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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 cause Golgi dispersion. Control cells (a and b) or IC truncation mutant expressing cells (c–f) induced for 2 d were double-labeled with a comitin mAb to localize the Golgi complex (a, c, and e) and DAPI to visualize the nucleus (b, d, and f). All three IC truncations produced dispersion of the Golgi complex; ICΔC shown (c and e). c and d show IC mutant cells with normal size, whereas e and f show IC mutants with the larger flattened morphology. Bars, 10 μm.
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Figure 6: IC mutants cause Golgi dispersion. Control cells (a and b) or IC truncation mutant expressing cells (c–f) induced for 2 d were double-labeled with a comitin mAb to localize the Golgi complex (a, c, and e) and DAPI to visualize the nucleus (b, d, and f). All three IC truncations produced dispersion of the Golgi complex; ICΔC shown (c and e). c and d show IC mutant cells with normal size, whereas e and f show IC mutants with the larger flattened morphology. Bars, 10 μm.

Mentions: Several studies have highlighted the importance of cytoplasmic dynein in Golgi apparatus positioning (Corthesy-Theulaz et al. 1992; Burkhardt et al. 1997; Harada et al. 1998). Therefore, Golgi complex localization is a good in vivo indicator of cytoplasmic dynein and dynactin function. Localization of the Golgi complex was detected by indirect immunofluorescence using a mAb against comitin (Weiner et al. 1993). In wild-type Dictyostelium cells, the Golgi complex appears as a compact perinuclear cluster whose center coincides with the MT organizing center (MTOC) (Fig. 6 a). In contrast, in all three IC mutants the Golgi complex was dispersed throughout the cytoplasm (Fig. 6c and Fig. e), supporting the idea that ICΔN and ICΔC expression disrupted dynein function.


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 cause Golgi dispersion. Control cells (a and b) or IC truncation mutant expressing cells (c–f) induced for 2 d were double-labeled with a comitin mAb to localize the Golgi complex (a, c, and e) and DAPI to visualize the nucleus (b, d, and f). All three IC truncations produced dispersion of the Golgi complex; ICΔC shown (c and e). c and d show IC mutant cells with normal size, whereas e and f show IC mutants with the larger flattened morphology. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: IC mutants cause Golgi dispersion. Control cells (a and b) or IC truncation mutant expressing cells (c–f) induced for 2 d were double-labeled with a comitin mAb to localize the Golgi complex (a, c, and e) and DAPI to visualize the nucleus (b, d, and f). All three IC truncations produced dispersion of the Golgi complex; ICΔC shown (c and e). c and d show IC mutant cells with normal size, whereas e and f show IC mutants with the larger flattened morphology. Bars, 10 μm.
Mentions: Several studies have highlighted the importance of cytoplasmic dynein in Golgi apparatus positioning (Corthesy-Theulaz et al. 1992; Burkhardt et al. 1997; Harada et al. 1998). Therefore, Golgi complex localization is a good in vivo indicator of cytoplasmic dynein and dynactin function. Localization of the Golgi complex was detected by indirect immunofluorescence using a mAb against comitin (Weiner et al. 1993). In wild-type Dictyostelium cells, the Golgi complex appears as a compact perinuclear cluster whose center coincides with the MT organizing center (MTOC) (Fig. 6 a). In contrast, in all three IC mutants the Golgi complex was dispersed throughout the cytoplasm (Fig. 6c and Fig. e), supporting the idea that ICΔN and ICΔC expression disrupted dynein function.

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