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

1997 ◽  
Vol 8 (10) ◽  
pp. 2077-2088 ◽  
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.

scholarly journals The Gene for the Intermediate Chain Subunit of Cytoplasmic Dynein Is Essential in Drosophila

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1211-1220 ◽  
Author(s):  
Kristin L M Boylan ◽  
Thomas S Hays
Keyword(s):  
Intracellular Transport ◽  
De Novo ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Wing Development ◽  
Dynein Motor ◽  
Dynein Intermediate Chain ◽  
Microtubule Motor ◽  
Intermediate Chain

Abstract The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued1, a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.

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

1995 ◽  
Vol 6 (6) ◽  
pp. 685-696 ◽  
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.

scholarly journals Is outer arm dynein intermediate chain 1 multifunctional?

1996 ◽  
Vol 7 (12) ◽  
pp. 1895-1907 ◽  
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.

scholarly journals p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

1996 ◽  
Vol 7 (5) ◽  
pp. 731-742 ◽  
Author(s):  
J H Tinsley ◽  
P F Minke ◽  
K S Bruno ◽  
M Plamann
Keyword(s):  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Structural Features ◽  
Gene Encoding ◽  
Nuclear Distribution ◽  
Transport Vesicles ◽  
Dynactin Complex ◽  
Intermediate Chains

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.

scholarly journals Interactions of LC8 with N-Terminal Segments of the Intermediate Chain of Cytoplasmic Dynein

2003 ◽  
Vol 3 ◽  
pp. 647-654 ◽  
Author(s):  
Afua Nyarko ◽  
Michael Hare ◽  
Moses Makokha ◽  
Elisar Barbar
Keyword(s):  
Limited Proteolysis ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Light Chains ◽  
Ordered Structure ◽  
Dynein Motor ◽  
Dynein Light Chains ◽  
Significant Conformational Change ◽  
Intermediate Chain

LC8, a highly conserved 10-kDa light chain, and IC74, a 74-kDa intermediate chain, are presumed to promote the assembly of the cytoplasmic dynein motor protein complex and to be engaged in the controlled binding and release of cargo. The interactions of LC8 from Drosophila melanogaster with constructs of IC74 were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Previously, we have performed limited proteolysis on the N-terminal domain of IC74, IC(1-289), when free and when bound to dynein light chains LC8 and Tctex-1[1]. We have also shown that upon addition of LC8, IC(1-289) undergoes a significant conformational change from a largely unfolded to a more ordered structure. The purpose of the work presented here is to determine whether residues 1-30 in IC74, predicted to be in a coiled coil, are involved in the stabilization of the protein upon binding to LC8. Constructs of IC74, IC(1-143), and IC(30-143) that include the LC8 binding site but with and without the first 30 residues were prepared, and their binding and protection patterns were compared to our previous results for IC(1-289). The results suggest that coiled coil residues 1-30 are not responsible for the increase in structure we observe when IC(1-289) binds to LC8.

scholarly journals Identification and developmental regulation of a neuron-specific subunit of cytoplasmic dynein.

1996 ◽  
Vol 7 (2) ◽  
pp. 331-343 ◽  
Author(s):  
K K Pfister ◽  
M W Salata ◽  
J F Dillman ◽  
E Torre ◽  
R J Lye
Keyword(s):  
Axonal Transport ◽  
Developmental Regulation ◽  
Cytoplasmic Dynein ◽  
Retrograde Axonal Transport ◽  
Protein Isoforms ◽  
Cultured Neurons ◽  
Developmentally Regulated ◽  
Time Period ◽  
Intermediate Chains

Cytoplasmic dynein is the microtubule minus-end-directed motor for the retrograde axonal transport of membranous organelles. Because of its similarity to the intermediate chains of flagellar dynein, the 74-kDa intermediate chain (IC74) subunit of dynein is thought to be involved in binding dynein to its membranous organelle cargo. Previously, we identified six isoforms of the IC74 cytoplasmic dynein subunit in the brain. We further demonstrated that cultured glia and neurons expressed different dynein IC74 isoforms and phospho-isoforms. Two isoforms were observed when dynein from glia was analyzed. When dynein from cultured neurons was analyzed, six IC74 isoforms were observed, although the relative amounts of the dynein isoforms from cultured neurons differed from those found in dynein from brain. To better understand the role of the neuronal IC74 isoforms and identify neuron-specific IC74 dynein subunits, the expression of the IC74 protein isoforms and mRNAs of various tissues were compared. As a result of this comparison, the identity of each of the isoform spots observed on two-dimensional gels was correlated with the products of each of the IC74 mRNAs. We also found that between the fifteenth day of gestation (E15) and the fifth day after birth (P5), the relative expression of the IC74 protein isoforms changes, demonstrating that the expression of IC74 isoforms is developmentally regulated in brain. During this time period, there is relatively little change in the abundance of the various IC74 mRNAs. The E15 to P5 time period is one of rapid process extension and initial pattern formation in the rat brain. This result indicates that the changes in neuronal IC74 isoforms coincide with neuronal differentiation, in particular the extension of processes. This suggests a role for the neuronal IC74 isoforms in the establishment or regulation of retrograde axonal transport.

scholarly journals Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

2011 ◽  
Vol 22 (15) ◽  
pp. 2690-2701 ◽  
Author(s):  
Melissa D. Stuchell-Brereton ◽  
Amanda Siglin ◽  
Jun Li ◽  
Jeffrey K. Moore ◽  
Shubbir Ahmed ◽  
...  
Keyword(s):  
Light Chain ◽  
Direct Evidence ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Dynein Light Chain ◽  
Spindle Positioning ◽  
Dynein Motor ◽  
Intermediate Chains ◽  
Activator Complex

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

scholarly journals Localization of Cytoplasmic Dynein Light-intermediate Chain mRNA in the Rat Testis Using In Situ Hybridization.

1998 ◽  
Vol 23 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Seishi Maeda ◽  
Sang-Yoon Nam ◽  
Masahiko Fujisawa ◽  
Nobuaki Nakamuta ◽  
Kenji Ogawa ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cytoplasmic Dynein ◽  
Rat Testis ◽  
Intermediate Chain

scholarly journals Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA

2015 ◽  
Author(s):  
John Salogiannis ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Coiled Coil ◽  
Leading Edge ◽  
Time Lapse ◽  
Early Endosome ◽  
Organelle Transport ◽  
The Novel ◽  
Coiled Coil Region ◽  
Early Endosomes

Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast to this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we find that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport where peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.

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