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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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(a) p22 binding to  the DIC column is blocked by  p150Glued. Two identical DIC  affinity columns were constructed. One column was  blocked with excess p150Glued  (fifth through seventh lanes),  whereas the other was  blocked with BSA as a control (second through fourth  lanes). Rat brain cytosol (1  ml) was then loaded (first  lane), and the 1 M NaCl eluates (fourth and seventh lanes)  were analyzed by Western  blotting. Amounts of sample  loaded in load and flow  through lanes are equivalent  (4 μl each), as are wash and  1 M eluate lanes (20 μl each  from a total of 50 μl TCA precipitate). The results show  that, like other dynactin subunits, p22 does not bind to a  DIC column that is preblocked with p150Glued. (b and  c) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations  were carried out using antibodies to p150Glued (1.5 ml),  p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were  eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150Glued precipitate and 2 μl of anti-p22 precipitate were  loaded and analyzed by SDS-PAGE (b) followed by Western blotting (c). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150Glued antibody.
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Figure 3: (a) p22 binding to the DIC column is blocked by p150Glued. Two identical DIC affinity columns were constructed. One column was blocked with excess p150Glued (fifth through seventh lanes), whereas the other was blocked with BSA as a control (second through fourth lanes). Rat brain cytosol (1 ml) was then loaded (first lane), and the 1 M NaCl eluates (fourth and seventh lanes) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150Glued. (b and c) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE (b) followed by Western blotting (c). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150Glued antibody.

Mentions: To further characterize the p22 polypeptide, we performed a column blocking experiment as well as immunoprecipitations and immunocytochemistry. We have previously demonstrated that a dynein intermediate chain affinity column retains the dynactin complex (Karki and Holzbaur, 1995) and that this binding can be specifically blocked by pretreating the column with exogenous p150Glued (Karki et al., 1997). We used this property of the dynein intermediate chain column to evaluate whether p22 behaved as a bona fide dynactin subunit. Fig. 3 a shows that p22 from brain cytosol binds to a dynein intermediate chain (DIC) column in a salt-dependent fashion as does p150Glued. This binding, however, is blocked by pretreating the column with a recombinant fragment corresponding to the NH2-terminal half of p150Glued. We have demonstrated that this blocking is specific since other DIC-binding proteins such as CKII are not blocked from binding to the affinity column by pretreatment with excess p150Glued (Karki et al., 1997).


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)

(a) p22 binding to  the DIC column is blocked by  p150Glued. Two identical DIC  affinity columns were constructed. One column was  blocked with excess p150Glued  (fifth through seventh lanes),  whereas the other was  blocked with BSA as a control (second through fourth  lanes). Rat brain cytosol (1  ml) was then loaded (first  lane), and the 1 M NaCl eluates (fourth and seventh lanes)  were analyzed by Western  blotting. Amounts of sample  loaded in load and flow  through lanes are equivalent  (4 μl each), as are wash and  1 M eluate lanes (20 μl each  from a total of 50 μl TCA precipitate). The results show  that, like other dynactin subunits, p22 does not bind to a  DIC column that is preblocked with p150Glued. (b and  c) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations  were carried out using antibodies to p150Glued (1.5 ml),  p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were  eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150Glued precipitate and 2 μl of anti-p22 precipitate were  loaded and analyzed by SDS-PAGE (b) followed by Western blotting (c). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150Glued antibody.
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Related In: Results  -  Collection

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Figure 3: (a) p22 binding to the DIC column is blocked by p150Glued. Two identical DIC affinity columns were constructed. One column was blocked with excess p150Glued (fifth through seventh lanes), whereas the other was blocked with BSA as a control (second through fourth lanes). Rat brain cytosol (1 ml) was then loaded (first lane), and the 1 M NaCl eluates (fourth and seventh lanes) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150Glued. (b and c) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE (b) followed by Western blotting (c). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150Glued antibody.
Mentions: To further characterize the p22 polypeptide, we performed a column blocking experiment as well as immunoprecipitations and immunocytochemistry. We have previously demonstrated that a dynein intermediate chain affinity column retains the dynactin complex (Karki and Holzbaur, 1995) and that this binding can be specifically blocked by pretreating the column with exogenous p150Glued (Karki et al., 1997). We used this property of the dynein intermediate chain column to evaluate whether p22 behaved as a bona fide dynactin subunit. Fig. 3 a shows that p22 from brain cytosol binds to a dynein intermediate chain (DIC) column in a salt-dependent fashion as does p150Glued. This binding, however, is blocked by pretreating the column with a recombinant fragment corresponding to the NH2-terminal half of p150Glued. We have demonstrated that this blocking is specific since other DIC-binding proteins such as CKII are not blocked from binding to the affinity column by pretreatment with excess p150Glued (Karki et al., 1997).

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