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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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Time course of MT and centrosome phenotypes. Mutant cells grown under repressed conditions were induced. Samples taken daily were fixed and stained with tubulin and centrosome-specific antibodies. The percentage of ICΔC cells (A) and ICΔN47 cells (C) showing normal MT organization, MT bundling, large centrosomes, multiple MT asters, or MTs without an organizing center on each day are presented. B shows the level of ICΔC mutant protein expression determined by densitometric analysis on Western blots of cell lysates.
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Figure 9: Time course of MT and centrosome phenotypes. Mutant cells grown under repressed conditions were induced. Samples taken daily were fixed and stained with tubulin and centrosome-specific antibodies. The percentage of ICΔC cells (A) and ICΔN47 cells (C) showing normal MT organization, MT bundling, large centrosomes, multiple MT asters, or MTs without an organizing center on each day are presented. B shows the level of ICΔC mutant protein expression determined by densitometric analysis on Western blots of cell lysates.

Mentions: To better understand the effect of IC mutations on MT organization and centrosome morphology, we determined MT and centrosome morphology at various timepoints after induction (Fig. 9). The phenotypes fell into one of five categories: (1) normal MT network; (2) bundled MTs; (3) large centrosomes; (4) multiple MT asters; and (5) disorganized MT without an obvious organizing center. Before induction (day 0), >90% of the ICΔC cells had normal MT networks and centrosomes (Fig. 9 A). 1 d after induction, the MT network was bundled in >50% of the cells, although the sizes of the centrosomes were normal. As induction proceeded, the cells, their MT networks, and their centrosomes became enlarged. On day 3 after induction, ∼60% of the cells had large MT bundles and ∼70% showed large centrosomes. The large MT networks were usually accompanied by large centrosomes and large nuclei. Less than 10% of the cells showed relatively normal MT networks. However after 4 d, the number of cells with normal MT networks began to increase. By day 6, 73% of the population had normal MTs and centrosomes.


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)

Time course of MT and centrosome phenotypes. Mutant cells grown under repressed conditions were induced. Samples taken daily were fixed and stained with tubulin and centrosome-specific antibodies. The percentage of ICΔC cells (A) and ICΔN47 cells (C) showing normal MT organization, MT bundling, large centrosomes, multiple MT asters, or MTs without an organizing center on each day are presented. B shows the level of ICΔC mutant protein expression determined by densitometric analysis on Western blots of cell lysates.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Time course of MT and centrosome phenotypes. Mutant cells grown under repressed conditions were induced. Samples taken daily were fixed and stained with tubulin and centrosome-specific antibodies. The percentage of ICΔC cells (A) and ICΔN47 cells (C) showing normal MT organization, MT bundling, large centrosomes, multiple MT asters, or MTs without an organizing center on each day are presented. B shows the level of ICΔC mutant protein expression determined by densitometric analysis on Western blots of cell lysates.
Mentions: To better understand the effect of IC mutations on MT organization and centrosome morphology, we determined MT and centrosome morphology at various timepoints after induction (Fig. 9). The phenotypes fell into one of five categories: (1) normal MT network; (2) bundled MTs; (3) large centrosomes; (4) multiple MT asters; and (5) disorganized MT without an obvious organizing center. Before induction (day 0), >90% of the ICΔC cells had normal MT networks and centrosomes (Fig. 9 A). 1 d after induction, the MT network was bundled in >50% of the cells, although the sizes of the centrosomes were normal. As induction proceeded, the cells, their MT networks, and their centrosomes became enlarged. On day 3 after induction, ∼60% of the cells had large MT bundles and ∼70% showed large centrosomes. The large MT networks were usually accompanied by large centrosomes and large nuclei. Less than 10% of the cells showed relatively normal MT networks. However after 4 d, the number of cells with normal MT networks began to increase. By day 6, 73% of the population had normal MTs and centrosomes.

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