Dynein heavy chain 64C
Dynactin - a link between Dynein and its cargo and/or the substratum:
Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of
action is unknown. The 50-kD subunit of dynactin (dynamitin) has been characterized, the effects of its
overexpression on mitosis has been analyzed in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved,
containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein
components in cultured vertebrate cells shows that both human and rat complexes are recruited to kinetochores during prometaphase, and
concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupts mitosis, causing cells to
accumulate in a prometaphase-like state. Chromosomes are condensed but unaligned, and spindles, while still bipolar,
are dramatically distorted. Sedimentation analysis reveals the dynactin complex to be dissociated in the transfected
cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores is markedly
diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic
dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo
evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both
dynactin and dynein in chromosome alignment and spindle organization (Echeverri, 1996).
Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian
cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from
prometaphase kinetochores, with consequent perturbation of mitosis. Based on these results, dynactin has been proposed to play a role in linking cytoplasmic
dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase
phenotype. In dynamitin-overexpressing cells, early endosomes , as well as late
endosomes and lysosomes, are redistributed to the
cell periphery. This redistribution is disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor
activity. The Golgi stack is
dramatically disrupted into scattered structures that colocalize with components of the intermediate compartment . The disrupted Golgi elements represent short stacks similar to those formed by
microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A are not inhibited by
dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment
reveals that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather,
remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing
centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of
dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role
for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that
dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding (Burkhardt, 1997).
Dynactin - a link between Dynein and its cargo and/or the substratum:
The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic
dynein motor machinery. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to
conventional actin and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize.
In vivo, all cytosolic Arp1 sediments at 20S suggesting that it assembles into oligomers, most likely dynactin - a multiprotein
complex known to contain eight or nine Arp1 monomers in a 37 nm filament. The uniform length of Arp1 polymers suggests a
novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping
protein at one end and a heterotetrameric protein complex containing the p62 subunit at the other. In the present study, the behavior of highly purified,
native Arp1 was analyzed. Arp1 polymerizes rapidly into short filaments that are similar, but not identical, in length to those in
dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional
actin filaments (Bingham, 1999).
Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the
protein, transient transfections were used to overexpress centractin in mammalian cells. The overexpressed
polypeptide forms filamentous structures that are significantly longer and more variable in length than those observed in the
native dynactin complex. The centractin filaments are distinct from conventional actin in subunit composition and pharmacology
as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment
with the drug cytochalasin D, and by the inability to bind phalloidin. The transfected cells were examined for evidence of specific
associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry the colocalization of Golgi marker proteins with the centractin polymers were observed. Additional immunocytochemical analysis using
antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) reveals that spectrin colocalizes with the
centractin filaments in transfected cells. Spectrin was present in dynactin-enriched cellular
fractions, is coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and is coeluted with dynactin
using affinity chromatography. Immunoprecipitations and affinity chromatography also reveal that actin is not a bona fide
component of dynactin. These results indicate that spectrin is associated with the dynactin complex. A model is suggested in which
dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a
spectrin-linked cytoskeletal network (Holleran, 1996).
Cytoplasmic dynein is a multisubunit, microtubule-dependent mechanochemical enzyme that has been proposed to function in a
variety of intracellular movements, including minus-end-directed transport of organelles. Dynein-mediated vesicle transport is
stimulated in vitro by addition of the Glued/dynactin complex raising the possibility that these two complexes interact in vivo. A class of phenotypically identical mutants of the filamentous fungus Neurospora crassa are defective in genes
encoding subunits of either cytoplasmic dynein or the Glued/dynactin complex. These mutants, defined as ropy, have curled
hyphae with abnormal nuclear distribution. ro-1 encodes the heavy chain of cytoplasmic dynein, while ro-4 encodes an
actin-related protein that is a probable homologue of the actin-related protein Arpl (formerly referred to as actin-RPV or centractin),
the major component of the glued/dynactin complex. The phenotypes of ro-1 and ro-4 mutants suggest that cytoplasmic dynein, as
well as the Glued/dynactin complex, are required to maintain uniform nuclear distribution in fungal hyphae. It is proposed that
cytoplasmic dynein maintains nuclear distribution through sliding of antiparallel microtubules emanating from neighboring spindle
pole bodies (Plamann, 1994).
Spindle orientation controls nuclear migration and segregation during mitosis. In yeast, defects in dynein and astral microtubules
lead to abnormal spindle orientation and nuclear migration. Dynactin complex is necessary for dynein-mediated vesicle motility in
vitro. The major polypeptide of dynactin complex is an actin-related protein in the family Arp1. In S. cerevisiae
a novel actin-related gene, ACT5, in the Arp1 family, has been identified. An act5 null mutant has defects in spindle orientation and nuclear migration,
as does overexpression of Act5p. The phenotype of a double mutant lacking dynein and Act5p is similar to that of single mutants.
Therefore, dynactin complex is in the same pathway as dynein and may be necessary for the action of dynein in vivo (Muhua, 1994).
Cytoplasmic dynein is a minus-end-directed microtubule
motor that participates in multiple cellular activities such
as organelle transport and mitotic spindle assembly.
To study the dynamic behavior of cytoplasmic dynein in
the filamentous fungus Aspergillus nidulans, the gene for the cytoplasmic dynein heavy
chain, nudA, was replaced with a gene encoding a green fluorescent
protein (GFP)-tagged chimera, GFP-nudA. The
GFP-NUDA fusion protein is fully functional in vivo:
strains expressing only the GFP-tagged nudA grow as
well as wild-type strains. Fluorescence microscopy
shows GFP-NUDA in comet-like structures that
move in the hyphae toward the growing tip. Retrograde
movement of some GFP-NUDA comets after they arrive
at the tip is also observed. These dynamics of
GFP-NUDA are not observed in cells treated with a
microtubule-destabilizing drug, benomyl, suggesting
they are microtubule-dependent. The rate of GFP-NUDA
tip-ward movement is similar to the rate of cytoplasmic
microtubule polymerization toward the hyphal tip,
suggesting that GFP-NUDA is associated and moving
with the polymerizing ends of microtubules. A mutation
in actin-related protein Arp1 of the dynactin complex
abolishes the presence of these dynamic GFP-NUDA
structures near the hyphal tip, suggesting a targeting
role of the dynactin complex (Xiang, 2000).
In filamentous fungi, genes encoding proteins in cytoplasmic dynein were originally identified as genes
required for nuclear distribution along the hyphae.
Nuclear migration may be mediated by interactions
between the hyphal cortex and astral microtubules from
the nuclear spindle pole body. In the budding yeast
S. cerevisiae, cytoplasmic dynein involved in spindle orientation is found along the entire length of astral microtubules, and a dynein heavy chain mutant shows
altered microtubule - cortex interactions and microtubule
dynamics.It is possible that the function of the proteins at the microtubule ends in A. nidulans is to interact
with the cortex and/or affect microtubule dynamics. In
mammalian cells, in addition to the CLIP-170-dynactin-dynein localization to the microtubule ends, the
adenomatous polyposis coli tumor suppressor protein
(APC) and its binding partner EB1 also locate to distal
ends of microtubules. EB1 has been shown to
interact with components of the dynein and dynactin
complexes, and a yeast homolog of EB1, Bim1p, also
locates at the distal ends of microtubules, and promotes
microtubule dynamics in G1 phase. It will be interesting to see whether cytoplasmic dynein in A.nidulans is
also involved in regulating microtubule dynamics for efficient retrograde transport and/or for other cellular
processes such as nuclear migration (Xiang, 2000).
Dynactin - a link between Dynein and its cargo and/or the substratum:
Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and
its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a
dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to
clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is
novel. p22 has been characterized as an integral component of dynactin by biochemical and immunocytochemical methods.
Affinity chromatography experiments indicate that p22 binds directly to the p150(Glued) subunit of dynactin.
Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to
the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to
other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing
cells; cytoplasmic dynein is also localized to these structures. Dynein/dynactin complexes may have a
novel function during cytokinesis (Karki, 1998).
A CLIP-170 dynactin subunit homolog in Drosophila
Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of
investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for
cellular organization and function. Molecules that may mediate coordination between the actin and microtubule
cytoskeletons are reported here. A 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F
unconventional myosin is described. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue
identified of cytoplasmic linker protein CLIP-170, a protein that links endocytic vesicles to microtubules. This
protein has been named D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment
with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they
have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a
number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same
particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this
localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein
in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important,
leads to the speculation that these two proteins may functionally link the actin and microtubule cytoskeletons (Lantz, 1998).
Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. A mammalian homolog of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein-dynactin interaction on the surface of membranous organelles, by associating with these complexes (Hoogenraad, 2001).
Bicaudal D is an evolutionarily conserved protein that is involved in dynein-mediated motility both in Drosophila and in mammals. The N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. The BICD2 N-terminal domain has been used as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. These findings provide a novel model system for dissection of the molecular mechanism of dynein motility (Hoogenraad, 2003).
In mammals, two homologs of Bicaudal D, BICD1 and BICD2, are present. Studies in cultured mammalian cells have shown that BICD proteins bind to the small GTPase Rab6, as well as to dynein and dynactin complexes, and therefore participate in recruitment of dynein motor to Rab6-positive membranes of the Golgi apparatus and cytoplasmic vesicles. However, in addition to BICD proteins, Rab6 GTPase can also interact directly with the p150Glued component of the dynactin complex. This raises the possibility that BICD acts as an accessory factor for the dynein motor, but is not sufficient by itself to recruit it to organelles (Hoogenraad, 2003 and references therein).
BICD proteins consist of several coiled-coil domains, and previous studies have demonstrated that while the C-terminal domain is responsible for interaction with membranes via Rab6, the N-terminal domain binds to cytoplasmic dynein. In addition, the N- and C-terminal domains of BICD can interact with each other. Based on these findings, it is proposed that when BICD binds to the cargo (cytoplasmic vesicle) via its C-terminal domain, the N-terminal domain of BICD2 becomes available for interaction with dynein motor, which, in its turn, would transport the vesicle. If this model is correct, tethering of the BICD N-terminus to membranous organelles, which are normally devoid of BICD (such as mitochondria or peroxisomes), should be sufficient to induce their transport by cytoplasmic dynein. In this study, this idea is tested, and it is shown that the N-terminal part of BICD2 protein is indeed a potent recruitment factor for dynein, and that it can act in different molecular contexts (Hoogenraad, 2003).
Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport. Dynactin can bind to vesicles and liposomes containing acidic phospholipids, but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion. In particular, Rab27a tethers melanosomes to the actin cytoskeleton. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule-dependent transport pathways through the Golgi and from endosomes to the Golgi. Dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes (Short, 2002).
Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, it has been demonstrated that mammalian LIS1 regulates dynein functions. NUDEL is a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. These findings point to cross-talk between two prominent pathways that regulate neuronal migration (Niethammer, 2000).
Mutations in mammalian Lis1 result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion (Sasaki, 2000).
Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), it was found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning (Shu, 2004).
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 unanswered. 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 (Liang, 2004).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
(4) Dynamitin subunit of Dynactin
(5) Arp1 subunit of Dynactin
(6) p22, the smallest dynactin subunit
Dynein heavy chain 64C:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.