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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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Molecular characterization of p22. (a) Northern blot  analysis of rat tissues. A 640-bp EcoRI/ApaI fragment of EST  clone 121348 was used to probe a multiple tissue Northern blot  (CLONTECH Laboratories). The position of the p22 mRNA  transcript, which corresponds approximately to 1 kb, is shown.  Note the higher levels of mRNA present in heart and pancreas  compared with the levels in the brain. (b)A human cDNA encoding p22 was fully sequenced from both directions. The resulting DNA and the predicted amino acid sequences are shown.  This clone has a complete open reading frame, the asterisk shows  the predicted translational start, and the filled square marks the  stop codon. The two underlined segments correspond to the two  nine-residue peptides that were microsequenced from the band  corresponding to the 22-kD polypeptide isolated from affinity-purified dynactin. Numbers to the right reflect the positions of  nucleotides, while the numbers to the left refer to the corresponding amino acid residues. (c) The predicted amino acid sequence was analyzed for secondary structure using the DNASTAR™ sequence analysis package. The analysis reveals that  p22 is primarily an α-helical protein with very little predicted  coiled coil. Most of the predicted turns of the protein occur between amino acid 120 and 155. The hydrophilicity plot predicts  that p22 is mostly hydrophilic except for a central 45–amino acid  residue stretch. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF082513.
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Figure 1: Molecular characterization of p22. (a) Northern blot analysis of rat tissues. A 640-bp EcoRI/ApaI fragment of EST clone 121348 was used to probe a multiple tissue Northern blot (CLONTECH Laboratories). The position of the p22 mRNA transcript, which corresponds approximately to 1 kb, is shown. Note the higher levels of mRNA present in heart and pancreas compared with the levels in the brain. (b)A human cDNA encoding p22 was fully sequenced from both directions. The resulting DNA and the predicted amino acid sequences are shown. This clone has a complete open reading frame, the asterisk shows the predicted translational start, and the filled square marks the stop codon. The two underlined segments correspond to the two nine-residue peptides that were microsequenced from the band corresponding to the 22-kD polypeptide isolated from affinity-purified dynactin. Numbers to the right reflect the positions of nucleotides, while the numbers to the left refer to the corresponding amino acid residues. (c) The predicted amino acid sequence was analyzed for secondary structure using the DNASTAR™ sequence analysis package. The analysis reveals that p22 is primarily an α-helical protein with very little predicted coiled coil. Most of the predicted turns of the protein occur between amino acid 120 and 155. The hydrophilicity plot predicts that p22 is mostly hydrophilic except for a central 45–amino acid residue stretch. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF082513.

Mentions: A multiple tissue mRNA blot as shown in Fig. 1 a was probed with a cDNA encoding p22 to determine the approximate size and tissue distribution of p22-encoding mRNA. Full-length p22 mRNA is ∼1 kb in length. While mRNAs encoding p22 are apparently ubiquitously expressed, expression levels are highest in muscle and pancreas, while lower levels were detected in brain. mRNAs encoding other dynactin subunits (Gill et al., 1991; Clark and Meyer, 1992; Echeverri et al., 1996) are also enriched in both cardiac and skeletal muscles, in addition to brain tissue. The significance of dynactin transcript enrichment in muscle tissues is not known.


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)

Molecular characterization of p22. (a) Northern blot  analysis of rat tissues. A 640-bp EcoRI/ApaI fragment of EST  clone 121348 was used to probe a multiple tissue Northern blot  (CLONTECH Laboratories). The position of the p22 mRNA  transcript, which corresponds approximately to 1 kb, is shown.  Note the higher levels of mRNA present in heart and pancreas  compared with the levels in the brain. (b)A human cDNA encoding p22 was fully sequenced from both directions. The resulting DNA and the predicted amino acid sequences are shown.  This clone has a complete open reading frame, the asterisk shows  the predicted translational start, and the filled square marks the  stop codon. The two underlined segments correspond to the two  nine-residue peptides that were microsequenced from the band  corresponding to the 22-kD polypeptide isolated from affinity-purified dynactin. Numbers to the right reflect the positions of  nucleotides, while the numbers to the left refer to the corresponding amino acid residues. (c) The predicted amino acid sequence was analyzed for secondary structure using the DNASTAR™ sequence analysis package. The analysis reveals that  p22 is primarily an α-helical protein with very little predicted  coiled coil. Most of the predicted turns of the protein occur between amino acid 120 and 155. The hydrophilicity plot predicts  that p22 is mostly hydrophilic except for a central 45–amino acid  residue stretch. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF082513.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132867&req=5

Figure 1: Molecular characterization of p22. (a) Northern blot analysis of rat tissues. A 640-bp EcoRI/ApaI fragment of EST clone 121348 was used to probe a multiple tissue Northern blot (CLONTECH Laboratories). The position of the p22 mRNA transcript, which corresponds approximately to 1 kb, is shown. Note the higher levels of mRNA present in heart and pancreas compared with the levels in the brain. (b)A human cDNA encoding p22 was fully sequenced from both directions. The resulting DNA and the predicted amino acid sequences are shown. This clone has a complete open reading frame, the asterisk shows the predicted translational start, and the filled square marks the stop codon. The two underlined segments correspond to the two nine-residue peptides that were microsequenced from the band corresponding to the 22-kD polypeptide isolated from affinity-purified dynactin. Numbers to the right reflect the positions of nucleotides, while the numbers to the left refer to the corresponding amino acid residues. (c) The predicted amino acid sequence was analyzed for secondary structure using the DNASTAR™ sequence analysis package. The analysis reveals that p22 is primarily an α-helical protein with very little predicted coiled coil. Most of the predicted turns of the protein occur between amino acid 120 and 155. The hydrophilicity plot predicts that p22 is mostly hydrophilic except for a central 45–amino acid residue stretch. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF082513.
Mentions: A multiple tissue mRNA blot as shown in Fig. 1 a was probed with a cDNA encoding p22 to determine the approximate size and tissue distribution of p22-encoding mRNA. Full-length p22 mRNA is ∼1 kb in length. While mRNAs encoding p22 are apparently ubiquitously expressed, expression levels are highest in muscle and pancreas, while lower levels were detected in brain. mRNAs encoding other dynactin subunits (Gill et al., 1991; Clark and Meyer, 1992; Echeverri et al., 1996) are also enriched in both cardiac and skeletal muscles, in addition to brain tissue. The significance of dynactin transcript enrichment in muscle tissues is not known.

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