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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 and actin polymerization are essential for vesicle neutralization. (a) Vesicle neutralization assay. Cells were loaded with FITC- and TRITC-dextran to distinguish between acidic and neutral vesicles. Neutral postlysosomes (arrows) are absent from WASH- cells and are rescued by expression of GFP-WASH but not GFP-WASHΔVCA (GFP is not seen under these conditions). (b) Latrunculin A completely but reversibly blocks the neutralization of vesicles. AX2 cells were loaded with a pulse of FITC-dextran, and the appearance of neutral vesicles was monitored in the presence or absence of 10 µg/ml latrunculin A. The figure shows measurements of Videos 1 and 2. The representative curves are from an experiment that was performed at least five times.
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fig3: WASH and actin polymerization are essential for vesicle neutralization. (a) Vesicle neutralization assay. Cells were loaded with FITC- and TRITC-dextran to distinguish between acidic and neutral vesicles. Neutral postlysosomes (arrows) are absent from WASH- cells and are rescued by expression of GFP-WASH but not GFP-WASHΔVCA (GFP is not seen under these conditions). (b) Latrunculin A completely but reversibly blocks the neutralization of vesicles. AX2 cells were loaded with a pulse of FITC-dextran, and the appearance of neutral vesicles was monitored in the presence or absence of 10 µg/ml latrunculin A. The figure shows measurements of Videos 1 and 2. The representative curves are from an experiment that was performed at least five times.

Mentions: When cells are loaded with a mixture of FITC- and TRITC-labeled dextran, neutral vesicles show yellow, but acidic vesicles are red because FITC loses fluorescence (Jenne et al., 1998). Most intracellular vesicles are acidic, but they neutralize as they mature to postlysosomes (Fig. 3 a, arrows). In wshA− cells, neutral postlysosomes were never seen. Again, this phenotype was fully rescued by GFP-WASH but not GFP-WASHΔVCA (Fig. 3 d). This suggests that wshA− cells cannot exocytose dextran because lysosomes are never able to neutralize and thus do not mature to postlysosomes. It also suggests that the role of WASH and the actin coat it generates is to mediate lysosome neutralization and maturation.


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 and actin polymerization are essential for vesicle neutralization. (a) Vesicle neutralization assay. Cells were loaded with FITC- and TRITC-dextran to distinguish between acidic and neutral vesicles. Neutral postlysosomes (arrows) are absent from WASH- cells and are rescued by expression of GFP-WASH but not GFP-WASHΔVCA (GFP is not seen under these conditions). (b) Latrunculin A completely but reversibly blocks the neutralization of vesicles. AX2 cells were loaded with a pulse of FITC-dextran, and the appearance of neutral vesicles was monitored in the presence or absence of 10 µg/ml latrunculin A. The figure shows measurements of Videos 1 and 2. The representative curves are from an experiment that was performed at least five times.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: WASH and actin polymerization are essential for vesicle neutralization. (a) Vesicle neutralization assay. Cells were loaded with FITC- and TRITC-dextran to distinguish between acidic and neutral vesicles. Neutral postlysosomes (arrows) are absent from WASH- cells and are rescued by expression of GFP-WASH but not GFP-WASHΔVCA (GFP is not seen under these conditions). (b) Latrunculin A completely but reversibly blocks the neutralization of vesicles. AX2 cells were loaded with a pulse of FITC-dextran, and the appearance of neutral vesicles was monitored in the presence or absence of 10 µg/ml latrunculin A. The figure shows measurements of Videos 1 and 2. The representative curves are from an experiment that was performed at least five times.
Mentions: When cells are loaded with a mixture of FITC- and TRITC-labeled dextran, neutral vesicles show yellow, but acidic vesicles are red because FITC loses fluorescence (Jenne et al., 1998). Most intracellular vesicles are acidic, but they neutralize as they mature to postlysosomes (Fig. 3 a, arrows). In wshA− cells, neutral postlysosomes were never seen. Again, this phenotype was fully rescued by GFP-WASH but not GFP-WASHΔVCA (Fig. 3 d). This suggests that wshA− cells cannot exocytose dextran because lysosomes are never able to neutralize and thus do not mature to postlysosomes. It also suggests that the role of WASH and the actin coat it generates is to mediate lysosome neutralization and maturation.

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