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Disease Models & Mechanisms Feb 2024Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are...
Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how kinesin motors contribute to cilia motility. Here, we show that Kif6 is a slow processive motor (12.2±2.0 nm/s) on microtubules in vitro and localizes to both the apical cytoplasm and the axoneme in ependymal cells, although it does not display processive movement in vivo. Using a mouse mutant that models a human Kif6 mutation in a proband displaying macrocephaly, hypotonia and seizures, we found that loss of Kif6 function causes decreased ependymal cilia motility and, subsequently, decreases fluid flow on the surface of brain ventricular walls. Disruption of Kif6 also disrupts orientation of cilia, formation of robust apical actin networks and stabilization of basal bodies at the apical surface. This suggests a role for the Kif6 motor protein in the maintenance of ciliary homeostasis within ependymal cells.
Topics: Humans; Brain; Cilia; Dyneins; Ependyma; Kinesins
PubMed: 38235522
DOI: 10.1242/dmm.050137 -
Environmental Health Perspectives Apr 1980All motile somatic cilia, including those of the human respiratory tract, are similar in ultrastructure in that they consist of an axenome of 9 + 2 microtubules... (Review)
Review
All motile somatic cilia, including those of the human respiratory tract, are similar in ultrastructure in that they consist of an axenome of 9 + 2 microtubules surrounded by a specialized extension of the cell membrane. The axonemal elements provide the ciliary motor, which is powered by ATP hydrolysis. In respiratory cilia, mutants occur where axonemal assembly is aberrant such that the doublet attachments known as arms (Afzelius and co-workers) or spokes (Sturgess) have been shown to be missing. These mutant cilia are apparently nonmotile. In model cilia, the arms are composed of dynein, a class of ATPase isoenzymes and associated polypeptides characterized byGibbons and colleagues. In negative stain preparations of arms, three subunits can be seen. In the presence of ATP, dynein functions with a set polarity to form transient cross-bridges that cause the microtubule doublets of the axoneme to slide relative to one another. After brief trypsin treatment, the axonemal microtubules slide almost completely apart with the arms of doublet n pushing doublet n + 1 in a tipward direction. To produce ciliary beating in vivo, sliding is carefully controlled and coordinated, in part probably by the spoke system. The ciliary membrane is responsible for maintaining the appropriate levels of ATP, Mg2+, and Ca2+, and Ca2+ (ca. 10(-7) M) around the axoneme. The beat of certain cilia--e.g., L cilia of mussel gills--can be arrested by increasing axonemal Ca2+ concentration, for example, in the presence of the ionophore A23187 and high external Ca2+. Although the net results of changes in axonemal Ca2+ concentration are not always complete stoppage of beat or of sliding, this ion is also part of the general behavioral control of ciliary motility.
Topics: Adenosine Triphosphate; Animals; Bivalvia; Calcimycin; Calcium; Cilia; Ciliophora; Dyneins; Humans; Kartagener Syndrome; Magnesium; Microtubules; Models, Biological; Movement; Sea Urchins
PubMed: 6447592
DOI: 10.1289/ehp.803577 -
Journal of Biochemistry Sep 2003The generation of a polarized microtubule organization is critically important for proper cellular functions, such as cell division, differentiation and migration.... (Review)
Review
The generation of a polarized microtubule organization is critically important for proper cellular functions, such as cell division, differentiation and migration. Microtubules themselves are highly dynamic structures, and this dynamic property is temporally and spatially regulated within cells, especially at their plus ends. To explain how microtubules set up and make contacts with cellular structures, a "search-and-capture" mechanism has been proposed, in which the microtubule plus ends dynamically search for and capture specific sites, such as mitotic kinetochores and cell cortex. To date, several classes of proteins have been shown to be associated with microtubule plus ends in a wide range of organisms from fungi to humans and to play critical roles in the "search-and-capture" mechanism. In this review, we overview our current understanding of the "plus-end-binding proteins" (+TIPs), including APC (adenomatous polyposis coli) tumor suppressor protein, cytoplasmic linker proteins (CLIPs), CLIP-associating proteins (CLASPs), cytoplasmic dynein/dynactin, and EB1, an APC-interacting protein.
Topics: Adenomatous Polyposis Coli Protein; Animals; Dynactin Complex; Dyneins; Kinetochores; Microtubule-Associated Proteins; Microtubules; Models, Biological; Spindle Apparatus
PubMed: 14561716
DOI: 10.1093/jb/mvg148 -
Advances in Experimental Medicine and... 2007Eukaryotic cilia and flagella are motile organelles built on a scaffold of doublet microtubules and powered by dynein ATPase motors. Some thirty years ago, two competing... (Review)
Review
Eukaryotic cilia and flagella are motile organelles built on a scaffold of doublet microtubules and powered by dynein ATPase motors. Some thirty years ago, two competing views were presented to explain how the complex machinery of these motile organelles had evolved. Overwhelming evidence now refutes the hypothesis that they are the modified remnants of symbiotic spirochaete-like prokaryotes, and supports the hypothesis that they arose from a simpler cytoplasmic microtubule-based intracellular transport system. However, because intermediate stages in flagellar evolution have not been found in living eukaryotes, a clear understanding of their early evolution has been elusive. Recent progress in understanding phylogenetic relationships among present day eukaryotes and in sequence analysis of flagellar proteins have begun to provide a clearer picture of the origins of doublet and triplet microtubules, flagellar dynein motors, and the 9+2 microtubule architecture common to these organelles. We summarize evidence that the last common ancestor of all eukaryotic organisms possessed a 9+2 flagellum that was used for gliding motility along surfaces, beating motility to generate fluid flow, and localized distribution of sensory receptors, and trace possible earlier stages in the evolution of these characteristics.
Topics: Animals; Cell Movement; Cilia; Dyneins; Eukaryotic Cells; Evolution, Molecular; Flagella; Kinesins; Microtubules; Models, Biological; Organelles; Phylogeny; Tubulin
PubMed: 17977465
DOI: 10.1007/978-0-387-74021-8_11 -
Biophysical Journal Apr 2020Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of a microtubule by ATP hydrolysis free energy. By employing its two heads (motor...
Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of a microtubule by ATP hydrolysis free energy. By employing its two heads (motor domains), cytoplasmic dynein exhibits various bipedal stepping motions: inchworm and hand-over-hand motions, as well as nonalternating steps of one head. However, the molecular basis to achieve such diverse stepping manners remains unclear because of the lack of an experimental method to observe stepping and the ATPase reaction of dynein simultaneously. Here, we propose a kinetic model for bipedal motions of cytoplasmic dynein and perform Gillespie Monte Carlo simulations that qualitatively reproduce most experimental data obtained to date. The model represents the status of each motor domain as five states according to conformation and nucleotide- and microtubule-binding conditions of the domain. In addition, the relative positions of the two domains were approximated by three discrete states. Accompanied by ATP hydrolysis cycles, the model dynein stochastically and processively moved forward in multiple steps via diverse pathways, including inchworm and hand-over-hand motions, similarly to experimental data. The model reproduced key experimental motility-related properties, including velocity and run length, as functions of the ATP concentration and external force, therefore providing a plausible explanation of how dynein achieves various stepping manners with explicit characterization of nucleotide states. Our model highlights the uniqueness of dynein in the coupling of ATPase with its movement during both inchworm and hand-over-hand stepping.
Topics: Adenosine Triphosphate; Cytoplasmic Dyneins; Dyneins; Hydrolysis; Kinetics; Microtubules
PubMed: 32272056
DOI: 10.1016/j.bpj.2020.03.012 -
JCI Insight Feb 2023Multiple morphological abnormalities of the sperm flagella (MMAF) are the most severe form of asthenozoospermia due to impaired axoneme structure in sperm flagella....
Multiple morphological abnormalities of the sperm flagella (MMAF) are the most severe form of asthenozoospermia due to impaired axoneme structure in sperm flagella. Dynein arms are necessary components of the sperm flagellar axoneme. In this study, we recruited 3 unrelated consanguineous Pakistani families with multiple MMAF-affected individuals, who had no overt ciliary symptoms. Whole-exome sequencing and Sanger sequencing identified 2 cilia and flagella associated protein 57 (CFAP57) loss-of-function mutations (c.2872C>T, p. R958*; and c.2737C>T, p. R913*) recessively segregating with male infertility. A mouse model mimicking the mutation (c.2872C>T) was generated and recapitulated the typical MMAF phenotype of CFAP57-mutated individuals. Both CFAP57 mutations caused loss of the long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals or from the Cfap57-mutant mouse model while the short transcript was not affected. Subsequent examinations of the spermatozoa from Cfap57-mutant mice revealed that CFAP57 deficiency disrupted the inner dynein arm (IDA) assembly in sperm flagella and that single-headed IDAs were more likely to be affected. Thus, our study identified 2 pathogenic mutations in CFAP57 in MMAF-affected individuals and reported a conserved and pivotal role for the long transcript-encoded CFAP57 in IDAs' assembly and male fertility.
Topics: Animals; Humans; Male; Mice; Cilia; Dyneins; Flagella; Semen; Microtubule-Associated Proteins; Loss of Function Mutation
PubMed: 36752199
DOI: 10.1172/jci.insight.166869 -
ELife Jun 2022Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered...
Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.
Topics: Animals; Chromosome Segregation; Dynactin Complex; Dyneins; Male; Meiosis; Mice; Microtubule-Associated Proteins; Microtubules
PubMed: 35703493
DOI: 10.7554/eLife.78201 -
Nature Communications Nov 2020Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein...
Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein's mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein's stepping cycle and underscores the complexity and crosstalk among the motor's multiple AAA+ domains.
Topics: AAA Domain; Adenosine Triphosphate; Cytoplasmic Dyneins; Hydrolysis; Microtubules; Movement; Mutagenesis; Optical Tweezers; Protein Binding; Protein Conformation; Protein Multimerization; Saccharomyces cerevisiae
PubMed: 33230227
DOI: 10.1038/s41467-020-19477-3 -
Nature Communications Dec 2017Cytoplasmic dynein is an enormous minus end-directed microtubule motor. Rather than existing as bare tracks, microtubules are bound by numerous microtubule-associated...
Cytoplasmic dynein is an enormous minus end-directed microtubule motor. Rather than existing as bare tracks, microtubules are bound by numerous microtubule-associated proteins (MAPs) that have the capacity to affect various cellular functions, including motor-mediated transport. One such MAP is She1, a dynein effector that polarizes dynein-mediated spindle movements in budding yeast. Here, we characterize the molecular basis by which She1 affects dynein, providing the first such insight into which a MAP can modulate motor motility. We find that She1 affects the ATPase rate, microtubule-binding affinity, and stepping behavior of dynein, and that microtubule binding by She1 is required for its effects on dynein motility. Moreover, we find that She1 directly contacts the microtubule-binding domain of dynein, and that their interaction is sensitive to the nucleotide-bound state of the motor. Our data support a model in which simultaneous interactions between the microtubule and dynein enables She1 to directly affect dynein motility.
Topics: Amino Acid Sequence; Dyneins; Microscopy, Fluorescence; Microtubule-Associated Proteins; Microtubules; Models, Biological; Models, Molecular; Molecular Motor Proteins; Myosin Heavy Chains; Myosin Type V; Protein Binding; Protein Domains; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 29247176
DOI: 10.1038/s41467-017-02004-2 -
Current Opinion in Cell Biology Feb 2009Molecular motors drive key biological processes such as cell division, intracellular organelle transport, and sperm propulsion and defects in motor function can give... (Review)
Review
Molecular motors drive key biological processes such as cell division, intracellular organelle transport, and sperm propulsion and defects in motor function can give rise to various human diseases. Two dimeric microtubule-based motor proteins, kinesin-1 and cytoplasmic dynein can take over one hundred steps without detaching from the track. In this review, we discuss how these processive motors coordinate the activities of their two identical motor domains so that they can walk along microtubules.
Topics: Cell Physiological Phenomena; Dyneins; Humans; Kinesins; Microtubules; Protein Structure, Tertiary
PubMed: 19179063
DOI: 10.1016/j.ceb.2008.12.002