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Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles.

Chen JL, Fucini RV, Lacomis L, Erdjument-Bromage H, Tempst P, Stamnes M - J. Cell Biol. (2005)

Bottom Line: Biol.Dynein recruitment was found to involve actin dynamics and dynactin.By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.

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Reclustering of Golgi membranes is sensitive to Cdc42 function. (A) NRK cells were transfected with a plasmid for the expression of myc-Cdc42(Q61L). The cells were treated with nocodazole and washed for the indicated times. The Golgi were labeled using an anti-GM130 antibody (red). Transfected cells (asterisks) were identified using an anti-myc antibody (green). (B) Before nocodazole treatment, NRK cells were transfected (asterisks) with HA–wild-type Cdc42 (WT), myc-Cdc42(Q61L), or HA-Cdc42(F28L) as indicated (green). The cells were allowed to recover for 60 min after the nocodazole washout as in A. The Golgi apparatus was labeled with anti-GM130 (red). Bar, 10 μm.
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fig4: Reclustering of Golgi membranes is sensitive to Cdc42 function. (A) NRK cells were transfected with a plasmid for the expression of myc-Cdc42(Q61L). The cells were treated with nocodazole and washed for the indicated times. The Golgi were labeled using an anti-GM130 antibody (red). Transfected cells (asterisks) were identified using an anti-myc antibody (green). (B) Before nocodazole treatment, NRK cells were transfected (asterisks) with HA–wild-type Cdc42 (WT), myc-Cdc42(Q61L), or HA-Cdc42(F28L) as indicated (green). The cells were allowed to recover for 60 min after the nocodazole washout as in A. The Golgi apparatus was labeled with anti-GM130 (red). Bar, 10 μm.

Mentions: Microtubules play an important role in Golgi localization within cells (Thyberg and Moskalewski, 1999). Depolymerizing microtubules with nocodazole leads to a dramatic dispersal of Golgi membranes. When nocodazole is removed, microtubules repolymerize and the dispersed Golgi recluster at the juxtanuclear MTOC via dynein-based translocation (Ho et al., 1989; Corthesy-Theulaz et al., 1992; Hafezparast et al., 2003). Because this processes involves dynein-based motility of coatomer-coated structures, we tested whether mutant Cdc42 affected it (Fig. 4).


Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles.

Chen JL, Fucini RV, Lacomis L, Erdjument-Bromage H, Tempst P, Stamnes M - J. Cell Biol. (2005)

Reclustering of Golgi membranes is sensitive to Cdc42 function. (A) NRK cells were transfected with a plasmid for the expression of myc-Cdc42(Q61L). The cells were treated with nocodazole and washed for the indicated times. The Golgi were labeled using an anti-GM130 antibody (red). Transfected cells (asterisks) were identified using an anti-myc antibody (green). (B) Before nocodazole treatment, NRK cells were transfected (asterisks) with HA–wild-type Cdc42 (WT), myc-Cdc42(Q61L), or HA-Cdc42(F28L) as indicated (green). The cells were allowed to recover for 60 min after the nocodazole washout as in A. The Golgi apparatus was labeled with anti-GM130 (red). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171931&req=5

fig4: Reclustering of Golgi membranes is sensitive to Cdc42 function. (A) NRK cells were transfected with a plasmid for the expression of myc-Cdc42(Q61L). The cells were treated with nocodazole and washed for the indicated times. The Golgi were labeled using an anti-GM130 antibody (red). Transfected cells (asterisks) were identified using an anti-myc antibody (green). (B) Before nocodazole treatment, NRK cells were transfected (asterisks) with HA–wild-type Cdc42 (WT), myc-Cdc42(Q61L), or HA-Cdc42(F28L) as indicated (green). The cells were allowed to recover for 60 min after the nocodazole washout as in A. The Golgi apparatus was labeled with anti-GM130 (red). Bar, 10 μm.
Mentions: Microtubules play an important role in Golgi localization within cells (Thyberg and Moskalewski, 1999). Depolymerizing microtubules with nocodazole leads to a dramatic dispersal of Golgi membranes. When nocodazole is removed, microtubules repolymerize and the dispersed Golgi recluster at the juxtanuclear MTOC via dynein-based translocation (Ho et al., 1989; Corthesy-Theulaz et al., 1992; Hafezparast et al., 2003). Because this processes involves dynein-based motility of coatomer-coated structures, we tested whether mutant Cdc42 affected it (Fig. 4).

Bottom Line: Biol.Dynein recruitment was found to involve actin dynamics and dynactin.By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.

Show MeSH
Related in: MedlinePlus