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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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ICΔN but not ICΔC cosediments with 20S dynein in sucrose gradients. Cell lysates were fractionated by centrifugation in 5–20% sucrose density gradient and equal volumes of the fractions separated by 7.5% SDS-PAGE. The positions of wild-type and mutant IC were detected with the IC144 antibody; IC mutants were also confirmed by 9E10 mAb that recognizes the myc tag (data not shown). Dynein HC, detected with NW127 antibody, sedimented in fractions 5–7. Cells analyzed are indicated on the left and the positions of relevant proteins are indicated on the right.
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Figure 4: ICΔN but not ICΔC cosediments with 20S dynein in sucrose gradients. Cell lysates were fractionated by centrifugation in 5–20% sucrose density gradient and equal volumes of the fractions separated by 7.5% SDS-PAGE. The positions of wild-type and mutant IC were detected with the IC144 antibody; IC mutants were also confirmed by 9E10 mAb that recognizes the myc tag (data not shown). Dynein HC, detected with NW127 antibody, sedimented in fractions 5–7. Cells analyzed are indicated on the left and the positions of relevant proteins are indicated on the right.

Mentions: To further investigate the dynein complex in IC mutant cells, cell lysates were fractionated on a 5–20% sucrose density gradient. Wild-type dynein migrated as a 20S complex containing both the HC and IC (Fig. 4). In ICΔC cells, ICΔC protein did not cosediment with the dynein HC and wild-type IC (Fig. 4), providing independent evidence that this mutant failed to associate with dynein. Also, since wild-type IC comigrated with HC, dynein complex was not affected by ICΔC expression. For both ICΔN47 and ICΔN106 cells, a significant amount of the mutant IC cosedimented with HC at the normal position for dynein. The ratio of mutant IC to wild-type IC in the dynein fractions was consistent with that in dynein IPs, with the majority of dynein complexes containing the truncated IC mutant. In ICΔN106, a large amount of the mutant IC migrated near the top of the gradient, most likely due to its high expression level.


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)

ICΔN but not ICΔC cosediments with 20S dynein in sucrose gradients. Cell lysates were fractionated by centrifugation in 5–20% sucrose density gradient and equal volumes of the fractions separated by 7.5% SDS-PAGE. The positions of wild-type and mutant IC were detected with the IC144 antibody; IC mutants were also confirmed by 9E10 mAb that recognizes the myc tag (data not shown). Dynein HC, detected with NW127 antibody, sedimented in fractions 5–7. Cells analyzed are indicated on the left and the positions of relevant proteins are indicated on the right.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: ICΔN but not ICΔC cosediments with 20S dynein in sucrose gradients. Cell lysates were fractionated by centrifugation in 5–20% sucrose density gradient and equal volumes of the fractions separated by 7.5% SDS-PAGE. The positions of wild-type and mutant IC were detected with the IC144 antibody; IC mutants were also confirmed by 9E10 mAb that recognizes the myc tag (data not shown). Dynein HC, detected with NW127 antibody, sedimented in fractions 5–7. Cells analyzed are indicated on the left and the positions of relevant proteins are indicated on the right.
Mentions: To further investigate the dynein complex in IC mutant cells, cell lysates were fractionated on a 5–20% sucrose density gradient. Wild-type dynein migrated as a 20S complex containing both the HC and IC (Fig. 4). In ICΔC cells, ICΔC protein did not cosediment with the dynein HC and wild-type IC (Fig. 4), providing independent evidence that this mutant failed to associate with dynein. Also, since wild-type IC comigrated with HC, dynein complex was not affected by ICΔC expression. For both ICΔN47 and ICΔN106 cells, a significant amount of the mutant IC cosedimented with HC at the normal position for dynein. The ratio of mutant IC to wild-type IC in the dynein fractions was consistent with that in dynein IPs, with the majority of dynein complexes containing the truncated IC mutant. In ICΔN106, a large amount of the mutant IC migrated near the top of the gradient, most likely due to its high expression level.

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