scholarly journals Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein

1994 ◽  
Vol 107 (3) ◽  
pp. 497-506 ◽  
Author(s):  
C.G. Wilkerson ◽  
S.M. King ◽  
G.B. Witman
Keyword(s):  
Heavy Chain ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Complete Sequence ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Microtubule Binding Domain ◽  
Helical Content ◽  
Axonemal Dynein ◽  
Dynein Heavy Chains

We report here the complete sequence of the gamma dynein heavy chain of the outer arm of the Chlamydomonas flagellum, and partial sequences for six other dynein heavy chains. The gamma dynein heavy chain sequence contains four P-loop motifs, one of which is the likely hydrolytic site based on its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein heavy chain sequences reveals regions that are highly conserved in all dynein heavy chains sequenced to date, regions that are conserved only among axonemal dynein heavy chains, and regions that are unique to individual dynein heavy chains. The presumed hydrolytic site is absolutely conserved among dyneins, two other P loops are highly conserved among cytoplasmic dynein heavy chains but not in axonemal dynein heavy chains, and the fourth P loop is invariant in axonemal dynein heavy chains but not in cytoplasmic dynein. One region that is very highly conserved in all dynein heavy chains is similar to a portion of the ATP-sensitive microtubule-binding domain of kinesin. Two other regions present in all dynein heavy chains are predicted to have high alpha-helical content and have a high probability of forming coiled-coil structures. Overall, the central one-third of the gamma dynein heavy chain is most conserved whereas the N-terminal one-third is least conserved; the fact that the latter region is divergent between the cytoplasmic dynein heavy chain and two different axonemal dynein heavy chains suggests that it is involved in chain-specific functions.

scholarly journals WDR92 is required for axonemal dynein heavy chain stability in cytoplasm

2019 ◽  
Vol 30 (15) ◽  
pp. 1834-1845 ◽  
Author(s):  
Ramila S. Patel-King ◽  
Miho Sakato-Antoku ◽  
Maya Yankova ◽  
Stephen M. King
Keyword(s):  
Heavy Chain ◽  
Beat Frequency ◽  
Gel Filtration ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Assembly Factor ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Phylogenetic Signature ◽  
Dynein Heavy Chains

WDR92 associates with a prefoldin-like cochaperone complex and known dynein assembly factors. WDR92 has been very highly conserved and has a phylogenetic signature consistent with it playing a role in motile ciliary assembly or activity. Knockdown of WDR92 expression in planaria resulted in ciliary loss, reduced beat frequency and dyskinetic motion of the remaining ventral cilia. We have now identified a Chlamydomonas wdr92 mutant that encodes a protein missing the last four WD repeats. The wdr92-1 mutant builds only ∼0.7-μm cilia lacking both inner and outer dynein arms, but with intact doublet microtubules and central pair. When cytoplasmic extracts prepared by freeze/thaw from a control strain were fractionated by gel filtration, outer arm dynein components were present in several distinct high molecular weight complexes. In contrast, wdr92-1 extracts almost completely lacked all three outer arm heavy chains, while the IFT dynein heavy chain was present in normal amounts. A wdr92-1 tpg1-2 double mutant builds ∼7-μm immotile flaccid cilia that completely lack dynein arms. These data indicate that WDR92 is a key assembly factor specifically required for the stability of axonemal dynein heavy chains in cytoplasm and suggest that cytoplasmic/IFT dynein heavy chains use a distinct folding pathway.

scholarly journals Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation.

1996 ◽  
Vol 7 (1) ◽  
pp. 71-79 ◽  
Author(s):  
K L Andrews ◽  
P Nettesheim ◽  
D J Asai ◽  
L E Ostrowski
Keyword(s):  
Cell Differentiation ◽  
Heavy Chain ◽  
Ciliated Cell ◽  
Northern Analysis ◽  
Ciliated Cells ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Cells In Culture ◽  
Axonemal Dynein ◽  
Dynein Heavy Chains

Axonemal dyneins are molecular motors that drive the beating of cilia and flagella. We report here the identification and partial cloning of seven unique axonemal dynein heavy chains from rat tracheal epithelial (RTE) cells. Combinations of axonemal-specific and degenerate primers to conserved regions around the catalytic site of dynein heavy chains were used to obtain cDNA fragments of rat dynein heavy chains. Southern analysis indicates that these are single copy genes, with one possible exception, and Northern analysis of RNA from RTE cells shows a transcript of approximately 15 kb for each gene. Expression of these genes was restricted to tissues containing axonemes (trachea, testis, and brain). A time course analysis during ciliated cell differentiation of RTE cells in culture demonstrated that the expression of axonemal dynein heavy chains correlated with the development of ciliated cells, while cytoplasmic dynein heavy chain expression remained constant. In addition, factors that regulate the development of ciliated cells in culture regulated the expression of axonemal dynein heavy chains in a parallel fashion. These are the first mammalian dynein heavy chain genes shown to be expressed specifically in axonemal tissues. Identification of the mechanisms that regulate the cell-specific expression of these axonemal dynein heavy chains will further our understanding of the process of ciliated cell differentiation.

scholarly journals Evidence for Four Cytoplasmic Dynein Heavy Chain Isoforms in Rat Testis

1998 ◽  
Vol 9 (2) ◽  
pp. 237-247 ◽  
Author(s):  
Peggy S. Criswell ◽  
David J. Asai
Keyword(s):  
Heavy Chain ◽  
Broad Band ◽  
Cytoplasmic Dynein ◽  
Peptide Sequence ◽  
Gene Encoding ◽  
Rat Testis ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Low Ionic Strength ◽  
Dynein Heavy Chains

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.

A novel cytoplasmic dynein heavy chain: expression of DHC1b in mammalian ciliated epithelial cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1891-1898 ◽  
Author(s):  
P.S. Criswell ◽  
L.E. Ostrowski ◽  
D.J. Asai
Keyword(s):  
Heavy Chain ◽  
Minor Component ◽  
Cytoplasmic Dynein ◽  
Heart Cells ◽  
Rat Tissues ◽  
Dynein Heavy Chain ◽  
Undifferentiated Cells ◽  
Tracheal Epithelial ◽  
Axonemal Dynein ◽  
Ciliated Epithelial Cells

Organisms that have cilia or flagella express over a dozen dynein heavy chain genes. Of these heavy chain genes, most appear to encode axonemal dyneins, one encodes conventional cytoplasmic dynein (MAP1C or DHC1a), and one, here referred to as DHC1b, encodes an unclassified heavy chain. Previous analysis of sea urchin DHC1b (Gibbons et al. (1994) Mol. Biol. Cell 5, 57–70) indicated that this isoform is either an axonemal dynein with an unusual protein sequence or a cytoplasmic dynein whose expression increases during ciliogenesis. In the present study, we examined the expression of DHC1b in rat tissues. The DHC1b gene is expressed in all tissues examined, including unciliated liver and heart cells. In contrast, rat axonemal dyneins are only expressed in tissues that produce cilia or flagella. In cultured rat tracheal epithelial (RTE) cells, DHC1b is expressed in undifferentiated cells and increases in expression during ciliogenesis. In contrast, the expression of conventional cytoplasmic dynein, DHC1a, does not change during RTE differentiation and axonemal dynein is not expressed until after differentiation commences. In order to examine the expression of DHC1b protein, we produced an isoform-specific antibody to a synthetic peptide derived from the rat DHC1b sequence. The antibody demonstrated that DHC1b is a relatively minor component of partially purified cytoplasmic dynein. Indirect immunofluorescence microscopy revealed that DHC1b is not detected in cilia and remains in the cytoplasm of ciliated RTE cells, often accumulating at the apical ends of the cells. These results suggest that DHC1b is a cytoplasmic dynein that may participate in intracellular trafficking in polarized cells.

scholarly journals The Third P-loop Domain in Cytoplasmic Dynein Heavy Chain Is Essential for Dynein Motor Function and ATP-sensitive Microtubule Binding

2003 ◽  
Vol 14 (4) ◽  
pp. 1355-1365 ◽  
Author(s):  
Andre Silvanovich ◽  
Min-gang Li ◽  
Madeline Serr ◽  
Sarah Mische ◽  
Thomas S. Hays
Keyword(s):  
Heavy Chain ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Sequence Comparisons ◽  
Microtubule Binding ◽  
Dynein Heavy Chain ◽  
Dynein Motor ◽  
Microtubule Binding Domain ◽  
Atp Binding And Hydrolysis ◽  
Loop Domains

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of theDrosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.

scholarly journals Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Samuel E Lacey ◽  
Shaoda He ◽  
Sjors HW Scheres ◽  
Andrew P Carter
Keyword(s):  
Conformational Changes ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Cross Sectional ◽  
Microtubule Binding ◽  
High Affinity ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Axonemal Dynein ◽  
Cilia And Flagella

Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

scholarly journals Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

2019 ◽  
Author(s):  
Samuel E. Lacey ◽  
Shaoda He ◽  
Sjors H. W. Scheres ◽  
Andrew P. Carter
Keyword(s):  
Conformational Changes ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Cross Sectional ◽  
Microtubule Binding ◽  
High Affinity ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Axonemal Dynein ◽  
Cilia And Flagella

AbstractDyneins are motor proteins responsible for transport in the cytoplasm and the beating of the axoneme in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a long coiled-coil stalk. Here we address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using the stand-alone Relion package. We show the majority of the MTBD is remarkably rigid upon binding, with the transition to the high affinity state controlled by the movement of a single helix at the MTBD interface. In addition DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that reaches over and contacts the adjacent protofilament. Unexpectedly we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

scholarly journals Gene Knockouts Reveal Separate Functions for Two Cytoplasmic Dyneins in Tetrahymena thermophila

1999 ◽  
Vol 10 (3) ◽  
pp. 771-784 ◽  
Author(s):  
Seungwon Lee ◽  
Julie C. Wisniewski ◽  
William L. Dentler ◽  
David J. Asai
Keyword(s):  
Division Of Labor ◽  
Heavy Chain ◽  
Phagocytic Activity ◽  
Cell Shape ◽  
Tetrahymena Thermophila ◽  
Cytoplasmic Dynein ◽  
Targeted Disruption ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Gene Knockouts

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophilapresents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of twoTetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, andDYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes

1994 ◽  
Vol 107 (3) ◽  
pp. 635-644 ◽  
Author(s):  
D.R. Mitchell ◽  
K.S. Brown
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Catalytic Domain ◽  
Nucleotide Binding ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Coding Region ◽  
Gamma Chain ◽  
Heavy Chains ◽  
Dynein Heavy Chains

We have sequenced genomic clones spanning the complete coding region of one heavy chain (beta) and the catalytic domain of a second (alpha) of the Chlamydomonas reinhardtii flagellar outer arm dynein ATPase. The beta heavy chain gene (ODA-4 locus) spans 20 kb, is divided into at least 30 exons, and encodes a predicted 520 kDa protein. Comparison with sea urchin beta dynein sequences reveals homology that extends throughout both proteins. Over the most conserved central catalytic region, the Chlamydomonas alpha and beta chains are equally divergent from the sea urchin beta chain (64% and 65% similarity, respectively), whereas the Chlamydomonas gamma chain is more divergent from urchin beta (54% similarity). The four glycine-rich loops identified as potential nucleotide-binding sites in other dynein heavy chains are also present in Chlamydomonas alpha and beta dyneins. Two of these four nucleotide-binding motifs are highly conserved among flagellar dyneins, but only the motif previously identified as the catalytic site in sea urchin dynein is highly conserved between flagellar and cytoplasmic dynein heavy chains. Predictions of secondary structure suggest that all dynein heavy chains possess three large domains, with the four nucleotide-binding consensus sequences located in a central 185 kDa domain that is bounded on both sides by regions that form multiple, short alpha-helical coiled-coils.

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