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.

Keywords: PIKfyve; TRP channel; vesicle fusion.

Fig. 1.

Colocalization of the ML1N-based PI(3,5)P₂ probe with endolysosomal markers. (A) Purified GST-GFP-ML1N*2 proteins bound strongly to PI(3,5)P₂-containing liposomes, but not to liposomes containing PI(3)P, PI(5)P, or PI(4,5)P₂. Liposomes, diluted to a final concentration of 20 μM total lipids with 1 μM tested phosphoinositide, were incubated with purified GST-fusion proteins for 20 min, centrifuged, and visualized by Western blot with GST antibodies. (B) GFP-ML1N*2 exhibited a vesicular location and significant colocalization with Lamp1-mCherry. Confocal images were taken 24 h posttransfection in COS1 cells that were cotransfected with Lamp1-mCherry and GFP-ML1N*2. (C) Charge-neutralizing mutations in the phosphoinositide-interacting domain (R⁴²Q/R⁴³Q/R⁴⁴Q/K⁵⁵Q/R⁵⁷Q/R⁶¹Q/K⁶²Q, abbreviated as 7Q; R⁶¹A/K⁶²A, abbreviated as 2A; also see ref. 21) selectively impaired or abolished the activation of TRPML1 by PI(3,5)P₂ (1 µM) in the whole-endolysosome configuration; enlarged endolysosomes were isolated from COS1 cells transfected with various TRPML1 constructs. In contrast, ML-SA1 readily activated wild-type (WT) TRPML1, TRPML1-7Q, and TRPML1-2A in the same endolysosomes. (D) The localization of the ML1N*2 probe to vacuolar membranes was dependent on the basic residues in its phosphoinositide-interacting domain. Both ML1N-7Q*2 and ML1N-2A*2 displayed a diffuse, cytosolic localization pattern. (E) Quantification of the colocalization index between ML1N*2 and Lamp1 (see SI Materials and Methods for the algorithm that was used to analyze the colocalization). (F) Colocalization of GFP-ML1N*2 with LysoTracker (red) in COS1 cells. (G) Colocalization of GFP-ML1N*2 with endogenous Lamp1 (recognized by a Lamp1 antibody) in MEF cells. (H–K) Colocalization analyses between ML1N*2 and other fluorescently tagged endolysosomal proteins/markers in COS1 or MEF cells that were transfected with the indicated constructs for 24 h. (H) Colocalization of mCherry-ML1N*2 with the early endosomal marker GFP-EEA1. (I) Colocalization of ML1N*2 with the PI(3)P probe GFP-Hrs-FYVE*2. (J) Colocalization of ML1N*2 with YFP-PIKfyve. (K) Quantitative analyses revealed that ML1N*2 exhibited various degrees of colocalization with YFP-PIKfyve, GFP-FYVE*2, and GFP-EEA1. Error bars represent SEM; numbers of data points are given in the text. The asterisk indicates P < 10⁻⁵. (Scale bar: 10 μm.)

Fig. 2.

Genetically or pharmacologically induced decreases in PI(3,5)P₂ levels diminish or abolish the vesicular localization of the ML1N*2 probe. (A) GFP-ML1N*2– and Lamp1-mCherry–transfected COS1 cells were treated with YM201636 (800 nM) overnight before confocal imaging analysis. Although Lamp1-positive compartments were dramatically enlarged, the GFP-ML1N*2 probe was primarily localized to the cytosol and was not found on enlarged vacuolar membranes. (B) The vesicular localization of the mCherry-ML1N*2 probe was significantly reduced in cells cotransfected with the 1-phosphatidylinositol-3-phosphate 5-kinase (Fab1)-DN construct. Images were taken 24 h posttransfection. (C) YM201636 treatment resulted in a progressive decrease in ML1N*2’s colocalization with Lamp1. Washout of the drug in the culture medium led to a complete recovery of the colocalization. (D–F) The ML1N*2 probe exhibited an apparent vesicular localization pattern in WT (D), but not in Vac14^−/− (E) or Fig4^−/− (F) MEF cells. (G) The colocalization index between ML1N*2 and Lamp1 was significantly reduced in Vac14^−/− and Fig4^−/− MEFs. Error bars represent SEM; numbers of data points are given in the text. The asterisk indicates P < 10⁻⁵. (Scale bar: 10 μm.)

Fig. 3.

Endolysosomal PI(3,5)P₂ levels are regulated by serum-derived factors. (A) In COS1 cells dually transfected with GFP-ML1N*2 and Lamp1-mCherry, serum starvation induced a progressive reduction in the colocalization of ML1N*2 and Lamp1. Readdition of complete medium rapidly (<2 h) restored the high degree of colocalization. (B) Serum-dependent colocalization of the ML1N*2 probe and Lamp1. Error bars represent SEM; numbers of data points are given in the text. The asterisk indicates P < 10⁻⁵. (Scale bar: 10 μm.)

Fig. 4.

PI(3,5)P₂ is transiently increased immediately before membrane fusion in LELs. (A–C) Time-lapse live imaging of COS1 cells that were dually transfected with GFP-ML1N*2 and Lamp1-mCherry. (A) Image of a COS1 cell immediately before membrane fusion between two Lamp1-positive vacuoles occurred (highlighted in the white box). (B) Time-dependent changes in the fluorescence intensities of GFP and mCherry for the selected region (white box, A). (C) Series of images of the selected region at corresponding time points as in B. For a 4-min track period, one fusion event between two vacuoles occurred between every 1 min and 1 min 15 s (D and E) GFP-ML1N*2 and Lamp1-mCherry dually transfected COS1 cells were treated with acetate Ringer’s solution for 30 min and then subjected to live imaging for 5–20 min after being returned to the normal Ringer’s solution. Approximately one-half of the vacuoles (45% of the 22 monitored events; D) exhibited a significant increase (>20% of the baseline membrane intensity) in their membranous PI(3,5)P₂ levels immediately before fusion, whereas the other half (55%; E) had little or no changes. The white and blue arrows mark vacuoles prefusion and postfusion, respectively. (F) Changes in vacuolar PI(3,5)P₂ levels in relation to fusion events. The fluorescent intensity of GFP-ML1N*2 was normalized to the cytosolic intensity at time 0, and two representative traces are shown; the vertical dotted line indicates the time when the fusion took place. The cytosolic intensity of the probe remained relatively constant over the time period of the live-imaging experiment. [Scale bar: 10 μm (for A) and 2 μm (for C and D).]