Lane JD et al. (2001), Apoptotic cleavage of cytoplasmic dynein interm...

XB-ART-8821

J Cell Biol 2001 Jun 25;1537:1415-26.

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Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

Lane JD , Vergnolle MA , Woodman PG , Allan VJ .

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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.

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Species referenced: Xenopus
Genes referenced: abl1 dctn1 dnai1 dync1li1

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	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 anti–CD-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, Casputin™. 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). (B–D) 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 vitro–translated caspase 2, whilst caspases 3 and 7 were tested using in vitro–translated PARP. (E) Native Xenopus egg cytoplasmic dynein–dynactin 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 anti–CD-LIC antibody, and immunoblotted with polyclonal anti–CD-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 3–6). The depleted cytosols (lanes 7 and 8) were then subjected to a further immunoprecipitation with anti–CD-LIC beads (lanes 9 and 10). All fractions were examined by silver stain for DHC (top) and immunoblotted with anti–CD-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 dynein–dynactin 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 dynein–dynactin (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 dynein–dynactin add-back experiment were immunoblotted using the antibodies shown (C, control cytosol and membranes; A, apoptotic cytosol and membranes). Anti–CD-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 (◂) 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 dynein–driven 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).

References [+] :

Addinall, Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes. 2001, Pubmed, Xenbase