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Nature Communications Mar 2024Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core...
Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.
Topics: Humans; Cilia; Microtubule Proteins; Axoneme; Microtubules; Centrioles
PubMed: 38538594
DOI: 10.1038/s41467-024-46737-3 -
Current Protocols in Protein Science Jun 2020Cilia and flagella play essential roles in environmental sensing, cell locomotion, and development. These organelles possess a central microtubule-based structure known...
Cilia and flagella play essential roles in environmental sensing, cell locomotion, and development. These organelles possess a central microtubule-based structure known as the axoneme, which serves as a scaffold and is crucial for the function of cilia. Despite their key roles, the biochemical and biophysical properties of the ciliary proteins are poorly understood. To address this issue, we have developed a novel method to purify functional tubulins from different parts of the axoneme, namely the central pair and B-tubule. We use the biflagellate green alga Chlamydomonas reinhardtii, a model organism for studying cilia due to the conserved structure of this organelle, availability of genetic tools and a large collection of mutant strains. Our method yields highly purified functional axonemal tubulins in sufficient quantities to be used for in vitro biochemical and biophysical studies, such as microtubule dynamic assays. It takes 7 to 8 days to grow enough cells; the isolation of the flagella and the purification of the axonemal tubulins require an additional two full days.© 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth and harvest of large volume of cell culture Support Protocol: Assembly of homemade concentration apparatus Basic Protocol 2: Isolation of flagella Basic Protocol 3: Tubulin extraction and purification.
Topics: Chlamydomonas reinhardtii; Cilia; Plant Proteins; Tubulin
PubMed: 32568459
DOI: 10.1002/cpps.107 -
PLoS Genetics Aug 2020The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An...
The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An axoneme is comprised of two central microtubules surrounded by nine doublets, the nexin-dynein regulatory complex, radial spokes, and dynein arms. Failure to properly assemble components of the axoneme in a sperm flagellum, leads to fertility alterations. To understand this process in detail, we have defined the function of an uncharacterized gene, Cfap97 domain containing 1 (Cfap97d1). This gene is evolutionarily conserved in mammals and multiple other species, including Chlamydomonas. We have used two independently generated Cfap97d1 knockout mouse models to study the gene function in vivo. Cfap97d1 is exclusively expressed in testes starting from post-natal day 20 and continuing throughout adulthood. Deletion of the Cfap97d1 gene in both mouse models leads to sperm motility defects (asthenozoospermia) and male subfertility. In vitro fertilization (IVF) of cumulus-intact oocytes with Cfap97d1 deficient sperm yielded few embryos whereas IVF with zona pellucida-free oocytes resulted in embryo numbers comparable to that of the control. Knockout spermatozoa showed abnormal motility characterized by frequent stalling in the anti-hook position. Uniquely, Cfap97d1 loss caused a phenotype associated with axonemal doublet heterogeneity linked with frequent loss of the fourth doublet in the sperm stored in the epididymis. This study demonstrates that Cfap97d1 is required for sperm flagellum ultra-structure maintenance, thereby playing a critical role in sperm function and male fertility in mice.
Topics: Animals; Axoneme; Chlamydomonas; Cilia; Cytoskeletal Proteins; Dyneins; Fertilization in Vitro; Humans; Infertility, Male; Male; Mice; Mice, Knockout; Sperm Motility; Sperm Tail; Spermatozoa; Testis
PubMed: 32785227
DOI: 10.1371/journal.pgen.1008954 -
The FEBS Journal Sep 2017Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play... (Review)
Review
Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play critical roles in motility, sensory reception, and signal transduction in virtually all eukaryotic cells. During IFT, anterograde kinesin-2 and retrograde IFT dynein motors drive the bidirectional transport of IFT trains that deliver cargo, for example, axoneme precursors such as tubulins as well as molecules of the signal transduction machinery, to their site of assembly within the cilium. Following its discovery in Chlamydomonas, IFT has emerged as a powerful model system for studying general principles of motor-dependent cargo transport and we now appreciate the diversity that exists in the mechanism of IFT within cilia of different cell types. The absence of heterotrimeric kinesin-2 function, for example, causes a complete loss of both IFT and cilia in Chlamydomonas, but following its loss in Caenorhabditis elegans, where its primary function is loading the IFT machinery into cilia, homodimeric kinesin-2-driven IFT persists and assembles a full-length cilium. Generally, heterotrimeric kinesin-2 and IFT dynein motors are thought to play widespread roles as core IFT motors, whereas homodimeric kinesin-2 motors are accessory motors that mediate different functions in a broad range of cilia, in some cases contributing to axoneme assembly or the delivery of signaling molecules but in many other cases their ciliary functions, if any, remain unknown. In this review, we focus on mechanisms of motor action, motor cooperation, and motor-dependent cargo delivery during IFT.
Topics: Animals; Axoneme; Basal Bodies; Biological Transport; Caenorhabditis elegans; Chlamydomonas; Cilia; Dyneins; Flagella; Gene Expression Regulation; Kinesins; Protein Multimerization; Signal Transduction; Tubulin
PubMed: 28342295
DOI: 10.1111/febs.14068 -
Cilia 2016Giardia lamblia is an intestinal parasitic protist that causes significant acute and chronic diarrheal disease worldwide. Giardia belongs to the diplomonads, a group of... (Review)
Review
Giardia lamblia is an intestinal parasitic protist that causes significant acute and chronic diarrheal disease worldwide. Giardia belongs to the diplomonads, a group of protists in the supergroup Excavata. Diplomonads are characterized by eight motile flagella organized into four bilaterally symmetric pairs. Each of the eight Giardia axonemes has a long cytoplasmic region that extends from the centrally located basal body before exiting the cell body as a membrane-bound flagellum. Each basal body is thus unique in its cytological position and its association with different cytoskeletal features, including the ventral disc, axonemes, and extra-axonemal structures. Inheritance of these unique and complex cytoskeletal elements is maintained through basal body migration, duplication, maturation, and their subsequent association with specific spindle poles during cell division. Due to the complex composition and inheritance of specific basal bodies and their associated structures, Giardia may require novel basal body-associated proteins. Thus, protists such as Giardia may represent an undiscovered source of novel basal body-associated proteins. The development of new tools that make Giardia genetically tractable will enable the composition, structure, and function of the eight basal bodies to be more thoroughly explored.
PubMed: 27379179
DOI: 10.1186/s13630-016-0042-4 -
Biomechanics and Modeling in... Apr 2020The fluctuating position of an optically trapped cilium tip under untreated and Taxol-treated conditions was used to characterize mechanical properties of the cilium...
The fluctuating position of an optically trapped cilium tip under untreated and Taxol-treated conditions was used to characterize mechanical properties of the cilium axoneme and its basal body by combining experimental, analytical, and computational tools. We provide, for the first time, evidence that the persistence length of a ciliary axoneme is length-dependent; longer cilia are stiffer than shorter cilia. We demonstrate that this apparent length dependence can be understood by a combination of modeling axonemal microtubules as anisotropic elastic shells and including actomyosin-driven stochastic basal body motion. Our results also demonstrate the possibility of using observable ciliary dynamics to probe interior cytoskeletal dynamics. It is hoped that our improved characterization of cilia will result in deeper understanding of the biological function of cellular flow sensing by this organelle.
Topics: Animals; Cilia; Dogs; Elastic Modulus; Image Processing, Computer-Assisted; Madin Darby Canine Kidney Cells; Microspheres; Models, Biological; Optical Tweezers; Paclitaxel; Stochastic Processes
PubMed: 31501964
DOI: 10.1007/s10237-019-01220-7 -
Cells Oct 2023Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At...
Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed to generate dynein holoenzymes and/or for their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation, which can generate degrons subject to the N-end rule, alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here, we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases, one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex is processed to yield an unmodified Pro residue, which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify and describe an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm.
Topics: Axonemal Dyneins; Microtubules; Chlamydomonas; Cilia; Axoneme
PubMed: 37887336
DOI: 10.3390/cells12202492 -
The Journal of Cell Biology Feb 2022Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate...
Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.
Topics: Animals; Axoneme; Consensus; Dyneins; Humans; Phenotype; Reference Standards; Terminology as Topic
PubMed: 35006274
DOI: 10.1083/jcb.202109014 -
ELife Apr 2021New mathematical model reveals how the flagella of some single-celled algae generate a lasso-like beat pattern that propels the cell through water.
New mathematical model reveals how the flagella of some single-celled algae generate a lasso-like beat pattern that propels the cell through water.
Topics: Euglena gracilis; Flagella
PubMed: 33899738
DOI: 10.7554/eLife.67701 -
Cell Discovery Jul 2021Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While...
Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked "density scissors" connect with one another to form a "scissors stack network (SSN)" plane oriented 45° to the axoneme axis; and ~370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with ~90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics.
PubMed: 34257277
DOI: 10.1038/s41421-021-00281-2