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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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BFA treatment of dynamitin overexpressing cells.  Transfected cells were treated with BFA for 20 min (A and B), or  they were treated and allowed to recover in the absence of drug  for 1 h (C and D) or for 10 min (E and F). Dynamitin overexpression did not prevent the redistribution of Golgi markers into the  ER. The fragmented Golgi phenotype was generated at early  times after BFA removal, without passage of marker proteins  through a juxtanuclear Golgi complex.
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Figure 8: BFA treatment of dynamitin overexpressing cells. Transfected cells were treated with BFA for 20 min (A and B), or they were treated and allowed to recover in the absence of drug for 1 h (C and D) or for 10 min (E and F). Dynamitin overexpression did not prevent the redistribution of Golgi markers into the ER. The fragmented Golgi phenotype was generated at early times after BFA removal, without passage of marker proteins through a juxtanuclear Golgi complex.

Mentions: Since the Golgi normally sits near the microtubule-organizing center (MTOC), while the ER is distributed throughout the cell, “retrograde” protein traffic from the Golgi to the ER represents movement toward microtubule plus ends, and this movement has been shown to depend at least in part on kinesin (Lippincott-Schwartz et al., 1995). To ask what effect overexpression of dynamitin has on this process, dynamitin-transfected cells were treated with BFA, which leads to a dramatic redistribution of Golgi proteins into the ER (Klausner et al., 1992). After treatment with BFA, ST was observed in a reticular pattern characteristic of ER labeling, both in control cells and in dynamitin overexpressors (Fig. 8, A and B). No differences were observed in the duration of treatment or in the dose of BFA required to induce ST redistribution. Upon removal of BFA, ST in untransfected cells returned to a juxtanuclear distribution, while in the dynamitin overexpressors, the dispersed punctate pattern was regenerated (Fig. 8, C and D). The formation of dispersed elements in dynamitin-overexpressing cells occurred within a few minutes of BFA washout, well before the reformation of a juxtanuclear Golgi complex in untransfected cells (Fig. 8, E and F). Thus, the progression of Golgi proteins from the ER into the dispersed Golgi elements can occur under conditions where dynactin function is disrupted, and this process does not rely on passage through an intact juxtanuclear Golgi compartment.


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)

BFA treatment of dynamitin overexpressing cells.  Transfected cells were treated with BFA for 20 min (A and B), or  they were treated and allowed to recover in the absence of drug  for 1 h (C and D) or for 10 min (E and F). Dynamitin overexpression did not prevent the redistribution of Golgi markers into the  ER. The fragmented Golgi phenotype was generated at early  times after BFA removal, without passage of marker proteins  through a juxtanuclear Golgi complex.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: BFA treatment of dynamitin overexpressing cells. Transfected cells were treated with BFA for 20 min (A and B), or they were treated and allowed to recover in the absence of drug for 1 h (C and D) or for 10 min (E and F). Dynamitin overexpression did not prevent the redistribution of Golgi markers into the ER. The fragmented Golgi phenotype was generated at early times after BFA removal, without passage of marker proteins through a juxtanuclear Golgi complex.
Mentions: Since the Golgi normally sits near the microtubule-organizing center (MTOC), while the ER is distributed throughout the cell, “retrograde” protein traffic from the Golgi to the ER represents movement toward microtubule plus ends, and this movement has been shown to depend at least in part on kinesin (Lippincott-Schwartz et al., 1995). To ask what effect overexpression of dynamitin has on this process, dynamitin-transfected cells were treated with BFA, which leads to a dramatic redistribution of Golgi proteins into the ER (Klausner et al., 1992). After treatment with BFA, ST was observed in a reticular pattern characteristic of ER labeling, both in control cells and in dynamitin overexpressors (Fig. 8, A and B). No differences were observed in the duration of treatment or in the dose of BFA required to induce ST redistribution. Upon removal of BFA, ST in untransfected cells returned to a juxtanuclear distribution, while in the dynamitin overexpressors, the dispersed punctate pattern was regenerated (Fig. 8, C and D). The formation of dispersed elements in dynamitin-overexpressing cells occurred within a few minutes of BFA washout, well before the reformation of a juxtanuclear Golgi complex in untransfected cells (Fig. 8, E and F). Thus, the progression of Golgi proteins from the ER into the dispersed Golgi elements can occur under conditions where dynactin function is disrupted, and this process does not rely on passage through an intact juxtanuclear Golgi compartment.

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