scholarly journals Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein.

1991 ◽  
Vol 115 (6) ◽  
pp. 1639-1650 ◽  
Author(s):  
S R Gill ◽  
T A Schroer ◽  
I Szilak ◽  
E R Steuer ◽  
M P Sheetz ◽  
...  
Keyword(s):  
Single Gene ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Protein Product ◽  
Structural Motif ◽  
Sequence Identity ◽  
Vesicle Movement ◽  
Dynactin Complex ◽  
Activator Complex

Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.

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 Dynactin Is Required for Microtubule Anchoring at Centrosomes

1999 ◽  
Vol 147 (2) ◽  
pp. 321-334 ◽  
Author(s):  
N.J. Quintyne ◽  
S.R. Gill ◽  
D.M. Eckley ◽  
C.L. Crego ◽  
D.A. Compton ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Cultured Fibroblasts ◽  
Cell Extracts ◽  
Dynein Motor ◽  
Mitotic Spindle Assembly ◽  
Dynactin Complex

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein–dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein–dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome–lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, γ tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin–cargo interactions.

scholarly journals Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules.

1996 ◽  
Vol 135 (2) ◽  
pp. 383-397 ◽  
Author(s):  
V Muresan ◽  
C P Godek ◽  
T S Reese ◽  
B J Schnapp
Keyword(s):  
Lipid Bilayers ◽  
Affinity Purification ◽  
Selective Extraction ◽  
Cytoplasmic Dynein ◽  
Isolated Populations ◽  
Kinesin Motor ◽  
Weakly Bound ◽  
Direction Of Movement ◽  
Vesicle Movement

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)-induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.

scholarly journals Functionally distinct isoforms of dynactin are expressed in human neurons.

1996 ◽  
Vol 7 (8) ◽  
pp. 1167-1180 ◽  
Author(s):  
M K Tokito ◽  
D S Howland ◽  
V M Lee ◽  
E L Holzbaur
Keyword(s):  
Cytoplasmic Dynein ◽  
Binding Motif ◽  
Binding Assays ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Human Neurons ◽  
Alternative Mrna Splicing ◽  
Associated Proteins ◽  
Dynactin Complex

P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.

scholarly journals The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

2017 ◽  
Author(s):  
Kuo-Fu Tseng ◽  
Pan Wang ◽  
Yuh-Ru Julie Lee ◽  
Joel Bowen ◽  
Allison M. Gicking ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Preprophase Band ◽  
Cytoplasmic Dynein ◽  
Coiled Coils ◽  
Land Plants ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microtubule Motor

AbstractIn animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

scholarly journals A new calcium-activated dynein adaptor protein, CRACR2a, regulates clathrin-independent endocytic traffic in T cells

2018 ◽  
Author(s):  
Yuxiao Wang ◽  
Walter Huynh ◽  
Taylor D. Skokan ◽  
Ronald D. Vale
Keyword(s):  
T Cells ◽  
Coiled Coil ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Cytoplasmic Dynein ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Organizing Center ◽  
Activated T Cells

AbstractCytoplasmic dynein is a microtubule minus-end-directed motor that transports numerous intracellular cargoes. Mammalian dynein transport is initiated by coiled-coil adaptor proteins that 1) join dynein and its co-factor dynactin into a complex capable of processive motility, and 2) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, which have a coiled-coil adaptor domain, a pair of EF hands, and a Rab GTPase domain fused into a single polypeptide. We find that CRACR2a-mediated dynein-dynactin motility is activated by calcium in vitro and in cells. In activated T cells, CRACR2a localizes to clathrin-independent endosomes that require microtubule-based transport to detach from the actin cortex and travel towards the microtubule organizing center. Together these results represent the first known examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a-dynein in regulation of endocytic trafficking in T cells.

scholarly journals BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

2012 ◽  
Vol 23 (21) ◽  
pp. 4226-4241 ◽  
Author(s):  
Daniël Splinter ◽  
David S. Razafsky ◽  
Max A. Schlager ◽  
Andrea Serra-Marques ◽  
Ilya Grigoriev ◽  
...  
Keyword(s):  
Cytoplasmic Dynein ◽  
Cellular Structures ◽  
Live Cells ◽  
Concerted Action ◽  
Membrane Tethering ◽  
Directed Motility ◽  
Cellular Motor ◽  
Dynactin Complex

Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.

scholarly journals Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi.

1994 ◽  
Vol 127 (1) ◽  
pp. 139-149 ◽  
Author(s):  
M Plamann ◽  
P F Minke ◽  
J H Tinsley ◽  
K S Bruno
Keyword(s):  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Related Protein ◽  
Nuclear Distribution ◽  
Spindle Pole Bodies ◽  
Fungal Hyphae ◽  
Genes Encoding ◽  
Dynactin Complex

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. We report here that 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. We propose that cytoplasmic dynein maintains nuclear distribution through sliding of antiparallel microtubules emanating from neighboring spindle pole bodies.

scholarly journals The Yeast Dynactin Complex Is Involved in Partitioning the Mitotic Spindle between Mother and Daughter Cells during Anaphase B

1998 ◽  
Vol 9 (7) ◽  
pp. 1741-1756 ◽  
Author(s):  
Jason A. Kahana ◽  
Gabriel Schlenstedt ◽  
Darren M. Evanchuk ◽  
John R. Geiser ◽  
M. Andrew Hoyt ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Cytoplasmic Dynein ◽  
Stable Complex ◽  
Complex Protein ◽  
Mother And Daughter ◽  
Green Fluorescent ◽  
Dynactin Complex

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene,NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150 glued . Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Δ strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

scholarly journals Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis.

1996 ◽  
Vol 132 (4) ◽  
pp. 617-633 ◽  
Author(s):  
C J Echeverri ◽  
B M Paschal ◽  
K T Vaughan ◽  
R B Vallee
Keyword(s):  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Cdna Clones ◽  
Sedimentation Analysis ◽  
Spindle Poles ◽  
Chromosome Alignment ◽  
Dynactin Complex

Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and analyzed the effects of its overexpression on mitosis 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 showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was 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.

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