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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 demonstrating immunolocalization of dynein and dynactin to the cleavage furrow and  to midbody of dividing cells. A Rat2 cell undergoing cytokinesis  is stained for p22 (a) and tubulin (b). A separate Rat2 cell visualized for both p22 and tubulin staining (c). A Ptk2 cell undergoing  cytokinesis stained for p22 shows the enrichment of this polypeptide at the cleavage furrow (d). Ptk2 cells were double-stained  with antitubulin and anti-p22 (e and f), anti-p150Glued (g and h), or  antidynein heavy chain (i and j). Note the prominent localization  of both dynein and dynactin at the midbodies revealed by yellow  spots. Occasionally, striking rings of p150Glued around the midbodies were observed (h, inset), which seemed to be persistent  even after the completion of cytokinesis (k, arrow). Bar in c is for  a–c; bar in j is for e–j. Bars, 5 μm.
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Figure 7: Epifluorescence micrographs demonstrating immunolocalization of dynein and dynactin to the cleavage furrow and to midbody of dividing cells. A Rat2 cell undergoing cytokinesis is stained for p22 (a) and tubulin (b). A separate Rat2 cell visualized for both p22 and tubulin staining (c). A Ptk2 cell undergoing cytokinesis stained for p22 shows the enrichment of this polypeptide at the cleavage furrow (d). Ptk2 cells were double-stained with antitubulin and anti-p22 (e and f), anti-p150Glued (g and h), or antidynein heavy chain (i and j). Note the prominent localization of both dynein and dynactin at the midbodies revealed by yellow spots. Occasionally, striking rings of p150Glued around the midbodies were observed (h, inset), which seemed to be persistent even after the completion of cytokinesis (k, arrow). Bar in c is for a–c; bar in j is for e–j. Bars, 5 μm.

Mentions: During our immunolocalization studies on PtK2 cells using antibodies against p22, we also noted a prominent localization of p22 to the midbodies of dividing cells (Fig. 7, e and f). This was a novel localization for a dynactin subunit. The localization of p22 to the cleavage furrow appears to occur early in cytokinesis (Figs. 7, a–d, and 6 q) and is reminiscent of the localization of actin to the contractile ring. To investigate whether other dynactin subunits also localized to the midbody, we analyzed cells stained with antibodies to the p150Glued subunit of dynactin and found that this polypeptide also was localized to this structure (Fig. 7, g and h). Occasionally, striking images were observed in which p150Glued formed what appears to be a ring around the midbody (Fig. 7 h, inset, and k). The dynactin rings that form around the midbody are persistent as evidenced by their continued presence even after cell division has been completed (Fig. 7 k).


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 demonstrating immunolocalization of dynein and dynactin to the cleavage furrow and  to midbody of dividing cells. A Rat2 cell undergoing cytokinesis  is stained for p22 (a) and tubulin (b). A separate Rat2 cell visualized for both p22 and tubulin staining (c). A Ptk2 cell undergoing  cytokinesis stained for p22 shows the enrichment of this polypeptide at the cleavage furrow (d). Ptk2 cells were double-stained  with antitubulin and anti-p22 (e and f), anti-p150Glued (g and h), or  antidynein heavy chain (i and j). Note the prominent localization  of both dynein and dynactin at the midbodies revealed by yellow  spots. Occasionally, striking rings of p150Glued around the midbodies were observed (h, inset), which seemed to be persistent  even after the completion of cytokinesis (k, arrow). Bar in c is for  a–c; bar in j is for e–j. Bars, 5 μm.
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Figure 7: Epifluorescence micrographs demonstrating immunolocalization of dynein and dynactin to the cleavage furrow and to midbody of dividing cells. A Rat2 cell undergoing cytokinesis is stained for p22 (a) and tubulin (b). A separate Rat2 cell visualized for both p22 and tubulin staining (c). A Ptk2 cell undergoing cytokinesis stained for p22 shows the enrichment of this polypeptide at the cleavage furrow (d). Ptk2 cells were double-stained with antitubulin and anti-p22 (e and f), anti-p150Glued (g and h), or antidynein heavy chain (i and j). Note the prominent localization of both dynein and dynactin at the midbodies revealed by yellow spots. Occasionally, striking rings of p150Glued around the midbodies were observed (h, inset), which seemed to be persistent even after the completion of cytokinesis (k, arrow). Bar in c is for a–c; bar in j is for e–j. Bars, 5 μm.
Mentions: During our immunolocalization studies on PtK2 cells using antibodies against p22, we also noted a prominent localization of p22 to the midbodies of dividing cells (Fig. 7, e and f). This was a novel localization for a dynactin subunit. The localization of p22 to the cleavage furrow appears to occur early in cytokinesis (Figs. 7, a–d, and 6 q) and is reminiscent of the localization of actin to the contractile ring. To investigate whether other dynactin subunits also localized to the midbody, we analyzed cells stained with antibodies to the p150Glued subunit of dynactin and found that this polypeptide also was localized to this structure (Fig. 7, g and h). Occasionally, striking images were observed in which p150Glued formed what appears to be a ring around the midbody (Fig. 7 h, inset, and k). The dynactin rings that form around the midbody are persistent as evidenced by their continued presence even after cell division has been completed (Fig. 7 k).

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