Phosphatidylinositols

The lipid extraction (adapted from Matyash et al. 23 (link)) was carried out in high grade polypropylene deep well plates. Fifty microliters of diluted plasma (50×) (equivalent of 1 μL of undiluted plasma) was mixed with 130 μL of ammonium bicarbonate solution and 810 μL of methyl tert-butyl ether/methanol (7:2, v/v) solution was added. Twenty-one microliters of internal standard mixture was pre-mixed with the organic solvents mixture. The internal standard mixture contained: 50 pmol of lysophasphatidylglycerol (LPG) 17:1, 50 pmol of lysophosphatic acid (LPA) 17:0, 500 pmol of phosphatidylcholine (PC) 17:0/17:0, 30 pmol of hexosylceramide (HexCer) 18:1;2/12:0, 50 pmol of phosphatidylserine (PS) 17:0/17:0, 50 pmol of phosphatidylglycerol (PG) 17:0/17:0, 50 pmol of phosphatic acid (PA) 17:0/17:0, 50 pmol of lysophposphatidylinositol (LPI 17:1), 50 pmol of lysophosphatidylserine (LPS) 17:1, 1 nmol cholesterol (Chol) D6, 100 pmol of diacylglycerol (DAG) 17:0/17:0, 50 pmol of triacylglycerol (TAG) 17:0/17:0/17:0, 50 pmol of ceramide (Cer) 18:1;2/17:0, 200 pmol of sphingomyelin (SM) 18:1;2/12:0, 50 pmol of lysophosphatidylcholine (LPC) 12:0, 30 pmol of lysophosphatidylethanolamine (LPE) 17:1, 50 pmol of phosphatidylethanolamine (PE) 17:0/17:0, 100 pmol of cholesterol ester (CE) 20:0, 50 pmol of phosphatidylinositol (PI) 16:0/16:0. The plate was then sealed with a teflon-coated lid, shaken at 4°C for 15 min, and spun down (3000 g, 5 min) to facilitate separation of the liquid phases and clean-up of the upper organic phase. Hundred microliters of the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. Dried lipids were re-suspended in 40 μL of 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, v/v/v) and the wells were sealed with an aluminum foil to avoid evaporation and contamination during infusion. All liquid handling steps were performed using Hamilton STARlet robotic platform with the Anti Droplet Control feature for organic solvents pipetting.

Lipid extracts were dissolved in 60 μl of chloroform/methanol (1:2, v/v) and subjected to mass spectrometric analysis using an LTQ Orbitrap XL instrument (Thermo Fisher Scientific) equipped with a TriVersa NanoMate (Advion Biosciences) as previously described [4 (link),7 (link)]. The 10:1-phase lipid extracts were analyzed by positive ion mode multiplexed FT MS analysis with scan ranges m/z 280-580 (monitoring lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species) and m/z 500-1200 (monitoring sphingomyelin (SM), ceramide (Cer), diacylglycerol (DAG), PC, ether-linked PC (PC O-), phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (PE O-) and triacylglycerol (TAG) species). The 2:1-phase lipid extracts were analyzed by negative ion mode multiplexed FT MS analysis with scan ranges m/z 370-660 (monitoring lysophosphatidic acid (LPA), lysophosphatidylserine (LPS) and lysophosphatidylinositol (LPI) species) and m/z 550-1700 (monitoring phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and sulfatide (SHexCer) species). All FT MS spectra were acquired in profile mode using a target mass resolution of 100,000 (fwhm), activation of isolation waveforms, automatic gain control at 1e6, max injection time at 250 ms and acquisition of 2 µscans.

Stock solutions (1–10 mg/mL)
of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS, Avanti Polar Lipids, Alabaster, AL, USA), and
ATTO 390-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
(Atto 390-DPPE, ATTO-TEC, Siegen, Germany) were prepared in chloroform. l-α-Phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P₂, brain porcine, Avanti Polar Lipids, Alabaster, AL, USA)
was freshly dissolved in chloroform/methanol/H₂O (10:20:8)
to a final concentration of 1 mg/mL. Lipid mixtures (0.4 mg) were
prepared in chloroform/methanol (10:1), and organic solvents were
evaporated with a nitrogen stream followed by 3 h in vacuum. The dried
lipid films were stored at 4 °C until needed.
Small unilamellar
vesicles (SUVs) were prepared by rehydrating a lipid film in spreading
buffer (50 mM KCl, 20 mM Na-citrate, 0.1 mM NaN₃, 0.1 mM
ethylenediaminetetraacetic acid (EDTA), pH 4.8),^{38 (link)} incubating for 30 min, subsequent vortexing (3 × 30
s at 5 min intervals), and a final sonification step for 30 min at
room temperature (cycle 4, 60%, Sonopuls HD2070, resonator cup; Bandelin,
Berlin, Germany). PtdIns[4,5]P₂ containing SUVs were used
immediately for the preparation of SLBs to avoid PtdIns[4,5]P₂ degradation.^{65 (link)}

Protein kinase A, lactate dehydrogenase, and phosphoenol-pyruvate were purchased from Roche CustomBiotech (Indianapolis, IN). Adenosine-5′-triphosphate disodium salt (ATP) ultrapure 98% was obtained from Alfa Aesar (Tewksbury, MA). Verapamil was acquired from Sigma Aldrich (Saint Louis, MO). n-dodecyl-β-D-maltopyranoside (DDM) was bought from Inalco S. p.A (Milano, Italy). Nicotinamide adenine dinucleotide (NADH) was purchased from Sigma-Aldrich (Burlington, MA).
E. coli polar lipids (polar extract) and synthetic lipids were acquired from Avanti (Alabaster, AL); these include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or 16:0-18:1 PC (POPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylinositol (POPI), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphate or 16:0 PA, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Sphingomyelin (SM) was >99% pure from porcine brain with major acyl chains of 18:0 (50%) and 21:1 (21%), and cardiolipin (CL) was from >99% bovine heart with major acyl chains of 18:2 (90%). All synthetic lipids, SM and CL had very low tryptophan fluorescence (ex/em 295/350 nm) if purchased as powder. Cholesterol (Chol) and cholesteryl hemisuccinate (CHS) were purchased from Anatrace (Maumee, OH).
General chemicals were at the highest grade from Thermo Fisher Scientific (Waltham, Massachusetts).

All intensity-based image processing and analysis were performed using ImageJ/(Fiji) software (Schindelin et al., 2012 (link)). The Imaris software (Bitplane AG, Switzerland) was specifically used to generate 3D projections and volume renderings. For analysis of the microtubule patterning, z-stacks were deconvolved with Huygens Essential version 22.04 (Scientific Volume Imaging, The Netherlands), using the CMLE algorithm, with Acuity: –60 and 20 iterations.
FLIM-based images were analyzed using LAS X SP8 Control Software (Leica Microsystems GmbH). Global fitting of the intensity decay profile using n-exponential reconvolution was performed to separate major fluorescent components within each channel and calculate their lifetime. The number of components (n) used for curve fitting was determined according to the evaluation of the chi-squared (χ2) value (Lakowicz, 2006 (link)) and a threshold of 30 photons was applied to generate the final images, unless otherwise stated. Components with lifetime value <1 ns representing autofluorescent species accumulating in plant cell walls (Donaldson, 2020 (link); Heskes et al., 2012 (link)) were subtracted from each channel.
All data on reconvolution of decay profiles of images presented in the article are provided as source files.
Since global fitting does not always allow to resolve and characterize the decay profile of pixels constituting minor populations within an image (Ranjit et al., 2018 (link)), the decay profile of fluorescent emission of regions of interest (ROIs) selected on membrane domains was additionally analyzed for the analysis and quantification of PI(4,5)P₂ membrane enrichment. n-exponential fitting of the decay curve was carried out to resolve lifetime values associated with each ROI (Figure 5—figure supplement 1). Reconvolution data from all images and selected ROIs analyzed are provided as source files. Further, the raw decay profile of each ROI was analyzed using the Phasor approach to obtain a Phasor fingerprint whose position describes the fluorescent emission at that region and reflects its composition with respect to the relative abundance of fluorescent species (Figure 5—figure supplement 1) (see Ranjit et al., 2018 (link) and Malacrida et al., 2021 (link) for a detailed description of the Phasor approach). Phasor plots were generated using a second harmonic, a threshold of 22 photons, and a median of 15. To visualize and compare the Phasor fingerprint of ROIs selected in multiple images from different genotypes, the center of mass of Phasor images depicting the pixel populations originating from each ROI was calculated images using ImageJ/(Fiji) (Schindelin et al., 2012 (link)) and the obtained XM and YM coordinates were plotted on a graph (Figure 5—figure supplement 1). A Mann-Whitney non-parametric test was performed to detect statistically significant differences between XM and YM values.
For the quantification of PI(4,5)P₂ and PI4P membrane enrichment, the mean fluorescence intensity of freehand ROIs drawn on the IT tip and on the plasma membrane of infected root hairs were measured on images of single fluorescent components from each channel, obtained from global fitting of the intensity decay profile (threshold of 20 photons) and subtraction of components with lifetime <1 ns (both channels) and >1.7 ns (channel 2). The mean intensity of a 2.5 × 2.5 μm square ROI outside of the infected root hair cell was measured as a background value, subtracted from each ROI and the mean gray value on the IT tip was divided by the mean gray value on the plasma membrane [(Int_{IT tip} - Int_bkgd) / (Int_PM- Int_bkgd)] for each component to obtain an index of the enrichment of each phosphoinositide at the IT tip relative to the plasma membrane. Statistically significant differences were detected using a Mann-Whitney non-parametric test.