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Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.

Carnell M, Zech T, Calaminus SD, Ura S, Hagedorn M, Johnston SA, May RC, Soldati T, Machesky LM, Insall RH - J. Cell Biol. (2011)

Bottom Line: Similar results occur when actin polymerization is blocked with latrunculin.V-ATPases are known to bind avidly to F-actin.Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, Scotland, UK.

ABSTRACT
WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

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WASH is required for exocytosis. (a) Parental AX2 (○) and wshA− (•) cells were incubated with FITC-dextran, and uptake was measured fluorimetrically. The inset shows a longer time course of the same data. (b and c) Parental AX2 (○), wshA− cells (•), and wshA− cells expressing GFP-WASH (△) and GFP-WASHΔVCA (▴) were loaded with FITC-dextran for 2 h and then washed. Expulsion of dextran was measured fluorimetrically. (d) Cells were grown for five generations in medium with 20% dextran. wshA− cells, but not the parental strain, grew large and accumulated multiple dense vesicles. Bars, 5 µm. (e) Cells were grown for five generations in medium with and without 20% dextran. wshA− growth is greatly slowed by the presence of indigestible dextran. (f and g) Transmission electron micrographs of parental AX2 (f) and wshA− (g) cells incubated overnight with BSA–colloidal gold and chased for 2 h. Cells were examined as described in Hagedorn et al. (2009). Arrowheads in g indicate vesicular structures containing gold particles. Bars, 2 µm. (h) C. neoformans–containing vesicles in macrophages are also coated in WASH. Cultured J774 cells (left) were allowed to phagocytose C. neoformans and were then fixed with 4% formaldehyde and stained with anti-WASH (middle) and phalloidin (right). Bar, 10 µm. (i) Impairment of C. neoformans exocytosis caused by WASHΔVCA expression. Cells were transfected with full-length WASH or WASHΔVCA, incubated with C. neoformans for 2 h, and then observed for 24 h. The difference is significant to P = 0.05 (Fisher’s exact test). Error bars in each case show the SD. n = 3 in each case in a–c and e.
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fig2: WASH is required for exocytosis. (a) Parental AX2 (○) and wshA− (•) cells were incubated with FITC-dextran, and uptake was measured fluorimetrically. The inset shows a longer time course of the same data. (b and c) Parental AX2 (○), wshA− cells (•), and wshA− cells expressing GFP-WASH (△) and GFP-WASHΔVCA (▴) were loaded with FITC-dextran for 2 h and then washed. Expulsion of dextran was measured fluorimetrically. (d) Cells were grown for five generations in medium with 20% dextran. wshA− cells, but not the parental strain, grew large and accumulated multiple dense vesicles. Bars, 5 µm. (e) Cells were grown for five generations in medium with and without 20% dextran. wshA− growth is greatly slowed by the presence of indigestible dextran. (f and g) Transmission electron micrographs of parental AX2 (f) and wshA− (g) cells incubated overnight with BSA–colloidal gold and chased for 2 h. Cells were examined as described in Hagedorn et al. (2009). Arrowheads in g indicate vesicular structures containing gold particles. Bars, 2 µm. (h) C. neoformans–containing vesicles in macrophages are also coated in WASH. Cultured J774 cells (left) were allowed to phagocytose C. neoformans and were then fixed with 4% formaldehyde and stained with anti-WASH (middle) and phalloidin (right). Bar, 10 µm. (i) Impairment of C. neoformans exocytosis caused by WASHΔVCA expression. Cells were transfected with full-length WASH or WASHΔVCA, incubated with C. neoformans for 2 h, and then observed for 24 h. The difference is significant to P = 0.05 (Fisher’s exact test). Error bars in each case show the SD. n = 3 in each case in a–c and e.

Mentions: We measured the rate of fluid phase uptake in wshA− cells using fluorescent dextran (Fig. 2 a). The initial rate of endocytosis was unaltered, although after incubations of >1 h, the wshA– cells accumulated significantly more dextran than the parent (Fig. 2 a, inset). Dextran is indigestible, and like the remains of food phagosomes (Clarke et al., 2002), it is normally exocytosed ∼1–2 h after passing through the endocytic and lysosomal pathways (Maniak, 2001; Neuhaus et al., 2002), leading to a plateau in accumulation. Therefore, this implies a defect in exocytosis rather than during earlier traffic. Thus, we measured the rate at which fluorescent dextran was expelled after loading. As shown in Fig. 2 b, whereas wild-type cells expelled nearly everything within 2 h, wshA− cells were completely unable to exocytose dextran (Fig. 2 b). Again, GFP-WASH rescued exocytosis near perfectly but had no effect when missing its VCA domain (Fig. 2 c). Thus, WASH-stimulated actin polymerization is essential for exocytosis. This has serious consequences to cells that encounter indigestible material (including, for example, bacterial cell walls; Clarke et al., 2002), presumably explaining the growth defect on bacteria. When cells were grown in 20% of 60-kD dextran, which barely affects normal cells, wshA− cells became extremely distended with large fluid-filled vesicles (Fig. 2 d) and divided far more slowly (Fig. 2 e).


Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.

Carnell M, Zech T, Calaminus SD, Ura S, Hagedorn M, Johnston SA, May RC, Soldati T, Machesky LM, Insall RH - J. Cell Biol. (2011)

WASH is required for exocytosis. (a) Parental AX2 (○) and wshA− (•) cells were incubated with FITC-dextran, and uptake was measured fluorimetrically. The inset shows a longer time course of the same data. (b and c) Parental AX2 (○), wshA− cells (•), and wshA− cells expressing GFP-WASH (△) and GFP-WASHΔVCA (▴) were loaded with FITC-dextran for 2 h and then washed. Expulsion of dextran was measured fluorimetrically. (d) Cells were grown for five generations in medium with 20% dextran. wshA− cells, but not the parental strain, grew large and accumulated multiple dense vesicles. Bars, 5 µm. (e) Cells were grown for five generations in medium with and without 20% dextran. wshA− growth is greatly slowed by the presence of indigestible dextran. (f and g) Transmission electron micrographs of parental AX2 (f) and wshA− (g) cells incubated overnight with BSA–colloidal gold and chased for 2 h. Cells were examined as described in Hagedorn et al. (2009). Arrowheads in g indicate vesicular structures containing gold particles. Bars, 2 µm. (h) C. neoformans–containing vesicles in macrophages are also coated in WASH. Cultured J774 cells (left) were allowed to phagocytose C. neoformans and were then fixed with 4% formaldehyde and stained with anti-WASH (middle) and phalloidin (right). Bar, 10 µm. (i) Impairment of C. neoformans exocytosis caused by WASHΔVCA expression. Cells were transfected with full-length WASH or WASHΔVCA, incubated with C. neoformans for 2 h, and then observed for 24 h. The difference is significant to P = 0.05 (Fisher’s exact test). Error bars in each case show the SD. n = 3 in each case in a–c and e.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: WASH is required for exocytosis. (a) Parental AX2 (○) and wshA− (•) cells were incubated with FITC-dextran, and uptake was measured fluorimetrically. The inset shows a longer time course of the same data. (b and c) Parental AX2 (○), wshA− cells (•), and wshA− cells expressing GFP-WASH (△) and GFP-WASHΔVCA (▴) were loaded with FITC-dextran for 2 h and then washed. Expulsion of dextran was measured fluorimetrically. (d) Cells were grown for five generations in medium with 20% dextran. wshA− cells, but not the parental strain, grew large and accumulated multiple dense vesicles. Bars, 5 µm. (e) Cells were grown for five generations in medium with and without 20% dextran. wshA− growth is greatly slowed by the presence of indigestible dextran. (f and g) Transmission electron micrographs of parental AX2 (f) and wshA− (g) cells incubated overnight with BSA–colloidal gold and chased for 2 h. Cells were examined as described in Hagedorn et al. (2009). Arrowheads in g indicate vesicular structures containing gold particles. Bars, 2 µm. (h) C. neoformans–containing vesicles in macrophages are also coated in WASH. Cultured J774 cells (left) were allowed to phagocytose C. neoformans and were then fixed with 4% formaldehyde and stained with anti-WASH (middle) and phalloidin (right). Bar, 10 µm. (i) Impairment of C. neoformans exocytosis caused by WASHΔVCA expression. Cells were transfected with full-length WASH or WASHΔVCA, incubated with C. neoformans for 2 h, and then observed for 24 h. The difference is significant to P = 0.05 (Fisher’s exact test). Error bars in each case show the SD. n = 3 in each case in a–c and e.
Mentions: We measured the rate of fluid phase uptake in wshA− cells using fluorescent dextran (Fig. 2 a). The initial rate of endocytosis was unaltered, although after incubations of >1 h, the wshA– cells accumulated significantly more dextran than the parent (Fig. 2 a, inset). Dextran is indigestible, and like the remains of food phagosomes (Clarke et al., 2002), it is normally exocytosed ∼1–2 h after passing through the endocytic and lysosomal pathways (Maniak, 2001; Neuhaus et al., 2002), leading to a plateau in accumulation. Therefore, this implies a defect in exocytosis rather than during earlier traffic. Thus, we measured the rate at which fluorescent dextran was expelled after loading. As shown in Fig. 2 b, whereas wild-type cells expelled nearly everything within 2 h, wshA− cells were completely unable to exocytose dextran (Fig. 2 b). Again, GFP-WASH rescued exocytosis near perfectly but had no effect when missing its VCA domain (Fig. 2 c). Thus, WASH-stimulated actin polymerization is essential for exocytosis. This has serious consequences to cells that encounter indigestible material (including, for example, bacterial cell walls; Clarke et al., 2002), presumably explaining the growth defect on bacteria. When cells were grown in 20% of 60-kD dextran, which barely affects normal cells, wshA− cells became extremely distended with large fluid-filled vesicles (Fig. 2 d) and divided far more slowly (Fig. 2 e).

Bottom Line: Similar results occur when actin polymerization is blocked with latrunculin.V-ATPases are known to bind avidly to F-actin.Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, Scotland, UK.

ABSTRACT
WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

Show MeSH
Related in: MedlinePlus