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American Journal of Respiratory Cell... Mar 2020
Topics: Axoneme; Cilia; Ciliary Motility Disorders; Humans; Microfilament Proteins; Mutation
PubMed: 31604022
DOI: 10.1165/rcmb.2019-0316ED -
Biophysical Journal Aug 2019Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella...
Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.
Topics: Adenosine Triphosphate; Axoneme; Binding Sites; Chlamydomonas reinhardtii; Dyneins; Fluorescence Recovery After Photobleaching; Kinetics; Mutation; Plant Proteins; Protein Binding
PubMed: 31400919
DOI: 10.1016/j.bpj.2019.07.004 -
Life Science Alliance Mar 2022Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins...
Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, collectively named the microtubule inner proteins (MIPs). We used cryoET to study axoneme DMT isolated from We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP RIB72A/B binds to the luminal wall of DMT by multiple DM10 domains. We found FAP115, an MIP-containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of A-tubule. It contacts both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. We observed substantial structure heterogeneity in DMT in an knockout strain, showing extensive structural defects beyond the FAP115-binding site. The defects propagate along the axoneme. Finally, by comparing DMT structures from and , we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.
Topics: Axoneme; Binding Sites; Microtubule Proteins; Microtubules; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Protozoan Proteins; Structure-Activity Relationship; Tetrahymena thermophila
PubMed: 34969817
DOI: 10.26508/lsa.202101225 -
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 -
ELife Nov 2022In most eukaryotic organisms, cilia and flagella perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has...
In most eukaryotic organisms, cilia and flagella perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has provided considerable insight into the morphology and function of flagellar structures, but studies have been limited to less than a dozen of the millions of known eukaryotic species. Ultrastructural information is particularly lacking for unicellular organisms in the Opisthokonta clade, leaving a sizeable gap in our understanding of flagella evolution between unicellular species and multicellular metazoans (animals). Choanoflagellates are important aquatic heterotrophs, uniquely positioned within the opisthokonts as the metazoans' closest living unicellular relatives. We performed cryo-focused ion beam milling and cryo-electron tomography on flagella from the choanoflagellate species . We show that the axonemal dyneins, radial spokes, and central pair complex in more closely resemble metazoan structures than those of unicellular organisms from other suprakingdoms. In addition, we describe unique features of flagella, including microtubule holes, microtubule inner proteins, and the flagellar vane: a fine, net-like extension that has been notoriously difficult to visualize using other methods. Furthermore, we report barb-like structures of unknown function on the extracellular surface of the flagellar membrane. Together, our findings provide new insights into choanoflagellate biology and flagella evolution between unicellular and multicellular opisthokonts.
Topics: Animals; Choanoflagellata; Cryoelectron Microscopy; Flagella; Axoneme; Cilia
PubMed: 36384644
DOI: 10.7554/eLife.78133 -
PLoS Biology Jul 2019The primary cilium is a central signaling hub in cell proliferation and differentiation and is built and disassembled every cell cycle in many animal cells. Disassembly...
The primary cilium is a central signaling hub in cell proliferation and differentiation and is built and disassembled every cell cycle in many animal cells. Disassembly is critically important, as misregulation or delay of cilia loss leads to cell cycle defects. The physical means by which cilia are lost are poorly understood but are thought to involve resorption of ciliary components into the cell body. To investigate cilium loss in mammalian cells, we used live-cell imaging to comprehensively characterize individual events. The predominant mode of cilium loss was rapid deciliation, in which the membrane and axoneme of the cilium was shed from the cell. Gradual resorption was also observed, as well as events in which a period of gradual resorption was followed by rapid deciliation. Deciliation resulted in intact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels of katanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes cilia loss by deciliation, independently of calcium. Together, these results suggest that mammalian ciliary loss involves a tunable decision between deciliation and resorption.
Topics: Animals; Axoneme; Calcium; Cell Cycle; Cell Differentiation; Cell Line; Cell Proliferation; Cilia; Green Fluorescent Proteins; Katanin; Membrane Proteins; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Signal Transduction
PubMed: 31314751
DOI: 10.1371/journal.pbio.3000381 -
ELife Dec 2023Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar...
Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of . A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.
Topics: Mice; Animals; Male; Humans; Sperm Motility; Semen; Axoneme; Sperm Tail; Proteins; Spermatozoa; Infertility, Male; Flagella; Mammals
PubMed: 38091523
DOI: 10.7554/eLife.90095 -
Proceedings of the National Academy of... Nov 2019Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of...
Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet-Biedl syndrome.
Topics: Animals; Axoneme; Bardet-Biedl Syndrome; Centrioles; Cilia; Cryoelectron Microscopy; Disease Models, Animal; Electron Microscope Tomography; Eye Proteins; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Microtubule-Associated Proteins; Microtubules; Multiprotein Complexes; Muscle Proteins; Nanotechnology; Photoreceptor Connecting Cilium; Qa-SNARE Proteins; Rod Cell Outer Segment; Single Molecule Imaging; Tumor Suppressor Proteins
PubMed: 31690665
DOI: 10.1073/pnas.1902003116 -
Signal Transduction and Targeted Therapy Jan 2023
Topics: Male; Humans; Animals; Mice; Dyneins; Microtubule-Associated Proteins; Cytoskeleton; Axoneme; Spermatogenesis
PubMed: 36627292
DOI: 10.1038/s41392-022-01293-4 -
Bioscience Reports Dec 2020PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl...
PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.
Topics: Acetylation; Animals; Asthenozoospermia; Axoneme; Case-Control Studies; Flagella; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Humans; Male; Microtubule-Associated Proteins; Protein Processing, Post-Translational; Rats, Sprague-Dawley; Sperm Motility; Spermatozoa; Tubulin
PubMed: 33200789
DOI: 10.1042/BSR20202442