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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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(a) Specificity of  the anti-p22 antibody. Rabbit  polyclonal antibodies raised  against human p22 were affinity purified with additional  steps to ensure specificity (see  Materials and Methods for details). The antibodies were  then tested for specificity using total cytosol (Cyt, 5 μl of  1:1 brain cytosol) or salt-extract  of microtubules from rat brain  (Ext, 20 μl of a 2 ml extract  from 10 rat brains). The Western blot on the right panel  shows that the p22 antibody  recognizes only one band corresponding to ∼22 kD. (b) p22 is enriched in ATP- and salt-extracts of brain microtubules. Rat brain cytosol was prepared by a low-speed centrifugation (LSS  and LSP, 4 μl each) followed by a high-speed centrifugation (HSS, 4 μl and HSP,  10 μl). Both the HSP and the LSP were resuspended to the original volumes. The  tubulin was polymerized from the cytosol by addition of taxol (Microtubules), and  the microtubules were subsequently extracted with Mg-ATP (ATP-extract, 1.7 ml)  or with NaCl (Salt-extract, 1.7 ml). Equivalent amounts of total microtubules (resuspended to the starting volume before centrifugation), ATP-extract, or salt-extract were loaded (corresponding to 10 μl of ATP-extract). The samples were  resolved by 10% SDS-PAGE and Coomassie blue stained (upper panel) or transferred onto Immobilon-P and probed with rabbit polyclonal anti-p22 as well as  anti-Arp1 antibodies (lower panels). Note that p22, like other dynactin subunits, is  associated with microtubules in an ATP- and salt-sensitive manner. (c) p22 exists  as a part of the 20-S dynactin complex. A total of 800 μl rat brain cytosol (prepared at 1:1, wt/vol) was layered on top of a 5–20% linear  sucrose density gradient and centrifuged for 18 h at 32K rpm at 4°C using a Beckman SW41.Ti rotor. 1-ml fractions were collected from  the bottom, and 12 μl of each fraction was analyzed by SDS-PAGE and Western blot using antibodies to p22, p50 (dynamitin), and  Arp1 (centractin). The p22 peak corresponds to Arp1 and p50 peak, indicating that p22 exists exclusively as a component of the 20-S dynactin complex. LSS, low-speed supernatant; LSP, low-speed pellet; HSS, high-speed supernatant; HSP, high-speed pellet.
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Figure 2: (a) Specificity of the anti-p22 antibody. Rabbit polyclonal antibodies raised against human p22 were affinity purified with additional steps to ensure specificity (see Materials and Methods for details). The antibodies were then tested for specificity using total cytosol (Cyt, 5 μl of 1:1 brain cytosol) or salt-extract of microtubules from rat brain (Ext, 20 μl of a 2 ml extract from 10 rat brains). The Western blot on the right panel shows that the p22 antibody recognizes only one band corresponding to ∼22 kD. (b) p22 is enriched in ATP- and salt-extracts of brain microtubules. Rat brain cytosol was prepared by a low-speed centrifugation (LSS and LSP, 4 μl each) followed by a high-speed centrifugation (HSS, 4 μl and HSP, 10 μl). Both the HSP and the LSP were resuspended to the original volumes. The tubulin was polymerized from the cytosol by addition of taxol (Microtubules), and the microtubules were subsequently extracted with Mg-ATP (ATP-extract, 1.7 ml) or with NaCl (Salt-extract, 1.7 ml). Equivalent amounts of total microtubules (resuspended to the starting volume before centrifugation), ATP-extract, or salt-extract were loaded (corresponding to 10 μl of ATP-extract). The samples were resolved by 10% SDS-PAGE and Coomassie blue stained (upper panel) or transferred onto Immobilon-P and probed with rabbit polyclonal anti-p22 as well as anti-Arp1 antibodies (lower panels). Note that p22, like other dynactin subunits, is associated with microtubules in an ATP- and salt-sensitive manner. (c) p22 exists as a part of the 20-S dynactin complex. A total of 800 μl rat brain cytosol (prepared at 1:1, wt/vol) was layered on top of a 5–20% linear sucrose density gradient and centrifuged for 18 h at 32K rpm at 4°C using a Beckman SW41.Ti rotor. 1-ml fractions were collected from the bottom, and 12 μl of each fraction was analyzed by SDS-PAGE and Western blot using antibodies to p22, p50 (dynamitin), and Arp1 (centractin). The p22 peak corresponds to Arp1 and p50 peak, indicating that p22 exists exclusively as a component of the 20-S dynactin complex. LSS, low-speed supernatant; LSP, low-speed pellet; HSS, high-speed supernatant; HSP, high-speed pellet.

Mentions: We confirmed the specificity of the affinity-purified polyclonal antibodies we have raised against p22 on a Western blot of rat brain cytosol (Fig. 2 a). As judged by a highly sensitive chemiluminescence detection system, anti-p22 antibody reacted with only a single band corresponding to an ∼22-kD polypeptide.


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)

(a) Specificity of  the anti-p22 antibody. Rabbit  polyclonal antibodies raised  against human p22 were affinity purified with additional  steps to ensure specificity (see  Materials and Methods for details). The antibodies were  then tested for specificity using total cytosol (Cyt, 5 μl of  1:1 brain cytosol) or salt-extract  of microtubules from rat brain  (Ext, 20 μl of a 2 ml extract  from 10 rat brains). The Western blot on the right panel  shows that the p22 antibody  recognizes only one band corresponding to ∼22 kD. (b) p22 is enriched in ATP- and salt-extracts of brain microtubules. Rat brain cytosol was prepared by a low-speed centrifugation (LSS  and LSP, 4 μl each) followed by a high-speed centrifugation (HSS, 4 μl and HSP,  10 μl). Both the HSP and the LSP were resuspended to the original volumes. The  tubulin was polymerized from the cytosol by addition of taxol (Microtubules), and  the microtubules were subsequently extracted with Mg-ATP (ATP-extract, 1.7 ml)  or with NaCl (Salt-extract, 1.7 ml). Equivalent amounts of total microtubules (resuspended to the starting volume before centrifugation), ATP-extract, or salt-extract were loaded (corresponding to 10 μl of ATP-extract). The samples were  resolved by 10% SDS-PAGE and Coomassie blue stained (upper panel) or transferred onto Immobilon-P and probed with rabbit polyclonal anti-p22 as well as  anti-Arp1 antibodies (lower panels). Note that p22, like other dynactin subunits, is  associated with microtubules in an ATP- and salt-sensitive manner. (c) p22 exists  as a part of the 20-S dynactin complex. A total of 800 μl rat brain cytosol (prepared at 1:1, wt/vol) was layered on top of a 5–20% linear  sucrose density gradient and centrifuged for 18 h at 32K rpm at 4°C using a Beckman SW41.Ti rotor. 1-ml fractions were collected from  the bottom, and 12 μl of each fraction was analyzed by SDS-PAGE and Western blot using antibodies to p22, p50 (dynamitin), and  Arp1 (centractin). The p22 peak corresponds to Arp1 and p50 peak, indicating that p22 exists exclusively as a component of the 20-S dynactin complex. LSS, low-speed supernatant; LSP, low-speed pellet; HSS, high-speed supernatant; HSP, high-speed pellet.
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Related In: Results  -  Collection

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Figure 2: (a) Specificity of the anti-p22 antibody. Rabbit polyclonal antibodies raised against human p22 were affinity purified with additional steps to ensure specificity (see Materials and Methods for details). The antibodies were then tested for specificity using total cytosol (Cyt, 5 μl of 1:1 brain cytosol) or salt-extract of microtubules from rat brain (Ext, 20 μl of a 2 ml extract from 10 rat brains). The Western blot on the right panel shows that the p22 antibody recognizes only one band corresponding to ∼22 kD. (b) p22 is enriched in ATP- and salt-extracts of brain microtubules. Rat brain cytosol was prepared by a low-speed centrifugation (LSS and LSP, 4 μl each) followed by a high-speed centrifugation (HSS, 4 μl and HSP, 10 μl). Both the HSP and the LSP were resuspended to the original volumes. The tubulin was polymerized from the cytosol by addition of taxol (Microtubules), and the microtubules were subsequently extracted with Mg-ATP (ATP-extract, 1.7 ml) or with NaCl (Salt-extract, 1.7 ml). Equivalent amounts of total microtubules (resuspended to the starting volume before centrifugation), ATP-extract, or salt-extract were loaded (corresponding to 10 μl of ATP-extract). The samples were resolved by 10% SDS-PAGE and Coomassie blue stained (upper panel) or transferred onto Immobilon-P and probed with rabbit polyclonal anti-p22 as well as anti-Arp1 antibodies (lower panels). Note that p22, like other dynactin subunits, is associated with microtubules in an ATP- and salt-sensitive manner. (c) p22 exists as a part of the 20-S dynactin complex. A total of 800 μl rat brain cytosol (prepared at 1:1, wt/vol) was layered on top of a 5–20% linear sucrose density gradient and centrifuged for 18 h at 32K rpm at 4°C using a Beckman SW41.Ti rotor. 1-ml fractions were collected from the bottom, and 12 μl of each fraction was analyzed by SDS-PAGE and Western blot using antibodies to p22, p50 (dynamitin), and Arp1 (centractin). The p22 peak corresponds to Arp1 and p50 peak, indicating that p22 exists exclusively as a component of the 20-S dynactin complex. LSS, low-speed supernatant; LSP, low-speed pellet; HSS, high-speed supernatant; HSP, high-speed pellet.
Mentions: We confirmed the specificity of the affinity-purified polyclonal antibodies we have raised against p22 on a Western blot of rat brain cytosol (Fig. 2 a). As judged by a highly sensitive chemiluminescence detection system, anti-p22 antibody reacted with only a single band corresponding to an ∼22-kD polypeptide.

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