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Journal of Thrombosis and Haemostasis :... Jul 2007The cellular and molecular basis of the intricate process by which megakaryocytes (MKs) form and release platelets remains poorly understood. Work has shown that... (Review)
Review
The cellular and molecular basis of the intricate process by which megakaryocytes (MKs) form and release platelets remains poorly understood. Work has shown that proplatelets, long cytoplasmic extensions made by mature MKs, are essential intermediates in platelet biogenesis. Microtubules are the main structural component of proplatelets and it is microtubule sliding, driven by dynein motors within cortical bundles, which elongates and thins proplatelets. Kinesin motors carry their cargo of platelet-specific granules and organelles into the proplatelets using the microtubule bundles as tracks. Extension of proplatelets is associated with repeated actin-dependent bending and bifurcation, which results in considerable amplification of free proplatelet ends. Large proplatelets, dissociated from the residual MK cell body, have the capacity to mature platelets. Only the ends of proplatelets form marginal microtubule coils similar to that observed in mature platelets, demonstrating that platelet formation completes primarily at proplatelet ends. Understanding the molecular basis of platelet formation requires detailed knowledge of how the MK microtubule machinery interacts to generate proplatelets and release platelets.
Topics: Blood Platelets; Cytoplasm; Dyneins; Humans; Microtubules
PubMed: 17635704
DOI: 10.1111/j.1538-7836.2007.02487.x -
The Journal of Cell Biology Mar 1983Dynein has been examined by scanning transmission electron microscopy (STEM). Samples of 30S dynein from tetrahymena cilia were applied to carbon films and either were...
Dynein has been examined by scanning transmission electron microscopy (STEM). Samples of 30S dynein from tetrahymena cilia were applied to carbon films and either were freeze- dried and examined as unstained, unfixed specimens, or were negatively stained with uranyl sulfate. A totally new image of the dynein molecule was revealed showing three globular heads connected by three separate strands to a common base. Two of the heads appeared to be identical and exhibited a diameter of 10 nm while the third head was somewhat larger (approximately 12 nm). The overall length of the particle was 35 nm. Mass analysis, based upon the integration of electron scattering intensities for unstained particles, gave a molecular weight of 1.95 (+/-)0.24) megadaltons. Mass per unit length analysis was performed using bovine brain microtubules decorated with dynein under conditions where the dynein shows a linear repeat of 24 nm with seven dynein molecules surrounding a microtubule made up of 14 protofilaments. Undecorated microtubules gave a molecular weight per unit length of 21,000+/-1,900 daltons/A, compared to a value of 84,400+/-2,200 daltons/A for the fully decorated microtubules. Taken together, these data give a molecular weight of 2.17 (+/- 0.14) megadaltons per dynein molecule, in agreement with measurements on the isolated particles. Mass analysis of individual globular heads observed in isolated particles gave a molecular weight distribution with a mean of 416+/- 76 kdaltons. These data could also be viewed as the sum of two populations of head with two-thirds of the heads at approximately 400 kdaltons and one-third at approximately 550 kdaltons, although more precise data will be required to distinguish two classes of heads with confidence. The mass of the dynein-microtubule complex as a function of distance from the midline of the particle was analysed to distinguish which end of the dynein molecule was bound to the microtubule. The projected mass distribution was consistent with a model where the three dynein heads were oriented toward the microtubule and clearly not consistent with the opposite orientation. These data indicate that the three globular heads form the ATP-sensitive site in this heterologous dynein-microtubule system and suggest that the rootlike base of the dynein molecule forms the structural attachment site to the A-subfiber of the outer doublet in cilia and flagella. The structure and function of the dynein are dicussed in terms of these new results.
Topics: Adenosine Triphosphatases; Animals; Binding Sites; Chemical Phenomena; Chemistry; Dyneins; Microscopy, Electron, Scanning; Microtubules; Molecular Weight; Protein Conformation; Structure-Activity Relationship; Tetrahymena
PubMed: 6220019
DOI: 10.1083/jcb.96.3.669 -
Science (New York, N.Y.) Mar 2022Natural dynein protein motors are reengineered to walk on specific artificial DNA tracks.
Natural dynein protein motors are reengineered to walk on specific artificial DNA tracks.
Topics: DNA; Dyneins; Shoes
PubMed: 35271310
DOI: 10.1126/science.abn9659 -
Microbiology and Molecular Biology... Mar 2016The ubiquitous biological nanomotors were classified into two categories in the past: linear and rotation motors. In 2013, a third type of biomotor, revolution without... (Review)
Review
The ubiquitous biological nanomotors were classified into two categories in the past: linear and rotation motors. In 2013, a third type of biomotor, revolution without rotation (http://rnanano.osu.edu/movie.html), was discovered and found to be widespread among bacteria, eukaryotic viruses, and double-stranded DNA (dsDNA) bacteriophages. This review focuses on recent findings about various aspects of motors, including chirality, stoichiometry, channel size, entropy, conformational change, and energy usage rate, in a variety of well-studied motors, including FoF1 ATPase, helicases, viral dsDNA-packaging motors, bacterial chromosome translocases, myosin, kinesin, and dynein. In particular, dsDNA translocases are used to illustrate how these features relate to the motion mechanism and how nature elegantly evolved a revolution mechanism to avoid coiling and tangling during lengthy dsDNA genome transportation in cell division. Motor chirality and channel size are two factors that distinguish rotation motors from revolution motors. Rotation motors use right-handed channels to drive the right-handed dsDNA, similar to the way a nut drives the bolt with threads in same orientation; revolution motors use left-handed motor channels to revolve the right-handed dsDNA. Rotation motors use small channels (<2 nm in diameter) for the close contact of the channel wall with single-stranded DNA (ssDNA) or the 2-nm dsDNA bolt; revolution motors use larger channels (>3 nm) with room for the bolt to revolve. Binding and hydrolysis of ATP are linked to different conformational entropy changes in the motor that lead to altered affinity for the substrate and allow work to be done, for example, helicase unwinding of DNA or translocase directional movement of DNA.
Topics: Animals; Bacteria; Bacterial Proteins; Bacteriophages; DNA Helicases; DNA-Directed RNA Polymerases; Dyneins; Flagella; Gene Expression; Humans; Kinesins; Models, Molecular; Myosins; Rotation; Viral Proteins
PubMed: 26819321
DOI: 10.1128/MMBR.00056-15 -
Cell Chemical Biology Oct 2021Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport....
Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.
Topics: Allosteric Regulation; Binding Sites; Catalytic Domain; Cryoelectron Microscopy; Dyneins; Humans; Molecular Docking Simulation; Mutagenesis, Site-Directed; Protein Binding; Pyrazoles; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Small Molecule Libraries
PubMed: 34015309
DOI: 10.1016/j.chembiol.2021.04.024 -
Bioarchitecture 2014Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying...
Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge.
Topics: Animals; Dyneins; Microtubules
PubMed: 24847718
DOI: 10.4161/bioa.29070 -
Nature Structural & Molecular Biology Apr 2015Cytoplasmic dynein associates with dynactin to drive cargo movement on microtubules, but the structure of the dynein-dynactin complex is unknown. Using electron...
Cytoplasmic dynein associates with dynactin to drive cargo movement on microtubules, but the structure of the dynein-dynactin complex is unknown. Using electron microscopy, we determined the organization of native bovine dynein, dynactin and the dynein-dynactin-microtubule quaternary complex. In the microtubule-bound complex, the dynein motor domains are positioned for processive unidirectional movement, and the cargo-binding domains of both dynein and dynactin are accessible.
Topics: Animals; Cattle; Cryoelectron Microscopy; Dynactin Complex; Dyneins; Microtubule-Associated Proteins; Microtubules; Models, Molecular
PubMed: 25751425
DOI: 10.1038/nsmb.2996 -
Experimental & Molecular Medicine Apr 2024Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, dynein has a... (Review)
Review
Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, dynein has a single isoform, which raises questions about its cargo specificity and regulatory mechanisms. The precision of dynein-mediated cargo transport is governed by a multitude of factors, including temperature, phosphorylation, the microtubule track, and interactions with a family of activating adaptor proteins. Activating adaptors are of particular importance because they not only activate the unidirectional motility of the motor but also connect a diverse array of cargoes with the dynein motor. Therefore, it is unsurprising that dysregulation of the dynein-activating adaptor transport machinery can lead to diseases such as spinal muscular atrophy, lower extremity, and dominant. Here, we discuss dynein motor motility within cells and in in vitro, and we present several methodologies employed to track the motion of the motor. We highlight several newly identified activating adaptors and their roles in regulating dynein. Finally, we explore the potential therapeutic applications of manipulating dynein transport to address diseases linked to dynein malfunction.
Topics: Humans; Cytoplasmic Dyneins; Animals; Biological Transport; Microtubules; Dyneins
PubMed: 38556551
DOI: 10.1038/s12276-024-01200-7 -
The Journal of Cell Biology Jan 2015Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is...
Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is crucial to know how the signal of MT binding is transmitted to the ATPase domain to enhance ATP hydrolysis. However, the molecular basis of signal transmission at the dynein-MT interface remains unclear. Scanning mutagenesis of tubulin identified two residues in α-tubulin, R403 and E416, that are critical for ATPase activation and directional movement of dynein. Electron cryomicroscopy and biochemical analyses revealed that these residues form salt bridges with the residues in the dynein MT-binding domain (MTBD) that work in concert to induce registry change in the stalk coiled coil and activate the ATPase. The R403-E3390 salt bridge functions as a switch for this mechanism because of its reversed charge relative to other residues at the interface. This study unveils the structural basis for coupling between MT binding and ATPase activation and implicates the MTBD in the control of directional movement.
Topics: Amino Acid Sequence; Animals; Conserved Sequence; Cryoelectron Microscopy; Dictyostelium; Dyneins; Enzyme Activation; Microtubules; Models, Molecular; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Protozoan Proteins; Sus scrofa
PubMed: 25583999
DOI: 10.1083/jcb.201407039 -
Methods in Molecular Biology (Clifton,... 2023Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules...
Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein's molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein's multiple AAA+ ATPase domains regulate dynein's attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.
Topics: Humans; Cytoplasmic Dyneins; Dyneins; Microtubules; Saccharomyces cerevisiae; Mutagenesis
PubMed: 36602689
DOI: 10.1007/978-1-0716-2958-1_14