Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.
Figure 1. CD-IC and p150Glued are cleaved during apoptosis. (A) Cytosols were made from untreated human HL60 cells (C), cells treated with anisomycin (A; 5 μg/ml) for 4 h, and cells treated with anisomycin and the cell-permeable capsase inhibitor zVAD.FMK (Z). Equal amounts of protein were analyzed by silver staining (left) and immunoblotting. The positions of full-length β-COP and p50 are shown (<). (B) HL60 cells were treated with 5 μg/ml anisomycin or 50 μM etoposide. Cell extracts prepared at the indicated times were immunoblotted using the antibodies shown. Samples treated for the maximum times with each agent in the presence of zVAD.FMK are also shown (Z). (C) A Xenopus egg extract was incubated with 10 μM cytochrome c ± the caspase inhibitor Ac-DEVD-CHO for the times indicated. Extracts were then analyzed by immunoblotting. In all panels, the antiâCD-IC antibody used was 70.1.
Figure 2. Cleavage of CD-IC takes place within its NH2-terminal p150Glued binding region. (A) Schematic diagram of cytoplasmic dynein and dynactin, showing the interaction between the NH2 terminus of CD-IC and p150Glued. (B) Representation of CD-IC showing the regions that interact with other components of the cytoplasmic dynein and dynactin complexes. (C) Immunoblot of a Xenopus egg extract that had been treated for the times indicated (in hours) with cytochrome c ± Ac-DEVD-CHO using monoclonal (70.1) and polyclonal antibodies to CD-IC. The positions of molecular weight markers are also shown.
Figure 3. Amino acid sequence of Xenopus laevis (Xl) CD-IC aligned with the NH2 terminus of rat (Rn) CD-IC1, 2A, 2B, and 2C. The locations of several potential caspase cleavage motifs are highlighted. In each of these, the crucial aspartic acid residue at position P1 (P4P3P2P1) of Xenopus CD-IC was altered by site-directed mutagenesis. The peptide sequence used for polyclonal antibody production is underlined.
Figure 4. Cleavage of Xenopus CD-IC in vitro occurs at DSGD99 and at another, unidentified site. (A) Immunoprecipitated [35S]methionine-labeled wild-type or mutated (DSGA99) CD-IC was incubated in apoptotic Xenopus egg cytosol for the times shown, with or without Ac-DEVD-CHO or the caspase 3 and 7 inhibitor, Casputinâ¢. Two cleavage products, C1 and C2, are indicated. Note that appearance of C1 is prevented by Casputin⢠and does not occur using CD-IC (DSGA99). (BâD) Immunoprecipitated [35S]methionine-labeled wild-type or mutated (DSGA99) CD-IC was incubated for 2 h at 30°C with recombinant caspases 2 (B), 3 (C), and 7 (D). Caspase 2 activity was confirmed by assessing cleavage of in vitroâtranslated caspase 2, whilst caspases 3 and 7 were tested using in vitroâtranslated PARP. (E) Native Xenopus egg cytoplasmic dyneinâdynactin was incubated with recombinant caspase 2 (50 nM) or caspase 3 (20 nM), ± Ac-DEVD-CHO for 3 h at 37°C. Proteins were analyzed after SDS-PAGE by immunoblotting (CD-IC was detected using polyclonal antiserum).
Figure 6. The cytoplasmic dynein complex is released from membranes during apoptosis in Xenopus egg extracts. Cytosol (Cyt) and membranes (Mem) were prepared from control or apoptotic extracts (C and A, respectively). Equal amounts of protein were loaded on SDS-PAGE gels, silver stained for total protein (A) and immunoblotted using the antibodies shown (B).
Figure 5. The fate of cytoplasmic dynein and dynactin complexes in apoptotic extracts. (A) Microtubule motors were isolated from control or apoptotic Xenopus egg cytosol by microtubule affinity, then separated on sucrose density gradients and analyzed by silver staining and immunoblotting (CD-IC was detected using 70.1). CD-HC was not seen in fraction 1 of the apoptotic gradient: the silver staining in that lane is artefactual. (B) The cytoplasmic dynein complex was immunoprecipitated from control (C) and apoptotic (A) egg cytosols using antiâCD-LIC antibody, and immunoblotted with polyclonal antiâCD-IC peptide antibody to detect cleaved (<<) and intact (<) CD-IC. (C) Control (C; lane 1) and apoptotic (A; lane 2) egg cytosols were depleted of any cytoplasmic dynein complex containing uncleaved CD-IC by three rounds of depletion using the monoclonal antibody, 1618, against the NH2-terminus of CD-IC (beads from only rounds 1 and 3 are shown; lanes 3â6). The depleted cytosols (lanes 7 and 8) were then subjected to a further immunoprecipitation with antiâCD-LIC beads (lanes 9 and 10). All fractions were examined by silver stain for DHC (top) and immunoblotted with antiâCD-IC peptide antibody to detect cleaved (<<) and uncleaved (<) CD-IC (bottom).
Figure 7. Motile properties of apoptotic and nonapoptotic membranes. (A) Membranes were prepared by flotation from control and apoptotic Xenopus egg extracts, then incubated in their respective cytosols for 45 min. They were then assayed for motility using a three way junction index (see Materials and Methods). (B) Control or apoptotic cytosols and membranes were mixed as indicated, and assayed for motility as above. Values are means ± SEM for three independent experiments. In each case, Ac-DEVD-CHO was added to a final concentration of 2 μM before motility assays. Representative VE-DIC fields (27 μm across) are also shown for each assay.
Figure 9. Addition of purified pig brain cytoplasmic dyneinâdynactin restores ER network formation. (A) Membranes were prepared by flotation from apoptotic egg extracts, and incubated for 45 min in apoptotic cytosol in the presence or absence of pig brain cytoplasmic dyneinâdynactin (CD). To avoid apoptotic cleavage of the added motor components, 2 μM Ac-DEVD-CHO was included in the motility assays. Values are means ± SEM for three independent experiments. (B) Total samples from a pig brain dyneinâdynactin add-back experiment were immunoblotted using the antibodies shown (C, control cytosol and membranes; A, apoptotic cytosol and membranes). AntiâCD-IC antibodies: i, 70.1; ii, anti-Xenopus CD-IC peptide antibody; iii, Xenopus-specific NH2-terminal antibody. The position of the added pig brain motor components (â) and the Xenopus p150Glued and CD-IC cleavage products (3) are shown. In contrast to Fig. 8, total samples, not re-isolated membranes, were analyzed.
Figure 8. Apoptotic membranes can recruit intact cytoplasmic dynein and dynactin. Membranes from control (C) or apoptotic (A) egg extracts were incubated in control or apoptotic cytosol for 1 h. Membranes were then recovered and equal protein samples were run on SDS-PAGE and immunoblotted (antiribophorin was used as a loading control).
Figure 10. Diagram showing the predicted effects of CD-IC and p150Glued cleavage on cytoplasmic dyneinâdriven organelle motility. Cleavage leads to the removal of the p150Glued-binding domain of CD-IC, rendering cytoplasmic dynein incapable of binding to, and hence moving, its cargoes. Cleavage of p150Glued, which occurs more slowly, removes the microtubule-binding capacity of the dynactin complex, which may ensure that cargoes cannot associate with microtubules. Our results suggest that p50 dynamitin is also partially released from cargo (not shown in model).
Addinall,
Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes.
2001, Pubmed,
Xenbase
Addinall,
Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes.
2001,
Pubmed
,
Xenbase
Allan,
Protein phosphatase 1 regulates the cytoplasmic dynein-driven formation of endoplasmic reticulum networks in vitro.
1995,
Pubmed
,
Xenbase
Allan,
Motor proteins: a dynamic duo.
1996,
Pubmed
Allan,
Organelle motility and membrane network formation in metaphase and interphase cell-free extracts.
1998,
Pubmed
,
Xenbase
Allan,
Cell cycle control of microtubule-based membrane transport and tubule formation in vitro.
1991,
Pubmed
,
Xenbase
Balch,
Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine.
1984,
Pubmed
Blocker,
Molecular requirements for bi-directional movement of phagosomes along microtubules.
1997,
Pubmed
Brancolini,
Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases.
1995,
Pubmed
Burkhardt,
Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.
1997,
Pubmed
Caulín,
Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis.
1997,
Pubmed
Chang,
Cyclooxygenase 2 promotes cell survival by stimulation of dynein light chain expression and inhibition of neuronal nitric oxide synthase activity.
2000,
Pubmed
Cohen,
Caspases: the executioners of apoptosis.
1997,
Pubmed
Cosulich,
Bcl-2 regulates activation of apoptotic proteases in a cell-free system.
1996,
Pubmed
,
Xenbase
Cosulich,
Cleavage of rabaptin-5 blocks endosome fusion during apoptosis.
1997,
Pubmed
,
Xenbase
Desagher,
Mitochondria as the central control point of apoptosis.
2000,
Pubmed
Devitt,
Human CD14 mediates recognition and phagocytosis of apoptotic cells.
1998,
Pubmed
De Vos,
Tumor necrosis factor induces hyperphosphorylation of kinesin light chain and inhibits kinesin-mediated transport of mitochondria.
2000,
Pubmed
De Vos,
The 55-kDa tumor necrosis factor receptor induces clustering of mitochondria through its membrane-proximal region.
1998,
Pubmed
Echeverri,
Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis.
1996,
Pubmed
Fadok,
A receptor for phosphatidylserine-specific clearance of apoptotic cells.
2000,
Pubmed
Gill,
Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein.
1991,
Pubmed
Hamon,
ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine.
2000,
Pubmed
Harada,
Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic dynein.
1998,
Pubmed
Hollenbeck,
Kinesin delivers: identifying receptors for motor proteins.
2001,
Pubmed
Karki,
Cytoplasmic dynein and dynactin in cell division and intracellular transport.
1999,
Pubmed
Karki,
Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex.
1995,
Pubmed
King,
The dynein microtubule motor.
2000,
Pubmed
King,
Dynactin increases the processivity of the cytoplasmic dynein motor.
2000,
Pubmed
Kluck,
Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.
1997,
Pubmed
,
Xenbase
Lane,
Microtubule-based endoplasmic reticulum motility in Xenopus laevis: activation of membrane-associated kinesin during development.
1999,
Pubmed
,
Xenbase
Lin,
Cytoplasmic dynein undergoes intracellular redistribution concomitant with phosphorylation of the heavy chain in response to serum starvation and okadaic acid.
1994,
Pubmed
Liu,
Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.
1996,
Pubmed
Machleidt,
Caspase 7-induced cleavage of kinectin in apoptotic cells.
1998,
Pubmed
Mancini,
Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis.
2000,
Pubmed
Martin,
Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.
1995,
Pubmed
McGrail,
Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.
1995,
Pubmed
Mills,
Extranuclear apoptosis. The role of the cytoplasm in the execution phase.
1999,
Pubmed
Muresan,
Dynactin-dependent, dynein-driven vesicle transport in the absence of membrane proteins: a role for spectrin and acidic phospholipids.
2001,
Pubmed
Murray,
Cell cycle extracts.
1991,
Pubmed
Newmeyer,
Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria.
1994,
Pubmed
,
Xenbase
Niclas,
Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.
1996,
Pubmed
,
Xenbase
Nicolás,
Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts.
1997,
Pubmed
,
Xenbase
O'Connor,
Bim: a novel member of the Bcl-2 family that promotes apoptosis.
1998,
Pubmed
Parnaik,
Differences between the clearance of apoptotic cells by professional and non-professional phagocytes.
2000,
Pubmed
Platt,
Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro.
1996,
Pubmed
Presley,
ER-to-Golgi transport visualized in living cells.
1997,
Pubmed
Puthalakath,
The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex.
1999,
Pubmed
Quintyne,
Dynactin is required for microtubule anchoring at centrosomes.
1999,
Pubmed
Ren,
CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis.
1995,
Pubmed
Roghi,
Dynamic association of cytoplasmic dynein heavy chain 1a with the Golgi apparatus and intermediate compartment.
1999,
Pubmed
,
Xenbase
Savill,
Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis.
1990,
Pubmed
Schafer,
Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin.
1994,
Pubmed
Steffen,
The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Susalka,
Distinct cytoplasmic dynein complexes are transported by different mechanisms in axons.
2000,
Pubmed
Swanton,
Human rabaptin-5 is selectively cleaved by caspase-3 during apoptosis.
1999,
Pubmed
,
Xenbase
Swanton,
Bcl-2 regulates a caspase-3/caspase-2 apoptotic cascade in cytosolic extracts.
1999,
Pubmed
Valetti,
Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170.
1999,
Pubmed
Vaughan,
Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued.
1995,
Pubmed
Waterman-Storer,
The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1).
1995,
Pubmed
Waterman-Storer,
The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport.
1997,
Pubmed
Whyte,
Impairment of function in aging neutrophils is associated with apoptosis.
1993,
Pubmed
Wyllie,
Cell death: the significance of apoptosis.
1980,
Pubmed
