scholarly journals Golgi Vesiculation and Lysosome Dispersion in Cells Lacking Cytoplasmic Dynein

1998 ◽  
Vol 141 (1) ◽  
pp. 51-59 ◽  
Author(s):  
A. Harada ◽  
Y. Takei ◽  
Y. Kanai ◽  
Y. Tanaka ◽  
S. Nonaka ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Gene Targeting ◽  
Heavy Chain ◽  
Golgi Complex ◽  
Major Part ◽  
Motor Protein ◽  
Cytoplasmic Dynein ◽  
Blastocyst Stage ◽  
Dynein Heavy Chain

Cytoplasmic dynein, a minus end–directed, microtubule-based motor protein, is thought to drive the movement of membranous organelles and chromosomes. It is a massive complex that consists of multiple polypeptides. Among these polypeptides, the cytoplasmic dynein heavy chain (cDHC) constitutes the major part of this complex. To elucidate the function of cytoplasmic dynein, we have produced mice lacking cDHC by gene targeting. cDHC−/− embryos were indistinguishable from cDHC+/−or cDHC+/+ littermates at the blastocyst stage. However, no cDHC−/− embryos were found at 8.5 d postcoitum. When cDHC−/− blastocysts were cultured in vitro, they showed interesting phenotypes. First, the Golgi complex became highly vesiculated and distributed throughout the cytoplasm. Second, endosomes and lysosomes were not concentrated near the nucleus but were distributed evenly throughout the cytoplasm. Interestingly, the Golgi “fragments” and lysosomes were still found to be attached to microtubules. These results show that cDHC is essential for the formation and positioning of the Golgi complex. Moreover, cDHC is required for cell proliferation and proper distribution of endosomes and lysosomes. However, molecules other than cDHC might mediate attachment of the Golgi complex and endosomes/lysosomes to microtubules.

scholarly journals Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

1993 ◽  
Vol 178 (1) ◽  
pp. 49-62 ◽  
Author(s):  
M J Fritzler ◽  
J C Hamel ◽  
R L Ochs ◽  
E K Chan
Keyword(s):  
Recombinant Proteins ◽  
Heavy Chain ◽  
Golgi Complex ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Cytoplasmic Dynein ◽  
In Vitro Translation ◽  
Rabbit Serum ◽  
Cell Extracts

Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.

Recombinant human cytoplasmic dynein heavy chain 1 and 2: Observation of dynein-2 motor activity in vitro

FEBS Letters ◽  
2011 ◽  
Vol 585 (15) ◽  
pp. 2419-2423 ◽  
Author(s):  
Muneyoshi Ichikawa ◽  
Yuta Watanabe ◽  
Takashi Murayama ◽  
Yoko Yano Toyoshima
Keyword(s):  
Motor Activity ◽  
Heavy Chain ◽  
Cytoplasmic Dynein ◽  
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 2M1448 Properties of human cytoplasmic dynein heavy chain 1 and 2(Molecular motor 3,The 48th Annual Meeting of the Biophysical Society of Japan)

2011 ◽  
Vol 51 (supplement) ◽  
pp. S97
Author(s):  
Ichikawa Muneyoshi ◽  
Yuta Watanabe ◽  
Takashi Murayama ◽  
Yoko Yano Toyoshima
Keyword(s):  
Annual Meeting ◽  
Heavy Chain ◽  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Dynein Heavy Chain ◽  
Biophysical Society

scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein–dynactin–cargo adaptor complexes

2017 ◽  
Vol 114 (9) ◽  
pp. E1597-E1606 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L. Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
In Vitro Motility

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein–dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo–motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

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.

scholarly journals A fertility region on the Y chromosome of Drosophila melanogaster encodes a dynein microtubule motor

1993 ◽  
Vol 90 (23) ◽  
pp. 11132-11136 ◽  
Author(s):  
J Gepner ◽  
T S Hays
Keyword(s):  
Drosophila Melanogaster ◽  
Y Chromosome ◽  
Heavy Chain ◽  
Male Fertility ◽  
Chromosome Region ◽  
Cytoplasmic Dynein ◽  
Dynein Heavy Chain ◽  
Microtubule Motor ◽  
Hydrolysis Of

A clone encoding a portion of the highly conserved ATP-binding domain of a dynein heavy-chain polypeptide was mapped to a region of the Drosophila melanogaster Y chromosome. Dyneins are large multisubunit enzymes that utilize the hydrolysis of ATP to move along microtubules. They were first identified as the motors that provide the force for flagellar and ciliary bending. Seven different dynein heavy-chain genes have been identified in D. melanogaster by PCR. In the present study, we demonstrate that one of the dynein genes, Dhc-Yh3, is located in Y chromosome region h3, which is contained within kl-5, a locus required for male fertility. The PCR clone derived from Dhc-Yh3 is 85% identical to the corresponding region of the beta heavy chain of sea urchin flagellar dynein but only 53% identical to a cytoplasmic dynein heavy chain from Drosophila. In situ hybridization to Drosophila testes shows Dhc-Yh3 is expressed in wild-type males but not in males missing the kl-5 region. These results are consistent with the hypothesis that the Y chromosome is needed for male fertility because it contains conventional genes that function during spermiogenesis.

scholarly journals Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

2018 ◽  
Vol 315 (5) ◽  
pp. E924-E948 ◽  
Author(s):  
Qing Wen ◽  
Elizabeth I. Tang ◽  
Wing-yee Lui ◽  
Will M. Lee ◽  
Chris K. C. Wong ◽  
...  
Keyword(s):  
Germ Cell ◽  
Heavy Chain ◽  
Sertoli Cells ◽  
Cytoplasmic Dynein ◽  
Seminiferous Epithelium ◽  
Organelle Transport ◽  
Cellular Events

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.

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