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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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Epifluorescence micrographs of Ptk2 cells overexpressing p22. Ptk2 cells were grown to 75% confluency and transfected  with cDNA encoding p22 fused to FLAG epitope at the NH2 terminus. Cells were fixed in 1 mM EGTA in MeOH and processed  for immunocytochemistry. The transfected cells were examined  by epifluorescence microscopy using double-label immunocytochemistry with anti-FLAG and anti-p150Glued (a and b), anti-p22 and antidynein intermediate chain antibodies (c and d), and  anti-p22 and anti-58K antibodies (e and f). No clear effects on the  distribution of p150Glued (a and b), dynein (c and d), or the Golgi  apparatus (e and f) were observed in p22-overexpressing cells.  Bar, 10 μm.
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Figure 9: Epifluorescence micrographs of Ptk2 cells overexpressing p22. Ptk2 cells were grown to 75% confluency and transfected with cDNA encoding p22 fused to FLAG epitope at the NH2 terminus. Cells were fixed in 1 mM EGTA in MeOH and processed for immunocytochemistry. The transfected cells were examined by epifluorescence microscopy using double-label immunocytochemistry with anti-FLAG and anti-p150Glued (a and b), anti-p22 and antidynein intermediate chain antibodies (c and d), and anti-p22 and anti-58K antibodies (e and f). No clear effects on the distribution of p150Glued (a and b), dynein (c and d), or the Golgi apparatus (e and f) were observed in p22-overexpressing cells. Bar, 10 μm.

Mentions: To investigate the intracellular role of p22, we used transient transfection assays. Mammalian PtK2 cells were transiently transfected with a cDNA construct encoding full-length human p22 or with a construct encoding FLAG-tagged human p22 under the control of CMV promoter. After fixation, the cells were processed for immunocytochemistry using antibodies against the FLAG epitope (M5; Eastman Kodak Corp.) and/or antibodies specific for p22. p22 was distributed throughout the cytoplasm in transfected cells expressing low to intermediate levels of the protein (Fig. 9, a, c, and e). In cells expressing very high levels of p22, the protein was found in aggregates throughout the cytoplasm (data not shown). We noted that overexpression of p22 in PtK2 cells, as well as in REF52 and Rat2 cells, caused a significant number of transfected cells to detach from the coverslips after 24 h of transfection followed by 24 h of washout, suggesting that high levels of overexpression of p22 are lethal to the cell. However, it is possible that this lethal effect might be a nonspecific result of protein aggregation.


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)

Epifluorescence micrographs of Ptk2 cells overexpressing p22. Ptk2 cells were grown to 75% confluency and transfected  with cDNA encoding p22 fused to FLAG epitope at the NH2 terminus. Cells were fixed in 1 mM EGTA in MeOH and processed  for immunocytochemistry. The transfected cells were examined  by epifluorescence microscopy using double-label immunocytochemistry with anti-FLAG and anti-p150Glued (a and b), anti-p22 and antidynein intermediate chain antibodies (c and d), and  anti-p22 and anti-58K antibodies (e and f). No clear effects on the  distribution of p150Glued (a and b), dynein (c and d), or the Golgi  apparatus (e and f) were observed in p22-overexpressing cells.  Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 9: Epifluorescence micrographs of Ptk2 cells overexpressing p22. Ptk2 cells were grown to 75% confluency and transfected with cDNA encoding p22 fused to FLAG epitope at the NH2 terminus. Cells were fixed in 1 mM EGTA in MeOH and processed for immunocytochemistry. The transfected cells were examined by epifluorescence microscopy using double-label immunocytochemistry with anti-FLAG and anti-p150Glued (a and b), anti-p22 and antidynein intermediate chain antibodies (c and d), and anti-p22 and anti-58K antibodies (e and f). No clear effects on the distribution of p150Glued (a and b), dynein (c and d), or the Golgi apparatus (e and f) were observed in p22-overexpressing cells. Bar, 10 μm.
Mentions: To investigate the intracellular role of p22, we used transient transfection assays. Mammalian PtK2 cells were transiently transfected with a cDNA construct encoding full-length human p22 or with a construct encoding FLAG-tagged human p22 under the control of CMV promoter. After fixation, the cells were processed for immunocytochemistry using antibodies against the FLAG epitope (M5; Eastman Kodak Corp.) and/or antibodies specific for p22. p22 was distributed throughout the cytoplasm in transfected cells expressing low to intermediate levels of the protein (Fig. 9, a, c, and e). In cells expressing very high levels of p22, the protein was found in aggregates throughout the cytoplasm (data not shown). We noted that overexpression of p22 in PtK2 cells, as well as in REF52 and Rat2 cells, caused a significant number of transfected cells to detach from the coverslips after 24 h of transfection followed by 24 h of washout, suggesting that high levels of overexpression of p22 are lethal to the cell. However, it is possible that this lethal effect might be a nonspecific result of protein aggregation.

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