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 Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte.

1994 ◽  
Vol 126 (6) ◽  
pp. 1475-1494 ◽  
Author(s):  
M Li ◽  
M McGrail ◽  
M Serr ◽  
T S Hays
Keyword(s):  
Heavy Chain ◽  
Maternal Effect ◽  
Nurse Cell ◽  
Cytoplasmic Dynein ◽  
Cdna Clones ◽  
Dynein Heavy Chain ◽  
Dynein Motor ◽  
Maternal Effect Gene ◽  
Microtubule Motor ◽  
Egg Chambers

The unidirectional movements of the microtubule-associated motors, dyneins, and kinesins, provide an important mechanism for the positioning of cellular organelles and molecules. An intriguing possibility is that this mechanism may underlie the directed transport and asymmetric positioning of morphogens that influence the development of multicellular embryos. In this report, we characterize the Drosophila gene, Dhc64C, that encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development with the highest levels being detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte.

Cytoplasmic Dynein Function Is Essential in Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 865-878 ◽  
Author(s):  
Janice Gepner ◽  
Min-gang Li ◽  
Susan Ludmann ◽  
Cynthia Kortas ◽  
Kristin Boylan ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Intracellular Transport ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Complementation Group ◽  
Transport Processes ◽  
Female Sterility ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Dynein Heavy Chain

Abstract The microtubule motor cytoplasmic dynein has been implicated in a variety of intracellular transport processes. We previously identified and characterized the Drosophila gene Dhc64C, which encodes a cytoplasmic dynein heavy chain. To investigate the function of the cytoplasmic dynein motor, we initiated a mutational analysis of the Dhc64C dynein gene. A small deletion that removes the chromosomal region containing the heavy chain gene was used to isolate EMS-induced lethal mutations that define at least eight essential genes in the region. Germline transformation with a Dhc64C transgene rescued 16 mutant alleles in the single complementation group that identifies the dynein heavy chain gene. All 16 alleles were hemizygous lethal, which demonstrates that the cytoplasmic dynein heavy chain gene Dhc64C is essential for Drosophila development. Furthermore, our failure to recover somatic clones of cells homozygous for a Dhc64C mutation indicates that cytoplasmic dynein function is required for cell viability in several Drosophila tissues. The intragenic complementation of dynein alleles reveals multiple mutant phenotypes including male and/or female sterility, bristle defects, and defects in eye development.

scholarly journals An alpha tubulin mutation suppresses nuclear migration mutations in Aspergillus nidulans.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1287-1298 ◽  
Author(s):  
D A Willins ◽  
X Xiang ◽  
N R Morris
Keyword(s):  
Aspergillus Nidulans ◽  
Heavy Chain ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Beta Subunit ◽  
Alpha Tubulin ◽  
Dynein Heavy Chain ◽  
Suppressor Mutations ◽  
Dynein Motor ◽  
Low Concentrations

Abstract Microtubules and cytoplasmic dynein, a microtubule-dependent motor, are required for nuclei to move along the hyphae of filamentous fungi. Nuclear migration in Aspergillus nidulans is blocked by heat-sensitive (hs-) mutations in the nudA gene, which encodes dynein heavy chain, and the nudF gene, which encodes a G protein beta-subunit-like protein. Hs- mutations in the nudC and nudG genes also prevent nuclear migration. We have isolated extragenic suppressor mutations that reverse the hs- phenotypes caused by these mutations. Here we show that one nudF suppressor also suppresses hs- mutations in nudA, nudC, and nudG and deletions in nudA and nudF. This suppressor mutation is in the tubA alpha tubulin gene, and its characteristics suggest that it destabilizes microtubules. The mutation alters microtubule staining and confers sensitivity to cold and benomyl, two treatments that destabilize microtubules. Treatment with low concentrations of benomyl also suppresses the hs- nudA, nudC, nudF, and nudG mutations and the nudA and nudF deletions. Suppression of the hs- nudA mutation and the nudA deletion is especially interesting because these strains lack active dynein heavy chain. Together, these results suggest that microtubule destabilization allows nuclei to migrate even in the absence of cytoplasmic dynein motor function.

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 Nudel functions in membrane traffic mainly through association with Lis1 and cytoplasmic dynein

2004 ◽  
Vol 164 (4) ◽  
pp. 557-566 ◽  
Author(s):  
Yun Liang ◽  
Wei Yu ◽  
Yan Li ◽  
Zhenye Yang ◽  
Xiumin Yan ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Neuronal Migration ◽  
Time Lapse ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Time Lapse Microscopy ◽  
Dynein Motor ◽  
Defective Mutant ◽  
Directed Motility

Nudel and Lis1 appear to regulate cytoplasmic dynein in neuronal migration and mitosis through direct interactions. However, whether or not they regulate other functions of dynein remains elusive. Herein, overexpression of a Nudel mutant defective in association with either Lis1 or dynein heavy chain is shown to cause dispersions of membranous organelles whose trafficking depends on dynein. In contrast, the wild-type Nudel and the double mutant that binds to neither protein are much less effective. Time-lapse microscopy for lysosomes reveals significant reduction in both frequencies and velocities of their minus end–directed motions in cells expressing the dynein-binding defective mutant, whereas neither the durations of movement nor the plus end–directed motility is considerably altered. Moreover, silencing Nudel expression by RNA interference results in Golgi apparatus fragmentation and cell death. Together, it is concluded that Nudel is critical for dynein motor activity in membrane transport and possibly other cellular activities through interactions with both Lis1 and dynein heavy chain.

Identification and molecular evolution of new dynein-like protein sequences in rat brain

1995 ◽  
Vol 108 (5) ◽  
pp. 1883-1893 ◽  
Author(s):  
Y. Tanaka ◽  
Z. Zhang ◽  
N. Hirokawa
Keyword(s):  
Rat Brain ◽  
Heavy Chain ◽  
Phylogenetic Trees ◽  
Cytoplasmic Dynein ◽  
Amino Acid Sequences ◽  
Adult Brain ◽  
Chain Gene ◽  
Evolutionary Analysis ◽  
Degenerate Primers ◽  
Dynein Heavy Chain

RT-PCR cloning was performed to find unknown members of the dynein superfamily expressed in rat brain. Six kinds of degenerate primers designed for the dynein catalytic domain consensuses were used for extensive PCR amplifications. We have sequenced 550 plasmid clones which turned out to include 13 kinds of new dynein-like sequences (DLP1-8, 9A/B, 10–12) and cytoplasmic dynein heavy chain. In these clones, alternative splicing was detected for a 105 nt-domain containing the CFDEFNRI consensus just downstream of the most N-terminal P-loop (DLP9A and 9B). By using these obtained sequences, initial hybridization studies were performed. Genomic Southern blotting showed each sequence corresponds to a single copy of the gene, while northern blotting of adult brain presented more than one band for some subtypes. We further accomplished molecular evolutionary analysis to recognize their phylogenetic origins for the axonemal and non-axonemal (cytoplasmic) functions. Different methods (UPGMA, NJ and MP) presented well coincident phylogenetic trees from 44 partial amino acid sequences of dynein heavy chain from various eukaryotes. The trunk for all the cytoplasmic dynein heavy chain homologues diverged directly from the root of the phylogenetic tree, suggesting that the first dynein gene duplication defined two distinct functions as respective subfamilies. Of particular interest, we found a duplication event of the cytoplasmic dynein heavy chain gene giving rise to another subtype, DLP4, located between the divergence of yeast and that of Dictyostelium. Such evolutionary topology builds up an inceptive hypothesis that there are at least two non-axonemal dynein heavy chains in mammals.

scholarly journals Cytoplasmic Dynein, the Dynactin Complex, and Kinesin Are Interdependent and Essential for Fast Axonal Transport

1999 ◽  
Vol 10 (11) ◽  
pp. 3717-3728 ◽  
Author(s):  
MaryAnn Martin ◽  
Stanley J. Iyadurai ◽  
Andrew Gassman ◽  
Joseph G. Gindhart ◽  
Thomas S. Hays ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Heavy Chain ◽  
Cytoplasmic Dynein ◽  
Genetic Interactions ◽  
Organelle Transport ◽  
Fast Axonal Transport ◽  
Anterograde Transport ◽  
Dynein Heavy Chain ◽  
Conventional Kinesin ◽  
Dynactin Complex

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150Glued(Glued) component of the dynactin complex with the use of genetic techniques in Drosophila.cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150Glued were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued orcDhc64C mutations were stronger than those betweenGlued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

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