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Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis.

Sekiya-Kawasaki M, Groen AC, Cope MJ, Kaksonen M, Watson HA, Zhang C, Shokat KM, Wendland B, McDonald KL, McCaffery JM, Drubin DG - J. Cell Biol. (2003)

Bottom Line: Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly.Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins.Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.

ABSTRACT
We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.

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Reversible actin clump formation from cortical actin patches upon inhibition of Prk1p activity. A and C are the selected frames from Video 1 and Video 2, respectively. (A–C) ark1Δ prk1-as1 cells expressing Abp1-GFP were imaged. (A and B) Cells before or after treatment with 1NA-PP1 for the indicated times. (A) The two Abp1 patches (top, arrowheads) or already formed clumps (bottom, arrowheads) fuse to become one entity (asterisks) upon addition of 1NA-PP1. (B) An Abp1 clump (arrowhead) moves from the daughter to the mother cell. (C) Cells were pretreated with 1NA-PP1 for 30 min, followed by washout of the inhibitor. An Abp1 clump at the neck (arrowhead) disappears during the time course. (D) ark1Δ prk1-as1 cells expressing Abp1-CFP (red) and Sla2-YFP (green) were imaged after a 10-min treatment with 1NA-PP1. Mock-treated cells are also shown. The actin clumps are marked with arrowheads. Exposure times for each panel: 1 s for A and C, 66 ms for B, and 300–700 ms for D. Strains: ark1Δ prk1-as1 ABP1-GFP, DDY2600 (A and C) and DDY2601 (B); ark1Δ prk1-as1 ABP1-CFP SLA2-YFP, DDY2603 (D). Bars, 5 μm.
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fig2: Reversible actin clump formation from cortical actin patches upon inhibition of Prk1p activity. A and C are the selected frames from Video 1 and Video 2, respectively. (A–C) ark1Δ prk1-as1 cells expressing Abp1-GFP were imaged. (A and B) Cells before or after treatment with 1NA-PP1 for the indicated times. (A) The two Abp1 patches (top, arrowheads) or already formed clumps (bottom, arrowheads) fuse to become one entity (asterisks) upon addition of 1NA-PP1. (B) An Abp1 clump (arrowhead) moves from the daughter to the mother cell. (C) Cells were pretreated with 1NA-PP1 for 30 min, followed by washout of the inhibitor. An Abp1 clump at the neck (arrowhead) disappears during the time course. (D) ark1Δ prk1-as1 cells expressing Abp1-CFP (red) and Sla2-YFP (green) were imaged after a 10-min treatment with 1NA-PP1. Mock-treated cells are also shown. The actin clumps are marked with arrowheads. Exposure times for each panel: 1 s for A and C, 66 ms for B, and 300–700 ms for D. Strains: ark1Δ prk1-as1 ABP1-GFP, DDY2600 (A and C) and DDY2601 (B); ark1Δ prk1-as1 ABP1-CFP SLA2-YFP, DDY2603 (D). Bars, 5 μm.

Mentions: To investigate actin patch dynamics as a function of Prk1p inhibition, we performed real-time analyses of ark1Δ prk1-as1 cells expressing Abp1-GFP (Fig. 2). Similar results were obtained with ark1Δ prk1-as3 cells (unpublished data). Abp1p is a component of cortical actin patches (Drubin et al., 1988). Within 1 to 2 min of 1NA-PP1 addition to ark1Δ prk1-as1 cells, Abp1-GFP patches aggregated into clumps (Fig. 2 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200305077/DC1). Once formed in the daughter cell, the actin clumps invariably moved toward the bud neck, and then into the mother cell (Fig. 2 B), consistent with the observation for fixed cells (Fig. 1 F), which exhibited an increase of mother clumps and a decrease of daughter clumps during the time course. The mechanistic basis for this movement is unknown, but does not seem to involve microtubules because the process was not sensitive to the microtubule-depolymerizing drug nocodazole (unpublished data). Within 1 min of Prk1p reactivation by inhibitor washout, the clumps disassembled, and the normal polarized distribution of Abp1 patches was restored (Fig. 2 C and Video 2). Rhodamine-phalloidin staining of fixed ark1Δ prk1-as1 cells confirmed that F-actin undergoes reversible aggregation upon 1NA-PP1 addition (unpublished data).


Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis.

Sekiya-Kawasaki M, Groen AC, Cope MJ, Kaksonen M, Watson HA, Zhang C, Shokat KM, Wendland B, McDonald KL, McCaffery JM, Drubin DG - J. Cell Biol. (2003)

Reversible actin clump formation from cortical actin patches upon inhibition of Prk1p activity. A and C are the selected frames from Video 1 and Video 2, respectively. (A–C) ark1Δ prk1-as1 cells expressing Abp1-GFP were imaged. (A and B) Cells before or after treatment with 1NA-PP1 for the indicated times. (A) The two Abp1 patches (top, arrowheads) or already formed clumps (bottom, arrowheads) fuse to become one entity (asterisks) upon addition of 1NA-PP1. (B) An Abp1 clump (arrowhead) moves from the daughter to the mother cell. (C) Cells were pretreated with 1NA-PP1 for 30 min, followed by washout of the inhibitor. An Abp1 clump at the neck (arrowhead) disappears during the time course. (D) ark1Δ prk1-as1 cells expressing Abp1-CFP (red) and Sla2-YFP (green) were imaged after a 10-min treatment with 1NA-PP1. Mock-treated cells are also shown. The actin clumps are marked with arrowheads. Exposure times for each panel: 1 s for A and C, 66 ms for B, and 300–700 ms for D. Strains: ark1Δ prk1-as1 ABP1-GFP, DDY2600 (A and C) and DDY2601 (B); ark1Δ prk1-as1 ABP1-CFP SLA2-YFP, DDY2603 (D). Bars, 5 μm.
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fig2: Reversible actin clump formation from cortical actin patches upon inhibition of Prk1p activity. A and C are the selected frames from Video 1 and Video 2, respectively. (A–C) ark1Δ prk1-as1 cells expressing Abp1-GFP were imaged. (A and B) Cells before or after treatment with 1NA-PP1 for the indicated times. (A) The two Abp1 patches (top, arrowheads) or already formed clumps (bottom, arrowheads) fuse to become one entity (asterisks) upon addition of 1NA-PP1. (B) An Abp1 clump (arrowhead) moves from the daughter to the mother cell. (C) Cells were pretreated with 1NA-PP1 for 30 min, followed by washout of the inhibitor. An Abp1 clump at the neck (arrowhead) disappears during the time course. (D) ark1Δ prk1-as1 cells expressing Abp1-CFP (red) and Sla2-YFP (green) were imaged after a 10-min treatment with 1NA-PP1. Mock-treated cells are also shown. The actin clumps are marked with arrowheads. Exposure times for each panel: 1 s for A and C, 66 ms for B, and 300–700 ms for D. Strains: ark1Δ prk1-as1 ABP1-GFP, DDY2600 (A and C) and DDY2601 (B); ark1Δ prk1-as1 ABP1-CFP SLA2-YFP, DDY2603 (D). Bars, 5 μm.
Mentions: To investigate actin patch dynamics as a function of Prk1p inhibition, we performed real-time analyses of ark1Δ prk1-as1 cells expressing Abp1-GFP (Fig. 2). Similar results were obtained with ark1Δ prk1-as3 cells (unpublished data). Abp1p is a component of cortical actin patches (Drubin et al., 1988). Within 1 to 2 min of 1NA-PP1 addition to ark1Δ prk1-as1 cells, Abp1-GFP patches aggregated into clumps (Fig. 2 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200305077/DC1). Once formed in the daughter cell, the actin clumps invariably moved toward the bud neck, and then into the mother cell (Fig. 2 B), consistent with the observation for fixed cells (Fig. 1 F), which exhibited an increase of mother clumps and a decrease of daughter clumps during the time course. The mechanistic basis for this movement is unknown, but does not seem to involve microtubules because the process was not sensitive to the microtubule-depolymerizing drug nocodazole (unpublished data). Within 1 min of Prk1p reactivation by inhibitor washout, the clumps disassembled, and the normal polarized distribution of Abp1 patches was restored (Fig. 2 C and Video 2). Rhodamine-phalloidin staining of fixed ark1Δ prk1-as1 cells confirmed that F-actin undergoes reversible aggregation upon 1NA-PP1 addition (unpublished data).

Bottom Line: Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly.Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins.Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.

ABSTRACT
We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.

Show MeSH
Related in: MedlinePlus