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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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Dictyostelium dynein antibodies. Dictyostelium whole cell extracts (lanes 1, 3, and 5) and purified dynein (lanes 2, 4, 6, and 7) were separated on 7.5% SDS-polyacrylamide gels. Gel strips were stained with Coomassie blue (lanes 1 and 2) and Western blots probed with anti-IC antibodies M4 (lanes 3 and 4), IC144 (lanes 5 and 6), or with anti-HC antibody NW127 (lane 7). Positions of dynein subunits are indicated on the right; molecular mass markers (in kD) are indicated on the left. The 55-kD doublet in lane 2 is contaminating tubulin.
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Figure 1: Dictyostelium dynein antibodies. Dictyostelium whole cell extracts (lanes 1, 3, and 5) and purified dynein (lanes 2, 4, 6, and 7) were separated on 7.5% SDS-polyacrylamide gels. Gel strips were stained with Coomassie blue (lanes 1 and 2) and Western blots probed with anti-IC antibodies M4 (lanes 3 and 4), IC144 (lanes 5 and 6), or with anti-HC antibody NW127 (lane 7). Positions of dynein subunits are indicated on the right; molecular mass markers (in kD) are indicated on the left. The 55-kD doublet in lane 2 is contaminating tubulin.

Mentions: Cytoplasmic dynein was isolated from vegetatively growing Dictyostelium cells (Koonce and McIntosh 1990). Dictyostelium dynein contains a 530-kD dynein HC, an 83-kD IC, and a 58-kD light IC (Fig. 1, lane 2). The 83-kD IC, purified from SDS-PAGE gels, was used to generate polyclonal antisera in mice. One of these antisera, M4, reacted specifically with an 83-kD protein on Western blots of Dictyostelium whole cell extract and with the IC of purified dynein (Fig. 1, lanes 3–4). M4 was used to screen a Dictyostelium cDNA expression library. A full-length clone, IC10, encodes a protein of 651–amino acids, predicted to have an Mr of 72 kD. For subsequent experiments, we generated additional Dictyostelium dynein antibodies: polyclonal rat antibody IC144 was raised against bacterially expressed IC (Fig. 1, lanes 5–6), and polyclonal rabbit antiserum NW127 was generated against SDS gel–purified dynein HC (Fig. 1, lane 7).


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)

Dictyostelium dynein antibodies. Dictyostelium whole cell extracts (lanes 1, 3, and 5) and purified dynein (lanes 2, 4, 6, and 7) were separated on 7.5% SDS-polyacrylamide gels. Gel strips were stained with Coomassie blue (lanes 1 and 2) and Western blots probed with anti-IC antibodies M4 (lanes 3 and 4), IC144 (lanes 5 and 6), or with anti-HC antibody NW127 (lane 7). Positions of dynein subunits are indicated on the right; molecular mass markers (in kD) are indicated on the left. The 55-kD doublet in lane 2 is contaminating tubulin.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Dictyostelium dynein antibodies. Dictyostelium whole cell extracts (lanes 1, 3, and 5) and purified dynein (lanes 2, 4, 6, and 7) were separated on 7.5% SDS-polyacrylamide gels. Gel strips were stained with Coomassie blue (lanes 1 and 2) and Western blots probed with anti-IC antibodies M4 (lanes 3 and 4), IC144 (lanes 5 and 6), or with anti-HC antibody NW127 (lane 7). Positions of dynein subunits are indicated on the right; molecular mass markers (in kD) are indicated on the left. The 55-kD doublet in lane 2 is contaminating tubulin.
Mentions: Cytoplasmic dynein was isolated from vegetatively growing Dictyostelium cells (Koonce and McIntosh 1990). Dictyostelium dynein contains a 530-kD dynein HC, an 83-kD IC, and a 58-kD light IC (Fig. 1, lane 2). The 83-kD IC, purified from SDS-PAGE gels, was used to generate polyclonal antisera in mice. One of these antisera, M4, reacted specifically with an 83-kD protein on Western blots of Dictyostelium whole cell extract and with the IC of purified dynein (Fig. 1, lanes 3–4). M4 was used to screen a Dictyostelium cDNA expression library. A full-length clone, IC10, encodes a protein of 651–amino acids, predicted to have an Mr of 72 kD. For subsequent experiments, we generated additional Dictyostelium dynein antibodies: polyclonal rat antibody IC144 was raised against bacterially expressed IC (Fig. 1, lanes 5–6), and polyclonal rabbit antiserum NW127 was generated against SDS gel–purified dynein HC (Fig. 1, lane 7).

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