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Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium.

Ma S, Triviños-Lagos L, Gräf R, Chisholm RL - J. Cell Biol. (1999)

Bottom Line: Biol.ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly.Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.

ABSTRACT
Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

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IC mutants alter MT networks, nuclear morphology, and DNA content. Vector controls (a and b) or cells expressing IC truncations (c–j) were stained with antitubulin mAb (a, c, e, g, and i) and DAPI (b, d, f, h, and j). ICΔC and ICΔN showed a similar range of phenotypes; ICΔC cells shown. d, f, h and j show altered nuclear morphology resulting from IC mutant expression. Mutant morphologies include collapsed MT networks forming bundles (c), unusually large MT network with large MTOC (e), multiple cytoplasmic asters (g), and poorly organized MTs lacking a visible organizing center (i). Bars, 10 μm. k shows a typical FACS® profile of control or ICΔC cells induced for 2 d. The x-axis shows the DNA content and y-axis shows the cell count. The dashed line represents controls and the shaded area represents ICΔC cells. 50,000 cells were analyzed for each sample.
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Figure 7: IC mutants alter MT networks, nuclear morphology, and DNA content. Vector controls (a and b) or cells expressing IC truncations (c–j) were stained with antitubulin mAb (a, c, e, g, and i) and DAPI (b, d, f, h, and j). ICΔC and ICΔN showed a similar range of phenotypes; ICΔC cells shown. d, f, h and j show altered nuclear morphology resulting from IC mutant expression. Mutant morphologies include collapsed MT networks forming bundles (c), unusually large MT network with large MTOC (e), multiple cytoplasmic asters (g), and poorly organized MTs lacking a visible organizing center (i). Bars, 10 μm. k shows a typical FACS® profile of control or ICΔC cells induced for 2 d. The x-axis shows the DNA content and y-axis shows the cell count. The dashed line represents controls and the shaded area represents ICΔC cells. 50,000 cells were analyzed for each sample.

Mentions: Consistent with the change in cell morphology in the IC mutants, we observed dramatic changes in the organization of the MT network. Cells induced for 3 d were analyzed by indirect immunofluorescence with tubulin antibody and DAPI staining of DNA. In wild-type cells (Fig. 7 a), interphase MTs formed extended radial arrays originating from the MTOC (Roos et al. 1984).


Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium.

Ma S, Triviños-Lagos L, Gräf R, Chisholm RL - J. Cell Biol. (1999)

IC mutants alter MT networks, nuclear morphology, and DNA content. Vector controls (a and b) or cells expressing IC truncations (c–j) were stained with antitubulin mAb (a, c, e, g, and i) and DAPI (b, d, f, h, and j). ICΔC and ICΔN showed a similar range of phenotypes; ICΔC cells shown. d, f, h and j show altered nuclear morphology resulting from IC mutant expression. Mutant morphologies include collapsed MT networks forming bundles (c), unusually large MT network with large MTOC (e), multiple cytoplasmic asters (g), and poorly organized MTs lacking a visible organizing center (i). Bars, 10 μm. k shows a typical FACS® profile of control or ICΔC cells induced for 2 d. The x-axis shows the DNA content and y-axis shows the cell count. The dashed line represents controls and the shaded area represents ICΔC cells. 50,000 cells were analyzed for each sample.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168085&req=5

Figure 7: IC mutants alter MT networks, nuclear morphology, and DNA content. Vector controls (a and b) or cells expressing IC truncations (c–j) were stained with antitubulin mAb (a, c, e, g, and i) and DAPI (b, d, f, h, and j). ICΔC and ICΔN showed a similar range of phenotypes; ICΔC cells shown. d, f, h and j show altered nuclear morphology resulting from IC mutant expression. Mutant morphologies include collapsed MT networks forming bundles (c), unusually large MT network with large MTOC (e), multiple cytoplasmic asters (g), and poorly organized MTs lacking a visible organizing center (i). Bars, 10 μm. k shows a typical FACS® profile of control or ICΔC cells induced for 2 d. The x-axis shows the DNA content and y-axis shows the cell count. The dashed line represents controls and the shaded area represents ICΔC cells. 50,000 cells were analyzed for each sample.
Mentions: Consistent with the change in cell morphology in the IC mutants, we observed dramatic changes in the organization of the MT network. Cells induced for 3 d were analyzed by indirect immunofluorescence with tubulin antibody and DAPI staining of DNA. In wild-type cells (Fig. 7 a), interphase MTs formed extended radial arrays originating from the MTOC (Roos et al. 1984).

Bottom Line: Biol.ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly.Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.

ABSTRACT
Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

Show MeSH
Related in: MedlinePlus