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Developmental Cell Aug 2022How synaptogenic signals trigger the targeted delivery of synaptic material is a fundamental question in neuroscience. In this issue of Developmental Cell,...
How synaptogenic signals trigger the targeted delivery of synaptic material is a fundamental question in neuroscience. In this issue of Developmental Cell, Balseiro-Gomez et al. identify a mechanism through which local synatogenic pathways control synaptic cargo delivery.
Topics: Cell Communication; Dyneins
PubMed: 35944475
DOI: 10.1016/j.devcel.2022.07.009 -
The Journal of Biological Chemistry Dec 1981Binding of 21 S dynein ATPase isolated from Tetrahymena cilia to B subfibers of microtubule doublets was used as a model system to study dynein-tubulin interactions and...
Binding of 21 S dynein ATPase isolated from Tetrahymena cilia to B subfibers of microtubule doublets was used as a model system to study dynein-tubulin interactions and their relationship to the microtubule-based sliding filament mechanism. Binding of 21 S dynein to both A and B microtubule subfibers is supported by monovalent as well as divalent ions. Monovalent cation chlorides support dynein binding to B subfibers with the specificity Li greater than Na congruent to K congruent to Rb congruent to Cs congruent to choline. The corresponding sodium or potassium halides follow the order F greater than Cl greater than Br greater than I. However, an optimal binding concentration of 40 mM KCl supports only 55% of the protein binding which takes place in 3 mM MgSO4 and does not stabilize dynein cross-bridges when whole axonemes are fixed for electron microscopy. Divalent metal ion chlorides (MgCl2, CaCl2, SrCl2, and BaCl2) have nearly equivalent effects at a concentration of 6 mM; all support about 140% of the binding observed in 6 mM MgSO4. The binding data suggest negative cooperativity or the presence of more than one class of dynein binding sites on the microtubule lattice. Low concentrations of MgATP2- induce dissociation of dynein bound to B subfibers in either 6 mM MgSO4 or 40 mM KCl. ADP, Pi, PPi, and AMP-PCH2P are unable to induce dynein dissociation, while AMP-PNHP and ATP4- both cause dynein release from B subfiber sites. The half-maximal sensitivities of the tubulin-dynein complex to MgATP2-, ATP4-, and adenylyl-imidodiphosphate (AMP.PNP) are 1.3 X 10(-8) M, 3.6 X 10(-5) M, and 4.7 X 10(-4) M respectively. Incubation of doublets or 21 S dynein in N-ethylmaleimide (NEM), which can inhibit active sliding, has no effect on either association of dynein with the B subfiber or on dissociation of the resulting dynein-B subfiber complex by MgATP2-.
Topics: Adenosine Triphosphatases; Animals; Cations, Divalent; Cations, Monovalent; Cilia; Dyneins; Kinetics; Microscopy, Electron; Microtubules; Molecular Weight; Protein Binding; Tetrahymena; Tubulin
PubMed: 6457836
DOI: No ID Found -
Journal of Cell Science Nov 2014Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT), and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we define for...
Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT), and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we define for the first time the composition of the human cytoplasmic dynein-2 complex. We show that the proteins encoded by the ciliopathy genes WDR34 and WDR60 are bona fide dynein-2 intermediate chains and are both required for dynein-2 function. In addition, we identify TCTEX1D2 as a unique dynein-2 light chain that is itself required for cilia function. We define several subunits common to both dynein-1 and dynein-2, including TCTEX-1 (also known as DYNLT1) and TCTEX-3 (also known as DYNLT3), roadblock-1 (also known as DYNLRB1) and roadblock-2 (also known as DYNLRB2), and LC8-1 and LC8-2 light chains (DYNLL1 and DYNLL2, respectively). We also find that NudCD3 associates with dynein-2 as it does with dynein-1. By contrast, the common dynein-1 regulators dynactin, LIS1 (also known as PAFAH1B1) and BICD2 are not found in association with dynein-2. These data explain why mutations in either WDR34 or WDR60 cause disease, as well as identifying TCTEX1D2 as a candidate ciliopathy gene.
Topics: Biological Transport; Cell Line; Cilia; Cytoplasmic Dyneins; Dyneins; Humans; Immunoprecipitation
PubMed: 25205765
DOI: 10.1242/jcs.159038 -
Biochimica Et Biophysica Acta.... Nov 2021The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores...
The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores attach to the spindle during mitosis. The formation of the Mad2-p31 complex correlates with the completion of spindle attachment and the entry into anaphase during mitosis. Herein, we showed that dynein intermediate chain 2c (DNCI2c)-a subunit of dynein motor protein-forms an immunocomplex with p31 during mitosis. DNCI2c-knockdown resulted in prolonged mitotic arrest in a Mad2-dependent manner. Furthermore, DNCI2c-knockdown-induced mitotic arrest was not rescued by p31 overexpression. However, the combination of p31 overexpression with the mitotic drug treatment reversed the mitotic arrest in DNCI2c-knockdown. Together, these results indicate that the DNCI2c-p31 complex plays an important role in exiting Mad2-dependent SAC.
Topics: Dyneins; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mad2 Proteins; Spindle Apparatus
PubMed: 34400173
DOI: 10.1016/j.bbamcr.2021.119120 -
Cell Nov 2014Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. Here, we report X-ray and electron microscopy (EM) structures of yeast dynein bound...
Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. Here, we report X-ray and electron microscopy (EM) structures of yeast dynein bound to different ATP analogs, which collectively provide insight into the roles of dynein's two major ATPase sites, AAA1 and AAA3, in the conformational change mechanism. ATP binding to AAA1 triggers a cascade of conformational changes that propagate to all six AAA domains and cause a large movement of the "linker," dynein's mechanical element. In contrast to the role of AAA1 in driving motility, nucleotide transitions in AAA3 gate the transmission of conformational changes between AAA1 and the linker, suggesting that AAA3 acts as a regulatory switch. Further structural and mutational studies also uncover a role for the linker in regulating the catalytic cycle of AAA1. Together, these results reveal how dynein's two major ATP-binding sites initiate and modulate conformational changes in the motor domain during motility.
Topics: Adenosine Triphosphate; Allosteric Regulation; Catalysis; Crystallography, X-Ray; Dictyostelium; Dyneins; Microscopy, Electron; Models, Molecular; Protein Structure, Tertiary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 25417161
DOI: 10.1016/j.cell.2014.10.018 -
The Journal of Biological Chemistry May 1988Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble...
Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.
Topics: Adenosine Triphosphatases; Alkaloids; Animals; Dyneins; Female; Isoenzymes; Kinetics; Microtubule-Associated Proteins; Microtubules; Ovum; Paclitaxel; Sea Urchins; Vanadates
PubMed: 2896199
DOI: No ID Found -
Science (New York, N.Y.) Mar 2011Dyneins are microtubule-based motor proteins that power ciliary beating, transport intracellular cargos, and help to construct the mitotic spindle. Evolved from...
Dyneins are microtubule-based motor proteins that power ciliary beating, transport intracellular cargos, and help to construct the mitotic spindle. Evolved from ring-shaped hexameric AAA-family adenosine triphosphatases (ATPases), dynein's large size and complexity have posed challenges for understanding its structure and mechanism. Here, we present a 6 angstrom crystal structure of a functional dimer of two ~300-kilodalton motor domains of yeast cytoplasmic dynein. The structure reveals an unusual asymmetric arrangement of ATPase domains in the ring-shaped motor domain, the manner in which the mechanical element interacts with the ATPase ring, and an unexpected interaction between two coiled coils that create a base for the microtubule binding domain. The arrangement of these elements provides clues as to how adenosine triphosphate-driven conformational changes might be transmitted across the motor domain.
Topics: Adenosine Triphosphate; Allosteric Regulation; Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Cytoplasmic Dyneins; Methionine; Microtubules; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Folding; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins
PubMed: 21330489
DOI: 10.1126/science.1202393 -
Nature Communications Feb 2020The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain...
The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain via a coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulate MTBD either in high-affinity or low-affinity states by introducing a disulfide bond to the stalk and analyze the resulting structures by NMR and cryo-EM. In the MT-unbound state, the affinity changes of MTBD are achieved by sliding of the stalk α-helix by a half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, MT binding induces further sliding of the stalk α-helix even without the disulfide bond, suggesting how the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.
Topics: Binding Sites; Cryoelectron Microscopy; Cytoplasm; Disulfides; Dyneins; Microtubules; Models, Molecular; Mutation; Nuclear Magnetic Resonance, Biomolecular; Protein Domains; Protein Structure, Quaternary; Protein Structure, Tertiary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tubulin
PubMed: 32098965
DOI: 10.1038/s41467-020-14842-8 -
Nature Cell Biology Dec 2010Cytoplasmic dynein drives vesicular transport from the periphery to the cell body of neurons. Missense mutations in the dynein tail domain cause neurodegenerative...
Cytoplasmic dynein drives vesicular transport from the periphery to the cell body of neurons. Missense mutations in the dynein tail domain cause neurodegenerative disease in mouse models. new data on the effect of one such dynein mutation provide insight into the intramolecular communication and flexible stepping of this essential cellular motor.
Topics: Animals; Dyneins; Humans; Models, Molecular; Mutation; Neurons
PubMed: 21102436
DOI: 10.1038/ncb1210-1126 -
Proceedings of the National Academy of... May 2015Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase...
Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.
Topics: Acetyltransferases; Anisotropy; Biomechanical Phenomena; Cytoplasm; DNA Primers; Dyneins; Green Fluorescent Proteins; Mechanotransduction, Cellular; Microtubules; Mutagenesis; Optical Tweezers; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 25941405
DOI: 10.1073/pnas.1417422112