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Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.

Burkhardt JK, Echeverri CJ, Nilsson T, Vallee RB - J. Cell Biol. (1997)

Bottom Line: In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery.These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment.These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

View Article: PubMed Central - PubMed

Affiliation: The University of Chicago, Department of Pathology, Chicago, Illinois 60637, USA. jburkhar@flowcity.bsd.uchicago.edu

ABSTRACT
Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617-634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

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Effects of dynamitin overexpression on cytoskeletal structures in COS-7  cells do not correlate with  Golgi disruption. (A–F)  COS-7 cells were transiently  transfected to coexpress  VSV-G–tagged ST with either myc-tagged dynamitin  (A–D), or β-galactosidase  (E and F). More than 90% of  overexpressing cells in cotransfected cultures were  found to express both transfected constructs. Double- labeling of antitubulin (B, D,  and F) with anti–VSV-G tag  (A, C, and E) revealed clear  Golgi disruptions in dynamitin-transfected cells with  normal radial microtubule arrays (A and B), as well as in  those showing less well- focused arrays (C and D).  Golgi distribution and microtubule organization in β-galactosidase–transfected cells (E  and F) were indistinguishable from those of control  untransfected cells. As seen  in HeLa cells, COS-7 cells  overexpressing myc-tagged  dynamitin alone and double-labeled with anti–myc tag (G)  and rhodamine phalloidin (H)  showed no apparent perturbations of the F-actin cytoskeleton.
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Figure 4: Effects of dynamitin overexpression on cytoskeletal structures in COS-7 cells do not correlate with Golgi disruption. (A–F) COS-7 cells were transiently transfected to coexpress VSV-G–tagged ST with either myc-tagged dynamitin (A–D), or β-galactosidase (E and F). More than 90% of overexpressing cells in cotransfected cultures were found to express both transfected constructs. Double- labeling of antitubulin (B, D, and F) with anti–VSV-G tag (A, C, and E) revealed clear Golgi disruptions in dynamitin-transfected cells with normal radial microtubule arrays (A and B), as well as in those showing less well- focused arrays (C and D). Golgi distribution and microtubule organization in β-galactosidase–transfected cells (E and F) were indistinguishable from those of control untransfected cells. As seen in HeLa cells, COS-7 cells overexpressing myc-tagged dynamitin alone and double-labeled with anti–myc tag (G) and rhodamine phalloidin (H) showed no apparent perturbations of the F-actin cytoskeleton.

Mentions: In interphase HeLa cells, we observed no gross effects of dynamitin overexpression on the organization of major cytoskeletal filament systems. As shown in Fig. 3, E and F the overall distribution of microtubules in dynamitin-overexpressing HeLa cells was similar to control untransfected cells, and there was no evidence of microtubule bundling. We also examined this question in COS-7 cells, which have microtubule arrays more clearly focused at the centrosome. We found that 85–90% of COS-7 cells cotransfected with dynamitin and VSV-G–tagged ST exhibited specific Golgi disruptions similar to those seen in HeLa cells. Up to half of these cells also exhibited a less focused microtubule organizing center, though the microtubule array often still appeared to radiate from the perinuclear area (Fig. 4 D). Golgi fragmentation and dispersal was observed in dynamitin-overexpressing cells with clearly radial microtubule arrays (Fig. 4, A and B), as well as in those with less well-organized microtubules (Fig. 4 C). No changes were observed in the sensitivity to nocodazole- induced microtubule depolymerization, nor in the rate of centrosome-based microtubule regrowth after nocodazole washout in either cell type (not shown). Also, no change was detected in the population of stable microtubules containing acetylated α-tubulin in HeLa cells. Similarly, we observed no gross changes in the actin cytoskeleton in response to dynamitin transfection. This was true in HeLa cells (Fig. 3, G and H), COS-7 cells (Fig. 4, G and H), and BHK cells (not shown).


Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.

Burkhardt JK, Echeverri CJ, Nilsson T, Vallee RB - J. Cell Biol. (1997)

Effects of dynamitin overexpression on cytoskeletal structures in COS-7  cells do not correlate with  Golgi disruption. (A–F)  COS-7 cells were transiently  transfected to coexpress  VSV-G–tagged ST with either myc-tagged dynamitin  (A–D), or β-galactosidase  (E and F). More than 90% of  overexpressing cells in cotransfected cultures were  found to express both transfected constructs. Double- labeling of antitubulin (B, D,  and F) with anti–VSV-G tag  (A, C, and E) revealed clear  Golgi disruptions in dynamitin-transfected cells with  normal radial microtubule arrays (A and B), as well as in  those showing less well- focused arrays (C and D).  Golgi distribution and microtubule organization in β-galactosidase–transfected cells (E  and F) were indistinguishable from those of control  untransfected cells. As seen  in HeLa cells, COS-7 cells  overexpressing myc-tagged  dynamitin alone and double-labeled with anti–myc tag (G)  and rhodamine phalloidin (H)  showed no apparent perturbations of the F-actin cytoskeleton.
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Related In: Results  -  Collection

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Figure 4: Effects of dynamitin overexpression on cytoskeletal structures in COS-7 cells do not correlate with Golgi disruption. (A–F) COS-7 cells were transiently transfected to coexpress VSV-G–tagged ST with either myc-tagged dynamitin (A–D), or β-galactosidase (E and F). More than 90% of overexpressing cells in cotransfected cultures were found to express both transfected constructs. Double- labeling of antitubulin (B, D, and F) with anti–VSV-G tag (A, C, and E) revealed clear Golgi disruptions in dynamitin-transfected cells with normal radial microtubule arrays (A and B), as well as in those showing less well- focused arrays (C and D). Golgi distribution and microtubule organization in β-galactosidase–transfected cells (E and F) were indistinguishable from those of control untransfected cells. As seen in HeLa cells, COS-7 cells overexpressing myc-tagged dynamitin alone and double-labeled with anti–myc tag (G) and rhodamine phalloidin (H) showed no apparent perturbations of the F-actin cytoskeleton.
Mentions: In interphase HeLa cells, we observed no gross effects of dynamitin overexpression on the organization of major cytoskeletal filament systems. As shown in Fig. 3, E and F the overall distribution of microtubules in dynamitin-overexpressing HeLa cells was similar to control untransfected cells, and there was no evidence of microtubule bundling. We also examined this question in COS-7 cells, which have microtubule arrays more clearly focused at the centrosome. We found that 85–90% of COS-7 cells cotransfected with dynamitin and VSV-G–tagged ST exhibited specific Golgi disruptions similar to those seen in HeLa cells. Up to half of these cells also exhibited a less focused microtubule organizing center, though the microtubule array often still appeared to radiate from the perinuclear area (Fig. 4 D). Golgi fragmentation and dispersal was observed in dynamitin-overexpressing cells with clearly radial microtubule arrays (Fig. 4, A and B), as well as in those with less well-organized microtubules (Fig. 4 C). No changes were observed in the sensitivity to nocodazole- induced microtubule depolymerization, nor in the rate of centrosome-based microtubule regrowth after nocodazole washout in either cell type (not shown). Also, no change was detected in the population of stable microtubules containing acetylated α-tubulin in HeLa cells. Similarly, we observed no gross changes in the actin cytoskeleton in response to dynamitin transfection. This was true in HeLa cells (Fig. 3, G and H), COS-7 cells (Fig. 4, G and H), and BHK cells (not shown).

Bottom Line: In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery.These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment.These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

View Article: PubMed Central - PubMed

Affiliation: The University of Chicago, Department of Pathology, Chicago, Illinois 60637, USA. jburkhar@flowcity.bsd.uchicago.edu

ABSTRACT
Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617-634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti-lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding.

Show MeSH
Related in: MedlinePlus