PI4P detection

PI4P was detected by four independent approaches: three based on the imaging of PI4P in living or fixed cells and one on a biochemical PI4P mass assay on isolated Golgi fractions using recombinant PIP5K1C.

PI4P was detected using three independent imaging approaches: staining of PI4P with a monoclonal anti-PI4P antibody (Echelon) following the protocol described by Hammond et al. (2009), detection of PI4P using two different genetically encoded probes (P4M and P4C, corresponding to the PI4P-binding domains of the two Legionella pneumophila effectors SidM and SidC, respectively; Hammond et al., 2014; Luo et al., 2015), and staining of PI4P in streptolysin O (SLO)–permeabilized cells with a recombinant PI4P probe produced in Escherichia coli (GST-FAPP PH domain) as previously described (Godi et al., 2004).

Hela cells were double stained for PI4P and for the TGN with anti-PI4P and anti-Golgin-97 antibodies, respectively, following the protocol of Hammond et al. (2009). Briefly, cells were fixed with 2% PFA for 15 min at RT, washed three times in PBS supplemented with 50 mM NH₄Cl, and permeabilized for 5 min by the addition of 20 µM digitonin in Buffer A (20 mM Pipes, pH 6.8, 137 mM NaCl, and 2.7 mM KCl) for 5 min. After three rinses in Buffer A, cells were blocked for 45 min with blocking solution (Buffer A supplemented with 5% [vol/vol] FBS and 50 mM NH₄Cl) and incubated with primary antibodies diluted in blocking solution for 1 h. Cells were then washed with Buffer A, incubated with fluorescence-conjugated secondary antibodies for 1 h, and finally postfixed for 5 min in 2% PFA. 10–15 fields (each containing 10–15 cells) were randomly sampled and imaged at the same microscope settings (i.e., laser power and detector amplification) and below pixel saturation. The mean intensity per cell was determined using the ImageJ software. For PI4P level assessment, a mask using the TGN marker Golgin-97 was generated for each cell, and the mean intensities of both PI4P and Golgin-97 were measured in those regions. After background subtraction, the PI4P values were normalized singularly using their own Golgin-97 values.

Both P4M and P4C, expressed for 16 h in HeLa cells, exhibited a clear Golgi localization in living cells, but only P4C preserved a strong association with the Golgi complex after fixation, consistent with its higher affinity for PI4P as compared with P4M (Luo et al., 2015). Quantification of Golgi PI4P detected with P4C was performed both in live cells by fluorescence loss in photobeaching (FLIP) and in fixed cells. FLIP was performed in cells expressing GFP-P4C by bleaching iteratively (100 times) the GFP-associated fluorescence in the entire cell area except for the Golgi area and monitoring the Golgi-associated fluorescence over time. The relative fluorescence intensity, expressed as a percentage of prebleaching fluorescence, was plotted. Reflecting the increase in Golgi PI4P, the depletion of VAPs and FAPP1 induced a slowdown of the FLIP-induced decay curves of P4C from the Golgi area. For quantification of P4C associated with the Golgi complex in fixed cells, HeLa cells expressing GFP-P4C were stained for a TGN marker (Golgin-97) used to generate a mask in the Golgi region. The mean intensity of GFP-P4C was measured in the Golgi region and normalized for the mean intensity of an equivalent area outside the Golgi region. Golgi PI4P was expressed as the ratio between GFP-P4C fluorescence intensity in the Golgi region and in the equivalent extra-Golgi area.

Quantification of the GST-FAPP PH domain on the Golgi complex was performed as described (Godi et al., 2004): cells were incubated with 1 U/ml SLO in SLO buffer (20 mM Hepes, pH 7.2, 110 mM Mg(OAc)₂, and 1 mM DTT) for 10 min at 4°C. Cells were then washed twice and incubated in permeabilization buffer (25 mM Hepes, pH 6.95, 125 mM KOAc, 2.5 mM Mg(OAc)₂, 10 mM glucose, 1 mM DTT, 1 mM ATP, 5 mM creatine phosphate, 7.3 U/ml creatine phosphokinase, and 1 mg/ml rat liver cytosol) for 5 min at 37°C and then in permeabilization buffer containing 1 µM of GST or GST–FAPP-PH for an additional 5 min. Cells were then fixed with 4% PFA and processed for immunofluorescence microscopy, as described above. Quantification was performed as described above for the PI4P antibody, with the exception that the Golgi marker used was Giantin instead of Golgin-97.

Golgi fractions, identified as fractions enriched for Golgi markers and devoid of PM markers, were isolated as described (Balch et al., 1984). Golgi fractions (60 µg) were washed with 0.2 M KCl in 20 mM Hepes, pH 7.4, and 10 mM MgCl₂ and centrifuged at 14,000 rpm for 20 min. Pellets were resuspended in 20 mM Hepes and incubated for 30 min at 32°C in reaction buffer (20 mM Hepes, pH 7.4, 10 mM MgCl₂, 40 µM ATP, and 200 µM sodium orthovanadate) with 2 µCi [γ-³²P]-ATP, in the presence or absence of 30 ng of recombinant PIP5K1C in a final reaction volume of 100 µl. The reaction was stopped by adding 20 µl 8 M HCl, and lipids were extracted with chloroform:methanol (1:1). The organic phase was dried and spotted on silica-gel TLC plates (Millipore), resolved with a mixture of chloroform:methanol:ammonium hydroxide:water (60:47:2:11 vol/vol/vol/vol), and analyzed by TLC. A sample where PIP5K1C was incubated with 3 μM porcine brain PI4P in reaction buffer with 2 µCi [γ-³²P]-ATP was taken as a reference for PI4,5P₂. The radioactive products were detected with a Typhoon TLA 9500.