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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 causes recycling of the V-ATPase. (a) Confocal imaging of wshA− cells expressing GFP-WASH and VatB-mRFP after endocytosis of 0.5-µm agarose beads. Frames are taken from Video 3. Insets show a magnified view of the vesicle on which WASH is acting. (b) Quantification of various vesicular proteins during postlysosome formation. Graphs show that the loss of V-ATPase immediately follows the arrival of WASH and coronin, whereas the previously described (Maniak, 2001) postlysosome marker vacuolin is present much earlier than neutralization and rises steadily. The representative curves are from an experiment that was performed at least five times. (c) Rapid widefield oblique illumination imaging of small vesicles containing both GFP-WASH and VatB-mRFP budding a single lysosome. Frames are taken from Video 4. Bar, 1 µm.
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fig4: WASH causes recycling of the V-ATPase. (a) Confocal imaging of wshA− cells expressing GFP-WASH and VatB-mRFP after endocytosis of 0.5-µm agarose beads. Frames are taken from Video 3. Insets show a magnified view of the vesicle on which WASH is acting. (b) Quantification of various vesicular proteins during postlysosome formation. Graphs show that the loss of V-ATPase immediately follows the arrival of WASH and coronin, whereas the previously described (Maniak, 2001) postlysosome marker vacuolin is present much earlier than neutralization and rises steadily. The representative curves are from an experiment that was performed at least five times. (c) Rapid widefield oblique illumination imaging of small vesicles containing both GFP-WASH and VatB-mRFP budding a single lysosome. Frames are taken from Video 4. Bar, 1 µm.

Mentions: The V-ATPase, which consists of a membrane channel (V0) and a cytoplasmic ATP-hydrolyzing proton pump (V1; Jefferies et al., 2008), mediates vesicle acidification. When it is removed, vesicles neutralize through proton leaks. It has been known for several years that subunits of V1 have a surprisingly high affinity for actin. Each V1 contains three B subunits, which have a submicromolar affinity for F-actin (Holliday et al., 2000), and the single C subunit also binds actin (Vitavska et al., 2003). Together, these imply an extremely high avidity for F-actin. We hypothesized that the WASH-derived F-actin coat on lysosomes was neutralizing them by binding and removing the V-ATPase. This was supported by coimmunoprecipitation from detergent lysates (Fig. S3). VatM and VatB subunits (from V0 and V1, respectively) were identified with 100% confidence from GFP-WASH pulldowns but not from GFP alone (Fig. S3). Thus, WASH can physically associate with V-ATPase either directly or as part of a larger protein assembly. However, our data suggest that this interaction results in V-ATPase recycling and is thus short lived. We simultaneously imaged VatB-RFP and GFP-WASH in cells that had been fed ∼2-µm indigestible agarose beads, which made lysosomes large enough that they could be imaged continuously. As seen in Fig. 4 a and Video 3, the V1 subunit began disappearing from the lysosome as soon as puncta of GFP-WASH appeared. Quantitation shows V-ATPase levels start to drop within 1 or 2 min of the arrival of WASH (Fig. 4 b). Unexpectedly, however, WASH was only present in puncta, whereas the V-ATPase was removed; all detectable V-ATPase had gone well before formation of the contiguous coat of WASH seen on postlysosomes.


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 causes recycling of the V-ATPase. (a) Confocal imaging of wshA− cells expressing GFP-WASH and VatB-mRFP after endocytosis of 0.5-µm agarose beads. Frames are taken from Video 3. Insets show a magnified view of the vesicle on which WASH is acting. (b) Quantification of various vesicular proteins during postlysosome formation. Graphs show that the loss of V-ATPase immediately follows the arrival of WASH and coronin, whereas the previously described (Maniak, 2001) postlysosome marker vacuolin is present much earlier than neutralization and rises steadily. The representative curves are from an experiment that was performed at least five times. (c) Rapid widefield oblique illumination imaging of small vesicles containing both GFP-WASH and VatB-mRFP budding a single lysosome. Frames are taken from Video 4. Bar, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: WASH causes recycling of the V-ATPase. (a) Confocal imaging of wshA− cells expressing GFP-WASH and VatB-mRFP after endocytosis of 0.5-µm agarose beads. Frames are taken from Video 3. Insets show a magnified view of the vesicle on which WASH is acting. (b) Quantification of various vesicular proteins during postlysosome formation. Graphs show that the loss of V-ATPase immediately follows the arrival of WASH and coronin, whereas the previously described (Maniak, 2001) postlysosome marker vacuolin is present much earlier than neutralization and rises steadily. The representative curves are from an experiment that was performed at least five times. (c) Rapid widefield oblique illumination imaging of small vesicles containing both GFP-WASH and VatB-mRFP budding a single lysosome. Frames are taken from Video 4. Bar, 1 µm.
Mentions: The V-ATPase, which consists of a membrane channel (V0) and a cytoplasmic ATP-hydrolyzing proton pump (V1; Jefferies et al., 2008), mediates vesicle acidification. When it is removed, vesicles neutralize through proton leaks. It has been known for several years that subunits of V1 have a surprisingly high affinity for actin. Each V1 contains three B subunits, which have a submicromolar affinity for F-actin (Holliday et al., 2000), and the single C subunit also binds actin (Vitavska et al., 2003). Together, these imply an extremely high avidity for F-actin. We hypothesized that the WASH-derived F-actin coat on lysosomes was neutralizing them by binding and removing the V-ATPase. This was supported by coimmunoprecipitation from detergent lysates (Fig. S3). VatM and VatB subunits (from V0 and V1, respectively) were identified with 100% confidence from GFP-WASH pulldowns but not from GFP alone (Fig. S3). Thus, WASH can physically associate with V-ATPase either directly or as part of a larger protein assembly. However, our data suggest that this interaction results in V-ATPase recycling and is thus short lived. We simultaneously imaged VatB-RFP and GFP-WASH in cells that had been fed ∼2-µm indigestible agarose beads, which made lysosomes large enough that they could be imaged continuously. As seen in Fig. 4 a and Video 3, the V1 subunit began disappearing from the lysosome as soon as puncta of GFP-WASH appeared. Quantitation shows V-ATPase levels start to drop within 1 or 2 min of the arrival of WASH (Fig. 4 b). Unexpectedly, however, WASH was only present in puncta, whereas the V-ATPase was removed; all detectable V-ATPase had gone well before formation of the contiguous coat of WASH seen on postlysosomes.

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