Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 10, Pages 3793-3798Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0611733104
Keywords
actin; dynamin; epsin; phosphoinositides; rapamycin
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Funding
- Intramural NIH HHS Funding Source: Medline
- NCI NIH HHS [P01 CA046128, CA 46128] Funding Source: Medline
- NIDA NIH HHS [DA 018343, P30 DA018343] Funding Source: Medline
- NIDDK NIH HHS [DK 45735, P30 DK045735] Funding Source: Medline
- NINDS NIH HHS [R01 NS036251, R37 NS036251, NS 36251] Funding Source: Medline
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Phosphaticlylinositol 4,5-bisphosphate [PI(4,5)P-2], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P-2 breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P-2 breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P-2 depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P-2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.
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