期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 35, 页码 E2316-E2323出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1211305109
关键词
endocytosis; polarity; rapamycin; ion channel; ruffles
资金
- National Institutes of Health (NIH) [NS36251, DK082700, NS08174]
- Ellison Foundation
- Simons Foundation
- Swedish Research Council
Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P-2 and PI(3,4,5)P-3, was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P-2 and PI (3,4,5)P-3 biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P-2 dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P-2 biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P-3 synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.
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