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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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Lsb6 is required for endosome motility. (A) Tracings of endosome paths in lsb6Δ cells that lack or carry a plasmid (pLSB6) expressing wild-type Lsb6. (B) Gallery of fluorescence micrographs derived from Video S6 and S7 that illustrate endosome position over time in lsb6Δ cells lacking or carrying pLsb6. PM, plasma membrane; V, vacuole. (C) Histogram showing the distribution of endosome speeds in lsb6Δ cells lacking or carrying pLsb6.
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fig2: Lsb6 is required for endosome motility. (A) Tracings of endosome paths in lsb6Δ cells that lack or carry a plasmid (pLSB6) expressing wild-type Lsb6. (B) Gallery of fluorescence micrographs derived from Video S6 and S7 that illustrate endosome position over time in lsb6Δ cells lacking or carrying pLsb6. PM, plasma membrane; V, vacuole. (C) Histogram showing the distribution of endosome speeds in lsb6Δ cells lacking or carrying pLsb6.

Mentions: Of the six single mutants and two double mutants analyzed, lsb6Δ mutants displayed significant impairment of endosome motility relative to wild-type cells. Endosomes in lsb6Δ and wild-type cells moved with average speeds of 0.09 ± 0.01 μm/s and 0.19 ± 0.02 μm/s, respectively (Table I). The average speed of endosome motility in lsb6Δ mutants was similar to that observed in cells expressing Las17 lacking its COOH-terminal WCA domain (las17ΔWCA, 0.09 ± 0.01 μm/s) or in wild-type cells treated with the actin-depolymerizing drug latrunculin A (0.08 ± 0.04 μm/s; Chang et al., 2003). In lsb6Δ mutants, the distribution of endosome speeds was shifted to lower values (Fig. 2 C), and endosomes moved along abnormally short paths that remained within the plane of focus for many seconds (Fig. 2 B). Impaired endosome motility in lsb6Δ mutants was rescued by expression of the wild-type LSB6 gene on a single-copy plasmid (compare Video S6 [endosome motility in an lsb6Δ cell] with Video S7 [endosome motility in an lsb6Δ cell + pLSB6]; videos available at http://www.jcb.org/cgi/content/full/jcb.200501086/DC1). These results were confirmed by performing automated particle tracking and analyzing the data in MSD plots (Fig. S2 A). These results indicated that Lsb6 is required for motility of Ste2-containing endosomes. This is the first phenotype caused by the absence of the sole type II PI 4-kinase of yeast.


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)

Lsb6 is required for endosome motility. (A) Tracings of endosome paths in lsb6Δ cells that lack or carry a plasmid (pLSB6) expressing wild-type Lsb6. (B) Gallery of fluorescence micrographs derived from Video S6 and S7 that illustrate endosome position over time in lsb6Δ cells lacking or carrying pLsb6. PM, plasma membrane; V, vacuole. (C) Histogram showing the distribution of endosome speeds in lsb6Δ cells lacking or carrying pLsb6.
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Related In: Results  -  Collection

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fig2: Lsb6 is required for endosome motility. (A) Tracings of endosome paths in lsb6Δ cells that lack or carry a plasmid (pLSB6) expressing wild-type Lsb6. (B) Gallery of fluorescence micrographs derived from Video S6 and S7 that illustrate endosome position over time in lsb6Δ cells lacking or carrying pLsb6. PM, plasma membrane; V, vacuole. (C) Histogram showing the distribution of endosome speeds in lsb6Δ cells lacking or carrying pLsb6.
Mentions: Of the six single mutants and two double mutants analyzed, lsb6Δ mutants displayed significant impairment of endosome motility relative to wild-type cells. Endosomes in lsb6Δ and wild-type cells moved with average speeds of 0.09 ± 0.01 μm/s and 0.19 ± 0.02 μm/s, respectively (Table I). The average speed of endosome motility in lsb6Δ mutants was similar to that observed in cells expressing Las17 lacking its COOH-terminal WCA domain (las17ΔWCA, 0.09 ± 0.01 μm/s) or in wild-type cells treated with the actin-depolymerizing drug latrunculin A (0.08 ± 0.04 μm/s; Chang et al., 2003). In lsb6Δ mutants, the distribution of endosome speeds was shifted to lower values (Fig. 2 C), and endosomes moved along abnormally short paths that remained within the plane of focus for many seconds (Fig. 2 B). Impaired endosome motility in lsb6Δ mutants was rescued by expression of the wild-type LSB6 gene on a single-copy plasmid (compare Video S6 [endosome motility in an lsb6Δ cell] with Video S7 [endosome motility in an lsb6Δ cell + pLSB6]; videos available at http://www.jcb.org/cgi/content/full/jcb.200501086/DC1). These results were confirmed by performing automated particle tracking and analyzing the data in MSD plots (Fig. S2 A). These results indicated that Lsb6 is required for motility of Ste2-containing endosomes. This is the first phenotype caused by the absence of the sole type II PI 4-kinase of yeast.

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