期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 52, 页码 21165-21170出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1311864110
关键词
TRP channel; PIKfyve; vesicle fusion
资金
- National Institutes of Heath [R01 NS062792, R01 GM50403]
Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P-2] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P-2 exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P-2-metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI (3,5)P-2, however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI (3,5)P-2 binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P-2-specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P-2. Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P-2 probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P-2-specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.
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