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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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Actin-associated membrane accumulation upon inhibition of Prk1p activity. (A) FM4-64 labeling of ark1Δ prk1-as3 cells expressing Abp1-GFP. Cells were treated with media containing FM4-64 and 1NA-PP1 in a flow chamber. FM4-64 (red) and Abp1-GFP (green) were visualized at 0 and 5 min. Mock-treated cells are also shown. (B–J) Conventional EM of cells post-treated with 1% tannic acid. (B) A wild-type cell shows normal, unremarkable morphology. (C–H) ark1Δ prk1Δ cells reveal electron-dense areas (encircled by dashed line in C) containing a heterogeneous population of ∼100-nm vesicles (shown at higher magnification in D and indicated by arrows in C and D), as well as numerous microfilament profiles (marked by arrowheads in D–H). (I and J) ark1Δ prk1-as3 cells treated with 1NA-PP1 for 10 min also reveal ∼100-nm vesicles and microfilament profiles (marked by arrowheads). For more examples, also see Fig. S1. (K–N) EM of cells fixed by high pressure freezing and freeze substitution. ark1Δ prk1Δ cells reveal ribosome exclusion areas (encircled by dashed line in K) containing ∼100-nm vesicles (some of which are indicated by arrows in K–N). Ribosomes appear as small, uniform, electron-dense spots distributed throughout most of the cytoplasm. (O–Q) Indirect immunogold labeling of ultrathin cryosections of ark1Δ prk1Δ cells demonstrates the localization of actin within a slightly electron-dense/vesicle-enriched region (encircled by dashed line in O). In many instances, vesicles/membranous elements are detected within actin-labeled region (arrows in O and Q; boxed areas P and Q). (R) Double-indirect immunogold labeling with ark1Δ prk1Δ SLA1-GFP cells reveals occasional Sla1p-containing vesicles (5-nm gold, arrowheads) amid a clustered/electron-dense actin region (10-nm gold). Strains: ark1Δ prk1-as3 ABP1-GFP, DDY2606 (A); Wild-type, DDY904 (B); ark1Δ prk1Δ, DDY2541 (C–H and O–Q) and DDY1564 (K–N); ark1Δ prk1-as3, DDY2597 (I and J); ark1Δ prk1Δ SLA1-GFP, DDY2611 (R). n, nucleus; V, vacuole; pm, plasma membrane. Bars: (A) 5 μm; (B) 1 μm; (C) 0.2 μm; (D) 0.5 μm; (E–J) 0.1 μm; (K) 0.5 μm; (L–R) 0.1 μm.
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fig4: Actin-associated membrane accumulation upon inhibition of Prk1p activity. (A) FM4-64 labeling of ark1Δ prk1-as3 cells expressing Abp1-GFP. Cells were treated with media containing FM4-64 and 1NA-PP1 in a flow chamber. FM4-64 (red) and Abp1-GFP (green) were visualized at 0 and 5 min. Mock-treated cells are also shown. (B–J) Conventional EM of cells post-treated with 1% tannic acid. (B) A wild-type cell shows normal, unremarkable morphology. (C–H) ark1Δ prk1Δ cells reveal electron-dense areas (encircled by dashed line in C) containing a heterogeneous population of ∼100-nm vesicles (shown at higher magnification in D and indicated by arrows in C and D), as well as numerous microfilament profiles (marked by arrowheads in D–H). (I and J) ark1Δ prk1-as3 cells treated with 1NA-PP1 for 10 min also reveal ∼100-nm vesicles and microfilament profiles (marked by arrowheads). For more examples, also see Fig. S1. (K–N) EM of cells fixed by high pressure freezing and freeze substitution. ark1Δ prk1Δ cells reveal ribosome exclusion areas (encircled by dashed line in K) containing ∼100-nm vesicles (some of which are indicated by arrows in K–N). Ribosomes appear as small, uniform, electron-dense spots distributed throughout most of the cytoplasm. (O–Q) Indirect immunogold labeling of ultrathin cryosections of ark1Δ prk1Δ cells demonstrates the localization of actin within a slightly electron-dense/vesicle-enriched region (encircled by dashed line in O). In many instances, vesicles/membranous elements are detected within actin-labeled region (arrows in O and Q; boxed areas P and Q). (R) Double-indirect immunogold labeling with ark1Δ prk1Δ SLA1-GFP cells reveals occasional Sla1p-containing vesicles (5-nm gold, arrowheads) amid a clustered/electron-dense actin region (10-nm gold). Strains: ark1Δ prk1-as3 ABP1-GFP, DDY2606 (A); Wild-type, DDY904 (B); ark1Δ prk1Δ, DDY2541 (C–H and O–Q) and DDY1564 (K–N); ark1Δ prk1-as3, DDY2597 (I and J); ark1Δ prk1Δ SLA1-GFP, DDY2611 (R). n, nucleus; V, vacuole; pm, plasma membrane. Bars: (A) 5 μm; (B) 1 μm; (C) 0.2 μm; (D) 0.5 μm; (E–J) 0.1 μm; (K) 0.5 μm; (L–R) 0.1 μm.

Mentions: Next, we investigated the fate of the endocytic membrane marker FM4-64 (Vida and Emr, 1995) in inhibitor-treated ark1Δ prk1-as3 cells (Fig. 4). FM4-64 was first incorporated into the plasma membrane (Fig. 4 A, 0 min). In mock-treated ark1Δ prk1-as3 or wild-type cells, this dye is later found in endosomes (Fig. 4 A, top, 5 min) and finally accumulates in vacuoles (Vida and Emr, 1995; unpublished data). In mock-treated cells, the endosomes seen at 5 min do not show significant colocalization with Abp1p (Fig. 4 A, top). Next, we treated ark1Δ prk1-as3 cells simultaneously with FM4-64 and 1NA-PP1. At 5 min, we found that FM4-64 staining colocalized with actin clumps (Fig. 4 A, bottom). The FM4-64 dye in actin clumps was later transported to vacuoles (after 15–20 min), although the kinetics were delayed compared with the wild type (10 min; unpublished data). These observations suggest that the block of Prk1p activity leads to accumulation of an endocytic compartment that associates with actin.


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)

Actin-associated membrane accumulation upon inhibition of Prk1p activity. (A) FM4-64 labeling of ark1Δ prk1-as3 cells expressing Abp1-GFP. Cells were treated with media containing FM4-64 and 1NA-PP1 in a flow chamber. FM4-64 (red) and Abp1-GFP (green) were visualized at 0 and 5 min. Mock-treated cells are also shown. (B–J) Conventional EM of cells post-treated with 1% tannic acid. (B) A wild-type cell shows normal, unremarkable morphology. (C–H) ark1Δ prk1Δ cells reveal electron-dense areas (encircled by dashed line in C) containing a heterogeneous population of ∼100-nm vesicles (shown at higher magnification in D and indicated by arrows in C and D), as well as numerous microfilament profiles (marked by arrowheads in D–H). (I and J) ark1Δ prk1-as3 cells treated with 1NA-PP1 for 10 min also reveal ∼100-nm vesicles and microfilament profiles (marked by arrowheads). For more examples, also see Fig. S1. (K–N) EM of cells fixed by high pressure freezing and freeze substitution. ark1Δ prk1Δ cells reveal ribosome exclusion areas (encircled by dashed line in K) containing ∼100-nm vesicles (some of which are indicated by arrows in K–N). Ribosomes appear as small, uniform, electron-dense spots distributed throughout most of the cytoplasm. (O–Q) Indirect immunogold labeling of ultrathin cryosections of ark1Δ prk1Δ cells demonstrates the localization of actin within a slightly electron-dense/vesicle-enriched region (encircled by dashed line in O). In many instances, vesicles/membranous elements are detected within actin-labeled region (arrows in O and Q; boxed areas P and Q). (R) Double-indirect immunogold labeling with ark1Δ prk1Δ SLA1-GFP cells reveals occasional Sla1p-containing vesicles (5-nm gold, arrowheads) amid a clustered/electron-dense actin region (10-nm gold). Strains: ark1Δ prk1-as3 ABP1-GFP, DDY2606 (A); Wild-type, DDY904 (B); ark1Δ prk1Δ, DDY2541 (C–H and O–Q) and DDY1564 (K–N); ark1Δ prk1-as3, DDY2597 (I and J); ark1Δ prk1Δ SLA1-GFP, DDY2611 (R). n, nucleus; V, vacuole; pm, plasma membrane. Bars: (A) 5 μm; (B) 1 μm; (C) 0.2 μm; (D) 0.5 μm; (E–J) 0.1 μm; (K) 0.5 μm; (L–R) 0.1 μm.
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fig4: Actin-associated membrane accumulation upon inhibition of Prk1p activity. (A) FM4-64 labeling of ark1Δ prk1-as3 cells expressing Abp1-GFP. Cells were treated with media containing FM4-64 and 1NA-PP1 in a flow chamber. FM4-64 (red) and Abp1-GFP (green) were visualized at 0 and 5 min. Mock-treated cells are also shown. (B–J) Conventional EM of cells post-treated with 1% tannic acid. (B) A wild-type cell shows normal, unremarkable morphology. (C–H) ark1Δ prk1Δ cells reveal electron-dense areas (encircled by dashed line in C) containing a heterogeneous population of ∼100-nm vesicles (shown at higher magnification in D and indicated by arrows in C and D), as well as numerous microfilament profiles (marked by arrowheads in D–H). (I and J) ark1Δ prk1-as3 cells treated with 1NA-PP1 for 10 min also reveal ∼100-nm vesicles and microfilament profiles (marked by arrowheads). For more examples, also see Fig. S1. (K–N) EM of cells fixed by high pressure freezing and freeze substitution. ark1Δ prk1Δ cells reveal ribosome exclusion areas (encircled by dashed line in K) containing ∼100-nm vesicles (some of which are indicated by arrows in K–N). Ribosomes appear as small, uniform, electron-dense spots distributed throughout most of the cytoplasm. (O–Q) Indirect immunogold labeling of ultrathin cryosections of ark1Δ prk1Δ cells demonstrates the localization of actin within a slightly electron-dense/vesicle-enriched region (encircled by dashed line in O). In many instances, vesicles/membranous elements are detected within actin-labeled region (arrows in O and Q; boxed areas P and Q). (R) Double-indirect immunogold labeling with ark1Δ prk1Δ SLA1-GFP cells reveals occasional Sla1p-containing vesicles (5-nm gold, arrowheads) amid a clustered/electron-dense actin region (10-nm gold). Strains: ark1Δ prk1-as3 ABP1-GFP, DDY2606 (A); Wild-type, DDY904 (B); ark1Δ prk1Δ, DDY2541 (C–H and O–Q) and DDY1564 (K–N); ark1Δ prk1-as3, DDY2597 (I and J); ark1Δ prk1Δ SLA1-GFP, DDY2611 (R). n, nucleus; V, vacuole; pm, plasma membrane. Bars: (A) 5 μm; (B) 1 μm; (C) 0.2 μm; (D) 0.5 μm; (E–J) 0.1 μm; (K) 0.5 μm; (L–R) 0.1 μm.
Mentions: Next, we investigated the fate of the endocytic membrane marker FM4-64 (Vida and Emr, 1995) in inhibitor-treated ark1Δ prk1-as3 cells (Fig. 4). FM4-64 was first incorporated into the plasma membrane (Fig. 4 A, 0 min). In mock-treated ark1Δ prk1-as3 or wild-type cells, this dye is later found in endosomes (Fig. 4 A, top, 5 min) and finally accumulates in vacuoles (Vida and Emr, 1995; unpublished data). In mock-treated cells, the endosomes seen at 5 min do not show significant colocalization with Abp1p (Fig. 4 A, top). Next, we treated ark1Δ prk1-as3 cells simultaneously with FM4-64 and 1NA-PP1. At 5 min, we found that FM4-64 staining colocalized with actin clumps (Fig. 4 A, bottom). The FM4-64 dye in actin clumps was later transported to vacuoles (after 15–20 min), although the kinetics were delayed compared with the wild type (10 min; unpublished data). These observations suggest that the block of Prk1p activity leads to accumulation of an endocytic compartment that associates with actin.

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