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A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Chang FS, Han GS, Carman GM, Blumer KJ - J. Cell Biol. (2005)

Bottom Line: Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility.Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis.Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

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Appending the WCA domain of Las17 to the endocytic cargo Ste2 rescues endosome motility in las17ΔWCA cells. Automated particle tracking was used to analyze endosome position over time. Ste2-GFP was used as an endosome marker in this and subsequent figures. The mean square displacement of endosomes (n = 50–100) over time was calculated for the indicated wild-type and mutant cells.
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fig1: Appending the WCA domain of Las17 to the endocytic cargo Ste2 rescues endosome motility in las17ΔWCA cells. Automated particle tracking was used to analyze endosome position over time. Ste2-GFP was used as an endosome marker in this and subsequent figures. The mean square displacement of endosomes (n = 50–100) over time was calculated for the indicated wild-type and mutant cells.

Mentions: To obtain and analyze endosome motility more quantitatively, we used an automated particle tracking program developed to study actin patch motility in yeast (Carlsson et al., 2002). The resultant data were plotted as the mean squared displacement (MSD) of endosomes (n = 50–150) over time. This analysis indicated that endosomes labeled with Ste2-GFP were poorly motile in las17ΔWCA cells carrying an empty vector or a plasmid overexpressing Hxt1-WCA, as indicated by relatively flat curves in MSD plots (Fig. 1). In contrast, endosomes in the las17ΔWCA mutant expressing Ste2-WCA exhibited wild-type motility, as indicated by significantly greater displacement over time. Because under these conditions Ste2 is recruited to endosomes whereas Hxt1 is not, these results suggested that actin polymerization on endosomes rather than the plasma membrane promotes motility.


A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Chang FS, Han GS, Carman GM, Blumer KJ - J. Cell Biol. (2005)

Appending the WCA domain of Las17 to the endocytic cargo Ste2 rescues endosome motility in las17ΔWCA cells. Automated particle tracking was used to analyze endosome position over time. Ste2-GFP was used as an endosome marker in this and subsequent figures. The mean square displacement of endosomes (n = 50–100) over time was calculated for the indicated wild-type and mutant cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171216&req=5

fig1: Appending the WCA domain of Las17 to the endocytic cargo Ste2 rescues endosome motility in las17ΔWCA cells. Automated particle tracking was used to analyze endosome position over time. Ste2-GFP was used as an endosome marker in this and subsequent figures. The mean square displacement of endosomes (n = 50–100) over time was calculated for the indicated wild-type and mutant cells.
Mentions: To obtain and analyze endosome motility more quantitatively, we used an automated particle tracking program developed to study actin patch motility in yeast (Carlsson et al., 2002). The resultant data were plotted as the mean squared displacement (MSD) of endosomes (n = 50–150) over time. This analysis indicated that endosomes labeled with Ste2-GFP were poorly motile in las17ΔWCA cells carrying an empty vector or a plasmid overexpressing Hxt1-WCA, as indicated by relatively flat curves in MSD plots (Fig. 1). In contrast, endosomes in the las17ΔWCA mutant expressing Ste2-WCA exhibited wild-type motility, as indicated by significantly greater displacement over time. Because under these conditions Ste2 is recruited to endosomes whereas Hxt1 is not, these results suggested that actin polymerization on endosomes rather than the plasma membrane promotes motility.

Bottom Line: Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility.Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis.Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

Show MeSH
Related in: MedlinePlus