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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 truncations produced centrosomes with increased core lengths and failure to separate. Wild-type (a) or ICΔC cells (b–h) induced for 2 d were analyzed for centrosome morphology by EM. All cells shown are in interphase. A wild-type centrosome (a) is indicated by the arrowhead. (b–d) ICΔC cells with centrosome cores longer than wild-type. (e–h) ICΔC cells showing paired centrosomes suggesting separation defects. N, nucleus. Bars, 200 nm.
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Figure 10: IC truncations produced centrosomes with increased core lengths and failure to separate. Wild-type (a) or ICΔC cells (b–h) induced for 2 d were analyzed for centrosome morphology by EM. All cells shown are in interphase. A wild-type centrosome (a) is indicated by the arrowhead. (b–d) ICΔC cells with centrosome cores longer than wild-type. (e–h) ICΔC cells showing paired centrosomes suggesting separation defects. N, nucleus. Bars, 200 nm.

Mentions: One striking phenotype of IC mutants was the presence of apparently larger centrosomes. Apparently large centrosomes in the light microscope could result from closely positioned, morphologically normal centrosomes produced by defective centrosome duplication or separation, or from the abnormal accumulation of centrosomal material. To distinguish these possibilities, we examined centrosome morphology at the ultrastructural level. The interphase Dictyostelium centrosome is a nucleus-associated body consisting of a rectangular, electron-dense core surrounded by an amorphous matrix or corona from which MTs radiate (Fig. 10 a) (Moens 1976; Kuriyama et al. 1982; Omura and Fukui 1985). The core is a tripartite structure of ∼280 × 220 × 130 nm in size (Ueda et al. 1999). As expected, the length of wild-type centrosome cores averaged 285 ± 45 nm, whereas the ICΔC mutants were substantially larger, averaging 387 ± 104 nm in length, with some nearly twice the length of wild-type (Fig. 10, b–d). The tripartite organization of the core and the corona of these long centrosomes appeared similar to wild-type. The observation of centrosomes twice the normal length in interphase cells suggests they may arise from failure of the centrosome replication cycle which normally doubles the length of centrosomes during prophase and then separates the two halves longitudinally to produce the spindle poles (Ueda et al. 1999).


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 truncations produced centrosomes with increased core lengths and failure to separate. Wild-type (a) or ICΔC cells (b–h) induced for 2 d were analyzed for centrosome morphology by EM. All cells shown are in interphase. A wild-type centrosome (a) is indicated by the arrowhead. (b–d) ICΔC cells with centrosome cores longer than wild-type. (e–h) ICΔC cells showing paired centrosomes suggesting separation defects. N, nucleus. Bars, 200 nm.
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Related In: Results  -  Collection

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Figure 10: IC truncations produced centrosomes with increased core lengths and failure to separate. Wild-type (a) or ICΔC cells (b–h) induced for 2 d were analyzed for centrosome morphology by EM. All cells shown are in interphase. A wild-type centrosome (a) is indicated by the arrowhead. (b–d) ICΔC cells with centrosome cores longer than wild-type. (e–h) ICΔC cells showing paired centrosomes suggesting separation defects. N, nucleus. Bars, 200 nm.
Mentions: One striking phenotype of IC mutants was the presence of apparently larger centrosomes. Apparently large centrosomes in the light microscope could result from closely positioned, morphologically normal centrosomes produced by defective centrosome duplication or separation, or from the abnormal accumulation of centrosomal material. To distinguish these possibilities, we examined centrosome morphology at the ultrastructural level. The interphase Dictyostelium centrosome is a nucleus-associated body consisting of a rectangular, electron-dense core surrounded by an amorphous matrix or corona from which MTs radiate (Fig. 10 a) (Moens 1976; Kuriyama et al. 1982; Omura and Fukui 1985). The core is a tripartite structure of ∼280 × 220 × 130 nm in size (Ueda et al. 1999). As expected, the length of wild-type centrosome cores averaged 285 ± 45 nm, whereas the ICΔC mutants were substantially larger, averaging 387 ± 104 nm in length, with some nearly twice the length of wild-type (Fig. 10, b–d). The tripartite organization of the core and the corona of these long centrosomes appeared similar to wild-type. The observation of centrosomes twice the normal length in interphase cells suggests they may arise from failure of the centrosome replication cycle which normally doubles the length of centrosomes during prophase and then separates the two halves longitudinally to produce the spindle poles (Ueda et al. 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.

Show MeSH
Related in: MedlinePlus