title: Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics.
abstract: Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] 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)P2 exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P2-metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI(3,5)P2, however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI(3,5)P2 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)P2-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)P2. Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P2 probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P2-specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.
authors: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109.
authors: Xinran
authors: X
authors: Li
authors: Xiang
authors: X
authors: Wang
authors: Xiaoli
authors: X
authors: Zhang
authors: Mingkun
authors: M
authors: Zhao
authors: Wai Lok
authors: WL
authors: Tsang
authors: Yanling
authors: Y
authors: Zhang
authors: Richard Gar Wai
authors: RG
authors: Yau
authors: Lois S
authors: LS
authors: Weisman
authors: Haoxing
authors: H
authors: Xu
mesh_terms: D005456:Fluorescent Dyes / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D057054:Molecular Imaging / Q000379:methods*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D050051:Transient Receptor Potential Channels / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C416674:MCOLN1 protein, human; D018129:Phosphatidylinositol Phosphates; D050051:Transient Receptor Potential Channels; C106336:phosphatidylinositol 3,5-diphosphate
keywords: PIKfyve; TRP channel; vesicle fusion