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Stephen M. King - One of the best experts on this subject based on the ideXlab platform.
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chlamydomonas dyx1c1 pf23 is essential for axonemal assembly and proper morphology of inner Dynein arms
PLOS Genetics, 2017Co-Authors: Ryosuke Yamamoto, Takahide Kon, Lea M Alford, Juyeon Hwang, Jagan M Obbineni, Takahiro Ide, Mikito Owa, Kazuo Inaba, Noliyanda James, Stephen M. KingAbstract:Cytoplasmic assembly of ciliary Dyneins, a process known as preassembly, requires numerous non-Dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner Dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner Dynein arms (IDA) as well as a fraction of the outer Dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary Dyneins are reduced compared to wild-type, which suggests an important role in Dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.
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the oligomeric outer Dynein arm assembly factor ccdc103 is tightly integrated within the ciliary axoneme and exhibits periodic binding to microtubules
Journal of Biological Chemistry, 2015Co-Authors: Stephen M. King, Ramila S PatelkingAbstract:CCDC103 is an ∼29-kDa protein consisting of a central RPAP3_C domain flanked by N- and C-terminal coiled coils. Defects in CCDC103 lead to primary ciliary dyskinesia caused by the loss of outer Dynein arms. This protein is present along the entire length of the ciliary axoneme and does not require other Dynein or docking complex components for its integration. Unlike other known Dynein assembly factors within the axoneme, CCDC103 is not solubilized by 0.6 M NaCl and requires more chaotropic conditions, such as 0.5 M KI. Alternatively, it can be extracted using 0.3% sarkosyl. CCDC103 forms stable dimers and other oligomers in solution through interactions involving the central domain. The smallest particle observed by dynamic light scattering has a hydrodynamic diameter of ∼25 nm. Furthermore, CCDC103 binds microtubules directly, forming ∼9-nm diameter particles that exhibit a 12-nm spacing on the microtubule lattice, suggesting that there may be two CCDC103 units per outer arm Dynein repeat. Although the outer Dynein arm docking complex is necessary to form arrays of Dyneins along microtubules, it is not sufficient to set up a single array in a precise location on each axonemal doublet. We propose that CCDC103 helps generate a high-affinity site on the doublets for outer arm assembly, either through direct interactions or indirectly, perhaps by modifying the underlying microtubule lattice.
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a solid state control system for Dynein based ciliary flagellar motility
Journal of Cell Biology, 2013Co-Authors: Stephen M. KingAbstract:Ciliary and flagellar beating requires the coordinated action of multiple Dyneins with different enzymatic and motor properties. In this issue, Yamamoto et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211048) identify the MIA (modifier of inner arms) complex within the Chlamydomonas reinhardtii axoneme that physically links to a known regulatory structure and provides a signaling conduit from the radial spokes to an inner arm Dynein essential for waveform determination.
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The Mammalian Cytoplasmic Dynein Complexes
2013Co-Authors: Kevin K Pfister, Stephen M. King, Paresh R Shah, Holger Hummerich, Andreas Russ, James Cotton, Azlina Ahmad Annuar, Elizabeth M. C. FisherAbstract:(A) Cytoplasmic Dynein. (Left panel) Polypeptides of immunoaffinity-purified rat brain cytoplasmic Dynein. Polypeptide mass (in kDa) is indicated on the right side of the gel, and the consensus family names are indicated on the left. (Right panel) Structural model for the association of the cytoplasmic Dynein complex subunits. The core of the cytoplasmic Dynein complex is made of two DYNC1H1 heavy chains which homodimerize via regions in their N-termini. The motor domains are at the C-termini of the heavy chains, the large globular heads of ~350 kDa that are composed of a ring of seven densities surrounding a central cavity; six of the densities are AAA domains (numbered 1–6). AAA domain 1 is the site of ATP hydrolysis. The microtubule-binding domain is a projection found on the opposite side of the ring between AAA domains 4 and 5. C is the C-terminus of the heavy chain that would form the 7th density. Two DYNC1I intermediate chains (IC74) and DYNC1LI light intermediate chains bind at overlapping regions of the N-terminus of the heavy chain, overlapping with the heavy chain dimerization domains. Dimers of the three light chain families; DYNLT, the Tctex1 light chains; DYNLRB, the Roadblock light chains; and DYNLL, the LC8 light chains, bind to the intermediate chain dimers.(B) Cytoplasmic Dynein 2 complex, structural model for subunit association. This Dynein complex has a unique role in IFT and is sometimes known as IFT Dynein. Structural predictions indicate that the heavy chain, DYNC2H1, is similar to the cytoplasmic and axonemal Dyneins. The only known subunit of this complex is a 33- to 47-kDa polypeptide, DYNC2LI1, which is related to the cytoplasmic Dynein light intermediate chains. No intermediate chain or light chains have yet been identified [16].
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integrated control of axonemal Dynein aaa motors
Journal of Structural Biology, 2012Co-Authors: Stephen M. KingAbstract:Axonemal Dyneins are AAA+ enzymes that convert ATP hydrolysis to mechanical work. This leads to the sliding of doublet microtubules with respect to each other and ultimately the generation of ciliary/flagellar beating. However, in order for useful work to be generated, the action of individual Dynein motors must be precisely controlled. In addition, cells modulate the motility of these organelles through a variety of second messenger systems and these signals too must be integrated by the Dynein motors to yield an appropriate output. This review describes the current status of efforts to understand Dynein control mechanisms and their connectivity focusing mainly on studies of the outer Dynein arm from axonemes of the unicellular biflagellate green alga Chlamydomonas.
Ritsu Kamiya - One of the best experts on this subject based on the ideXlab platform.
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The outer Dynein armdocking complex: composition and characterization of a subunit (oda1) necessary for outer arm assembly. Mol Biol Cell 2002
2016Co-Authors: Saeko Takada, Ritsu Kamiya, Ken-ichi Wakabayashi, Curtis G. Wilkerson, George B WitmanAbstract:To learn more about how Dyneins are targeted to specific sites in the flagellum, we have investigated a factor necessary for binding of outer arm Dynein to the axonemal microtubules of Chlamydomonas. This factor, termed the outer Dynein arm-docking complex (ODA-DC), previously was shown to be missing from axonemes of the outer Dynein armless mutants oda1 and oda3. We have now partially purified the ODA-DC, determined that it contains equimolar amounts of Mr 105,000 and 70,000 proteins plus a third protein of Mr 25,000, and found that it is associated with the isolated outer arm in a 1:1 molar ratio. We have cloned a full-length cDNA encoding the Mr 70,000 protein; the sequence predicts a 62.5-kDa protein with potential homologs in higher ciliated organisms, including humans. Sequencing of corresponding cDNA from strain oda1 revealed it has a mutation resulting in a stop codon just downstream of the initiator ATG; thus, it is unable to make the full-length Mr 70,000 protein. These results demonstrate that the ODA1 gene encodes the Mr70,000 protein, and that the protein is essential for assembly of the ODA-DC and the outer Dynein arm onto the doublet microtubule
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the mia complex is a conserved and novel Dynein regulator essential for normal ciliary motility
Journal of Cell Biology, 2013Co-Authors: Ryosuke Yamamoto, Ritsu Kamiya, Toshiki Yagi, Haruaki Yanagisawa, Maureen Wirschell, Laura A. Fox, Kangkang Song, Masafumi Hirono, Daniela NicastroAbstract:Axonemal Dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 Dynein and structurally appeared to connect with the nexin–Dynein regulatory complex. In axonemes from mutants that lack both the outer Dynein arms and the MIA complex, I1 Dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 Dynein. The MIA complex appears to regulate I1 Dynein and possibly outer arm Dyneins, which are both essential for normal motility.
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Tubulin polyglutamylation regulates flagellar motility by controlling a specific inner-arm Dynein that interacts with the Dynein regulatory complex.
Cytoskeleton, 2012Co-Authors: Tomohiro Kubo, Toshiki Yagi, Ritsu KamiyaAbstract:The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm Dynein) is completely nonmotile. Thus, tubulin polyglutamylation must be important for the functioning of inner-arm Dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack Dynein "e," one of the seven species of major inner-arm Dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm Dynein species. This suggests that Dynein e is the main target of motility regulation by tubulin polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin polyglutamylation also affects the Dynein regulatory complex, a Dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.
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discrete pih proteins function in the cytoplasmic preassembly of different subsets of axonemal Dyneins
Journal of Cell Biology, 2010Co-Authors: Ryosuke Yamamoto, Masafumi Hirono, Ritsu KamiyaAbstract:Axonemal Dyneins are preassembled in the cytoplasm before being transported into cilia and flagella. Recently, PF13/KTU, a conserved protein containing a PIH (protein interacting with HSP90) domain, was identified as a protein responsible for Dynein preassembly in humans and Chlamydomonas reinhardtii. This protein is involved in the preassembly of outer arm Dynein and some inner arm Dyneins, possibly as a cofactor of molecular chaperones. However, it is not known which factors function in the preassembly of other inner arm Dyneins. Here, we analyzed a novel C. reinhardtii mutant, ida10, and found that another conserved PIH family protein, MOT48, is responsible for the formation of another subset of inner arm Dyneins. A variety of organisms with motile cilia and flagella typically have three to four PIH proteins, including potential homologues of MOT48 and PF13/KTU, whereas organisms without them have no, or only one, such protein. These findings raise the possibility that multiple PIH proteins are commonly involved in the preassembly of different subsets of axonemal Dyneins.
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novel 44 kilodalton subunit of axonemal Dynein conserved from chlamydomonas to mammals
Eukaryotic Cell, 2008Co-Authors: Ryosuke Yamamoto, Haruaki Yanagisawa, Toshiki Yagi, Ritsu KamiyaAbstract:Cilia and flagella have multiple Dyneins in their inner and outer arms. Chlamydomonas inner-arm Dynein contains at least seven major subspecies (Dynein a to Dynein g), of which all but Dynein f (also called Dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of Dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this Dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of Dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm Dynein d and its subunit organization are widely conserved.
Ahmet Yildiz - One of the best experts on this subject based on the ideXlab platform.
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Cargo adaptors regulate stepping and force generation of mammalian Dynein-dynactin.
Nature chemical biology, 2019Co-Authors: Mohamed M. Elshenawy, John T. Canty, Liya Oster, Scott C. Blanchard, Luke S Ferro, Zhou Zhou, Ahmet YildizAbstract:Cytoplasmic Dynein is an ATP-driven motor that transports intracellular cargos along microtubules. Dynein adopts an inactive conformation when not attached to a cargo, and motility is activated when Dynein assembles with dynactin and a cargo adaptor. It was unclear how active Dynein-dynactin complexes step along microtubules and transport cargos under tension. Using single-molecule imaging, we showed that Dynein-dynactin advances by taking 8 to 32-nm steps toward the microtubule minus end with frequent sideways and backward steps. Multiple Dyneins collectively bear a large amount of tension because the backward stepping rate of Dynein is insensitive to load. Recruitment of two Dyneins to dynactin increases the force generation and the likelihood of winning against kinesin in a tug-of-war but does not directly affect velocity. Instead, velocity is determined by cargo adaptors and tail-tail interactions between two closely packed Dyneins. Our results show that cargo adaptors modulate Dynein motility and force generation for a wide range of cellular functions.
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Lis1 activates Dynein motility by pairing it with dynactin
2019Co-Authors: Mohamed M. Elshenawy, Janina Baumbach, Simon L Bullock, Emre Kusakci, Sara Volz, Ahmet YildizAbstract:Abstract Lissencephaly-1 (Lis1) is a key cofactor for Dynein-mediated intracellular transport towards the minus-ends of microtubules (MTs). It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian Dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual Dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 binding releases Dynein from its auto-inhibited state and thereby promotes the formation of an active complex with dynactin. Lis1 also favors recruitment of two Dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. These results provide a mechanistic explanation for why Lis1 is required for efficient transport of many Dynein-associated cargoes in cells.
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kinesin 1 and Dynein use distinct mechanisms to bypass obstacles
bioRxiv, 2019Co-Authors: Luke S Ferro, Mohamed M. Elshenawy, Sinan Can, Meghan A Turner, Ahmet YildizAbstract:Abstract Kinesin-1 and cytoplasmic Dynein are microtubule (MT) motors that transport intracellular cargos. It remains unclear how these motors move along MTs densely coated with obstacles of various sizes in the cytoplasm. Here, we tested the ability of single and multiple motors to bypass synthetic obstacles on MTs in vitro. Contrary to previous reports, we found that mammalian Dynein is highly capable of bypassing obstacles. Unlike Dynein, single kinesin motors stall in the presence of obstacles, consistent with their inability to take sideways steps to neighboring protofilaments. Kinesins overcome this limitation when working in teams, bypassing obstacles as effectively as multiple Dyneins. Cargos driven by multiple kinesin or Dyneins are also capable of rotating around the MT to bypass large obstacles. These results suggest that multiplicity of motors is required not only for transporting cargos over long distances and generating higher forces, but also for maneuvering of the cargos on obstacle-coated MT surfaces.
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directionality of Dynein is controlled by the angle and length of its stalk
Nature, 2019Co-Authors: Sinan Can, Andrew P Carter, Samuel E Lacey, Mert Gur, Ahmet YildizAbstract:The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all Dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of Dynein by engineering a Saccharomyces cerevisiae Dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of Dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the Dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the Dynein stalk are fully conserved among species, our findings provide an explanation for why all Dyneins move towards the minus end of the microtubule.
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Cryo-EM shows how dynactin recruits two Dyneins for faster movement
Nature, 2018Co-Authors: L Urnavicius, Mohamed M. Elshenawy, Carina Motz, Ahmet Yildiz, Clinton K. Lau, Edgar Morales-rios, Andrew P CarterAbstract:Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link Dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second Dynein to dynactin. Whereas BICD2 is biased towards recruiting a single Dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two Dyneins. We find that the shift towards a double Dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a Dynein tail–dynactin–BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two Dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of Dyneins and regulate the motile properties of the Dynein–dynactin transport machine. In eukaryotic cells, the cytoplasmic protein Dynein-1 (Dynein) is the main transporter of cargoes towards the minus ends of microtubules—tube-like components of the cytoskeleton that, together with motor proteins, move material within the cell. Dynein is converted into a highly processive motor protein through binding both to its cofactor dynactin and to a cargo adaptor, such as BICD2, BICDR1 or HOOK3. BICD2 preferentially binds to one molecule of Dynein and one molecule of dynactin. Using cryo-electron microscopy Andrew Carter and team show that, in contrast, BICDR1 and HOOK3 bind to a complex of two Dyneins, which are brought together by a dynactin scaffold. The authors' single-molecule studies highlight that this configuration increases both the force and speed of the motor complex. Cryo-electron microscopy and single-molecule studies reveal that the adaptors BICDR1 and HOOK3 recruit two Dynein molecules to dynactin and thereby increase the force and speed of the Dynein–dynactin microtubule motor.
Andrew P Carter - One of the best experts on this subject based on the ideXlab platform.
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shulin packages axonemal outer Dynein arms for ciliary targeting
bioRxiv, 2020Co-Authors: G R Mali, Clinton K. Lau, Abid F Ali, Farida Begum, Mark Skehel, Andrew P CarterAbstract:The main force generators in eukaryotic cilia and flagella are axonemal outer Dynein arms (ODAs). During cilio-genesis, these [~]1.8 MDa complexes are assembled in the cytoplasm and targeted to cilia via an unknown mechanism. Here we use the ciliate Tetrahymena to identify two novel factors (Q22YU3 and Q22MS1) which bind ODAs in the cytoplasm and are required for their delivery to cilia. We show that Q22YU3, which we name Shulin, locks the ODA motor domains into a closed conformation and inhibits motor activity. Cryo-EM reveals how Shulin stabilizes this compact form of ODAs by binding to the Dynein tails. Our findings provide a molecular explanation for how newly assembled Dyneins are packaged for delivery to the cilia.
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directionality of Dynein is controlled by the angle and length of its stalk
Nature, 2019Co-Authors: Sinan Can, Andrew P Carter, Samuel E Lacey, Mert Gur, Ahmet YildizAbstract:The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all Dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of Dynein by engineering a Saccharomyces cerevisiae Dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of Dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the Dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the Dynein stalk are fully conserved among species, our findings provide an explanation for why all Dyneins move towards the minus end of the microtubule.
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Cryo-EM shows how dynactin recruits two Dyneins for faster movement
Nature, 2018Co-Authors: L Urnavicius, Mohamed M. Elshenawy, Carina Motz, Ahmet Yildiz, Clinton K. Lau, Edgar Morales-rios, Andrew P CarterAbstract:Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link Dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second Dynein to dynactin. Whereas BICD2 is biased towards recruiting a single Dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two Dyneins. We find that the shift towards a double Dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a Dynein tail–dynactin–BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two Dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of Dyneins and regulate the motile properties of the Dynein–dynactin transport machine. In eukaryotic cells, the cytoplasmic protein Dynein-1 (Dynein) is the main transporter of cargoes towards the minus ends of microtubules—tube-like components of the cytoskeleton that, together with motor proteins, move material within the cell. Dynein is converted into a highly processive motor protein through binding both to its cofactor dynactin and to a cargo adaptor, such as BICD2, BICDR1 or HOOK3. BICD2 preferentially binds to one molecule of Dynein and one molecule of dynactin. Using cryo-electron microscopy Andrew Carter and team show that, in contrast, BICDR1 and HOOK3 bind to a complex of two Dyneins, which are brought together by a dynactin scaffold. The authors' single-molecule studies highlight that this configuration increases both the force and speed of the motor complex. Cryo-electron microscopy and single-molecule studies reveal that the adaptors BICDR1 and HOOK3 recruit two Dynein molecules to dynactin and thereby increase the force and speed of the Dynein–dynactin microtubule motor.
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Cryo-EM shows how dynactin recruits two Dyneins for faster movement
2017Co-Authors: L Urnavicius, Mohamed M. Elshenawy, Carina Motz, Ahmet Yildiz, Clinton K. Lau, Edgar Morales-rios, Andrew P CarterAbstract:Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link Dynein/dynactin to distinct cargos. Here we use electron microscopy (EM) and single molecule studies to show that adaptors can recruit a second Dynein to dynactin. Whereas BICD2 is biased toward recruiting a single Dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two. We find that the shift toward a double Dynein complex increases both force and speed. A 3.5 A cryo-EM reconstruction of a Dynein tail/dynactin/BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two Dyneins side by side. Our work provides a structural basis for how diverse adaptors recruit different numbers of Dyneins and regulate the motile properties of the Dynein/dynactin transport machine.
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the mammalian Dynein dynactin complex is a strong opponent to kinesin in a tug of war competition
Nature Cell Biology, 2016Co-Authors: Vladislav Belyy, Andrew P Carter, Max A Schlager, Helen Foster, Armando Reimer, Ahmet YildizAbstract:Kinesin and Dynein motors transport intracellular cargos bidirectionally by pulling them in opposite directions along microtubules, through a process frequently described as a 'tug of war'. While kinesin produces 6 pN of force, mammalian Dynein was found to be a surprisingly weak motor (0.5-1.5 pN) in vitro, suggesting that many Dyneins are required to counteract the pull of a single kinesin. Mammalian Dynein's association with dynactin and Bicaudal-D2 (BICD2) activates its processive motility, but it was unknown how this affects Dynein's force output. Here, we show that formation of the Dynein-dynactin-BICD2 (DDB) complex increases human Dynein's force production to 4.3 pN. An in vitro tug-of-war assay revealed that a single DDB successfully resists a single kinesin. Contrary to previous reports, the clustering of many Dyneins is not required to win the tug of war. Our work reveals the key role of dynactin and a cargo adaptor protein in shifting the balance of forces between Dynein and kinesin motors during intracellular transport.
Ryosuke Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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Mutations in PIH proteins MOT48, TWI1 and PF13 define common and unique steps for preassembly of each, different ciliary Dynein.
'Public Library of Science (PLoS)', 2020Co-Authors: Ryosuke Yamamoto, Winfield S Sale, Toshiki Yagi, Shiho Yanagi, Masahito Nagao, Yuya Yamasaki, Yui Tanaka, Takahide KonAbstract:Ciliary Dyneins are preassembled in the cytoplasm before being transported into cilia, and a family of proteins containing the PIH1 domain, PIH proteins, are involved in the assembly process. However, the functional differences and relationships between members of this family of proteins remain largely unknown. Using Chlamydomonas reinhardtii as a model, we isolated and characterized two novel Chlamydomonas PIH preassembly mutants, mot48-2 and twi1-1. A new allele of mot48 (ida10), mot48-2, shows large defects in ciliary Dynein assembly in the axoneme and altered motility. A second mutant, twi1-1, shows comparatively smaller defects in motility and Dynein assembly. A double mutant mot48-2; twi1-1 displays greater reduction in motility and in Dynein assembly compared to each single mutant. Similarly, a double mutant twi1-1; pf13 also shows a significantly greater defect in motility and Dynein assembly than either parent mutant. Thus, MOT48 (IDA10), TWI1 and PF13 may define different steps, and have partially overlapping functions, in a pathway required for ciliary Dynein preassembly. Together, our data suggest the three PIH proteins function in preassembly steps that are both common and unique for different ciliary Dyneins
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chlamydomonas dyx1c1 pf23 is essential for axonemal assembly and proper morphology of inner Dynein arms
PLOS Genetics, 2017Co-Authors: Ryosuke Yamamoto, Takahide Kon, Lea M Alford, Juyeon Hwang, Jagan M Obbineni, Takahiro Ide, Mikito Owa, Kazuo Inaba, Noliyanda James, Stephen M. KingAbstract:Cytoplasmic assembly of ciliary Dyneins, a process known as preassembly, requires numerous non-Dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner Dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner Dynein arms (IDA) as well as a fraction of the outer Dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary Dyneins are reduced compared to wild-type, which suggests an important role in Dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.
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the ciliary inner Dynein arm i1 Dynein is assembled in the cytoplasm and transported by ift before axonemal docking
Cytoskeleton, 2014Co-Authors: Rasagnya Viswanadha, Ryosuke Yamamoto, Maureen Wirschell, Emily L Hunter, Lea M Alford, Susan K Dutcher, Winfield S SaleAbstract:To determine mechanisms of assembly of ciliary Dyneins, we focused on the Chlamydomonas inner Dynein arm, I1 Dynein, also known as Dynein f. I1 Dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 Dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 Dynein preassembly in the cytoplasm. Unlike I1 Dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 Dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 Dynein mutant cells. Rescue of I1 Dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 Dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 Dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 Dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 Dynein is dependent on kinesin-2 activity. Thus, I1 Dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme.
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the mia complex is a conserved and novel Dynein regulator essential for normal ciliary motility
Journal of Cell Biology, 2013Co-Authors: Ryosuke Yamamoto, Ritsu Kamiya, Toshiki Yagi, Haruaki Yanagisawa, Maureen Wirschell, Laura A. Fox, Kangkang Song, Masafumi Hirono, Daniela NicastroAbstract:Axonemal Dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 Dynein and structurally appeared to connect with the nexin–Dynein regulatory complex. In axonemes from mutants that lack both the outer Dynein arms and the MIA complex, I1 Dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 Dynein. The MIA complex appears to regulate I1 Dynein and possibly outer arm Dyneins, which are both essential for normal motility.
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discrete pih proteins function in the cytoplasmic preassembly of different subsets of axonemal Dyneins
Journal of Cell Biology, 2010Co-Authors: Ryosuke Yamamoto, Masafumi Hirono, Ritsu KamiyaAbstract:Axonemal Dyneins are preassembled in the cytoplasm before being transported into cilia and flagella. Recently, PF13/KTU, a conserved protein containing a PIH (protein interacting with HSP90) domain, was identified as a protein responsible for Dynein preassembly in humans and Chlamydomonas reinhardtii. This protein is involved in the preassembly of outer arm Dynein and some inner arm Dyneins, possibly as a cofactor of molecular chaperones. However, it is not known which factors function in the preassembly of other inner arm Dyneins. Here, we analyzed a novel C. reinhardtii mutant, ida10, and found that another conserved PIH family protein, MOT48, is responsible for the formation of another subset of inner arm Dyneins. A variety of organisms with motile cilia and flagella typically have three to four PIH proteins, including potential homologues of MOT48 and PF13/KTU, whereas organisms without them have no, or only one, such protein. These findings raise the possibility that multiple PIH proteins are commonly involved in the preassembly of different subsets of axonemal Dyneins.