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PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin-actin interactions.

Hong NH, Qi A, Weaver AM - J. Cell Biol. (2015)

Bottom Line: These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin.Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes.These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

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Affiliation: Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232.

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Inhibition of PI(3,5)P2 production leads to accumulation of cortactin and actin at LE membrane. (A) Representative images showing cortactin (green) and actin (red) localization on Rab7+ endosomes (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control in control and cortactin–KD MDA-MB-231 cells. Bars, 20 µm (3 µm for magnifications). (B) Immunoblot of cortactin expression and β-actin loading control in MDA-MB-231 cells. (C–E) Images were analyzed for percentage of colocalization of cortactin or actin with Rab7 or Rab7 area per cell. 3 independent experiments, n ≥ 55 cells in each condition. (F and G) Number and intensity of actin dots on Rab7+ endosomes. More than 170 vesicles from 12–19 cells analyzed for each condition. Data shown as box and whiskers plots, with the box indicating the 25th and 75th percentiles, solid line indicating the median, and whiskers indicating the 95% confidence intervals. ****, P < 0.0001.
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fig3: Inhibition of PI(3,5)P2 production leads to accumulation of cortactin and actin at LE membrane. (A) Representative images showing cortactin (green) and actin (red) localization on Rab7+ endosomes (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control in control and cortactin–KD MDA-MB-231 cells. Bars, 20 µm (3 µm for magnifications). (B) Immunoblot of cortactin expression and β-actin loading control in MDA-MB-231 cells. (C–E) Images were analyzed for percentage of colocalization of cortactin or actin with Rab7 or Rab7 area per cell. 3 independent experiments, n ≥ 55 cells in each condition. (F and G) Number and intensity of actin dots on Rab7+ endosomes. More than 170 vesicles from 12–19 cells analyzed for each condition. Data shown as box and whiskers plots, with the box indicating the 25th and 75th percentiles, solid line indicating the median, and whiskers indicating the 95% confidence intervals. ****, P < 0.0001.

Mentions: A central function of PIs on membranes is recruitment of cytosolic proteins, including cytoskeletal proteins (Janmey and Lindberg, 2004). To investigate whether PI(3,5)P2 recruits cortactin to endosomes, we prevented cellular PI(3,5)P2 synthesis by knocking down PIKfyve expression with siRNA in MDA-MB-231 cancer cells (Fig. 2 D). Consistent with previous studies, inhibition of PIKfyve led to enlargement of late endosomes marked by Rab7 (Fig. 2 E; Nicot et al., 2006). Surprisingly, silencing of PIKfyve led to a marked increase in cortactin localization to Rab7+ late endosomes rather than the expected decrease (Fig. 2, E and F). Likewise, treatment of MDA-MB-231 or SCC61 cells with the specific PIKfyve inhibitor drugs YM201636 (Ikonomov et al., 2006; Jefferies et al., 2008; de Lartigue et al., 2009) or apilimod (Cai et al., 2013) phenocopied the effect of PIKfyve siRNA (Fig. 3, A and C; Fig. S2, A–D; and Video 1). Despite inducing enlargement of individual endosomes, YM201636 drug treatment had no significant effect on the total Rab7+ endosome area per cell (Fig. 3 E), suggesting that the increased overlap between cortactin and Rab7+ area per cell (Fig. 3 C) is not a secondary effect of endosomal enlargement. Immunofluorescence of YM201636-treated cells using early (EEA1) and late endosomal (Rab7) markers confirmed that the majority of the enlarged vesicular compartments were Rab7+ with only a few endosomes positive for both EEA1 and Rab7, reflecting perturbation in late endosomal maturation and/or trafficking (Fig. S2 E; Rutherford et al., 2006). These data indicate that PI(3,5)P2 does not function to recruit cortactin to late endosomes but may instead regulate its activity by removing it from the late endosome.


PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin-actin interactions.

Hong NH, Qi A, Weaver AM - J. Cell Biol. (2015)

Inhibition of PI(3,5)P2 production leads to accumulation of cortactin and actin at LE membrane. (A) Representative images showing cortactin (green) and actin (red) localization on Rab7+ endosomes (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control in control and cortactin–KD MDA-MB-231 cells. Bars, 20 µm (3 µm for magnifications). (B) Immunoblot of cortactin expression and β-actin loading control in MDA-MB-231 cells. (C–E) Images were analyzed for percentage of colocalization of cortactin or actin with Rab7 or Rab7 area per cell. 3 independent experiments, n ≥ 55 cells in each condition. (F and G) Number and intensity of actin dots on Rab7+ endosomes. More than 170 vesicles from 12–19 cells analyzed for each condition. Data shown as box and whiskers plots, with the box indicating the 25th and 75th percentiles, solid line indicating the median, and whiskers indicating the 95% confidence intervals. ****, P < 0.0001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Inhibition of PI(3,5)P2 production leads to accumulation of cortactin and actin at LE membrane. (A) Representative images showing cortactin (green) and actin (red) localization on Rab7+ endosomes (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control in control and cortactin–KD MDA-MB-231 cells. Bars, 20 µm (3 µm for magnifications). (B) Immunoblot of cortactin expression and β-actin loading control in MDA-MB-231 cells. (C–E) Images were analyzed for percentage of colocalization of cortactin or actin with Rab7 or Rab7 area per cell. 3 independent experiments, n ≥ 55 cells in each condition. (F and G) Number and intensity of actin dots on Rab7+ endosomes. More than 170 vesicles from 12–19 cells analyzed for each condition. Data shown as box and whiskers plots, with the box indicating the 25th and 75th percentiles, solid line indicating the median, and whiskers indicating the 95% confidence intervals. ****, P < 0.0001.
Mentions: A central function of PIs on membranes is recruitment of cytosolic proteins, including cytoskeletal proteins (Janmey and Lindberg, 2004). To investigate whether PI(3,5)P2 recruits cortactin to endosomes, we prevented cellular PI(3,5)P2 synthesis by knocking down PIKfyve expression with siRNA in MDA-MB-231 cancer cells (Fig. 2 D). Consistent with previous studies, inhibition of PIKfyve led to enlargement of late endosomes marked by Rab7 (Fig. 2 E; Nicot et al., 2006). Surprisingly, silencing of PIKfyve led to a marked increase in cortactin localization to Rab7+ late endosomes rather than the expected decrease (Fig. 2, E and F). Likewise, treatment of MDA-MB-231 or SCC61 cells with the specific PIKfyve inhibitor drugs YM201636 (Ikonomov et al., 2006; Jefferies et al., 2008; de Lartigue et al., 2009) or apilimod (Cai et al., 2013) phenocopied the effect of PIKfyve siRNA (Fig. 3, A and C; Fig. S2, A–D; and Video 1). Despite inducing enlargement of individual endosomes, YM201636 drug treatment had no significant effect on the total Rab7+ endosome area per cell (Fig. 3 E), suggesting that the increased overlap between cortactin and Rab7+ area per cell (Fig. 3 C) is not a secondary effect of endosomal enlargement. Immunofluorescence of YM201636-treated cells using early (EEA1) and late endosomal (Rab7) markers confirmed that the majority of the enlarged vesicular compartments were Rab7+ with only a few endosomes positive for both EEA1 and Rab7, reflecting perturbation in late endosomal maturation and/or trafficking (Fig. S2 E; Rutherford et al., 2006). These data indicate that PI(3,5)P2 does not function to recruit cortactin to late endosomes but may instead regulate its activity by removing it from the late endosome.

Bottom Line: These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin.Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes.These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232.

Show MeSH
Related in: MedlinePlus