; Cilia; Drosophila; Dyneins; Flagella; Insect Proteins; Insecta; Male; Microscopy, Electron; Movement; Sperm Tail; Tubulin
PubMed: 18703405
DOI: 10.1016/S1937-6448(08)00804-6 -
Methods in Molecular Biology (Clifton,... 2020Microtubules can be detected in light microscopes, but the limited resolution of these instruments means that the polymers appear as lines whose width is defined by the...
Microtubules can be detected in light microscopes, but the limited resolution of these instruments means that the polymers appear as lines whose width is defined by the diffraction of light. Much important work on microtubule dynamics has been accomplished by light microscopy, but the details of microtubule end structure are not accessible in such studies. Slight variations in fluorescence intensity, etc. have been used to comment on the structure of dynamic ends, and the combination of light microscopy with laser tweezers has provided insight into aspects of microtubule elongation. However, for views that reveal structural details of the pathways for microtubule growth and shortening, electron microscopy has been of great value. Here, we describe methods for using electron microscopes to look at the ends of microtubules as they grow and shrink, both in vivo and in vitro. The key problems to be overcome for ultrastructural study of microtubule dynamics are those of reliable sample preparation. Dynamic microtubules are labile and can therefore be modified by preparative methods. Our chapter follows the premise that rapid freezing, which converts sample water into vitreous ice, is the best approach for sample preparation. Therefore, all of the methods described involve finding optimal conditions for sample vitrification, and then getting the frozen sample into a form suitable for electron microscopy. We also posit that the end of a microtubule must be considered in three dimensions, so we employ electron tomography as a way to get the necessary information. The methods described for the study of microtubules in cells employ rapid freezing, freeze-substitution fixation, plastic embedding, serial sectioning, and tomography of stained samples. The methods for following microtubule growth in vitro employ sample preparation on holy grids, blotting, and plunge-freezing, followed by electron cryo-tomography. Quantification of structure from both approaches is accomplished by segmentation and analysis of graphic models.
Topics: Axoneme; Cryoelectron Microscopy; Image Processing, Computer-Assisted; Microscopy, Electron; Microtubules; Neurons; Protein Binding; Protein Multimerization; Tubulin
PubMed: 31879906
DOI: 10.1007/978-1-0716-0219-5_13 -
FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction.Nature Communications Aug 2023Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules....
Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify MIP candidates. Using RNAi knockdown together with fitting of AlphaFold models into cryoET maps, we demonstrate that one of these candidates, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention.
Topics: Cilia; Flagella; Microtubules; Acclimatization; Axoneme; Microtubule Proteins
PubMed: 37633952
DOI: 10.1038/s41467-023-40230-z -
The Journal of Biological Chemistry 2021INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates...
INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates the phosphoinositide composition of membranes. Mutations to or loss of INPP5E is associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans-a syndromic ciliopathy affecting multiple tissues including the brain, liver, kidney, and retina. In contrast to other primary cilia, photoreceptor INPP5E is prominently expressed in the inner segment and connecting cilium and absent in the outer segment, which is a modified primary cilium dedicated to phototransduction. To investigate how loss of INPP5e causes retina degeneration, we generated mice with a retina-specific KO (Inpp5e;Six3Cre, abbreviated as Inpp5e). These mice exhibit a rapidly progressing rod-cone degeneration resembling Leber congenital amaurosis that is nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. Although P10 mutant outer segments contain structural and phototransduction proteins, axonemal structure and disc membranes fail to form. Connecting cilia of Inpp5e rods display accumulation of intraflagellar transport particles A and B at their distal ends, suggesting disrupted intraflagellar transport. Although INPP5E ablation may not prevent delivery of outer segment-specific proteins by means of the photoreceptor secretory pathway, its absence prevents the assembly of axonemal and disc components. Herein, we suggest a model for INPP5E-Leber congenital amaurosis, proposing how deletion of INPP5E may interrupt axoneme extension and disc membrane elaboration.
Topics: Animals; Axoneme; Eye Proteins; Mice; Mice, Knockout; Morphogenesis; Phosphoric Monoester Hydrolases; Protein Transport; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinal Rod Photoreceptor Cells
PubMed: 33711342
DOI: 10.1016/j.jbc.2021.100529 -
Clinical Genetics Dec 2023Asthenozoospermia (AZS) is the primary cause of infertility in males. The radial spoke (RS) is an axonemal structure, connecting the peripheral doublet microtubules with...
Asthenozoospermia (AZS) is the primary cause of infertility in males. The radial spoke (RS) is an axonemal structure, connecting the peripheral doublet microtubules with the central pair of microtubules. This T-shaped multiprotein complex functions as a mechanochemical sensor to promote sperm motility. LRRC23 is a novel subunit of the RS complex that is necessary for flagellar assembly and movement in mice. However, the importance of LRRC23 in modulating RS formation in humans remains unclear. Here, we identified a homozygous nonsense mutation in LRRC23 (c.376C>T:p. Arg126X) in an infertile AZS patient whose parents were consanguineous. We verified the adversity of this novel mutation because of its ability to disrupt LRRC23 synthesis and impair RSs integrity. Furthermore, we demonstrated an interaction between LRRC23 and RSPH3 in vitro, indicating that LCCR23 is associated with RS in humans. Meanwhile, the LRRC23-mutant patient had a good prognosis following intracytoplasmic sperm injection. This study provides strong preliminary evidence that LRRC23 defects are potential causative factors of AZS in humans, which expands our knowledge for improved genetic counseling and better reproductive recommendations for patients with AZS.
Topics: Male; Humans; Animals; Mice; Asthenozoospermia; Sperm Motility; Semen; Infertility, Male; Axoneme; Spermatozoa
PubMed: 37804054
DOI: 10.1111/cge.14433 -
Human Reproduction Update 2008Sperm hyperactivation is critical to fertilization, because it is required for penetration of the zona pellucida. Hyperactivation may also facilitate release of sperm... (Review)
Review
BACKGROUND
Sperm hyperactivation is critical to fertilization, because it is required for penetration of the zona pellucida. Hyperactivation may also facilitate release of sperm from the oviductal storage reservoir and may propel sperm through mucus in the oviductal lumen and the matrix of the cumulus oophorus. Hyperactivation is characterized by high amplitude, asymmetrical flagellar bending.
METHODS
This is a review of the original literature on the mechanisms that regulate hyperactivation, including physiological factors and signaling pathways.
RESULTS
Computer-assisted semen analysis systems can be used to identify hyperactivated sperm by setting minimum thresholds for curvilinear velocity (VSL) and lateral head movement and a maximum threshold for path linearity. Hyperactivation is triggered by a rise in flagellar Ca(2+) resulting from influx primarily through plasma membrane CatSper channels and possibly also by release of Ca(2+) from a store in the redundant nuclear envelope. It requires increased pH and ATP production. The physiological signals that trigger the rise in Ca(2+) remain elusive, but there is evidence that the increased Ca(2+) acts through a calmodulin/calmodulin kinase pathway. Hyperactivation is considered part of the capacitation process; however, the regulatory pathway that triggers hyperactivation can operate independently from that which prepares sperm to undergo the acrosome reaction. Hyperactivation may be modulated by chemotactic signals to turn sperm toward the oocyte.
CONCLUSIONS
Little is known about exactly what triggers hyperactivation in human sperm. This information could enable clinicians to develop reliable fertility assays to assess normal hyperactivation in human sperm samples.
Topics: Adenosine Triphosphate; Animals; Axoneme; Calcium; Calcium Signaling; Cell Membrane; Chemotactic Factors; Cyclic AMP; Fertility; Flagella; Humans; Hydrogen-Ion Concentration; Male; Sperm Capacitation; Sperm Motility; Spermatozoa
PubMed: 18653675
DOI: 10.1093/humupd/dmn029 -
Nature Communications Oct 2014Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named...
Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.
Topics: Adaptor Proteins, Signal Transducing; Axoneme; Centrioles; Cilia; Cullin Proteins; Humans; Proteasome Endopeptidase Complex; Ubiquitin
PubMed: 25270598
DOI: 10.1038/ncomms6081