Dynein heavy chain 64C
Cloning of Dynein heavy chains Seven Drosophila dynein heavy chain genes have been characterized. Sequence analysis of partial clones reveals that
each encodes a highly conserved portion of the putative hydrolytic ATP-binding site that
includes a consensus phosphate-binding (P-loop) motif. One of the clones is derived from Dhc64C, a Drosophila
cytoplasmic dynein heavy chain gene that shows extensive amino acid identity to cytoplasmic
dynein isoforms found in other organisms. Two other Drosophila dynein clones are 85 and 90% identical at the
amino acid level to the corresponding region of the beta heavy chain of sea urchin axonemal dynein. Probes
for all seven of the dynein-related sequences hybridize to transcripts that are the appropriate size
(approximately 14 kilobases) to encode the characteristic high molecular weight dynein heavy chain
polypeptides. The Dhc64C transcript is readily detected in RNA from ovaries, embryos, and testes.
Transcripts from five of the six remaining genes are also detected in tissues other
than testes, but in much lesser amounts. All but one of the dynein transcripts are expressed at comparable levels in testes, suggesting
their participation in flagellar axoneme assembly and motility (Rassmuson, 1994).
Dhc-Yh3, another of the Drosophila dynein genes, is located in Y chromosome region h3. This region 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 (Gepner, 1993).
To understand the contradiction between megabase-sized lampbrush loops and putative protein encoding genes both associated
with the loci of Y chromosomal fertility genes of Drosophila on the molecular level, PCR-mediated cloning was used to identify and
isolate the cDNA sequence of the Y chromosomal Drosophila hydei gene DhDhc7(Y). Alignment of the sequences of the putative
protein DhDhc7(Y) and the outer arm dynein beta heavy chain protein DYH2 of Tripneustes gratilla shows homology over the
entire length of the protein chains. Therefore the proteins can be assumed to fulfill orthologous functions within the sperm tail
axonemes of both species. Functional dynein beta heavy chain molecules, however, are necessary for the assembly and attachment
of outer dynein arms within the sperm tail axoneme. Localization of DhDhc7(Y) to the fertility factor Threads, comprising at least
5.1 Mb of transcriptionally active repetitive DNA, results from an infertile Threads- mutant where large clusters of Threads
specifically transcribed satellites and parts of DhDhc7(Y) encoding sequences are missing simultaneously. Consequently, the
complete lack of the outer dynein arms in Threads- males most probably causes sperm immotility and hence infertility of the fly.
Moreover, preliminary sequence analysis and several other features support the hypothesis that DhDhc7(Y) on the lampbrush
loops Threads in D. hydei and Dhc-Yh3 on the lampbrush loops kl-5 in Drosophila melanogaster on the heterochromatic Y
chromosome of both species might indeed code for orthologous dynein beta heavy chain proteins (Kurek, 1998).
Dynein structure Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. This study report X-ray and electron microscopy (EM) structures of yeast dynein bound to different ATP analogs, which collectively provide insight into the roles of dynein's two major ATPase sites, AAA1 and AAA3, in the conformational change mechanism. ATP binding to AAA1 triggers a cascade of conformational changes that propagate to all six AAA domains and cause a large movement of the "linker," dynein's mechanical element. In contrast to the role of AAA1 in driving motility, nucleotide transitions in AAA3 gate the transmission of conformational changes between AAA1 and the linker, suggesting that AAA3 acts as a regulatory switch. Further structural and mutational studies also uncover a role for the linker in regulating the catalytic cycle of AAA1. Together, these results reveal how dynein's two major ATP-binding sites initiate a
A 27-kDa protein has been isolated that binds to cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this
polypeptide yielded a sequence which completely matched the predicted sequence of the beta subunit of casein kinase II, a
highly conserved serine/threonine kinase. Affinity chromatography using a dynein column indicates that both the alpha and beta
subunits of casein kinase II are retained by the column from rat brain cytosol. Although dynactin is also bound to the column,
casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it binds to a
dynein intermediate chain column which has been preblocked with excess p150(Glued), a treatment that inhibits the binding of
dynactin from cytosol. Bacterially expressed and purified rat dynein intermediate chain can be phosphorylated by casein kinase
II in vitro. Further, native cytoplasmic dynein purified from rat brain can also be phosphorylated by casein kinase II in vitro.
It is proposed that CKII may be involved in the regulation of dynein function possibly by altering its cargo specificity or its
ability to interact with dynactin (Karki, 1997).
Dynein mutation
The development of characteristic visceral asymmetries along the left-right (LR) axis in an initially bilaterally symmetrical
embryo is an essential feature of vertebrate patterning. The allelic mouse mutations inversus viscerum (iv) and legless (lgl)
produce LR inversion, or situs inversus, in half of live-born homozygotes. This suggests that the iv gene product drives correct
LR determination, and in its absence this process is randomized. These mutations provide tools for studying the development
of LR-handed asymmetry and provide mouse models of human lateralization defects. At the molecular level, the normally LR
asymmetric expression patterns of nodal and lefty are randomized in iv/iv embryos, suggesting that iv functions early in the
genetic hierarchy of LR specification. An axonemal dynein heavy-chain gene,
left/right-dynein (lrd), is mutated in both lgl and iv. lrd is expressed in the node of the embryo at embryonic day 7.5,
consistent with its having a role in LR development. These findings indicate that dynein, a microtubule-based motor, is involved
in the determination of LR-handed asymmetry and provide insight into the early molecular mechanisms of this process (Supp, 1997).
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, mice lacking cDHC were produced by gene targeting. cDHC-/- embryos are indistinguishable from cDHC+/-or
cDHC+/+ littermates at the blastocyst stage. However, no cDHC-/- embryos are found at 8.5 d postcoitum. When cDHC-/-
blastocysts are cultured in vitro, they show interesting phenotypes. First, the Golgi complex becomes highly vesiculated and
distributed throughout the cytoplasm. Second, endosomes and lysosomes are not concentrated near the nucleus but are
distributed evenly throughout the cytoplasm. Interestingly, the Golgi "fragments" and lysosomes are 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 (Harada, 1998).
Most dynein heavy chains
(DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this
isoform, Chlamydomonas cDNA clones encoding a portion of DHC1b were isolated, and these clones were used to identify a
Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi
apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal
body pool to the flagella. Rafts are particles that normally move up and down the flagella in a process known as
intraflagellar transport (IFT), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits
apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT.
Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and
ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably
because it is the motor for retrograde IFT (Pazour, 1999).
Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and
spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal,
severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate
the complete range of dynein function in A. nidulans, synthetic lethal mutations were sought that significantly reduced growth
in the absence of dynein but have little effect on their own. Nineteen sld (synthetic lethality without dynein) mutations were isolated in nine
different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six
other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and,
thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing
drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously
replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from
Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic
genes, indicates that A. nidulans dynein plays a role in mitosis. A model for dynein motor action in A. nidulans is presented that
can explain dynein involvement in both mitosis and nuclear distribution (Efimov, 1998).
Dynein light chains The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. A new gene required for nuclear migration, nudG, is described which encodes a homologue of the "8-kD" cytoplasmic dynein
light chain (CDLC). The temperature sensitive nudG8 mutation inhibits nuclear migration and growth at
restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of
the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally
located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain
(CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex
with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed
with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG
mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates
that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather
than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact (Beckwith, 1998).
Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of
intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, the first non-metazoan dynein light chain 1, SLC1, has been identified in the yeast Saccharomyces cerevisiae. The amino acid sequence of
the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1. The SLC1
gene lies adjacent to the YAP2 (= CAD1) transcription unit. The SLC1 coding sequence is split by two introns and its mRNA is
detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate
that SLC1 function is not essential for cell viability. Furthermore, double mutants, defective for SLC1 and the
kinesin-related CIN8 genes are non-lethal. The redundancy of SLC1 function in yeast contrasts with the cell death caused by
loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster (Dick, 1996a).
To date, much attention has been focused on the heavy and intermediate chains of the multisubunit cytoplasmic dynein
complex; however, little is known about the localization or function of dynein light chains. Tctex-1,
a light chain of cytoplasmic dynein, localizes predominantly to the Golgi apparatus in interphase fibroblasts.
Immunofluorescent staining reveals striking juxtanuclear staining characteristic of the Golgi apparatus as well as nuclear
envelope and punctate cytoplasmic staining that often decorates microtubules. Tctex-1 colocalization with Golgi compartment
markers, its distribution upon treatment with various pharmacological agents, and the cofractionation of Tctex-1-associated
membranes with Golgi membranes are all consistent with a Golgi localization. The distribution of Tctex-1 in interphase cells
only partially overlaps with the dynein intermediate chain and p150(Glued) upon immunofluorescence, but most of Tctex-1 is
redistributed onto mitotic spindles along with other dynein/dynactin subunits. There is a subset of Tctex-1 not associated with the intermediate chain at steady state; the converse also appears
to be true. Distinct populations of dynein complexes are likely to exist, and such diversity may occur in part at the level of their
light chain compositions (Tai, 1998).
Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some
appear to play a critical role in developmentally programmed cell death. The regulation of the proapoptotic
activity of the BH3-only protein Bim was examined. In healthy cells, most Bim molecules are bound to LC8 cytoplasmic dynein light
chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupt the
interaction between LC8 and the dynein motor complex. This frees Bim to translocate together with LC8 to Bcl-2 and to
neutralize its antiapoptotic activity. This process does not require caspase activity and therefore constitutes an initiating event
in apoptosis signaling (Puthalakah, 1999).
Coordinated microtubule and microfilament changes are essential for the
morphological development of neurons; however, little is know about the
underlying molecular machinery linking these two cytoskeletal systems.
Similarly, the indispensable role of RhoGTPase family proteins has been
demonstrated, but it is unknown how their activities are specifically regulated
in different neurites. The cytoplasmic dynein light chain Tctex-1 (Drosophila homolog CG7276) is shown to play a key role in multiple steps of hippocampal neuron
development, including initial neurite sprouting, axon specification, and later
dendritic elaboration. The neuritogenic effects elicited by Tctex-1 are
independent from its cargo adaptor role for dynein motor transport. Finally, the
data suggest that the selective high level of Tctex-1 at the growth cone of
growing axons drives fast neurite extension by modulating actin dynamics and
also Rac1 activity (Chuang, 2005).
A study of migrating fibroblasts suggests that microtubule growth activates Rac1
and hence actin polymerization. The proteins
preferentially associated with the plus-end of growing microtubules thus are
good candidates to regulate Rac1 activity. Tctex-1 is also highly concentrated
at microtubule plus ends. It is thus conceivable that Tctex-1
dissociates from the dynein complex near the microtubule growing end, perhaps
via phosphorylation at Thr94, and locally activates Rac1. The data show that the
ectopically expressed unphosphorylated mimic T94A mutant is distributed in both
the dynein complex and complex-free pools, but it has no axogenic effect. It is
therefore likely that both the phosphorylation at Thr94 and its dissociation
from the dynein complex are required for the 'activation' of Tctex-1 for its
neuritic role (Chuang, 2005 and references therein).
The mitogen-activated protein kinase p38 plays a critical role in inflammation, cell cycle progression, differentiation, and apoptosis. The activity of p38 is stimulated by a variety of extracellular stimuli, such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), and subjected to regulation by other intracellular signaling pathways, including the cyclic AMP (cAMP) pathway. Yet the underlying mechanism by which cAMP inhibits p38 activation is unknown. This study shows that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of p38 activation. cAMP inhibits p38 activation via the protein kinase A-CREB pathway. The inhibition is mediated by the CREB target gene Dlc, whose protein product, DLC, interferes with the formation of the MKK3/6-p38 complex, thereby suppressing p38 phosphorylation activation by MKK3/6. The inhibition of p38 activation by cAMP leads to suppression of NF-kappaB activity and promotion of apoptosis in response to TNF-alpha. Thus, these results identify DLC as a novel inhibitor of the p38 pathway and provide a molecular mechanism by which cAMP suppresses p38 activation and promotes apoptosis (Zhang, 2006).
Dynein intermediate chains Cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying
polypeptides of unknown function. The most prominent of these is a 74-kD electrophoretic species which can be resolved as
two to three bands by SDS-PAGE. A series of overlapping rat brain cDNAs encoding the 74-kD species were selected.
The deduced sequence of a full-length cDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine
peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain cDNA together with the
sequence of a partially matching tryptic peptide indicate the existence of at least three isoforms of the 74-kD cytoplasmic
dynein subunit. Comparison with known sequences reveals that the carboxyl-terminal half of the polypeptide is 26.4%
identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70-kD intermediate chain of flagellar outer
arm dynein. Monoclonal antibody indicates a widespread tissue distribution,
as expected for a cytoplasmic dynein subunit. Nonetheless, the antibody recognizes a 67-kD species in ram sperm flagella and
pig tracheal cilia, supporting the existence of distinct but related cytoplasmic and axonemal polypeptides in mammals. In view
of evidence for a role for the ODA6 gene product in anchoring flagellar dynein to the A subfiber microtubule in the axoneme, an analogous role for the 74-kD polypeptide, perhaps in mediating the interaction of cytoplasmic dynein with
membranous organelles and kinetochores, is predicted (Paschal, 1992).
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. Monoclonal antibodies to ICs map the ICs to the base of the motor. Because the ICs have been
implicated in targeting the motor to cargo, a test was performed to see whether antibodies to the intermediate chain can block the
function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes are incubated with either one of the monoclonal
antibodies (m74-1, m74-2), neither organelle movement nor network formation is observed. Network formation and membrane
transport is blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect is
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 is no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of
cytoplasmic dynein from the membrane. A panel of dynein IC truncation mutants were used and the epitopes of both
antibodies were mapped to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column
binding assay, both antibodies inhibit the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued
interaction is required for the proper attachment of cytoplasmic dynein to membranes (Steffen, 1998).
Membrane proteins in growth cone-enriched and growth cone-non-enriched fractions prepared from neonatal mouse brain were
separated by lectin-affinity and ion exchange chromatographies, 2D-PAGE, and SDS-PAGE. Partial amino acid sequences of
the proteins concentrated in the growth cone-enriched fraction were determined. One such protein, gmp23-48k,
corresponds to the 50-kDa subunit (p50) of the dynactin complex. An antibody raised against gmp23-48k strongly reacts with
growth cones of differentiated neuronal precursor cells. Immunoblot analyses reveals that gmp23-48k was present both in
membrane and in soluble fractions of neonatal brain. However, the amount of gmp23-48k in the membrane fraction greatly
decreases in adult brain. These results suggest a special role of membrane-associated gmp23-48k/p50 in synapse formation
during brain development (Abe, 1997).
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Dynein heavy chain 64C:
Biological Overview
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| Developmental Biology
| Effects of Mutation
| References
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