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Proceedings of the National Academy of... Aug 2022Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome...
Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome footprinting, we performed global translatome profiling during cilia regeneration in and uncovered that flagellar genes undergo an early transcriptional activation but late translational repression. This pattern guided our identification of sphingolipid metabolism enzymes, including serine palmitoyltransferase (SPT), as essential regulators for ciliogenesis. Cryo-electron tomography showed that ceramide loss abnormally increased the membrane-axoneme distance and generated bulged cilia. We found that ceramides interact with intraflagellar transport (IFT) particle proteins that IFT motors transport along axoneme microtubules (MTs), suggesting that ceramide-IFT particle-IFT motor-MT interactions connect the ciliary membrane with the axoneme to form rod-shaped cilia. SPT-deficient vertebrate cells were defective in ciliogenesis, and SPT mutations from patients with hereditary sensory neuropathy disrupted cilia, which could be restored by sphingolipid supplementation. These results reveal a conserved role of sphingolipid in cilium formation and link compromised sphingolipid production with ciliopathies.
Topics: Axoneme; Ceramides; Chlamydomonas; Cilia; Flagella; Protein Transport; Regeneration; Sphingolipids
PubMed: 35895683
DOI: 10.1073/pnas.2201096119 -
Frontiers in Cell and Developmental... 2021In one of the first examples of how mechanics can inform axonemal mechanism, Machin's study in the 1950s highlighted that observations of sperm motility cannot be... (Review)
Review
In one of the first examples of how mechanics can inform axonemal mechanism, Machin's study in the 1950s highlighted that observations of sperm motility cannot be explained by molecular motors in the cell membrane, but would instead require motors distributed along the flagellum. Ever since, mechanics and hydrodynamics have been recognised as important in explaining the dynamics, regulation, and guidance of sperm. More recently, the digitisation of sperm videomicroscopy, coupled with numerous modelling and methodological advances, has been bringing forth a new era of scientific discovery in this field. In this review, we survey these advances before highlighting the opportunities that have been generated for both recent research and the development of further open questions, in terms of the detailed characterisation of the sperm flagellum beat and its mechanics, together with the associated impact on cell behaviour. In particular, diverse examples are explored within this theme, ranging from how collective behaviours emerge from individual cell responses, including how these responses are impacted by the local microenvironment, to the integration of separate advances in the fields of flagellar analysis and flagellar mechanics.
PubMed: 34354994
DOI: 10.3389/fcell.2021.710825 -
National Science Review Sep 2023Multiple morphological abnormalities of the sperm flagella (MMAF) are one of the major causes of male infertility and are characterized by multiple defects. In this...
Multiple morphological abnormalities of the sperm flagella (MMAF) are one of the major causes of male infertility and are characterized by multiple defects. In this study, we found that the coiled-coil domain-containing 189 () gene was predominantly expressed in mouse testes and that inactivation of the gene caused male infertility. Histological studies revealed that most sperm from mice carried coiled, curved or short flagella, which are typical MMAF phenotypes. Immunoelectron microscopy showed that the CCDC189 protein was located at the radial spoke of the first peripheral microtubule doublet in the sperm axoneme. A CCDC189-interacting protein, CABCOCO1 (ciliary-associated calcium-binding coiled-coil protein 1), was discovered via co-immunoprecipitation and mass spectrometry, and inactivation of caused malformation of sperm flagella, which was consistent with findings obtained with mice. Further studies revealed that inactivation of CCDC189 caused downregulation of CABCOCO1 protein expression and that both CCDC189 and CABCOCO1 interacted with the radial-spoke-specific protein RSPH1 and intraflagellar transport proteins. This study demonstrated that is a radial-spoke-associated protein and is involved in sperm flagellum formation through its interactions with CABCOCO1 and intraflagellar transport proteins.
PubMed: 37601242
DOI: 10.1093/nsr/nwad181 -
Open Biology Nov 2023Spermatid elongation is a crucial event in the late stage of spermatogenesis in the testis, eventually leading to the formation of mature sperm after meiosis. During... (Review)
Review
Spermatid elongation is a crucial event in the late stage of spermatogenesis in the testis, eventually leading to the formation of mature sperm after meiosis. During spermatogenesis, significant structural and morphological changes take place in a cluster of post-meiotic germ cells, which are enclosed in a microenvironment surrounded by somatic cyst cells. Microtubule-based axoneme assembly, formation of individualization complexes and mitochondria maintenance are key processes involved in the differentiation of elongated spermatids. They provide important structural foundations for accessing male fertility. How these structures are constructed and maintained are basic questions in the testis. Although the roles of several genes in different structures during the development of elongated spermatids have been elucidated, the relationships between them have not been widely studied. In addition, the genetic basis of spermatid elongation and the regulatory mechanisms involved have not been thoroughly investigated. In the present review, we focus on current knowledge with regard to spermatid axoneme assembly, individualization complex and mitochondria maintenance. We also touch upon promising directions for future research to unravel the underlying mechanisms of spermatid elongation in the testis.
Topics: Animals; Male; Spermatids; Testis; Drosophila; Drosophila melanogaster; Drosophila Proteins; Semen; Spermatogenesis
PubMed: 37935354
DOI: 10.1098/rsob.230136 -
The Journal of Clinical Investigation Aug 2020Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins...
Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.
Topics: Abnormalities, Multiple; Acetylation; Animals; Armadillo Domain Proteins; CRISPR-Cas Systems; Cerebellum; Cilia; Disease Models, Animal; Eye Abnormalities; Humans; Kidney Diseases, Cystic; Peptides; Retina; Zebrafish; Zebrafish Proteins
PubMed: 32453716
DOI: 10.1172/JCI131656 -
BMC Biology May 2023The axonemal microtubules of primary cilium undergo a conserved protein posttranslational modification (PTM) - polyglutamylation. This reversible procedure is processed...
BACKGROUND
The axonemal microtubules of primary cilium undergo a conserved protein posttranslational modification (PTM) - polyglutamylation. This reversible procedure is processed by tubulin tyrosine ligase-like polyglutamylases to form secondary polyglutamate side chains, which are metabolized by the 6-member cytosolic carboxypeptidase (CCP) family. Although polyglutamylation modifying enzymes have been linked to ciliary architecture and motility, it was unknown whether they also play a role in ciliogenesis.
RESULTS
In this study, we found that CCP5 expression is transiently downregulated upon the initiation of ciliogenesis, but recovered after cilia are formed. Overexpression of CCP5 inhibited ciliogenesis, suggesting that a transient downregulation of CCP5 expression is required for ciliation initiation. Interestingly, the inhibitory effect of CCP5 on ciliogenesis does not rely on its enzyme activity. Among other 3 CCP members tested, only CCP6 can similarly suppress ciliogenesis. Using CoIP-MS analysis, we identified a protein that potentially interacts with CCP - CP110, a known negative regulator of ciliogenesis, whose degradation at the distal end of mother centriole permits cilia assembly. We found that both CCP5 and CCP6 can modulate CP110 level. Particularly, CCP5 interacts with CP110 through its N-terminus. Loss of CCP5 or CCP6 led to the disappearance of CP110 at the mother centriole and abnormally increased ciliation in cycling RPE-1 cells. Co-depletion of CCP5 and CCP6 synergized this abnormal ciliation, suggesting their partially overlapped function in suppressing cilia formation in cycling cells. In contrast, co-depletion of the two enzymes did not further increase the length of cilia, although CCP5 and CCP6 differentially regulate polyglutamate side-chain length of ciliary axoneme and both contribute to limiting cilia length, suggesting that they may share a common pathway in cilia length control. Through inducing the overexpression of CCP5 or CCP6 at different stages of ciliogenesis, we further demonstrated that CCP5 or CCP6 inhibited cilia formation before ciliogenesis, while shortened the length of cilia after cilia formation.
CONCLUSION
These findings reveal the dual role of CCP5 and CCP6. In addition to regulating cilia length, they also retain CP110 level to suppress cilia formation in cycling cells, pointing to a novel regulatory mechanism for ciliogenesis mediated by demodifying enzymes of a conserved ciliary PTM, polyglutamylation.
Topics: HEK293 Cells; Humans; Carboxypeptidases; Microtubule-Associated Proteins; Cilia; Microtubules
PubMed: 37226238
DOI: 10.1186/s12915-023-01622-1 -
Nature Communications Dec 2023Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian...
Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian hosts. To better understand the 3D organisation of male gametogenesis, we used serial block face scanning electron microscopy (SBF-SEM) and serial-section cellular electron tomography (ssET) of P. berghei microgametocytes to examine key structures during male gamete formation. Our data reveals an elaborate organisation of axonemes coiling around the nucleus in opposite directions forming a central axonemal band in microgametocytes. Furthermore, we discover the nucleus of microgametes to be tightly coiled around the axoneme in a complex structure whose formation starts before microgamete emergence during exflagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlight some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite.
Topics: Male; Animals; Plasmodium berghei; Haploidy; Germ Cells; Anopheles; Flagella; Mammals
PubMed: 38092766
DOI: 10.1038/s41467-023-43877-w -
The Journal of Cell Biology Nov 2023Cilia are essential organelles that protrude from the cell body. Cilia are made of a microtubule-based structure called the axoneme. In most types of cilia, the ciliary...
Cilia are essential organelles that protrude from the cell body. Cilia are made of a microtubule-based structure called the axoneme. In most types of cilia, the ciliary tip is distinct from the rest of the cilium. Here, we used cryo-electron tomography and subtomogram averaging to obtain the structure of the ciliary tip of the ciliate Tetrahymena thermophila. We show that the microtubules at the tip are highly crosslinked with each other and stabilized by luminal proteins, plugs, and cap proteins at the plus ends. In the tip region, the central pair lacks typical projections and twists significantly. By analyzing cells lacking a ciliary tip-enriched protein CEP104/FAP256 by cryo-electron tomography and proteomics, we discovered candidates for the central pair cap complex and explained the potential functions of CEP104/FAP256. These data provide new insights into the function of the ciliary tip and the mechanisms of ciliary assembly and length regulation.
Topics: Axoneme; Cilia; Microtubules; Tetrahymena thermophila
PubMed: 37756660
DOI: 10.1083/jcb.202301129 -
Nature Communications Apr 2023Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of...
Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.
Topics: Tetrahymena thermophila; Cryoelectron Microscopy; Microtubules; Axoneme; Cytoskeleton; Cilia; Microtubule Proteins; Tetrahymena
PubMed: 37061538
DOI: 10.1038/s41467-023-37868-0 -
Proceedings of the National Academy of... Aug 2022Cilia are microtubule-based organelles that power cell motility and regulate sensation and signaling, and abnormal ciliary structure and function cause various...
Cilia are microtubule-based organelles that power cell motility and regulate sensation and signaling, and abnormal ciliary structure and function cause various ciliopathies. Cilium formation and maintenance requires intraflagellar transport (IFT), during which the kinesin-2 family motor proteins ferry IFT particles carrying axonemal precursors such as tubulins into cilia. Tubulin dimers are loaded to IFT machinery through an interaction between tubulin and the IFT-74/81 module; however, little is known of how tubulins are unloaded when arriving at the ciliary tip. Here, we show that the ciliary kinase DYF-5/MAK phosphorylates multiple sites within the tubulin-binding module of IFT-74, reducing the tubulin-binding affinity of IFT-74/81 approximately sixfold. Ablation or constitutive activation of IFT-74 phosphorylation abnormally elongates or shortens sensory cilia in neurons. We propose that DYF-5/MAK-dependent phosphorylation plays a fundamental role in ciliogenesis by regulating tubulin unloading.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cilia; Mitogen-Activated Protein Kinases; Phosphorylation; Tubulin
PubMed: 35969738
DOI: 10.1073/pnas.2207134119