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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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Dynamics of p22 localization through mitosis. Ptk2 cells  were grown to 75% confluency and methanol fixed as in Fig. 5  and subsequently stained with anti-p22 polyclonal antibodies.  The p22 localization was visualized by FITC-conjugated anti–rabbit antibodies (left column). The same cells were also stained with  Hoechst 33258 (bis-benzimide) to facilitate chromosome visualization (right column). The kinetochore localization appears immediately after nuclear envelope breakdown in prophase (a–f),  and persists through metaphase (g–l), anaphase (m–p), and cytokinesis (q and r). Note the dramatic localization of p22 at the  cleavage furrow during cytokinesis (q). Bar, 5 μm.
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Figure 6: Dynamics of p22 localization through mitosis. Ptk2 cells were grown to 75% confluency and methanol fixed as in Fig. 5 and subsequently stained with anti-p22 polyclonal antibodies. The p22 localization was visualized by FITC-conjugated anti–rabbit antibodies (left column). The same cells were also stained with Hoechst 33258 (bis-benzimide) to facilitate chromosome visualization (right column). The kinetochore localization appears immediately after nuclear envelope breakdown in prophase (a–f), and persists through metaphase (g–l), anaphase (m–p), and cytokinesis (q and r). Note the dramatic localization of p22 at the cleavage furrow during cytokinesis (q). Bar, 5 μm.

Mentions: Immunocytochemistry with antibodies to the dynactin subunits p150Glued, Arp1, and p50 has shown that dynactin has a prominent perinuclear distribution. Immunostaining is concentrated at the centrosome, although a more diffuse punctate distribution is found throughout the cytoplasm. Immunolocalization studies using anti-p22 antibodies to label interphase PtK2 cells also results in perinuclear, centrosomal, and punctate cytoplasmic distributions, the latter indicative of a vesicular association (Fig. 5, a and b). Furthermore, as has been previously shown for the p50 (dynamitin) subunit of dynactin, p22 also localizes to the kinetochores of dividing cells (Figs. 5, c–e, and 6). However, this localization of p22 is visible early in metaphase and is persistent until late anaphase (Fig. 6), in contrast to the staining previously described for dynamitin, which was found at the kinetochores only up to the alignment of chromosomes at the metaphase plate (Echeverri et al., 1996).


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)

Dynamics of p22 localization through mitosis. Ptk2 cells  were grown to 75% confluency and methanol fixed as in Fig. 5  and subsequently stained with anti-p22 polyclonal antibodies.  The p22 localization was visualized by FITC-conjugated anti–rabbit antibodies (left column). The same cells were also stained with  Hoechst 33258 (bis-benzimide) to facilitate chromosome visualization (right column). The kinetochore localization appears immediately after nuclear envelope breakdown in prophase (a–f),  and persists through metaphase (g–l), anaphase (m–p), and cytokinesis (q and r). Note the dramatic localization of p22 at the  cleavage furrow during cytokinesis (q). Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132867&req=5

Figure 6: Dynamics of p22 localization through mitosis. Ptk2 cells were grown to 75% confluency and methanol fixed as in Fig. 5 and subsequently stained with anti-p22 polyclonal antibodies. The p22 localization was visualized by FITC-conjugated anti–rabbit antibodies (left column). The same cells were also stained with Hoechst 33258 (bis-benzimide) to facilitate chromosome visualization (right column). The kinetochore localization appears immediately after nuclear envelope breakdown in prophase (a–f), and persists through metaphase (g–l), anaphase (m–p), and cytokinesis (q and r). Note the dramatic localization of p22 at the cleavage furrow during cytokinesis (q). Bar, 5 μm.
Mentions: Immunocytochemistry with antibodies to the dynactin subunits p150Glued, Arp1, and p50 has shown that dynactin has a prominent perinuclear distribution. Immunostaining is concentrated at the centrosome, although a more diffuse punctate distribution is found throughout the cytoplasm. Immunolocalization studies using anti-p22 antibodies to label interphase PtK2 cells also results in perinuclear, centrosomal, and punctate cytoplasmic distributions, the latter indicative of a vesicular association (Fig. 5, a and b). Furthermore, as has been previously shown for the p50 (dynamitin) subunit of dynactin, p22 also localizes to the kinetochores of dividing cells (Figs. 5, c–e, and 6). However, this localization of p22 is visible early in metaphase and is persistent until late anaphase (Fig. 6), in contrast to the staining previously described for dynamitin, which was found at the kinetochores only up to the alignment of chromosomes at the metaphase plate (Echeverri et al., 1996).

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