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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 cultured Ptk2  cells stained with the p22-specific antibody UP831 (a  and c–e) and antitubulin antibody (b). Ptk2 cells were  seeded on 18 mm × 18 mm  glass coverslips and grown to  75% confluency. The cells  were then rapidly fixed in  −20°C 100% methanol and  1 mm EGTA for 5 min and  processed for immunocytochemistry. (a) An interphase Ptk2 cell stained with  rabbit polyclonal anti-p22  antibody. (b) The same cell  in a stained with anti–α-tubulin antibody (Sigma Chemical Co., St. Louis, MO) to reveal microtubules. Note the  prominent localization of p22  at the perinuclear and centrosomal regions. Prominent  localization of p22 was also observed at the kinetochores at various stages of mitosis (c, prometaphase; d, anaphase A; e, late anaphase).  Bar in b is for a and b; bar in e is for c–e. Bars, 5 μm.
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Figure 5: Epifluorescence micrographs of cultured Ptk2 cells stained with the p22-specific antibody UP831 (a and c–e) and antitubulin antibody (b). Ptk2 cells were seeded on 18 mm × 18 mm glass coverslips and grown to 75% confluency. The cells were then rapidly fixed in −20°C 100% methanol and 1 mm EGTA for 5 min and processed for immunocytochemistry. (a) An interphase Ptk2 cell stained with rabbit polyclonal anti-p22 antibody. (b) The same cell in a stained with anti–α-tubulin antibody (Sigma Chemical Co., St. Louis, MO) to reveal microtubules. Note the prominent localization of p22 at the perinuclear and centrosomal regions. Prominent localization of p22 was also observed at the kinetochores at various stages of mitosis (c, prometaphase; d, anaphase A; e, late anaphase). Bar in b is for a and b; bar in e is for c–e. Bars, 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)

Epifluorescence micrographs of cultured Ptk2  cells stained with the p22-specific antibody UP831 (a  and c–e) and antitubulin antibody (b). Ptk2 cells were  seeded on 18 mm × 18 mm  glass coverslips and grown to  75% confluency. The cells  were then rapidly fixed in  −20°C 100% methanol and  1 mm EGTA for 5 min and  processed for immunocytochemistry. (a) An interphase Ptk2 cell stained with  rabbit polyclonal anti-p22  antibody. (b) The same cell  in a stained with anti–α-tubulin antibody (Sigma Chemical Co., St. Louis, MO) to reveal microtubules. Note the  prominent localization of p22  at the perinuclear and centrosomal regions. Prominent  localization of p22 was also observed at the kinetochores at various stages of mitosis (c, prometaphase; d, anaphase A; e, late anaphase).  Bar in b is for a and b; bar in e is for c–e. Bars, 5 μm.
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getmorefigures.php?uid=PMC2132867&req=5

Figure 5: Epifluorescence micrographs of cultured Ptk2 cells stained with the p22-specific antibody UP831 (a and c–e) and antitubulin antibody (b). Ptk2 cells were seeded on 18 mm × 18 mm glass coverslips and grown to 75% confluency. The cells were then rapidly fixed in −20°C 100% methanol and 1 mm EGTA for 5 min and processed for immunocytochemistry. (a) An interphase Ptk2 cell stained with rabbit polyclonal anti-p22 antibody. (b) The same cell in a stained with anti–α-tubulin antibody (Sigma Chemical Co., St. Louis, MO) to reveal microtubules. Note the prominent localization of p22 at the perinuclear and centrosomal regions. Prominent localization of p22 was also observed at the kinetochores at various stages of mitosis (c, prometaphase; d, anaphase A; e, late anaphase). Bar in b is for a and b; bar in e is for c–e. Bars, 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