Limits...
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.

Show MeSH

Related in: MedlinePlus

The effects of dynamitin overexpression are mimicked  by microinjection of a function-blocking DIC antibody. HeLa  cells were injected with a mixture of anti-DIC and fluorescent  dextran, incubated for 4 h at 37°C, and then labeled for sialyltransferase (B) or LAMP (D). FITC-dextran-injected cells are  shown in A and C (Note that the FITC signal was weak; the apparent punctate stain in A is an artifact of the strong LAMP fluorescence.) Anti-DIC disrupted the Golgi and peripheralized the  lysosomes in a manner strikingly similar to dynamitin overexpression. (E and F) Quantitative comparison of the Golgi (E) and the  lysosome (F) phenotypes resulting from microinjection and transfection. Cells were either injected with antibodies (E, right-hand  portion; F, open symbols), or transfected with dynamitin or β-galactosidase (E, left-hand portion; F, closed symbols), and individual  cells positive for the transfected proteins or for injected dextran  were scored for the severity of the organelle redistribution, as described in Materials and Methods. For transfected cells, 50–60  cells were scored in each of three independent experiments. Data  represent average scores ± 1 SD. For injected cells, data represent an average of 60–70 cells accumulated from two independent  experiments. The effects of interfering with dynein and with dynactin were strikingly similar, and they were specific to these protein complexes.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139801&req=5

Figure 10: The effects of dynamitin overexpression are mimicked by microinjection of a function-blocking DIC antibody. HeLa cells were injected with a mixture of anti-DIC and fluorescent dextran, incubated for 4 h at 37°C, and then labeled for sialyltransferase (B) or LAMP (D). FITC-dextran-injected cells are shown in A and C (Note that the FITC signal was weak; the apparent punctate stain in A is an artifact of the strong LAMP fluorescence.) Anti-DIC disrupted the Golgi and peripheralized the lysosomes in a manner strikingly similar to dynamitin overexpression. (E and F) Quantitative comparison of the Golgi (E) and the lysosome (F) phenotypes resulting from microinjection and transfection. Cells were either injected with antibodies (E, right-hand portion; F, open symbols), or transfected with dynamitin or β-galactosidase (E, left-hand portion; F, closed symbols), and individual cells positive for the transfected proteins or for injected dextran were scored for the severity of the organelle redistribution, as described in Materials and Methods. For transfected cells, 50–60 cells were scored in each of three independent experiments. Data represent average scores ± 1 SD. For injected cells, data represent an average of 60–70 cells accumulated from two independent experiments. The effects of interfering with dynein and with dynactin were strikingly similar, and they were specific to these protein complexes.

Mentions: When the dynamitin-overexpressing cells were labeled with anti-LAMP antibody, a dramatic redistribution of lysosomes and late endosomes was revealed. In control β-galactosidase–transfected cells (Fig. 2, I and J), late endocytic organelles were clustered in the juxtanuclear region. This pattern was also observed in untransfected controls (see Figs. 5 D and 10 D). After dynamitin transfection, however, late endocytic organelles were shifted away from the cell center, accumulating at the extreme peripheral processes of the cell (Fig. 2, G and H). In contrast to early endosomes, which became enriched in the cell periphery, the entire population of late endosomes and lysosomes was often found just under the cell surface. Although it was particularly noticeable in elongated cells (e.g., Fig. 2 H), this phenotype was observed in cells having a variety of different shapes (see Fig. 2 H, top cell, and Fig. 5 D). The observed redistribution of endocytic organelles resembled the effects of serum deprivation or cytoplasmic acidification (Heuser, 1989), but was much more dramatic. Indeed, when cells overexpressing dynamitin were incubated in acetate-Ringers solution to acidify the cytoplasm, the effect was the same as with dynamitin alone, and the net inward movement that normally occurs upon realkalinization was completely blocked (data 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)

The effects of dynamitin overexpression are mimicked  by microinjection of a function-blocking DIC antibody. HeLa  cells were injected with a mixture of anti-DIC and fluorescent  dextran, incubated for 4 h at 37°C, and then labeled for sialyltransferase (B) or LAMP (D). FITC-dextran-injected cells are  shown in A and C (Note that the FITC signal was weak; the apparent punctate stain in A is an artifact of the strong LAMP fluorescence.) Anti-DIC disrupted the Golgi and peripheralized the  lysosomes in a manner strikingly similar to dynamitin overexpression. (E and F) Quantitative comparison of the Golgi (E) and the  lysosome (F) phenotypes resulting from microinjection and transfection. Cells were either injected with antibodies (E, right-hand  portion; F, open symbols), or transfected with dynamitin or β-galactosidase (E, left-hand portion; F, closed symbols), and individual  cells positive for the transfected proteins or for injected dextran  were scored for the severity of the organelle redistribution, as described in Materials and Methods. For transfected cells, 50–60  cells were scored in each of three independent experiments. Data  represent average scores ± 1 SD. For injected cells, data represent an average of 60–70 cells accumulated from two independent  experiments. The effects of interfering with dynein and with dynactin were strikingly similar, and they were specific to these protein complexes.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: The effects of dynamitin overexpression are mimicked by microinjection of a function-blocking DIC antibody. HeLa cells were injected with a mixture of anti-DIC and fluorescent dextran, incubated for 4 h at 37°C, and then labeled for sialyltransferase (B) or LAMP (D). FITC-dextran-injected cells are shown in A and C (Note that the FITC signal was weak; the apparent punctate stain in A is an artifact of the strong LAMP fluorescence.) Anti-DIC disrupted the Golgi and peripheralized the lysosomes in a manner strikingly similar to dynamitin overexpression. (E and F) Quantitative comparison of the Golgi (E) and the lysosome (F) phenotypes resulting from microinjection and transfection. Cells were either injected with antibodies (E, right-hand portion; F, open symbols), or transfected with dynamitin or β-galactosidase (E, left-hand portion; F, closed symbols), and individual cells positive for the transfected proteins or for injected dextran were scored for the severity of the organelle redistribution, as described in Materials and Methods. For transfected cells, 50–60 cells were scored in each of three independent experiments. Data represent average scores ± 1 SD. For injected cells, data represent an average of 60–70 cells accumulated from two independent experiments. The effects of interfering with dynein and with dynactin were strikingly similar, and they were specific to these protein complexes.
Mentions: When the dynamitin-overexpressing cells were labeled with anti-LAMP antibody, a dramatic redistribution of lysosomes and late endosomes was revealed. In control β-galactosidase–transfected cells (Fig. 2, I and J), late endocytic organelles were clustered in the juxtanuclear region. This pattern was also observed in untransfected controls (see Figs. 5 D and 10 D). After dynamitin transfection, however, late endocytic organelles were shifted away from the cell center, accumulating at the extreme peripheral processes of the cell (Fig. 2, G and H). In contrast to early endosomes, which became enriched in the cell periphery, the entire population of late endosomes and lysosomes was often found just under the cell surface. Although it was particularly noticeable in elongated cells (e.g., Fig. 2 H), this phenotype was observed in cells having a variety of different shapes (see Fig. 2 H, top cell, and Fig. 5 D). The observed redistribution of endocytic organelles resembled the effects of serum deprivation or cytoplasmic acidification (Heuser, 1989), but was much more dramatic. Indeed, when cells overexpressing dynamitin were incubated in acetate-Ringers solution to acidify the cytoplasm, the effect was the same as with dynamitin alone, and the net inward movement that normally occurs upon realkalinization was completely blocked (data 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