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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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Disruption of endocytic organelles by dynamitin overexpression. HeLa  cells were transfected with  dynamitin (A–H) or β-galactosidase (I and J). Transfected cells are identified by  labeling with antidynamitin  (C and G), anti–β-galactosidase (I), or by the characteristic dynamitin-induced fragmentation of the Golgi (A  and E). A and B show double  labeling for ST and TGN38,  respectively. In transfected  cells, one pool of TGN38  colocalizes with ST (A and B,  arrows), while another shifts  to the periphery (B, arrowheads). (C–F) Effect of dynamitin on TfR distribution.  TfR-labeled endosomes (D  and F) are lost from the juxtanuclear recycling compartment, and in some cells, they  accumulate at the tips of cell  processes (arrows). This peripheral distribution is only  infrequently observed in untransfected cells (broad arrow, F; see also Table I).  Double labeling for TfR (F)  and TGN38 (E) reveals that  the two markers accumulate  at overlapping peripheral  sites (arrows). (H and J) The  distribution of LAMP-1 in  dynamitin overexpressors  and control transfectants, respectively. Note the extreme  shift of LAMP-positive late  endosomes and lysosomes  into the periphery. The cell  elongation shown in H was  sometimes observed in dynamitin-overexpressing cells,  though in HeLa cells, this effect was infrequent and was  not required for maximal organelle redistribution.
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Figure 2: Disruption of endocytic organelles by dynamitin overexpression. HeLa cells were transfected with dynamitin (A–H) or β-galactosidase (I and J). Transfected cells are identified by labeling with antidynamitin (C and G), anti–β-galactosidase (I), or by the characteristic dynamitin-induced fragmentation of the Golgi (A and E). A and B show double labeling for ST and TGN38, respectively. In transfected cells, one pool of TGN38 colocalizes with ST (A and B, arrows), while another shifts to the periphery (B, arrowheads). (C–F) Effect of dynamitin on TfR distribution. TfR-labeled endosomes (D and F) are lost from the juxtanuclear recycling compartment, and in some cells, they accumulate at the tips of cell processes (arrows). This peripheral distribution is only infrequently observed in untransfected cells (broad arrow, F; see also Table I). Double labeling for TfR (F) and TGN38 (E) reveals that the two markers accumulate at overlapping peripheral sites (arrows). (H and J) The distribution of LAMP-1 in dynamitin overexpressors and control transfectants, respectively. Note the extreme shift of LAMP-positive late endosomes and lysosomes into the periphery. The cell elongation shown in H was sometimes observed in dynamitin-overexpressing cells, though in HeLa cells, this effect was infrequent and was not required for maximal organelle redistribution.

Mentions: Scoring of Golgi disruption and lysosome peripheralization was performed in a blind study. Cells were transfected with dynamitin or β-galactosidase, and were double labeled for the transfected proteins and either ST (anti-G tag) or LAMP. Without knowing which transfected protein was being scored, cells that were positive for the transfected protein were first scored arbitrarily as either “bright” or “dim” (corresponding to “high” and “moderate” overexpressors respectively), and then the phenotype of the Golgi or lysosomes was assessed. Cells exhibiting either one or two elongated juxtanuclear Golgi structures were scored as “contiguous,” those with a small number (three to five) of large Golgi elements as “partially fragmented,” and cells with numerous ST positive structures scattered throughout the cytoplasm were scored as “fragmented.” According to this system, the two transfected cells in Fig. 1 B would be scored as “fragmented,” while all of the untransfected cells would be scored as unfragmented. For lysosomes, cells were scored on a scale of 1–5, with a score of 1 given to cells where nearly all LAMP-positive structures were clustered in the juxtanuclear region, 2 to cells where lysosomes were primarily concentrated in the juxtanuclear region, 3 where lysosomes were spread, evenly or unevenly, through all parts of the cell, 4 where lysosomes were primarily concentrated in the peripheral cell processes, and 5 where lysosomes were almost exclusively concentrated in the peripheral processes. The cells shown in Fig. 2 H would receive scores of 4 and 5 on this scale. In each case, 50–60 cells were scored in each of three independent experiments. Data represent an average ± 1 SD.


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)

Disruption of endocytic organelles by dynamitin overexpression. HeLa  cells were transfected with  dynamitin (A–H) or β-galactosidase (I and J). Transfected cells are identified by  labeling with antidynamitin  (C and G), anti–β-galactosidase (I), or by the characteristic dynamitin-induced fragmentation of the Golgi (A  and E). A and B show double  labeling for ST and TGN38,  respectively. In transfected  cells, one pool of TGN38  colocalizes with ST (A and B,  arrows), while another shifts  to the periphery (B, arrowheads). (C–F) Effect of dynamitin on TfR distribution.  TfR-labeled endosomes (D  and F) are lost from the juxtanuclear recycling compartment, and in some cells, they  accumulate at the tips of cell  processes (arrows). This peripheral distribution is only  infrequently observed in untransfected cells (broad arrow, F; see also Table I).  Double labeling for TfR (F)  and TGN38 (E) reveals that  the two markers accumulate  at overlapping peripheral  sites (arrows). (H and J) The  distribution of LAMP-1 in  dynamitin overexpressors  and control transfectants, respectively. Note the extreme  shift of LAMP-positive late  endosomes and lysosomes  into the periphery. The cell  elongation shown in H was  sometimes observed in dynamitin-overexpressing cells,  though in HeLa cells, this effect was infrequent and was  not required for maximal organelle redistribution.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139801&req=5

Figure 2: Disruption of endocytic organelles by dynamitin overexpression. HeLa cells were transfected with dynamitin (A–H) or β-galactosidase (I and J). Transfected cells are identified by labeling with antidynamitin (C and G), anti–β-galactosidase (I), or by the characteristic dynamitin-induced fragmentation of the Golgi (A and E). A and B show double labeling for ST and TGN38, respectively. In transfected cells, one pool of TGN38 colocalizes with ST (A and B, arrows), while another shifts to the periphery (B, arrowheads). (C–F) Effect of dynamitin on TfR distribution. TfR-labeled endosomes (D and F) are lost from the juxtanuclear recycling compartment, and in some cells, they accumulate at the tips of cell processes (arrows). This peripheral distribution is only infrequently observed in untransfected cells (broad arrow, F; see also Table I). Double labeling for TfR (F) and TGN38 (E) reveals that the two markers accumulate at overlapping peripheral sites (arrows). (H and J) The distribution of LAMP-1 in dynamitin overexpressors and control transfectants, respectively. Note the extreme shift of LAMP-positive late endosomes and lysosomes into the periphery. The cell elongation shown in H was sometimes observed in dynamitin-overexpressing cells, though in HeLa cells, this effect was infrequent and was not required for maximal organelle redistribution.
Mentions: Scoring of Golgi disruption and lysosome peripheralization was performed in a blind study. Cells were transfected with dynamitin or β-galactosidase, and were double labeled for the transfected proteins and either ST (anti-G tag) or LAMP. Without knowing which transfected protein was being scored, cells that were positive for the transfected protein were first scored arbitrarily as either “bright” or “dim” (corresponding to “high” and “moderate” overexpressors respectively), and then the phenotype of the Golgi or lysosomes was assessed. Cells exhibiting either one or two elongated juxtanuclear Golgi structures were scored as “contiguous,” those with a small number (three to five) of large Golgi elements as “partially fragmented,” and cells with numerous ST positive structures scattered throughout the cytoplasm were scored as “fragmented.” According to this system, the two transfected cells in Fig. 1 B would be scored as “fragmented,” while all of the untransfected cells would be scored as unfragmented. For lysosomes, cells were scored on a scale of 1–5, with a score of 1 given to cells where nearly all LAMP-positive structures were clustered in the juxtanuclear region, 2 to cells where lysosomes were primarily concentrated in the juxtanuclear region, 3 where lysosomes were spread, evenly or unevenly, through all parts of the cell, 4 where lysosomes were primarily concentrated in the peripheral cell processes, and 5 where lysosomes were almost exclusively concentrated in the peripheral processes. The cells shown in Fig. 2 H would receive scores of 4 and 5 on this scale. In each case, 50–60 cells were scored in each of three independent experiments. Data represent an average ± 1 SD.

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