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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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Structure and expression of dynein IC truncation mutants. (A) Diagram of predicted IC domain structure in relation to the IC truncations used in this study. ICΔC consists of amino acids 1–278 of the IC sequence. ICΔN106 deletes the NH2-terminal 106 amino acids, whereas ICΔN47 removes the NH2-terminal 47 amino acids. (B) IC truncation mutants are expressed at high levels in wild-type Dictyostelium. Whole cell lysates (2 × 105 cells) were separated on a 7.5% SDS-PAGE gel and the blot probed with IC144 antibody. Control was wild-type cells transformed with pVEII vector alone. ICΔN47a and ICΔN47b are two independent cell lines that differ in their mutant protein expression level. The lower level of ICΔN47 expression in ICΔN74a allows visualization of both endogenous and mutant IC.
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Figure 2: Structure and expression of dynein IC truncation mutants. (A) Diagram of predicted IC domain structure in relation to the IC truncations used in this study. ICΔC consists of amino acids 1–278 of the IC sequence. ICΔN106 deletes the NH2-terminal 106 amino acids, whereas ICΔN47 removes the NH2-terminal 47 amino acids. (B) IC truncation mutants are expressed at high levels in wild-type Dictyostelium. Whole cell lysates (2 × 105 cells) were separated on a 7.5% SDS-PAGE gel and the blot probed with IC144 antibody. Control was wild-type cells transformed with pVEII vector alone. ICΔN47a and ICΔN47b are two independent cell lines that differ in their mutant protein expression level. The lower level of ICΔN47 expression in ICΔN74a allows visualization of both endogenous and mutant IC.

Mentions: To functionally define the domains of dynein IC and to generate cell lines with defective dynein function, we overexpressed different IC domains in wild-type Dictyostelium. We designed several dynein IC truncation mutants that deleted or disrupted the hypothetical HC binding domain or the predicted dynactin binding domain (Fig. 2 A). ICΔC lacks the COOH-terminal 373 amino acids, whereas ICΔN47 and ICΔN106 lack the NH2-terminal 47 or 106 residues, respectively. Mutant IC expression was controlled by a discoidin promoter, whose activity can be repressed by addition of folate to the medium (Blusch et al. 1992). This system makes it possible to conditionally express potentially deleterious mutants in Dictyostelium. Mutant protein expression was assessed using Western blots of whole cell lysates from cells induced for 3 d with IC-specific IC144 antibody (Fig. 2 B). For all three mutants, the mutant expression level was >10 times that of the endogenous wild-type IC.


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)

Structure and expression of dynein IC truncation mutants. (A) Diagram of predicted IC domain structure in relation to the IC truncations used in this study. ICΔC consists of amino acids 1–278 of the IC sequence. ICΔN106 deletes the NH2-terminal 106 amino acids, whereas ICΔN47 removes the NH2-terminal 47 amino acids. (B) IC truncation mutants are expressed at high levels in wild-type Dictyostelium. Whole cell lysates (2 × 105 cells) were separated on a 7.5% SDS-PAGE gel and the blot probed with IC144 antibody. Control was wild-type cells transformed with pVEII vector alone. ICΔN47a and ICΔN47b are two independent cell lines that differ in their mutant protein expression level. The lower level of ICΔN47 expression in ICΔN74a allows visualization of both endogenous and mutant IC.
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Related In: Results  -  Collection

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

Figure 2: Structure and expression of dynein IC truncation mutants. (A) Diagram of predicted IC domain structure in relation to the IC truncations used in this study. ICΔC consists of amino acids 1–278 of the IC sequence. ICΔN106 deletes the NH2-terminal 106 amino acids, whereas ICΔN47 removes the NH2-terminal 47 amino acids. (B) IC truncation mutants are expressed at high levels in wild-type Dictyostelium. Whole cell lysates (2 × 105 cells) were separated on a 7.5% SDS-PAGE gel and the blot probed with IC144 antibody. Control was wild-type cells transformed with pVEII vector alone. ICΔN47a and ICΔN47b are two independent cell lines that differ in their mutant protein expression level. The lower level of ICΔN47 expression in ICΔN74a allows visualization of both endogenous and mutant IC.
Mentions: To functionally define the domains of dynein IC and to generate cell lines with defective dynein function, we overexpressed different IC domains in wild-type Dictyostelium. We designed several dynein IC truncation mutants that deleted or disrupted the hypothetical HC binding domain or the predicted dynactin binding domain (Fig. 2 A). ICΔC lacks the COOH-terminal 373 amino acids, whereas ICΔN47 and ICΔN106 lack the NH2-terminal 47 or 106 residues, respectively. Mutant IC expression was controlled by a discoidin promoter, whose activity can be repressed by addition of folate to the medium (Blusch et al. 1992). This system makes it possible to conditionally express potentially deleterious mutants in Dictyostelium. Mutant protein expression was assessed using Western blots of whole cell lysates from cells induced for 3 d with IC-specific IC144 antibody (Fig. 2 B). For all three mutants, the mutant expression level was >10 times that of the endogenous wild-type IC.

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