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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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p22 does not associate with actomyosin network at the midbody. Rapidly growing Ptk2 cells were  methanol fixed as described  previously and double immunostained for nonmuscle myosin II and for p150Glued (a–c)  or for actin and for p22 (d–f).  Anti–myosin II (mAb 1670;  Chemicon International, Inc.,  Temecula, CA) and antiactin antibodies (C4; Boehringer Mannheim Corp., Indianapolis, IN) were mouse  monoclonal and were visualized by FITC-conjugated  anti–mouse secondaries (a  and d), whereas anti-p150Glued and anti-p22 antibodies were rabbit polyclonal and were visualized by Texas red–conjugated anti–rabbit  secondaries (b and e). Superimposition of green and red channels shows that neither actin nor myosin II localize to the midbody where  dynactin is prominently localized (arrows, c and f). Bar, 5 μm.
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Figure 8: p22 does not associate with actomyosin network at the midbody. Rapidly growing Ptk2 cells were methanol fixed as described previously and double immunostained for nonmuscle myosin II and for p150Glued (a–c) or for actin and for p22 (d–f). Anti–myosin II (mAb 1670; Chemicon International, Inc., Temecula, CA) and antiactin antibodies (C4; Boehringer Mannheim Corp., Indianapolis, IN) were mouse monoclonal and were visualized by FITC-conjugated anti–mouse secondaries (a and d), whereas anti-p150Glued and anti-p22 antibodies were rabbit polyclonal and were visualized by Texas red–conjugated anti–rabbit secondaries (b and e). Superimposition of green and red channels shows that neither actin nor myosin II localize to the midbody where dynactin is prominently localized (arrows, c and f). Bar, 5 μm.

Mentions: Potentially, dynactin may localize to the cleavage furrow and subsequently to the midbody of dividing cells by its association with an actomyosin-based network, a microtubule-based network, or a direct association with the membrane-rich cortex. As a first step toward discriminating between these possibilities, we performed double immunocytochemistry on Ptk2 cells using anti-p150Glued and anti–myosin II (Fig. 8, a–c) or anti-actin and anti-p22 (Fig. 8, d–f) antibodies. The results show that at the midbody where prominent localization of dynactin subunits is observed, neither myosin II nor actin was colocalized at the level of resolution of immunofluorescence microscopy. In contrast, microtubules are localized at the midbody (see Fig. 7, e–j), supporting the possibility that dynactin is associated with the microtubular network and not the actomyosin network. The nature of dynactin's association with the membrane cortex at the cleavage furrow and the midbody still remains to be determined. Potentially, dynactin may be associated with short actin filaments at the cellular cortex that were not resolved in these studies.


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)

p22 does not associate with actomyosin network at the midbody. Rapidly growing Ptk2 cells were  methanol fixed as described  previously and double immunostained for nonmuscle myosin II and for p150Glued (a–c)  or for actin and for p22 (d–f).  Anti–myosin II (mAb 1670;  Chemicon International, Inc.,  Temecula, CA) and antiactin antibodies (C4; Boehringer Mannheim Corp., Indianapolis, IN) were mouse  monoclonal and were visualized by FITC-conjugated  anti–mouse secondaries (a  and d), whereas anti-p150Glued and anti-p22 antibodies were rabbit polyclonal and were visualized by Texas red–conjugated anti–rabbit  secondaries (b and e). Superimposition of green and red channels shows that neither actin nor myosin II localize to the midbody where  dynactin is prominently localized (arrows, c and f). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132867&req=5

Figure 8: p22 does not associate with actomyosin network at the midbody. Rapidly growing Ptk2 cells were methanol fixed as described previously and double immunostained for nonmuscle myosin II and for p150Glued (a–c) or for actin and for p22 (d–f). Anti–myosin II (mAb 1670; Chemicon International, Inc., Temecula, CA) and antiactin antibodies (C4; Boehringer Mannheim Corp., Indianapolis, IN) were mouse monoclonal and were visualized by FITC-conjugated anti–mouse secondaries (a and d), whereas anti-p150Glued and anti-p22 antibodies were rabbit polyclonal and were visualized by Texas red–conjugated anti–rabbit secondaries (b and e). Superimposition of green and red channels shows that neither actin nor myosin II localize to the midbody where dynactin is prominently localized (arrows, c and f). Bar, 5 μm.
Mentions: Potentially, dynactin may localize to the cleavage furrow and subsequently to the midbody of dividing cells by its association with an actomyosin-based network, a microtubule-based network, or a direct association with the membrane-rich cortex. As a first step toward discriminating between these possibilities, we performed double immunocytochemistry on Ptk2 cells using anti-p150Glued and anti–myosin II (Fig. 8, a–c) or anti-actin and anti-p22 (Fig. 8, d–f) antibodies. The results show that at the midbody where prominent localization of dynactin subunits is observed, neither myosin II nor actin was colocalized at the level of resolution of immunofluorescence microscopy. In contrast, microtubules are localized at the midbody (see Fig. 7, e–j), supporting the possibility that dynactin is associated with the microtubular network and not the actomyosin network. The nature of dynactin's association with the membrane cortex at the cleavage furrow and the midbody still remains to be determined. Potentially, dynactin may be associated with short actin filaments at the cellular cortex that were not resolved in these studies.

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