Journal: The Journal of Cell Biology

Article Title: Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

doi: 10.1083/jcb.201009119

Figure Lengend Snippet: 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-null ( wshA − ) cell lines fixed and stained with phalloidin. WASH-null 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.

Article Snippet: For qualitative high speed acquisition, cells were imaged on a TE2000-U widefield microscope with a 100× 1.49 NA objective, with illumination provided by 473- and 561-nm solid-state lasers (Deepstar; Omicron Laserage Laserprodukte GmbH).

Techniques: Staining, Expressing

Journal: The Journal of Cell Biology

Article Title: Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

doi: 10.1083/jcb.201009119

Figure Lengend Snippet: 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 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.

Article Snippet: For qualitative high speed acquisition, cells were imaged on a TE2000-U widefield microscope with a 100× 1.49 NA objective, with illumination provided by 473- and 561-nm solid-state lasers (Deepstar; Omicron Laserage Laserprodukte GmbH).

Techniques: Imaging, Expressing, Marker, Neutralization