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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 localizes to postlysosomes and is required for postlysosomal actin coats. (a) Domain architecture of WASP subfamilies. (b) Deconvolved widefield images of parent (AX2) and WASH- (wshA−) cell lines fixed and stained with phalloidin. WASH- cells lack large F-actin–coated vesicles (arrows in AX2). (c) Loss of actin-coated vesicles. Parental (n = 82) and wshA− (n = 79) cells were fixed and stained with phalloidin. Vesicles were counted in all planes of focus; macropinosomes were excluded. Error bars represent SEM. (d) Expression of GFP-WASH (middle panel and green) in fixed, phalloidin-stained (left panel and red) wshA− cells. (e) Expression of GFP-WASHΔVCA (green) in fixed, phalloidin-stained (left panel and red) wshA− cells. Bars, 10 µm.
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fig1: WASH localizes to postlysosomes and is required for postlysosomal actin coats. (a) Domain architecture of WASP subfamilies. (b) Deconvolved widefield images of parent (AX2) and WASH- (wshA−) cell lines fixed and stained with phalloidin. WASH- cells lack large F-actin–coated vesicles (arrows in AX2). (c) Loss of actin-coated vesicles. Parental (n = 82) and wshA− (n = 79) cells were fixed and stained with phalloidin. Vesicles were counted in all planes of focus; macropinosomes were excluded. Error bars represent SEM. (d) Expression of GFP-WASH (middle panel and green) in fixed, phalloidin-stained (left panel and red) wshA− cells. (e) Expression of GFP-WASHΔVCA (green) in fixed, phalloidin-stained (left panel and red) wshA− cells. Bars, 10 µm.

Mentions: The D. discoideum WASH is well conserved, containing all the domains seen in human and D. melanogaster (Fig. 1 a), and, like WASP and SCAR, is encoded by a single gene. We generated several disruptants of the gene (wshA) in an AX2 background, giving identical phenotypes. wshA− mutants grew at near-normal rates in liquid medium, though we observed slower growth on bacteria (Fig. S1 a). They also showed no defects in cell migration, moving, if anything, faster than parental cells (Fig. S1 b). When wshA− cells were fixed and stained with phalloidin, there were also no visible changes in pseudopods or filopods at the leading edge. However, the large F-actin–coated intracellular vesicles seen in most normal cells were entirely absent from wshA− cells (Fig. 1 b). These do not colocalize with WASP or SCAR and are distinct from early endocytic actin structures such as phagosomes and macropinosomes, which are seen at normal levels. They have been previously observed using GFP fused to the actin-binding proteins ABP120 (Lee and Knecht, 2002) and coronin (Drengk et al., 2003) some time before indigestible material is exocytosed (Rauchenberger et al., 1997). These vesicles are present in >95% of normal cells but completely lost from wshA− cells (Fig. 1 c).


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 localizes to postlysosomes and is required for postlysosomal actin coats. (a) Domain architecture of WASP subfamilies. (b) Deconvolved widefield images of parent (AX2) and WASH- (wshA−) cell lines fixed and stained with phalloidin. WASH- cells lack large F-actin–coated vesicles (arrows in AX2). (c) Loss of actin-coated vesicles. Parental (n = 82) and wshA− (n = 79) cells were fixed and stained with phalloidin. Vesicles were counted in all planes of focus; macropinosomes were excluded. Error bars represent SEM. (d) Expression of GFP-WASH (middle panel and green) in fixed, phalloidin-stained (left panel and red) wshA− cells. (e) Expression of GFP-WASHΔVCA (green) in fixed, phalloidin-stained (left panel and red) wshA− cells. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105540&req=5

fig1: WASH localizes to postlysosomes and is required for postlysosomal actin coats. (a) Domain architecture of WASP subfamilies. (b) Deconvolved widefield images of parent (AX2) and WASH- (wshA−) cell lines fixed and stained with phalloidin. WASH- cells lack large F-actin–coated vesicles (arrows in AX2). (c) Loss of actin-coated vesicles. Parental (n = 82) and wshA− (n = 79) cells were fixed and stained with phalloidin. Vesicles were counted in all planes of focus; macropinosomes were excluded. Error bars represent SEM. (d) Expression of GFP-WASH (middle panel and green) in fixed, phalloidin-stained (left panel and red) wshA− cells. (e) Expression of GFP-WASHΔVCA (green) in fixed, phalloidin-stained (left panel and red) wshA− cells. Bars, 10 µm.
Mentions: The D. discoideum WASH is well conserved, containing all the domains seen in human and D. melanogaster (Fig. 1 a), and, like WASP and SCAR, is encoded by a single gene. We generated several disruptants of the gene (wshA) in an AX2 background, giving identical phenotypes. wshA− mutants grew at near-normal rates in liquid medium, though we observed slower growth on bacteria (Fig. S1 a). They also showed no defects in cell migration, moving, if anything, faster than parental cells (Fig. S1 b). When wshA− cells were fixed and stained with phalloidin, there were also no visible changes in pseudopods or filopods at the leading edge. However, the large F-actin–coated intracellular vesicles seen in most normal cells were entirely absent from wshA− cells (Fig. 1 b). These do not colocalize with WASP or SCAR and are distinct from early endocytic actin structures such as phagosomes and macropinosomes, which are seen at normal levels. They have been previously observed using GFP fused to the actin-binding proteins ABP120 (Lee and Knecht, 2002) and coronin (Drengk et al., 2003) some time before indigestible material is exocytosed (Rauchenberger et al., 1997). These vesicles are present in >95% of normal cells but completely lost from wshA− cells (Fig. 1 c).

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