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 Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

2003 ◽  
Vol 14 (12) ◽  
pp. 5089-5097 ◽  
Author(s):  
Stephen J. King ◽  
Christa L. Brown ◽  
Kerstin C. Maier ◽  
Nicholas J. Quintyne ◽  
Trina A. Schroer
Keyword(s):  
Cytoplasmic Dynein ◽  
Binding Assays ◽  
Microtubule Organization ◽  
Binding Domains ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Dynein Intermediate Chain ◽  
Transient Overexpression

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

scholarly journals Specific enzymatic dephosphorylation of the retinoblastoma protein.

1993 ◽  
Vol 13 (1) ◽  
pp. 367-372 ◽  
Author(s):  
J W Ludlow ◽  
C L Glendening ◽  
D M Livingston ◽  
J A DeCarprio
Keyword(s):  
Phosphatase Activity ◽  
Large Population ◽  
Mitotic Cell ◽  
Phosphatase Inhibitors ◽  
Cell Extracts ◽  
Protein Phosphatase Inhibitors ◽  
Cycle Dependent

The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.

scholarly journals Lissencephaly-1 is a context-dependent regulator of the human dynein complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Janina Baumbach ◽  
Andal Murthy ◽  
Mark A McClintock ◽  
Carly I Dix ◽  
Ruta Zalyte ◽  
...  
Keyword(s):  
Complex Formation ◽  
Binding Protein ◽  
Cytoplasmic Dynein ◽  
Cargo Transport ◽  
Dynein Motor ◽  
Context Dependent ◽  
Shed Light ◽  
Dynamic Microtubule

The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.

C-terminal domain phosphorylation of ERK3 controlled by Cdk1 and Cdc14 regulates its stability in mitosis

2010 ◽  
Vol 428 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Pierre-Luc Tanguay ◽  
Geneviève Rodier ◽  
Sylvain Meloche
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mitotic Cell ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Cycle Dependent

ERK3 (extracellular-signal-regulated kinase 3) is an atypical MAPK (mitogen-activated protein kinase) that is suggested to play a role in cell-cycle progression and cellular differentiation. However, it is not known whether the function of ERK3 is regulated during the cell cycle. In the present paper, we report that ERK3 is stoichiometrically hyperphosphorylated during entry into mitosis and is dephosphorylated at the M→G1 transition. The phosphorylation of ERK3 is associated with the accumulation of the protein in mitosis. In vitro phosphorylation of a series of ERK3-deletion mutants by mitotic cell extracts revealed that phosphorylation is confined to the unique C-terminal extension of the protein. Using MS analysis, we identified four novel phosphorylation sites, Ser684, Ser688, Thr698 and Ser705, located at the extreme C-terminus of ERK3. All four sites are followed by a proline residue. We have shown that purified cyclin B-Cdk1 (cyclindependent kinase 1) phosphorylates these sites in vitro and demonstrate that Cdk1 acts as a major Thr698 kinase in vivo. Reciprocally, we found that the phosphatases Cdc14A and Cdc14B (Cdc is cell-division cycle) bind to ERK3 and reverse its C-terminal phosphorylation in mitosis. Importantly, alanine substitution of the four C-terminal phosphorylation sites markedly decreased the half-life of ERK3 in mitosis, thereby linking phosphorylation to the stabilization of the kinase. The results of the present study identify a novel regulatory mechanism of ERK3 that operates in a cell-cycle-dependent manner.

scholarly journals Specific enzymatic dephosphorylation of the retinoblastoma protein

1993 ◽  
Vol 13 (1) ◽  
pp. 367-372
Author(s):  
J W Ludlow ◽  
C L Glendening ◽  
D M Livingston ◽  
J A DeCarprio
Keyword(s):  
Phosphatase Activity ◽  
Large Population ◽  
Mitotic Cell ◽  
Phosphatase Inhibitors ◽  
Cell Extracts ◽  
Protein Phosphatase Inhibitors ◽  
Cycle Dependent

The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.

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 Complete Loss of Ndel1 Results in Neuronal Migration Defects and Early Embryonic Lethality

2005 ◽  
Vol 25 (17) ◽  
pp. 7812-7827 ◽  
Author(s):  
Shinji Sasaki ◽  
Daisuke Mori ◽  
Kazuhito Toyo-oka ◽  
Amy Chen ◽  
Lisa Garrett-Beal ◽  
...  
Keyword(s):  
Neuronal Migration ◽  
Granule Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Protein Product ◽  
Microtubule Organization ◽  
Hypomorphic Alleles

ABSTRACT Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1 −/− mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1 −/− blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1 +/− mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1 cko/− ) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1 cko/+ -Ndel1 +/− mice or Lis1 +/− -Ndel1 +/− mice displayed more severe neuronal migration defects than Lis1 cko/+ -Ndel1 +/ + mice or Lis1 +/− -Ndel1 +/+ mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of β-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.

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.

Overexpression of normal and mutant Arp1alpha (centractin) differentially affects microtubule organization during mitosis and interphase

1999 ◽  
Vol 112 (20) ◽  
pp. 3507-3518 ◽  
Author(s):  
I.B. Clark ◽  
D.I. Meyer
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Mutant Proteins ◽  
Cell Extracts ◽  
Dynactin Complex

Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin's most abundant component, Arp1alpha was transiently overexpressed in mammalian cells. Arp1alpha overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1alpha in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1alpha that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1alpha proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1alpha, NuMA localization remained unaffected. Although expression of mutant Arp1alpha proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1alpha interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1alpha mutants. We conclude that the amino acids altered in the Arp1alpha mutant proteins participate in stabilizing interactions between overexpressed Arp1alpha and components of the endogenous dynactin complex as well as the NuMA protein.

scholarly journals Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.

1995 ◽  
Vol 131 (2) ◽  
pp. 411-425 ◽  
Author(s):  
M McGrail ◽  
J Gepner ◽  
A Silvanovich ◽  
S Ludmann ◽  
M Serr ◽  
...  
Keyword(s):  
Temporal Distribution ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Gene Products ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Dynein Motor ◽  
Associated Proteins

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.

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