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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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Cytoskeletal structures and certain organelles are not  grossly affected by dynamitin overexpression in HeLa cells.  HeLa cells transiently transfected with dynamitin were labeled  with anti-dynamitin antibody (A, C, E, and G), and were double  labeled with organelle markers. (B) Protein disulfide isomerase  labeling the ER. (D) Antimitochondria antibody. (F) Anti–tubulin–labeling microtubules. (H) Bodipy phallicidin labeling filamentous actin. E and F show projections of confocal images to  better show the microtubule arrays. The apparent microtubule  staining pattern in E is an artifact resulting from the much stronger microtubule fluorescence.
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Figure 3: Cytoskeletal structures and certain organelles are not grossly affected by dynamitin overexpression in HeLa cells. HeLa cells transiently transfected with dynamitin were labeled with anti-dynamitin antibody (A, C, E, and G), and were double labeled with organelle markers. (B) Protein disulfide isomerase labeling the ER. (D) Antimitochondria antibody. (F) Anti–tubulin–labeling microtubules. (H) Bodipy phallicidin labeling filamentous actin. E and F show projections of confocal images to better show the microtubule arrays. The apparent microtubule staining pattern in E is an artifact resulting from the much stronger microtubule fluorescence.

Mentions: Whereas dynamitin overexpression had pronounced effects on some membrane organelles, effects on other organelles were not detectable. Labeling of cells with anti– protein disulfide isomerase revealed no obvious changes in the distribution of the ER (Fig. 3, A and B). Similarly, no clear perturbation was observed in the distribution of mitochondria (Fig. 3, C and D) or peroxisomes (not shown), even in the strongest dynamitin overexpressors where effects on other organelles were prominent. Taken together, these results show that the effects of dynamitin overexpression are specific for a subset of membrane organelles that includes the intermediate compartment, Golgi complex, early and late endosomes, and lysosomes. Moreover, the differences in the response among the affected organelles indicates organelle-specific differences in the way their positioning and movement are controlled.


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)

Cytoskeletal structures and certain organelles are not  grossly affected by dynamitin overexpression in HeLa cells.  HeLa cells transiently transfected with dynamitin were labeled  with anti-dynamitin antibody (A, C, E, and G), and were double  labeled with organelle markers. (B) Protein disulfide isomerase  labeling the ER. (D) Antimitochondria antibody. (F) Anti–tubulin–labeling microtubules. (H) Bodipy phallicidin labeling filamentous actin. E and F show projections of confocal images to  better show the microtubule arrays. The apparent microtubule  staining pattern in E is an artifact resulting from the much stronger microtubule fluorescence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139801&req=5

Figure 3: Cytoskeletal structures and certain organelles are not grossly affected by dynamitin overexpression in HeLa cells. HeLa cells transiently transfected with dynamitin were labeled with anti-dynamitin antibody (A, C, E, and G), and were double labeled with organelle markers. (B) Protein disulfide isomerase labeling the ER. (D) Antimitochondria antibody. (F) Anti–tubulin–labeling microtubules. (H) Bodipy phallicidin labeling filamentous actin. E and F show projections of confocal images to better show the microtubule arrays. The apparent microtubule staining pattern in E is an artifact resulting from the much stronger microtubule fluorescence.
Mentions: Whereas dynamitin overexpression had pronounced effects on some membrane organelles, effects on other organelles were not detectable. Labeling of cells with anti– protein disulfide isomerase revealed no obvious changes in the distribution of the ER (Fig. 3, A and B). Similarly, no clear perturbation was observed in the distribution of mitochondria (Fig. 3, C and D) or peroxisomes (not shown), even in the strongest dynamitin overexpressors where effects on other organelles were prominent. Taken together, these results show that the effects of dynamitin overexpression are specific for a subset of membrane organelles that includes the intermediate compartment, Golgi complex, early and late endosomes, and lysosomes. Moreover, the differences in the response among the affected organelles indicates organelle-specific differences in the way their positioning and movement are controlled.

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