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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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Time-lapse analysis of living COS-7 cells during recovery from BFA directly demonstrates dynamitin-induced inhibition of ER-to-Golgi transport. COS-7 cultures were transiently  transfected with a GFP–NAGT-I fusion protein either alone (A–E)  or with myc-tagged dynamitin (F–J). More than 90% of overexpressing cells in fixed, cotransfected cultures were found to express both transfected constructs. After a 20-min exposure to  BFA, the drug was washed out and the GFP–NAGT-I fluorescence was recorded by time-lapse microscopy at 3- to 7-s intervals. (A–E) Cytoplasmic region beneath the nucleus with reforming Golgi complex at upper left corner. (F–J) Cytoplasmic region  between nucleus at upper left corner and cell periphery at lower  right corner. Control cells typically exhibited rapid centripetal  movements of newly formed GFP–NAGT-I–positive vesicles  (A–E, arrows; arrowhead indicates static vesicle for reference),  leading to the reformation of a perinuclear Golgi complex (A–E,  upper left corner). Similar labeled vesicles appeared throughout  the cytoplasm of dynamitin-transfected cells, but no sustained  movements were observed in any direction, as seen in F–J. Note  partial alignments of vesicles in F–J, forming short linear arrays  suggestive of interactions with microtubules. Bars, 1 μm.
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Figure 9: Time-lapse analysis of living COS-7 cells during recovery from BFA directly demonstrates dynamitin-induced inhibition of ER-to-Golgi transport. COS-7 cultures were transiently transfected with a GFP–NAGT-I fusion protein either alone (A–E) or with myc-tagged dynamitin (F–J). More than 90% of overexpressing cells in fixed, cotransfected cultures were found to express both transfected constructs. After a 20-min exposure to BFA, the drug was washed out and the GFP–NAGT-I fluorescence was recorded by time-lapse microscopy at 3- to 7-s intervals. (A–E) Cytoplasmic region beneath the nucleus with reforming Golgi complex at upper left corner. (F–J) Cytoplasmic region between nucleus at upper left corner and cell periphery at lower right corner. Control cells typically exhibited rapid centripetal movements of newly formed GFP–NAGT-I–positive vesicles (A–E, arrows; arrowhead indicates static vesicle for reference), leading to the reformation of a perinuclear Golgi complex (A–E, upper left corner). Similar labeled vesicles appeared throughout the cytoplasm of dynamitin-transfected cells, but no sustained movements were observed in any direction, as seen in F–J. Note partial alignments of vesicles in F–J, forming short linear arrays suggestive of interactions with microtubules. Bars, 1 μm.

Mentions: Recovery from BFA treatment was also observed by time-lapse microscopy in living COS-7 cells transfected with a NAGT-I–GFP fusion protein, either alone or combined with dynamitin. Control cells in these experiments clearly showed the appearance of NAGT-I–GFP–labeled vesicles throughout the cytoplasm within a few minutes after BFA washout, followed by their rapid centripetal movements along linear tracks consistent with microtubules, leading to the reformation of a perinuclear Golgi complex after 20–30 min (Fig. 9, A–E). In cells overexpressing dynamitin, similar labeled vesicles also reappeared throughout the cytoplasm after BFA washout, but these exhibited no sustained linear movements in any direction (Fig. 9, F–J). It was noted that many of these vesicles did align to form short linear arrays suggestive of static interactions with microtubules (Fig. 9, F–J). These data directly demonstrate that dynamitin overexpression inhibits microtubule-based ER-to-Golgi transport, resulting in the accumulation of Golgi-destined components in the periphery, most likely at their sites of emergence from the ER.


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)

Time-lapse analysis of living COS-7 cells during recovery from BFA directly demonstrates dynamitin-induced inhibition of ER-to-Golgi transport. COS-7 cultures were transiently  transfected with a GFP–NAGT-I fusion protein either alone (A–E)  or with myc-tagged dynamitin (F–J). More than 90% of overexpressing cells in fixed, cotransfected cultures were found to express both transfected constructs. After a 20-min exposure to  BFA, the drug was washed out and the GFP–NAGT-I fluorescence was recorded by time-lapse microscopy at 3- to 7-s intervals. (A–E) Cytoplasmic region beneath the nucleus with reforming Golgi complex at upper left corner. (F–J) Cytoplasmic region  between nucleus at upper left corner and cell periphery at lower  right corner. Control cells typically exhibited rapid centripetal  movements of newly formed GFP–NAGT-I–positive vesicles  (A–E, arrows; arrowhead indicates static vesicle for reference),  leading to the reformation of a perinuclear Golgi complex (A–E,  upper left corner). Similar labeled vesicles appeared throughout  the cytoplasm of dynamitin-transfected cells, but no sustained  movements were observed in any direction, as seen in F–J. Note  partial alignments of vesicles in F–J, forming short linear arrays  suggestive of interactions with microtubules. Bars, 1 μm.
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Related In: Results  -  Collection

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Figure 9: Time-lapse analysis of living COS-7 cells during recovery from BFA directly demonstrates dynamitin-induced inhibition of ER-to-Golgi transport. COS-7 cultures were transiently transfected with a GFP–NAGT-I fusion protein either alone (A–E) or with myc-tagged dynamitin (F–J). More than 90% of overexpressing cells in fixed, cotransfected cultures were found to express both transfected constructs. After a 20-min exposure to BFA, the drug was washed out and the GFP–NAGT-I fluorescence was recorded by time-lapse microscopy at 3- to 7-s intervals. (A–E) Cytoplasmic region beneath the nucleus with reforming Golgi complex at upper left corner. (F–J) Cytoplasmic region between nucleus at upper left corner and cell periphery at lower right corner. Control cells typically exhibited rapid centripetal movements of newly formed GFP–NAGT-I–positive vesicles (A–E, arrows; arrowhead indicates static vesicle for reference), leading to the reformation of a perinuclear Golgi complex (A–E, upper left corner). Similar labeled vesicles appeared throughout the cytoplasm of dynamitin-transfected cells, but no sustained movements were observed in any direction, as seen in F–J. Note partial alignments of vesicles in F–J, forming short linear arrays suggestive of interactions with microtubules. Bars, 1 μm.
Mentions: Recovery from BFA treatment was also observed by time-lapse microscopy in living COS-7 cells transfected with a NAGT-I–GFP fusion protein, either alone or combined with dynamitin. Control cells in these experiments clearly showed the appearance of NAGT-I–GFP–labeled vesicles throughout the cytoplasm within a few minutes after BFA washout, followed by their rapid centripetal movements along linear tracks consistent with microtubules, leading to the reformation of a perinuclear Golgi complex after 20–30 min (Fig. 9, A–E). In cells overexpressing dynamitin, similar labeled vesicles also reappeared throughout the cytoplasm after BFA washout, but these exhibited no sustained linear movements in any direction (Fig. 9, F–J). It was noted that many of these vesicles did align to form short linear arrays suggestive of static interactions with microtubules (Fig. 9, F–J). These data directly demonstrate that dynamitin overexpression inhibits microtubule-based ER-to-Golgi transport, resulting in the accumulation of Golgi-destined components in the periphery, most likely at their sites of emergence from the ER.

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