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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 -
Cell Nov 2023To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization,...
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
Topics: Animals; Male; Mice; Axoneme; Cryoelectron Microscopy; Flagella; Microtubules; Semen; Spermatozoa; Proteome
PubMed: 37865089
DOI: 10.1016/j.cell.2023.09.017 -
Seminars in Cell & Developmental Biology Jan 2023Ciliogenesis is a complex multistep process used to describe assembly of cilia and flagella. These organelles play essential roles in motility and signaling on the... (Review)
Review
Ciliogenesis is a complex multistep process used to describe assembly of cilia and flagella. These organelles play essential roles in motility and signaling on the surface of cells. Cilia are built at the distal ends of centrioles through the formation of an axoneme that is surrounded by the ciliary membrane. As is the case in the biogenesis of other cellular organelles, regulators of membrane trafficking play essential roles in ciliogenesis, albeit with a unique feature that membranes are organized around microtubule-based structures. Membrane association with the distal end of the centriole is a critical initiating step for ciliogenesis. Studies of this process in different cell types suggests that a singular mechanism may not be utilized to initiate cilium assembly. In this review, we focus on recent insights into cilium biogenesis and the roles membrane trafficking regulators play in described ciliogenesis mechanisms with relevance to human disease.
Topics: Humans; Centrioles; Axoneme; Cilia; Microtubules; Flagella
PubMed: 35351373
DOI: 10.1016/j.semcdb.2022.03.021 -
ACS Synthetic Biology Apr 2022Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages of anchoring enzymes to a stable...
Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages of anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. However, these nanoparticles are limited in size, which constrains the packaging and the accessibility of the proteins. An axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living cells, they become incorporated into linear arrays, which have the advantages of high protein loading capacity and single-step purification with retention of biomass. The arrays can be isolated as membrane-enclosed vesicles or as exposed protein arrays. The approach is demonstrated for both a fluorescent protein and an enzyme (beta-lactamase), showing that incorporation into axonemes retains protein function in a stable, easily isolated array form.
Topics: Axoneme; Chlamydomonas reinhardtii; Flagella
PubMed: 35271249
DOI: 10.1021/acssynbio.1c00439 -
Cytoskeleton (Hoboken, N.J.) Apr 2010Axonemal dyneins have been demonstrated to monitor the mechanical state of the axoneme and must also alter activity in response to various signaling pathways. The... (Review)
Review
Axonemal dyneins have been demonstrated to monitor the mechanical state of the axoneme and must also alter activity in response to various signaling pathways. The central pair/radial spoke systems are clearly involved in controlling inner dynein arm function; however, the mechanisms by which the outer dynein arm transduces regulatory signals appear quite distinct at the molecular level. In Chlamydomonas, these regulatory components include thioredoxins involved in response to redox changes, molecules that tether the gamma heavy-chain motor unit to the A-tubule of the outer doublet and a Ca(2+)-binding protein that controls the structure of the gamma heavy-chain N-terminal domain. Together, these studies now suggest that the gamma heavy chain acts as a key regulatory node for controlling outer arm function in response to alterations in curvature and ligand binding. Furthermore, they allow us to propose a testable molecular mechanism by which altered Ca(2+) levels might lead to a change in ciliary waveform by controlling whether one heavy chain of outer arm dynein acts as a microtubule translocase or as an ATP-dependent brake that limits the amount of interdoublet sliding.
Topics: Animals; Axoneme; Calcium; Calcium Signaling; Dyneins
PubMed: 20186692
DOI: 10.1002/cm.20445 -
Zoological Science Apr 2021The ultrastructural features of axoneme organization within the cytoplasm and exflagellation were investigated in detail in microgametes of a malaria parasite, by...
The ultrastructural features of axoneme organization within the cytoplasm and exflagellation were investigated in detail in microgametes of a malaria parasite, by electron and fluorescence microscopy. The kinetosomes (basal bodies) of the microgamete were characterized by an electron dense mass in which singlet microtubules (MTs) were embedded. Around the kinetosomes, several singlet and doublet MTs were recognized in transverse sections. Incomplete doublets with growing B-tubule were also observed. As precursors of the axoneme, arrays of over three doublets showed a tendency to encircle the central pair MTs. Some of the doublet MTs were already equipped with inner and outer dynein arms. In the microgamete, which lacks an intraflagellar transport (IFT) system, self-assembly of microtubular and associated components appeared to proceed stepwise from singlet MTs through arrays of one to nine doublet MTs, surrounding the central pair, to form the complete axoneme in a quite short time. At exflagellation, some extra doublets were occasionally included between the axoneme and the flagellar membrane. At high magnification, the outer dynein arm of the microgamete had a pistol-like shape representing a three-headed dynein molecule like that of other Alveolata.
Topics: Animals; Axoneme; Dyneins; Female; Gametogenesis; Germ Cells; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron; Microscopy, Fluorescence; Plasmodium berghei
PubMed: 33812358
DOI: 10.2108/zs200064 -
Cellular and Molecular Life Sciences :... May 2023The sperm flagellum is a specialized type of motile cilium composed of a typical "9 + 2" axonemal structure with peri-axonemal structures, such as outer dense fibers...
The sperm flagellum is a specialized type of motile cilium composed of a typical "9 + 2" axonemal structure with peri-axonemal structures, such as outer dense fibers (ODFs). This flagellar arrangement is crucial for sperm movement and fertilization. However, the association of axonemal integrity with ODFs remains poorly understood. Here, we demonstrate that mouse BBOF1 could interact with both MNS1, an axonemal component, and ODF2, an ODF protein, and is required for sperm flagellar axoneme maintenance and male fertility. BBOF1 is expressed exclusively in male germ cells from the pachytene stage onwards and is detected in sperm axoneme fraction. Spermatozoa derived from Bbof1-knockout mice exhibit a normal morphology, however, reduced motility due to the absence of certain microtubule doublets, resulting in the failure to fertilize mature oocytes. Furthermore, BBOF1 is found to interact with ODF2 and MNS1 and is also required for their stability. Our findings in mice suggest that Bbof1 could also be essential for human sperm motility and male fertility, thus is a novel potential candidate gene for asthenozoospermia diagnosis.
Topics: Animals; Male; Mice; Axoneme; Fertility; Heat-Shock Proteins; Infertility, Male; Mice, Knockout; Semen; Sperm Motility; Spermatozoa
PubMed: 37198331
DOI: 10.1007/s00018-023-04800-0 -
Cell Motility and the Cytoskeleton Mar 2008The relationship between the primary structure of the beta-tubulin C-terminal tail (CTT) and axoneme structure and function is explored using the...
The relationship between the primary structure of the beta-tubulin C-terminal tail (CTT) and axoneme structure and function is explored using the spermatogenesis-specific beta2-tubulin of Drosophila. We previously showed that all beta-tubulins used for motile 9 + 2 axonemes contain a conserved sequence motif in the proximal part of the CTT, the beta-tubulin axoneme motif. The differential ability of tubulin isoforms and abilities of beta2-tubulin C-terminal truncations to form axonemes led us to hypothesize that the axoneme motif is essential for axoneme formation and the distal half of the CTT was less important. The studies we report here indicate that it is not that simple. Unexpectedly, some changes in the core sequence of the axoneme motif did not disrupt formation of motile axonemes. And, while deletion of the distal CTT did not disrupt the ability to produce functional sperm [Popodi et al., Cell Motil Cytoskeleton 2005;62:48-64], changing the amino acid sequence in this region can. Thus both regions are important. The deep conservation of the axoneme motif in all eukaryotic groups implies that the presence of the sequence motif confers a functional advantage. The central pair is the axoneme structure most sensitive to perturbations in tubulin molecules; we hypothesize central pair assembly is facilitated by the presence of this motif. Our data reveal that beta2-tubulin has robust properties for axoneme assembly, and that axonemal specializations are embedded in both the CTT and the body of the beta2 molecule.
Topics: Amino Acid Sequence; Animals; Axoneme; Dimerization; Drosophila melanogaster; Fertility; Male; Mitosis; Molecular Sequence Data; Mutant Proteins; Mutation; Protein Processing, Post-Translational; Sperm Motility; Spermatids; Testis; Tubulin
PubMed: 18157906
DOI: 10.1002/cm.20256 -
JCI Insight May 2023Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a...
Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus-based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.
Topics: Animals; Mice; Leber Congenital Amaurosis; Axoneme; Eye Proteins; Photoreceptor Cells
PubMed: 37071472
DOI: 10.1172/jci.insight.169162 -
Archives of Biochemistry and Biophysics Jun 2011Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for... (Review)
Review
Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.
Topics: Animals; Axoneme; Cilia; Conserved Sequence; Humans; Movement; Phosphoric Monoester Hydrolases; Protein Kinases
PubMed: 21513695
DOI: 10.1016/j.abb.2011.04.003