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Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells.

Karki S, LaMonte B, Holzbaur EL - J. Cell Biol. (1998)

Bottom Line: Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis.Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures.We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150(Glued) subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.

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p22 binds to the  p150Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains  was incubated with PBS  alone (control) or with recombinant p50 (dynamitin) and  subjected to linear density sucrose gradient (5–20%) for  18 h at 4°C in a Beckman  SW41.Ti rotor at 32K rpm.  Approximately 0.9-ml fractions were collected, and the  fractions were analyzed by  SDS-PAGE followed by  Western blotting using antibodies to p150Glued, Arp1, p50, and p22. In a, all subunits probed peak exclusively  at fraction 5, corresponding to the 20-S peak. However, incubation of ATP-extract  with recombinant p50 partially disrupts the dynactin complex as indicated by the  presence of p150Glued at fractions 9–11 (b). Interestingly, p22 is also found at the  second peak at fraction 10. Note that the heavy p50 staining in b is due to excess  recombinant p50 used for dynactin disruption. (c) A p150Glued affinity column and  a BSA control column were constructed and loaded with in vitro–translated and  radio-labeled recombinant p22. The columns were extensively washed and eluted  with 1 M NaCl. The loaded material (load), flow-through (F.T.), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.
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Figure 4: p22 binds to the p150Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains was incubated with PBS alone (control) or with recombinant p50 (dynamitin) and subjected to linear density sucrose gradient (5–20%) for 18 h at 4°C in a Beckman SW41.Ti rotor at 32K rpm. Approximately 0.9-ml fractions were collected, and the fractions were analyzed by SDS-PAGE followed by Western blotting using antibodies to p150Glued, Arp1, p50, and p22. In a, all subunits probed peak exclusively at fraction 5, corresponding to the 20-S peak. However, incubation of ATP-extract with recombinant p50 partially disrupts the dynactin complex as indicated by the presence of p150Glued at fractions 9–11 (b). Interestingly, p22 is also found at the second peak at fraction 10. Note that the heavy p50 staining in b is due to excess recombinant p50 used for dynactin disruption. (c) A p150Glued affinity column and a BSA control column were constructed and loaded with in vitro–translated and radio-labeled recombinant p22. The columns were extensively washed and eluted with 1 M NaCl. The loaded material (load), flow-through (F.T.), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.

Mentions: To better define the relative position of p22 in the context of the dynactin complex, we disrupted the dynactin complex using excess recombinant p50 (dynamitin). p50 has been previously shown to dissociate the p150Glued sidearm from the centractin filament at the base of dynactin when overexpressed in cultured mammalian cells (Echeverri et al., 1996). We incubated excess recombinant p50 with a sample (ATP-extract) enriched in dynactin, and then sedimented the reaction through a linear sucrose gradient. Gradient fractions were analyzed by Western blotting with antibodies to dynactin subunits. In control reactions, dynactin subunits sedimented as a single peak near 20 S (Fig. 4 a). After incubation with excess p50, dynactin was partially disrupted (Fig. 4 b) as compared with the control (compare p150 in a and b). When the fractions were probed with an antibody to p22, in addition to the peak at 20 S we observed a peak in p22 immunoreactivity in fractions 9–11 (Fig. 4 b). Immunoblot analysis of these same fractions with antibodies to p150Glued and to p50 indicated that each of these polypeptides comigrated with p22 after disruption of the dynactin complex with excess recombinant dynamitin.


Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells.

Karki S, LaMonte B, Holzbaur EL - J. Cell Biol. (1998)

p22 binds to the  p150Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains  was incubated with PBS  alone (control) or with recombinant p50 (dynamitin) and  subjected to linear density sucrose gradient (5–20%) for  18 h at 4°C in a Beckman  SW41.Ti rotor at 32K rpm.  Approximately 0.9-ml fractions were collected, and the  fractions were analyzed by  SDS-PAGE followed by  Western blotting using antibodies to p150Glued, Arp1, p50, and p22. In a, all subunits probed peak exclusively  at fraction 5, corresponding to the 20-S peak. However, incubation of ATP-extract  with recombinant p50 partially disrupts the dynactin complex as indicated by the  presence of p150Glued at fractions 9–11 (b). Interestingly, p22 is also found at the  second peak at fraction 10. Note that the heavy p50 staining in b is due to excess  recombinant p50 used for dynactin disruption. (c) A p150Glued affinity column and  a BSA control column were constructed and loaded with in vitro–translated and  radio-labeled recombinant p22. The columns were extensively washed and eluted  with 1 M NaCl. The loaded material (load), flow-through (F.T.), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.
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Related In: Results  -  Collection

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Figure 4: p22 binds to the p150Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains was incubated with PBS alone (control) or with recombinant p50 (dynamitin) and subjected to linear density sucrose gradient (5–20%) for 18 h at 4°C in a Beckman SW41.Ti rotor at 32K rpm. Approximately 0.9-ml fractions were collected, and the fractions were analyzed by SDS-PAGE followed by Western blotting using antibodies to p150Glued, Arp1, p50, and p22. In a, all subunits probed peak exclusively at fraction 5, corresponding to the 20-S peak. However, incubation of ATP-extract with recombinant p50 partially disrupts the dynactin complex as indicated by the presence of p150Glued at fractions 9–11 (b). Interestingly, p22 is also found at the second peak at fraction 10. Note that the heavy p50 staining in b is due to excess recombinant p50 used for dynactin disruption. (c) A p150Glued affinity column and a BSA control column were constructed and loaded with in vitro–translated and radio-labeled recombinant p22. The columns were extensively washed and eluted with 1 M NaCl. The loaded material (load), flow-through (F.T.), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.
Mentions: To better define the relative position of p22 in the context of the dynactin complex, we disrupted the dynactin complex using excess recombinant p50 (dynamitin). p50 has been previously shown to dissociate the p150Glued sidearm from the centractin filament at the base of dynactin when overexpressed in cultured mammalian cells (Echeverri et al., 1996). We incubated excess recombinant p50 with a sample (ATP-extract) enriched in dynactin, and then sedimented the reaction through a linear sucrose gradient. Gradient fractions were analyzed by Western blotting with antibodies to dynactin subunits. In control reactions, dynactin subunits sedimented as a single peak near 20 S (Fig. 4 a). After incubation with excess p50, dynactin was partially disrupted (Fig. 4 b) as compared with the control (compare p150 in a and b). When the fractions were probed with an antibody to p22, in addition to the peak at 20 S we observed a peak in p22 immunoreactivity in fractions 9–11 (Fig. 4 b). Immunoblot analysis of these same fractions with antibodies to p150Glued and to p50 indicated that each of these polypeptides comigrated with p22 after disruption of the dynactin complex with excess recombinant dynamitin.

Bottom Line: Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis.Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures.We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150(Glued) subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.

Show MeSH
Related in: MedlinePlus