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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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KD of PIKfyve expression leads to accumulation of cortactin and actin at LE membrane. (A) Representative images of endogenous cortactin (red) localized at discrete subdomains of hVac14-EGFP+ (green) vesicular structures in SCC61 (left) and HeLa (right) cells. Bars: (main panels) 20 µm; (enlarged views) 3 µm. n = 3 independent experiments for each cell line. (B) Representative images of mCherry-ML1N*2 (red) and endogenous cortactin (blue) localization to EGFP-Rab7+ (green) endosomes. Small white boxes are enlarged on the right. Bars, 20 µm (3 µm for magnifications). (C) Magnifications of boxed areas labeled with asterisks in B show colocalization of mCherry-ML1N*2 (red) with EGFP-Rab7 (green). Bars, 5 µm. (D) Immunoblot of PIKfyve expression in PIKfyve-KD MDA-MB-231 cells. NTC, nontargeting control. (E) Immunofluorescence of PIKfyve siRNA-treated cells using antibodies recognizing cortactin (green), actin (red), and Rab7 (blue). Bars, 20 µm (3 µm for magnifications). (F) Percentage of colocalization of cortactin or actin with Rab7. 3 independent experiments, n ≥ 61 cells in each condition. ****, P < 0.0001.
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fig2: KD of PIKfyve expression leads to accumulation of cortactin and actin at LE membrane. (A) Representative images of endogenous cortactin (red) localized at discrete subdomains of hVac14-EGFP+ (green) vesicular structures in SCC61 (left) and HeLa (right) cells. Bars: (main panels) 20 µm; (enlarged views) 3 µm. n = 3 independent experiments for each cell line. (B) Representative images of mCherry-ML1N*2 (red) and endogenous cortactin (blue) localization to EGFP-Rab7+ (green) endosomes. Small white boxes are enlarged on the right. Bars, 20 µm (3 µm for magnifications). (C) Magnifications of boxed areas labeled with asterisks in B show colocalization of mCherry-ML1N*2 (red) with EGFP-Rab7 (green). Bars, 5 µm. (D) Immunoblot of PIKfyve expression in PIKfyve-KD MDA-MB-231 cells. NTC, nontargeting control. (E) Immunofluorescence of PIKfyve siRNA-treated cells using antibodies recognizing cortactin (green), actin (red), and Rab7 (blue). Bars, 20 µm (3 µm for magnifications). (F) Percentage of colocalization of cortactin or actin with Rab7. 3 independent experiments, n ≥ 61 cells in each condition. ****, P < 0.0001.

Mentions: The enzyme complex that synthesizes PI(3,5)P2 localizes to both early and late endosomes and consists of the scaffold protein Vac14, the lipid kinase PIKfyve (which converts PI(3)P to PI(3,5)P2), and the counteracting lipid phosphatase Fig4 (Rudge et al., 2004; Ikonomov et al., 2006; Nicot et al., 2006; Rusten et al., 2006; Rutherford et al., 2006). To determine whether cortactin and the PI(3,5)P2-synthesizing enzyme complex are likely to be present in the same compartment, we immunolocalized cortactin with Vac14. Immunostaining of SCC61 and HeLa cells transiently expressing hVac14-EGFP (Jin et al., 2008) with antibodies against cortactin revealed that cortactin is indeed present on Vac14+ vesicular structures (Fig. 2 A). To further determine whether cortactin is present at PI(3,5)P2-containing endosomes, we localized cortactin with a specific probe for PI(3,5)P2, mCherry-ML1N*2 (Li et al., 2013), and with the late endosomal marker Rab7. As expected, mCherry-ML1N*2 and Rab7 were present together in the same endosomal populations with virtually all ML1N*7+ endosomes containing Rab7+ puncta or rings (Fig. 2, B and C). Consistent with the role of PI(3,5)P2 in late endosomal maturation (de Lartigue et al., 2009; Dove et al., 2009), individual endosomes had different proportions of Rab7 or ML1N*2 positivity (Fig. 2 C). Cortactin localized to some of these Rab7-ML1N*2+ endosomes (Fig. 2 B).


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

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

KD of PIKfyve expression leads to accumulation of cortactin and actin at LE membrane. (A) Representative images of endogenous cortactin (red) localized at discrete subdomains of hVac14-EGFP+ (green) vesicular structures in SCC61 (left) and HeLa (right) cells. Bars: (main panels) 20 µm; (enlarged views) 3 µm. n = 3 independent experiments for each cell line. (B) Representative images of mCherry-ML1N*2 (red) and endogenous cortactin (blue) localization to EGFP-Rab7+ (green) endosomes. Small white boxes are enlarged on the right. Bars, 20 µm (3 µm for magnifications). (C) Magnifications of boxed areas labeled with asterisks in B show colocalization of mCherry-ML1N*2 (red) with EGFP-Rab7 (green). Bars, 5 µm. (D) Immunoblot of PIKfyve expression in PIKfyve-KD MDA-MB-231 cells. NTC, nontargeting control. (E) Immunofluorescence of PIKfyve siRNA-treated cells using antibodies recognizing cortactin (green), actin (red), and Rab7 (blue). Bars, 20 µm (3 µm for magnifications). (F) Percentage of colocalization of cortactin or actin with Rab7. 3 independent experiments, n ≥ 61 cells in each condition. ****, P < 0.0001.
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fig2: KD of PIKfyve expression leads to accumulation of cortactin and actin at LE membrane. (A) Representative images of endogenous cortactin (red) localized at discrete subdomains of hVac14-EGFP+ (green) vesicular structures in SCC61 (left) and HeLa (right) cells. Bars: (main panels) 20 µm; (enlarged views) 3 µm. n = 3 independent experiments for each cell line. (B) Representative images of mCherry-ML1N*2 (red) and endogenous cortactin (blue) localization to EGFP-Rab7+ (green) endosomes. Small white boxes are enlarged on the right. Bars, 20 µm (3 µm for magnifications). (C) Magnifications of boxed areas labeled with asterisks in B show colocalization of mCherry-ML1N*2 (red) with EGFP-Rab7 (green). Bars, 5 µm. (D) Immunoblot of PIKfyve expression in PIKfyve-KD MDA-MB-231 cells. NTC, nontargeting control. (E) Immunofluorescence of PIKfyve siRNA-treated cells using antibodies recognizing cortactin (green), actin (red), and Rab7 (blue). Bars, 20 µm (3 µm for magnifications). (F) Percentage of colocalization of cortactin or actin with Rab7. 3 independent experiments, n ≥ 61 cells in each condition. ****, P < 0.0001.
Mentions: The enzyme complex that synthesizes PI(3,5)P2 localizes to both early and late endosomes and consists of the scaffold protein Vac14, the lipid kinase PIKfyve (which converts PI(3)P to PI(3,5)P2), and the counteracting lipid phosphatase Fig4 (Rudge et al., 2004; Ikonomov et al., 2006; Nicot et al., 2006; Rusten et al., 2006; Rutherford et al., 2006). To determine whether cortactin and the PI(3,5)P2-synthesizing enzyme complex are likely to be present in the same compartment, we immunolocalized cortactin with Vac14. Immunostaining of SCC61 and HeLa cells transiently expressing hVac14-EGFP (Jin et al., 2008) with antibodies against cortactin revealed that cortactin is indeed present on Vac14+ vesicular structures (Fig. 2 A). To further determine whether cortactin is present at PI(3,5)P2-containing endosomes, we localized cortactin with a specific probe for PI(3,5)P2, mCherry-ML1N*2 (Li et al., 2013), and with the late endosomal marker Rab7. As expected, mCherry-ML1N*2 and Rab7 were present together in the same endosomal populations with virtually all ML1N*7+ endosomes containing Rab7+ puncta or rings (Fig. 2, B and C). Consistent with the role of PI(3,5)P2 in late endosomal maturation (de Lartigue et al., 2009; Dove et al., 2009), individual endosomes had different proportions of Rab7 or ML1N*2 positivity (Fig. 2 C). Cortactin localized to some of these Rab7-ML1N*2+ endosomes (Fig. 2 B).

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