Is outer arm dynein intermediate chain 1 multifunctional?

K Ogawa; H Takai; A Ogiwara; E Yokota; T Shimizu; K Inaba; H Mohri

doi:10.1091/mbc.7.12.1895

Is outer arm dynein intermediate chain 1 multifunctional?

Molecular Biology of the Cell ◽

10.1091/mbc.7.12.1895 ◽

1996 ◽

Vol 7 (12) ◽

pp. 1895-1907 ◽

Cited By ~ 65

Author(s):

K Ogawa ◽

H Takai ◽

A Ogiwara ◽

E Yokota ◽

T Shimizu ◽

...

Keyword(s):

Sea Urchin ◽

Related Species ◽

Repetitive Sequences ◽

Middle Part ◽

Affinity Column ◽

Terminal Part ◽

Related Part ◽

Dynein Intermediate Chain ◽

Intermediate Chains ◽

Intermediate Chain

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.

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Interspecies conservation of outer arm dynein intermediate chain sequences defines two intermediate chain subclasses.

Molecular Biology of the Cell ◽

10.1091/mbc.6.6.685 ◽

1995 ◽

Vol 6 (6) ◽

pp. 685-696 ◽

Cited By ~ 37

Author(s):

K Ogawa ◽

R Kamiya ◽

C G Wilkerson ◽

G B Witman

Keyword(s):

Sea Urchin ◽

Related Species ◽

Cytoplasmic Dynein ◽

Cross Reactivity ◽

The Other ◽

Immunological Analysis ◽

Dynein Intermediate Chain ◽

Structural Differences ◽

Intermediate Chains ◽

Intermediate Chain

Immunological analysis showed that antibodies against the intermediate chains (ICs) IC2 and IC3 of sea urchin outer arm dynein specifically cross-reacted with intermediate chains IC78 and IC69, respectively, of Chlamydomonas outer arm dynein. In contrast, no specific cross-reactivity with any Chlamydomonas outer arm polypeptide was observed using antibody against IC1 of sea urchin outer arm dynein. To learn more about the relationships between the different ICs, overlapping cDNAs encoding all of IC2 and IC3 of sea urchin were isolated and sequenced. Comparison of these sequences with those previously obtained for the Chlamydomonas ICs revealed that, although all four chains are homologous, sea urchin IC2 is much more closely related to Chlamydomonas IC78 (45.8% identity), and sea urchin IC3 is much more closely related to Chlamydomonas IC69 (48.5% identity), than either sea urchin chain is related to the other (23.5% identity). For homologous pairs, the similarities extend throughout the full lengths of the chains. Regions of similarity between all four ICs and the IC (IC74) of cytoplasmic dynein, located in the C-terminal halves of the chains, are due primarily to conservation of the WD repeats present in all of these ICs. This is the first demonstration that structural differences between individual ICs within an outer arm dynein have been highly conserved in the dyneins of distantly related species. The results provide a basis for the subclassification of these chains.

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The M r 140,000 Intermediate Chain ofChlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

Molecular Biology of the Cell ◽

10.1091/mbc.9.12.3335 ◽

1998 ◽

Vol 9 (12) ◽

pp. 3335-3349 ◽

Cited By ~ 69

Author(s):

Pinfen Yang ◽

Winfield S. Sale

Keyword(s):

Fusion Protein ◽

Protein A ◽

Dynein Intermediate Chain ◽

In Vitro Reconstitution ◽

Unique Site ◽

Intermediate Chains ◽

Β Sheet ◽

Chemical Cross Linking ◽

Intermediate Chain

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonasflagella. Immunofluorescent microscopy of Chlamydomonascells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.

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A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

Molecular Biology of the Cell ◽

10.1091/mbc.5.9.1051 ◽

1994 ◽

Vol 5 (9) ◽

pp. 1051-1063 ◽

Cited By ~ 20

Author(s):

C Gagnon ◽

D White ◽

P Huitorel ◽

J Cosson

Keyword(s):

Sea Urchin ◽

Cortical Microtubule ◽

Marginal Effect ◽

Microtubule Network ◽

Lytechinus Pictus ◽

Oxyrrhis Marina ◽

Dynein Intermediate Chain ◽

Immotile Spermatozoa ◽

Intermediate Chain

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.

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The Involvement of the Intermediate Chain of Cytoplasmic Dynein in Binding the Motor Complex to Membranous Organelles ofXenopus Oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.8.10.2077 ◽

1997 ◽

Vol 8 (10) ◽

pp. 2077-2088 ◽

Cited By ~ 84

Author(s):

Walter Steffen ◽

Sher Karki ◽

Kevin T. Vaughan ◽

Richard B. Vallee ◽

Erika L.F. Holzbaur ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Intracellular Transport ◽

Network Formation ◽

Coiled Coil ◽

Cytoplasmic Dynein ◽

Antibody Concentration ◽

Affinity Column ◽

Organelle Movement ◽

Intermediate Chains ◽

Intermediate Chain

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74–1, m74–2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 μg/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74–1 and m74–2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Gluedbinding domain. In an IC affinity column binding assay, both antibodies inhibited the IC–p150Glued interaction. Thus these findings demonstrate that direct IC–p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

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Structure of Tctex-1 and Its Interaction with Cytoplasmic Dynein Intermediate Chain

Journal of Biological Chemistry ◽

10.1074/jbc.m011358200 ◽

2001 ◽

Vol 276 (17) ◽

pp. 14067-14074 ◽

Cited By ~ 85

Author(s):

Yu-Keung Mok ◽

Kevin W.-H. Lo ◽

Mingjie Zhang

Keyword(s):

Cytoplasmic Dynein ◽

Dynein Intermediate Chain ◽

Intermediate Chain

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PLAC-24 Is a Cytoplasmic Dynein-Binding Protein That Is Recruited to Sites of Cell-Cell Contact

Molecular Biology of the Cell ◽

10.1091/mbc.02-02-0011 ◽

2002 ◽

Vol 13 (5) ◽

pp. 1722-1734 ◽

Cited By ~ 16

Author(s):

Sher Karki ◽

Lee A. Ligon ◽

Jamison DeSantis ◽

Mariko Tokito ◽

Erika L. F. Holzbaur

Keyword(s):

Epithelial Cells ◽

Recent Observation ◽

Polyclonal Antibodies ◽

Adherens Junction ◽

Cytoplasmic Dynein ◽

Cell Contact ◽

Dynein Intermediate Chain ◽

Cellular Cytoskeleton ◽

Cell Cell ◽

Intermediate Chain

We screened for polypeptides that interact specifically with dynein and identified a novel 24-kDa protein (PLAC-24) that binds directly to dynein intermediate chain (DIC). PLAC-24 is not a dynactin subunit, and the binding of PLAC-24 to the dynein intermediate chain is independent of the association between dynein and dynactin. Immunocytochemistry using PLAC-24–specific polyclonal antibodies revealed a punctate perinuclear distribution of the polypeptide in fibroblasts and isolated epithelial cells. However, as epithelial cells in culture make contact with adjacent cells, PLAC-24 is specifically recruited to the cortex at sites of contact, where the protein colocalizes with components of the adherens junction. Disruption of the cellular cytoskeleton with latrunculin or nocodazole indicates that the localization of PLAC-24 to the cortex is dependent on intact actin filaments but not on microtubules. Overexpression of β-catenin also leads to a loss of PLAC-24 from sites of cell-cell contact. On the basis of these data and the recent observation that cytoplasmic dynein is also localized to sites of cell-cell contact in epithelial cells, we propose that PLAC-24 is part of a multiprotein complex localized to sites of intercellular contact that may function to tether microtubule plus ends to the actin-rich cellular cortex.

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Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.107.5.1793 ◽

1988 ◽

Vol 107 (5) ◽

pp. 1793-1797 ◽

Cited By ~ 65

Author(s):

W S Sale ◽

L A Fox

Keyword(s):

Ionic Strength ◽

Sea Urchin ◽

Heavy Chain ◽

Glass Surface ◽

Functional Assay ◽

Atpase Subunits ◽

Low Ionic Strength ◽

Intermediate Chains ◽

Sea Urchin Sperm

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.

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Somatic CRISPR–Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia

The Journal of Cell Biology ◽

10.1083/jcb.201411041 ◽

2015 ◽

Vol 208 (6) ◽

pp. 683-692 ◽

Cited By ~ 41

Author(s):

Wenjing Li ◽

Peishan Yi ◽

Guangshuo Ou

Keyword(s):

Caenorhabditis Elegans ◽

Intraflagellar Transport ◽

Cytoplasmic Dynein ◽

Regulatory Mechanisms ◽

Light Chains ◽

Induced Mutations ◽

Functional Studies ◽

Cilium Formation ◽

Dynein Intermediate Chain ◽

Intermediate Chain

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2–driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT–dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

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Evolutionary characterization of a Y chromosomal sequence conserved in the genus Mus

Genetics Research ◽

10.1017/s001667230002752x ◽

1988 ◽

Vol 52 (2) ◽

pp. 145-150 ◽

Cited By ~ 5

Author(s):

Yutaka Nishioka

Keyword(s):

Related Species ◽

Rattus Norvegicus ◽

Phylogenetic Relationships ◽

Repetitive Sequence ◽

Repetitive Sequences ◽

Closely Related Species ◽

Specific Hybridization ◽

Male Specific ◽

Chromosomal Sequence

SummaryThe extent of accumulation of mouse Y chromosomal repetitive sequences generally correlates with the known phylogenetic relationships in the genus Mus. However, we describe here a M. musculus Y chromosomal repetitive sequence, designated as ACClfl, whose accumulation patterns among eight Mus species do not correspond to their phylogenetic relationships. Although male-specific hybridization bands were present in all the species examined, significant accumulation (> 200 copies) in the Y chromosomes was found in M. minutoides (subgenus Nannomys), M. pahari (subgenus Coelomys) and M. saxicola (subgenus Pyromys) as well as in the three closely related species M. hortulanus, M. musculus and M. spretus that belong to the subgenus Mus. Unexpectedly, the Y chromosomes of M. caroli and M. cookii (both subgenus Mus) had considerably reduced amounts of ACClfl-related sequences. Furthermore, in rats (Rattus norvegicus) the major accumulation sites appear to be autosomal. These observations suggest that caution must be taken in the interpretation of data obtained with repetitive sequences that have evolved quickly.

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