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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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ICΔC binds dynactin but not dynein, whereas ICΔN associates with dynein but associates poorly with dynactin. Protein samples of cell lysates (WC) or immunoprecipitates (IP) from ICΔC cells, ICΔN cells, or vector controls were probed with IC144 antibody. Dynein was immunoprecipitated using the NW127 HC antibody (HC IP) and dynactin was immunoprecipitated using the capping protein β antibody R18 (CP IP). The faint double bands at the lower part of the CP IP lanes in control and ICΔC panels are Ig HCs.
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Figure 3: ICΔC binds dynactin but not dynein, whereas ICΔN associates with dynein but associates poorly with dynactin. Protein samples of cell lysates (WC) or immunoprecipitates (IP) from ICΔC cells, ICΔN cells, or vector controls were probed with IC144 antibody. Dynein was immunoprecipitated using the NW127 HC antibody (HC IP) and dynactin was immunoprecipitated using the capping protein β antibody R18 (CP IP). The faint double bands at the lower part of the CP IP lanes in control and ICΔC panels are Ig HCs.

Mentions: From wild-type Dictyostelium lysates, the endogenous IC coprecipitated with both NW127 and R18 antibody (Fig. 3), suggesting that wild-type IC associates with both dynein and dynactin complexes. However, each of the IC truncations was deficient in association with one of the two complexes. Although a significant amount of ICΔC was detected in the capping protein IP, very little was detectable in dynein HC IP (Fig. 3). This suggests that the COOH-terminal deletion abolished the ability of IC to bind to dynein HC while preserving its dynactin binding activity. In contrast, both ICΔN106 and ICΔN47 mutants bound dynein HC well but associated poorly with dynactin. The ratio of ICΔN to endogenous IC associated with dynactin is greatly reduced when compared with their ratio in cell lysates (Fig. 3). This suggests that the NH2-terminal IC region is crucial for dynactin association and is consistent with previous studies that mapped the p150 binding activity to the NH2-terminal region (Vaughan and Vallee 1995). ICΔN47 showed better HC binding than ICΔN106, indicating that whereas the COOH-terminal conserved region of IC is required for dynein HC association, the binding is stabilized by residues that extend into the NH2-terminal domain.


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)

ICΔC binds dynactin but not dynein, whereas ICΔN associates with dynein but associates poorly with dynactin. Protein samples of cell lysates (WC) or immunoprecipitates (IP) from ICΔC cells, ICΔN cells, or vector controls were probed with IC144 antibody. Dynein was immunoprecipitated using the NW127 HC antibody (HC IP) and dynactin was immunoprecipitated using the capping protein β antibody R18 (CP IP). The faint double bands at the lower part of the CP IP lanes in control and ICΔC panels are Ig HCs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168085&req=5

Figure 3: ICΔC binds dynactin but not dynein, whereas ICΔN associates with dynein but associates poorly with dynactin. Protein samples of cell lysates (WC) or immunoprecipitates (IP) from ICΔC cells, ICΔN cells, or vector controls were probed with IC144 antibody. Dynein was immunoprecipitated using the NW127 HC antibody (HC IP) and dynactin was immunoprecipitated using the capping protein β antibody R18 (CP IP). The faint double bands at the lower part of the CP IP lanes in control and ICΔC panels are Ig HCs.
Mentions: From wild-type Dictyostelium lysates, the endogenous IC coprecipitated with both NW127 and R18 antibody (Fig. 3), suggesting that wild-type IC associates with both dynein and dynactin complexes. However, each of the IC truncations was deficient in association with one of the two complexes. Although a significant amount of ICΔC was detected in the capping protein IP, very little was detectable in dynein HC IP (Fig. 3). This suggests that the COOH-terminal deletion abolished the ability of IC to bind to dynein HC while preserving its dynactin binding activity. In contrast, both ICΔN106 and ICΔN47 mutants bound dynein HC well but associated poorly with dynactin. The ratio of ICΔN to endogenous IC associated with dynactin is greatly reduced when compared with their ratio in cell lysates (Fig. 3). This suggests that the NH2-terminal IC region is crucial for dynactin association and is consistent with previous studies that mapped the p150 binding activity to the NH2-terminal region (Vaughan and Vallee 1995). ICΔN47 showed better HC binding than ICΔN106, indicating that whereas the COOH-terminal conserved region of IC is required for dynein HC association, the binding is stabilized by residues that extend into the NH2-terminal domain.

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