The surfactant concentrations varied from 0.2 mmol/L to 3.0 mmol/L. All of the hydrogels presented viscous behavior (G″ > G′). Hydrogels containing AOT (c > 2.0 mmol/L) led to cryogels with the lowest compressive modulus (13 ± 1 kPa), the highest specific surface area (2.31 m2/g), the lowest thermal conductivity (0.030 W/(m·°C)), and less hygroscopic walls. The addition of Kolliphor® EL to the hydrogels yielded the stiffest cryogels (320 ± 32 kPa) with the lowest specific surface area (1.11 m2/g) and the highest thermal conductivity (0.055 W/(m·°C)). Density functional theory (DFT) calculations indicated an interaction energy of −31.8 kcal/mol due to the interaction between the AOT sulfonate group and the HPMC hydroxyl group and the hydrogen bond between the AOT carbonyl group and the HPMC hydroxyl group. The interaction energy between the HPMC hydroxyl group and the Kolliphor® EL hydroxyl group was calculated as −7.91 kcal/mol. A model was proposed to describe the effects of AOT or Kolliphor® EL on the microstructures and the mechanical/thermal properties of HPMC cryogels.The mechanical and thermal properties of cryogels depend on their microstructure. In this study, the microstructure of hydroxypropyl methylcellulose (HPMC) cryogels was modified by the addition of ionic (bis (2-ethylhexyl) sodium sulfosuccinate, AOT) and non-ionic (Kolliphor There are different methods for preparing 3D porous polymeric structures. One of them is the addition of porogenic agents to the polymerization process or to the polymer system. The choice of porogens requires a careful analysis on their chemical structure, physical state, solubility, and miscibility with other chemical constituents  . Other sructures . Cryogelructures . On the −3 and surface area larger than several hundreds of m2 g−1. For instance, chitosan aerogels presented a thermal conductivity of 0.022 W/(m K) and a specific surface area of 545 of m2 g−1 [2 based aerogels presented a thermal conductivity of (0.022–0.025 W m−1 K−1) [−1 K−1) and density values below 0.15 g cm−3 [Polysaccharide-based porous structures are sustainable materials because they stem from renewable sources, they are biodegradable and nontoxic, and their preparation does not generate hazardous waste. Aerogels and cryogels have interconnected micro- and macropores and low density and low thermal conductivity, respectively. Recently, Zou and Budtova presented an overview on the correlation between the thermal conductivity, density, and surface area of polysaccharide-based aerogels and cryogels reported in the literature . They cof m2 g−1 . Pectin-m−1 K−1) , whereas5 g cm−3 . However5 g cm−3 ; the dis5 g cm−3 .co-polyethylene glycol diacrylate) [One strategy to control the microstructure of cryogels and therefore their properties is the addition of surfactants to the hydrogel precursor. Ni et al. prepared Konjac glucomannan-based cryogels from hydrogel precursors containing different glyceryl monostearate contents . A high crylate) . However® EL (Polyoxyl-35 castor oil), a non-ionic surfactant with long alkyl chains that is frequently used as emulsifier in cosmetic and drug formulations [Hydroxypropyl methylcellulose (HPMC) is a cellulose ether with amphiphilic character controlled by the degree of substitution (DS) of the methyl groups and the molar substitution (MS) of the hydroxypropyl groups in the repeated unit . Due itsulations , or dioculations , to HPMC3 (AOT5) to 39 ± 5 kg/m3 (K7.25). The gel content, which indicates the efficiency of the esterification reaction between citric acid carboxyl groups and HPMC hydroxyl groups, decreased from 86 ± 1% (HPMC~K0.5) to 61 ± 1% (K7.25) as the Kolliphor® EL concentration in the precursor hydrogels increased. This finding indicates that the Kolliphor® EL hydroxyl groups might compete with the HPMC hydroxyl groups for esterification with the citric acid carboxylic acid groups [−1 assigned as the ester -C=O stretching vibrations in all of the cryogels evidenced crosslinking between HPMC and citric acid [−1.The sample codes and their compositions are listed in d groups . In the ric acid ,22. Noteric acid ,22, but Y) at which the material starts to plastically deform for all of the cryogels. All of the cryogels prepared with Kolliphor® EL were considerably stiffer than pure HPMC cryogels. On the other hand, the addition of a low content (less than 1.0 mM) of AOT in the hydrogel precursors resulted in the cryogels being slightly stiffer than HPMC cryogels, whereas the addition of a higher content (more than 2.0 mM) of AOT yielded very soft cryogels. AOT7.25 presented a mean E value one order of magnitude smaller than that of the HPMC cryogel. A plausible explanation for this effect is that the addition of AOT yielded cryogels with thinner cell walls than those of pure HPMC cryogels.® EL stiffened the HPMC cell walls, whereas the presence of AOT (at concentration higher than the cmc) yielded softer cryogels.The type or the amount of surfactant had no substantial effect on the rheological behavior of the precursor hydrogel at 25.0 ± 0.5 °C. All of the hydrogels showed viscous behavior (G″ > G′) , in agre® EL presented large pores in a lesser amount, implying cell wall thickening. On the other hand, the cryogels prepared with AOT presented small pores in larger quantities, implying cell wall thinning. These trends explain the high values of Young’s moduli determined for cryogels prepared with Kolliphor and the low values of Young’s moduli determined for cryogels prepared with AOT , and av2 g−1 and ~1.8 m2 g−1 determined for AOT and HPMC are of the same order of magnitude of those determined for cellulose nanofibrils/nonionic polyoxamer-based foams [® EL hydroxyl groups compete with the HPMC hydroxyl groups for esterification with citric acid. One should notice that the connectivity density correlates to the amount of the connected polymeric material to form the cell walls [ed foams  and nanoed foams ,28. Furtll walls . Cryogelll walls  might ink) values obtained using the THS method at 23.7 ± 0.7 °C and 60% relative humidity. The linear region between 46 s and 164 s of each curve was fitted to linear regression  although there was no statistically significant difference (pn = 3) among this k value and those determined for the AOT2.75, AOT5, K0.5, and K7.25 cryogels; it was statically different from the HPMC (0.038 W m−1 °C−1) cryogel. Cellulose nanofibrils crosslinked with 1,2,3,4-butanetetracarboxylic acid and N-methylol dimethylphosphonopropionamide led to sponges with a k value (0.0326 W m−1 °C−1) and an E value of ~5 kPa [k value (0.033± 0.001 W m−1 °C−1) and E value of ~13 ± 1 kPa. The highest k values were observed for K2.75 (0.055 W m−1 °C−1), K5 (0.045 W m−1 °C−1), and AOT0.5 (0.043 W m−1 °C−1); similar values were reported for nanofibrillated cellulose sponges [gression ; the lowf ~5 kPa ; these p sponges . Materia sponges , becausek values obtained are consistent with the range observed by Jimenez-Saelices et al. [The s et al. . The heall voids .3). Among all samples, AOT7.25 presented the smallest lowest k value because it contains the largest surface area (Asp) and the smallest pore size ; the compressive modulus can be tuned by the amount of AOT added to the precursor hydrogel, keeping low thermal conductivity. Cryogels prepared with Kolliphor® EL occupied a region (magenta) characteristic of higher compressive modulus and thermal conductivity values; the thermal conductivity can be tuned by the content of Kolliphor® EL in the precursor hydrogel.® EL, and AOT was evaluated using TG/DTG curves  between 30 °C  of the HPMC cryogels, K0.5, AOT 0.5 (lowest surfactant concentration), and K7.25 (highest surfactant concentration) were similar, ranging from 32 ± 1 ggwater/g . However® EL system, the calculated interaction energy was −7.91 kcal/mol. ® EL, where only one H bond between the Kolliphor® EL hydroxyl and the HPMC hydroxyl groups drives the interaction.Density Functional Theory (DFT) calculations were performed in order to gain insight into the molecular interactions between the surfactants and the HPMC in vacuum, which might be correlated to the molecular interactions in the cryogels. The optimized geometries indicated an interaction energy of −31.8 kcal/mol for the HPMC–AOT system, driven by the strong interaction between the AOT sulfonate group (ion) and the HPMC hydroxyl group (dipole) and the hydrogen bond between the AOT carbonyl group and the HPMC hydroxyl group, as depicted in n < 20) [The relation between the microstructure and the mechanical/thermal properties of HPMC cryogels could be tuned by the concentration of AOT added to the precursor HPMC hydrogels; the highest AOT concentrations (AOT5 or AOT7.25) led to soft cryogels with a relatively low thermal conductivity, which were associated to a microstructure of many small pores and thin cell walls. Furthermore, these cryogels were less hydrophilic than the control (HPMC cryogel). AOT is frequently used as an emulsifier in water/oil emulsion, building reverse micelles, where the aggregation number is usually low (n < 20) . In HPMCn < 20) . Based on < 20) . Therefo® EL added to the precursor hydrogels led to cryogels with a high compressive modulus and high thermal conductivity compared to HPMC cryogels. DFT calculations evidenced that the interactions between Kolliphor® EL and HPMC are not as strong as those between AOT and HPMC are. The presence of the Kollipho® EL micelles [The concentration of Kolliphormicelles  possibly−1 °C−1) and low hygroscopicity. The interactions between Kolliphor® EL and HPMC are weak; the Kolliphor® EL micelles probably increased the ice crystal size, yielding stiffer cryogels. The stiffest cryogels (E = 55 kPa) presented the highest thermal conductivity value (0.055 W m−1 °C−1). By changing the type and concentration of the surfactant in the precursor hydrogels, a wide range of compressive modulus and thermal conductivity values could be achieved  cryogels with relatively low thermal conductivity , dioctyl sulfosuccinate salt sodium or AOT , citric acid , and sodium hypophosphite  were used as received. MilliQ water was employed in all experiments. The critical micelle concentration (cmc) of AOT and Kolliphor® EL in water were determined as 1.9 mM and 0.02 mM, respectively, at 21 ± 1 °C  of 1.5 and a molar substitution (MS) of 0.75 (USP 1828) was kindly supplied by The Dow Chemical Company ; the viscometric average molar mass  and sodium hypophosphite  were then added at 1.0 g·L−1 and 0.5 g·L−1; these concentrations were chosen based on previous reports [−1 and was homogenized for 1 h. The resulting hydrogels were coded as HPMC , K0.5 and AOT0.5 (0.2 mM), K2.75 and AOT2.75 (1.0 mM), K5 and AOT5 (2.0 mM), and K7.25 and AOT7.25 (3.0 mM), as shown in ® EL in all gels was above of its cmc (0.02 m)M, whereas the concentration of AOT in the AOT0.5 and AOT2.75 gels was below its cmc, and in the AOT5 and AOT7.25 gels, it was above its cmc. Aqueous solutions of AOT and Kolliphor reports . After 3The precursor hydrogels were transferred to acrylic molds of different geometries: A , B , C , D , and E . The molds were covered in order to avoid significant volume variation due to ice crystallization during freezing. They were kept inside three Styrofoam boxes  for 24 h® rheometer from Anton Paar at 25.0 ± 0.5 °C under a dry nitrogen atmosphere. A cone and plate geometry (angle of 0.992°) with a gap size of 0.101 mm and a diameter of 50 mm was used. For DSST, pure HPMC (blank), K7.25, and AOT7.25 hydrogels were tested at the frequencies of 1 Hz and 10 Hz, measuring G′, G″ as a function of the strain amplitude (Dynamic strain sweep tests (DSST), dynamic time sweep tests (DTST), and small amplitude oscillatory shear (SAOS) tests were performed for the hydrated samples using a stress-controlled MCR 501mplitude . In orde at 0.5% . The obt−1 in order to remove unreacted molecules. The cryogels were then frozen and freeze-dried again. The apparent density (apρ) of six dried cryogels (mold B) was determined at 23 ± 1 °C and relative air humidity of 65 ± 10%. The ratio between their masses  until the rinsing water achieved the conductivity of ~5 µS·cmTo determine the gel content (percentage of crosslinked HPMC chains), the cryogels (mold C) were swollen with MilliQ water, freeze-dried, and weighed (waterm) divided by driedm, as shown by Equation (3): The swelling degree (SD) was determined for HPMC, K0.5, AOT0.5, K7.25, and AOT7.25 (mold C) samples using a force tensiometer Krüss K100 at 21.0 ± 0.5 °C as the mass of sorbed MilliQ water (pH 5.5) at equilibrium (®) with a diamond crystal and an accumulation of 128 scans (2 cm−1 resolution). Thermogravimetric analyses (TGA) were performed for the HPMC, K0.5, AOT0.5, K7.25, and AOT7.25 samples (mold C) in alumina crucibles using the Netzsch STA 449 F3 Jupiter (Netzsch®) equipment under a N2 atmosphere. The temperature program had three segments: 1 (from 30 °C to 200 °C at a heating rate of 20 °C/min), 2 (from 200 °C to 30 °C at a cooling rate of −20 °C/min), and 3 (from 30 °C to 500 °C at a heating rate of 10 °C/min). The first run up to 200 °C or well below the degradation point was performed in order to remove thermal history and moisture. Afterwards, the second run was performed slowly at the heating rate of 10 °C/min to observe the actual thermal stability in TGA and its char yield.FTIR-ATR vibrational spectra of the HPMC, K0.5, AOT0.5, K7.25, and AOT7.25 samples (mold C) were obtained using an Alpha FTIR-ATR spectrometer  method ,39,40,41.5 kg/m3 ). The co® in order to obtain the electric power supplied to the strip over time. An Arduino Uno R3 board was used to transfer the acquired data to the computer .All measurements of thermal conductivity were performed with a low cost (~USD 100) homemade device. It comprises an isothermal aluminum case with a NiCr resistive strip (2.0 mm × 0.1 mm × 110.0 mm) positioned at the center of the case, and one of the sample faces was against the strip. One piece of PU foam (reference) was placed against the other side of the strip, and another piece of PU foam was placed against the sample, as shown in computer b. The te−1 and at an air temperature of 23 ± 1 °C (60% relative humidity). The samples remained close to the dynamometer under 60% relative humidity for 24 h as recommended by ISO 3386-1 [The compressive tests were performed in quadruplicate for the cryogel samples (mold B) using an Impac Digital Dynamometer IP-90DI with a 1 kN load cell at strain rate of 0.01 sO 3386-1 . ® software were the specific area 3); and average pore size. The pore size distributions in the HPMC, K7.25, and AOT7.25 samples were calculated with CTan® software.VOI (volume of interest) is the reconstructed volume from which the software performed the calculations, VOI, and ρ is the material density, which was considered as 34 kg/m3 (Freeze-dried cryogels (mold D) coated with a gold layer (~10 nm) were analyzed in a JEOL Neoscope JCM-5000 microscope operating at voltage of 10 kV. The cryogels (mold E) were analyzed using X-ray microtomography (micro-CT) with Bruker Skyscan 1272 equipment with the X-ray source operating at 20 kV and a current of 175 μA. The samples were rotated 0.6° per step, obtaining a resolution of 4 μm. The NRecon  and CTVOx  software programs were used for the 3D reconstruction from cross-sectional images and for the visualization and image acquisition, respectively. Specific surface area values of volumes of interest (VOI) were calculated using the software CTan . The analyses of the VOIs with the CTan34 kg/m3 .® EL–HPMC in vacuum. For the calculations, an HPMC tetramer containing 6 -CH3, 3 -CH2-CHOH-CH3, and 5 OH groups and one third of the Kolliphor® EL molecule were drawn (the -O-CH2- bond from the precursor glycerol was considered as -O-CH3), as shown in Density Functional Theory (DFT) calculations were performed using the software Gaussian 09  with the


Eighty lactating cows were randomly allocated to one of four treatment groups:1) CON (control), received 5 mL of saline solution on d 6 and 14 post AI; 2) IP4 (injection of P4), received 125 mg of P4 im on d 6 and 14 post AI; 3) CIDR, received acontrolled internal drug release insert containing 1.38g of P4 from d 6 to 20 postAI; and 4) FM (Flunixin Meglumine), received 0.625 g of Flunixin Meglumine, a nonsteroidalanti-inflammatory drug, twice daily on d 15 and 19 post AI. Blood samples were taken on d 0, 6,14, 17 and 20 post AI to determine P4 concentrations. Transrectal palpation wasperformed between 40 and 45 d post AI to determine pregnancy status. All treatment groups  resulted in greater serum P4 concentration on d 17 and 20 postAI compared to CON (P < 0.05). Cows given a CIDR insert had greater concentrations of P4 on d 17 and 20 than IP4 and FM cows (P < 0.05). However, no significant differencewas found between IP4 and FM groups for serum P4 concentrations. The P/AI was greater(P < 0.05) in CIDR-treated cows  than CON , and intermediate in IP4 and FM  cows. In summary, treatment with exogenous P4 (i.e.CIDR and IP4) or FM increased serum P4 concentrations in lactating dairy cows.However, results suggest that only CIDR administration would improve P/AI.The objective of this study was to determine the effect of post AI administration of exogenousprogesterone (P Cows treated with P4 during metaestrusor during the time of pregnancy recognition were 1.71 and 1.4 times, respectively, most likelyto become pregnant than untreated control cows.Early embryonic development and establishment of pregnancy in cattle depends upon progesterone(P2α (PGF2α) at the late luteal stage  is initiated by the release of uterine prostaglandinF4 or a PGF2α synthesis inhibitor agent on serum P4concentrations in lactating dairy cows. A secondary objective was to determine whether postAI administration of exogenous P4 or a PGF2α synthesis inhibitoragent would improve P/AI. Our hypothesis was that post AI administration of exogenous P4 or a PGF2α synthesis inhibitor agent would increase serum P4 concentrations and the proportion of cows becoming pregnant after AI.The primary objective of this study was to evaluate the effect of post AI administration of exogenousPThis study was carried out on a commercial dairy farm located near Kermanshah, Iran. All animalexperimental procedures were reviewed and approved by the Iranian Ministry of Agriculture. Eighty multiparous Holstein dairy cows  were used in thepresent study. Cows were fed a TMR  formulated for a lactating dairy cow of650 kg body weight, producing 35 kg of 3.5% fat milk per day, according to NRC (2001). The TMR wasoffered thrice daily and cows had free access to water. Estrus detection was performed by twotechnicians every 3 h for at least 20 min during each observation. Estrus was confirmed by transrectalpalpation before AI as previously described  on d 6 and 14 post AI. Cows in theCIDR group received a controlled internal drug release insert containing 1.38g of P4 from d 6 to 20 post AI. Cows in the FM group received0.625 g of Flunixin Meglumine twice daily on d 15 and 19 post AI. All treatments were carried out at approximately 0007 h.The day of AI was considered as day 0 of the study and cows (n = 20/group) were randomly allocatedto one of four treatment groups . Cows in the CON group received 5 mLof saline solution on d 6 and 14 post AI. Cows in IP4 group received 125 mg of P4. The serum P4 concentration wasdetermined using a commercial ELISA kit  as described previously(Blood samples (~10 mL) were collected from coccygeal vein into evacuated tubes  on d 0, 6, 14, 17 and 20 post AI in all cows approximatelyat 1100 h. Blood samples were left undisturbed for approximately 2 h at room temperature to allowclot formation and then centrifuged at 2000 × g for 10 min, serum harvested and frozenat -20°C until assayed for PIllustration of experimental procedures and treatments during the study are shown inData were analyzed using SAS software . A probabilityof 0.05 or less was considered statistically significant, and a probability between 0.051 and0.1 was considered a tendency.4 data was analyzed with the MIXED procedure of SAS using the following model:i,j, k,j and sampling day kanda priori todetermine the differences between treatment groups . Pregnancy per AI (P/AI) was comparedamong all experimental groups by using logistic procedure of SAS.Serum P4 concentrations at d 0 and 6 post AI did not differ (P > 0.05) among treatmentgroups. However, cows in the IP4, CIDR and FM groups tended to have greater (P = 0.06) serum P4 concentration at d 14 post AI and had greater (P < 0.01) serum P4 concentrationat d 17 and 20 post AI compared to cows in the CON group. In addition, cows given a CIDR had greater(P < 0.01) concentrations of P4 in serum at d 17 and 20 post AI than IP4- or FM-treatedanimals. There was no significant difference for serum P4 concentration betweenIP4 and FM during the entire study.The serum P4 concentrations in all cows during the entire study are presented and displayed in4 concentrations in pregnant (4 concentrations on d 0 and 6 postAI did not differ (P > 0.05) among treatment groups in pregnant and non-pregnant cows. Similarly,serum P4 concentrations on d 14, 17 and 20 post AI were not significantly different(P > 0.05) among treatment groups in non-pregnant cows. However, in pregnant cows, serumP4 concentrations on d 14 post AI were greatest (P < 0.05) in CIDR group comparedto CON, IP4 or FM. Moreover, pregnant cows in IP4 and FM groups had greater (P < 0.05) serum P4 concentrations than pregnant cows in CON group. However, P4 concentrationsdid not differ between IP4 and FM groups (P < 0.05). On d 17 post AI, serum P4 concentrationswere significantly greater in CIDR group (P < 0.01) compared to CON, IP4 or FM. InterestinglyP4 concentrations did not differ between CON and FM groups. However, pregnant cowsin the IP4 group had greater (P < 0.01) P4 concentrations than pregnant cows inthe CON group. Cows in the CIDR group also had the greatest serum P4 concentrations on d 20 postAI (P < 0.01). In addition, cows in FM and IP4 groups had greater P4 concentrationsthan cows in CON group (P < 0.01). However, serum P4 concentrations did not differbetween IP4 and FM groups (P > 0.05).We also compared the serum Pregnant  and nonregnant  cows amThe P/AI for CON, IP4, CIDR and FM groups were 25% (5/20), 40% (8/20), 55% (11/20) and 35% (7/20),respectively. There was a significant difference for P/AI between CIDR and CON groups (P = 0.03).However, P/AI did not differ between IP4 and FM groups (P = 0.58) or when both groups were comparedwith CON (P = 0.17).4 is one of the main reasons of low fertility in lactating dairy cows in CIDR-treated cows.The pharmacokinetics of P4 following intramuscular injections nor theireffect on serum P4 concentrations and PR/AI has been reported in lactating dairycows. However,4 in lactating dairy cows given an intravenousinjection of 100 mg P4. Plasma P4 reached a concentration of 140 ng/mL1 min after treatment and dropped to baseline concentrations within 2 h after treatment (4 concentrations increased from ∼3 (day 6 post AI)to ∼5 ng/mL (day 14 post AI) and from ∼5 (day 14 post AI) to∼ 9 ng/mL (day20 post AI) in cows given P4 im.Neither the pharmacokinetics of P2αsecretion (2 (PGE2), which is considered a potent luteoprotectivefactor  which may act on differentprostaglandin pathways (i.e. COX-1 and COX-2). Flunixin Meglumine is both a COX-1 and COX-2inhibitor but is more selective for COX-1; it has an elimination half-life of 3 to 8 h  that were supplemented with P4 during maternal recognitionof pregnancy were 1.6 times more likely to become pregnant than the control (no P4treatment) herd mates.A research group from Spain has investigated the effect of PpostAI . In a m4 on fertility was not the primary objective of thisstudy, which was under powered to detect a 14% difference in P/AI among treatments. However,our results are in agreement with others and support the notion that P4 supplementationduring early and late diestrus has a positive effect on P/AI in lactating dairy cows. In addition,numerically more FM cows (35%) become pregnant compared to control cows (25%), but the differencein P/AI was not statistically significant. Current research has not investigated the effectof administration of FM on P/AI in lactating dairy cows, but several reports have been publishedregarding the effect of FM on P/AI in dairy heifers. In agreement with our results, dairy heifers(n = 2325) treated twice with 400 mg of FM im at d 15 and 16 post AI had similar P/AI compared to untreatedheifers (59.4 vs 59.5%)  or Flunixin Meglumine  increased serum P4 concentrationscompared to treatment with saline solution (control). However, results suggest that only CIDRadministration would improve P/AI in lactating dairy cows.In summary, treatment with exogenous P


Plasmodium falciparum, carrying various pfmdr1 gene mutations, were analysed for their transport characteristics of Fluo-4 in isolated DVs of parasites. To obtain quantitative estimates for PfMDR1 DV surface expression, PfMDR1 protein amounts on each strain’s DV membrane were evaluated by quantitative ELISA. Fluo-4, acting as a substrate for PfMDR1, was applied in DV uptake assays (‘reverse Ca2+ imaging’). Viable DVs were isolated from trophozoite stages with preserved PfMDR1 activity. This newly developed assay enabled us to measure the number of Fluo-4 molecules actively transported into isolated DVs per PfMDR1 molecule. The drug-resistant strain Dd2 presented the highest transport rates, followed by K1 and the drug-sensitive strain 3D7, compatible with their copy numbers. With this assay, an evaluation of the probability of resistance formation for newly developed drugs can be implemented in early stages of drug development.Drug resistance often emerges from mutations in solute transporters. Single amino acid exchanges may alter functionality of transporters with ‘de novo’ ability to transport drugs away from their site of action. The PfMDR1 transporter (or P-glycoprotein 1) is located in the membrane of the digestive vacuole (DV), functions as an ATP-dependent pump, and transports substrates into the DV. In this study, four strains of  Plasmodium falciparum  is most prevalent on the African continent and, as the cause of malaria, continues to be a major cause of death, in particular in children under the age of five [ of five . Drug re of five ,3. Most  of five  or metab of five . These a of five .During its blood cycle, the malaria parasite infects human red blood cells (RBCs) and successfully evades the human immune system. For nutrient uptake within the confined RBC milieu, the pathogen builds up a transport system and digests the host’s haemoglobin in the digestive vacuole (DV), producing the bio-inert crystalline hemozoin. Since intermediates of the haemoglobin digestion pathway in the DV are toxic for the parasite , drugs aA homologue of the parasitic PfMDR1 is a pleiotropic human ABC transporter that is associated with drug resistance in cancer cells ,10 and nThe DV is highly shielded in its multi-compartment environment. It is surrounded by the RBC membrane, the parasitophorous vacuolar membrane, and the parasite membrane a. Due toin situ assessment of PfMDR1 transport rates is precariously hampered by the aforementioned conditions. In one of our previous studies, we developed a ‘reverse Ca2+ imaging’ approach using Fluo-4 as a readout transport substrate for PfMDR1 rather than monitoring Ca2+ ions within a DV environment that was previously shown to be relatively stable in free Ca2+ levels across Plasmodium strains [P. falciparum parasites. This system, however, may still be prone to underlying differences in buffer capacitance within the respective compartments to decisively influence observed Fluo-4 transport rates in the intact iRBC system over a putative isolated DV approach.In particular,  strains ,13. ThisP. falciparum parasites and validate their viability by showing intact ATP-dependence of PfMDR1 transport using isolated DVs. Two drug sensitive strains, 3D7 and HB3, and two drug resistant strains, K1 and Dd2, of P. falciparum were characterized. All strains carry different pfmdr1 gene mutations at distinct localizations in the amino acid sequence. As a special feature, Dd2 possesses two copy numbers of mutated pfmdr1  was considered, followed by PfMDR1 mass calculation per DV, with the final calculation of the number of transport protein on one µm surface a.E. coli with a His-tag and purified by Ni-NTA affinity purification . As the overall absorbance of hemozoin seems to differ between the strains a, potent Dd2 DVs b. It appOf great importance is emphasizing the active transport of Fluo-4 by PfMDR1. This is shown by the difference in signal data found without ATP (-ATP) or with ATP (+ATP) present b. This a2+ concentrations were generated   b.2+ indicator, its signal intensities increase with a rise in free Ca2+ present in the sample. This increase is recognized in the standard curves -1-piperazineethanesulfonic acid (HEPES) and 24 mM NaHCO3 supplemented with 0.1 mM hypoxanthine, 25 μg/mL gentamicin and 0.5% AlbuMAX® I [v/v) A+ erythrocytes. Cultures were kept in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 37 °C. Under these conditions, the Dd2 strain has a cycle length of 42 h (hours), 3D7 44 h, HB3 45 h, and K1 47 h to fulfil one asexual replication cycle. Dd2 and 3D7 strains were synchronized twice with 5% Sorbitol in a time interval of 11 h, whereas HB3 and K1 need an interval of 8 h to reach approximately same yields as Dd2 and 3D7 in the final purification step. The 48-h replications factor for Dd2 and 3D7 strains is about 8, whereas HB3 and K1 reproduce approximately 5 times in the same time frame. For DV isolation, trophozoite stages of Dd2 were harvested 53 h after the second synchronization and 3D7 55 h later. HB3 was harvested after 62 h, and K1 64 h after the second synchronization. The parasitaemia reached 5–6% when DV isolation was performed.The four buMAX® I  containiTrophozoite isolation: A 1 mL sized iRBC culture pellet  was centrifuged in one 50 mL tube at 800× g for 3 min. The cell pellet was washed once in 20 mL phosphate-buffered saline (PBS) pH 7.2 and centrifuged again at 800× g for 3 min. Erythrocyte membranes were permeabilized by 10 min exposure to saponin (0.03% w/v) in 40 mL PBS. The tube was inverted and vortexed after 5 min incubation time for 10 sec. Isolated trophozoites were centrifuged with 2000× g at 4 °C for 5 min, and the supernatant (SN) was discarded. This step was repeated once, and cells were resuspended thoroughly in 10 mL PBS. Cells were counted in a Neubaur counting chamber. Counting cells at this point, instead of counting isolated DVs later, is more accurate because isolated DVs often stick together, which hinders precise counting. Remaining cells were stored on ice during counting, then isolated trophozoites were centrifuged at 2000× g for 5 min, and the SN was discarded.DV isolation . The mixture was incubated for 4 min on ice. Then 1 mL UB containing 1 mg/mL bovine serum albumin (BSA) was added and centrifuged immediately at 4000× g at 4 °C for 5 min. The pellet was washed twice with 2 mL UB/1 mg/mL BSA and resuspended in 400 µL UB/1 mg/mL BSA/30% Glycerine containing 160 × 106 DVs, respectively. Aliquots were frozen at −80 °C.pired by ): For thFor diameter and surface measurements, remaining DVs from Fluo-4 uptake assays were reused. Consequently, these organelles already contain Fluo-4. To prove PfMDR1 accessibility and facilitate optical DV diameter measurements with Nikon NIS elements software, DVs were indirectly labelled with PfMDR1 antibodies without organelle perforation or fixation.6 DVs were centrifuged for 2.5 min at 8000× g and resuspended in 1 mL UB. Organelles were blocked in 3% BSA/UB for 1 h at room temperature (RT). After centrifugation (settings as above), the DV pellet was resuspended and incubated with the primary antibody anti-PfMDR1-ct 1:200 in 500 µL 3% BSA/PBS at 4°C overnight. The next day, the DVs were centrifuged and washed three times in 1 mL UB. The organelles were then incubated with the secondary anti-rabbit antibody-Alexa 594  for 2 h at room temperature (RT). Finally, the labelled DVs were washed three times in PBS, resuspended in 200 µL UB, and DV diameters (d) were measured with 1000x magnification using a 100× oil immersion objective with an Eclipse Ti microscope . The organelles were confined between a microscopic slide and a coverslip (1.5H) in UB buffer. For each parasite strain, 50 DVs were examined. The surface area (A) of spherical DVs was calculated with the formula A = π·d2.Approx. 200 × 10E. coli. The antibody was generated in rabbits immunized with a 17 kDa recombinant PfMDR1-ct protein with a GST-tag [To generate a standard curve in the ELISA assay with defined signals on known numbers of PfMDR1 (ID: PF3D7_0523000) molecules, a recombinant protein that covers the C-terminal (ct) directed target sequence of the anti-PfMDR1-ct-antibody was produced in  GST-tag . To avoiGeneration of PfMDR1-ct-His in E. coli: The following primers were implemented to multiply copies of the target gene by PCR: Fwd-BamHI + NcoI: 5′-TAA TAA GGA TCC ATG GTA GTT ACT CAA GAA CCC ATG TTA TTT AAT ATG; and Rev-XhoI: 5′-TAA TAA CTC GAG TTT AGC TAA TTT TAC ATA TTT TTT ATA TAT TCC ATC TTG-3′. First, the PCR product was cloned into pBluescript II SK (+) plasmid and then into the final His-tag containing pET-21d(+) vector. The protein was produced in E. coli BL21 (DE3) bacteria. Protein expression was induced with 0.05 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 18 h at 18 °C.Protein purification: As the major portion of the protein was insoluble, the following nickel-charged nitrilotriacetic acid (Ni-NTA) agarose purification was implemented under denaturing conditions. The lysis buffer with additives (LB+1) contains 50 mM Tris, 300 mM NaCl, 1 mM Benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 8 M urea at pH 8. An E. coli cell pellet of 100 mL bacteria culture was lysed with 5 mL LB+ and sonicated on ice water. After 10 min centrifugation at 18,000× g at 4 °C, the SN was mixed with 1 mL Ni-NTA agarose. Previously, 2 mL 50% agarose was washed once with 10 mL water and once with 10 mL WB . For binding of proteins to the agarose column, the SN was restocked with 50 mM imidazol and loaded. The agarose-protein complex was washed three times with 8 mL WB. PfMDR1-ct-His-proteins were eluted seven times with 500 µL elution buffer (WB with 500 mM imidazol) each. Because the elutions showed highest purity (data not shown), all seven elutions were combined and stored in aliquots at −80 °C.6 DV aliquot was taken from −80 °C and thawed in a 37 °C water bath for 2 min. The DVs were then put on ice and centrifuged at 8000× g for 3 min at 4 °C. The supernatant was discarded, and DVs were resuspended in 400 µL LB+2 . Then, the lysate was sonicated and centrifuged at 18,000× g for 10 min at 4 °C. The supernatant was implemented for lysis efficiency experiments together with total DV lysate in Western blot assays -N,N,N′,N′-tetraacetic acid (EGTA) was implemented for free Ca2+ control. As the ATPase of PfMDR1 is at risk of being inhibited by EGTA concentrations higher than 0.07 mM [2+ containing buffers were prepared with 0.01 mM EGTA, solely in plastic containers. Because the stability of free Ca2+ concentrations in this buffer is limited, strict incubation and preparation times must be followed. For this study, buffers were always prepared one day before the assay and used only on this day. Defined free Ca2+ concentrations were measured before assay implementation with a Fluo-4 calibration curve created with Calcium Calibration Buffer Kit #1 . Two Fluo-4 incubation buffers (IB), one at pH 7.2 and one at pH 5.2 were required. They consist of 3 mM MgCl2, 100 mM KCl, 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 50 mM Tris, without or with 2 mM ATP , 0.525 µM (0.525Ca2+), or 0.1 µM (0.1Ca2+). For 0.1 µM free Ca2+ buffer, no CaCl2 was added, this was the lowest Ca2+ concentration that could be reached with 0.01 mM EGTA. The assay was implemented in 0.5 mL tubes and measured in white polypropylene 96-well U-plates . 0.07 mM , Ca2+ cooFisher, ), withouDV preparation: In the same way as described for the ELISA, one DV aliquot from -80 °C was taken and thawed in a 37 °C water bath for 2 min. As many DVs as needed for the assay were combined and centrifuged at 8000× g for 3 min at 4 °C. DVs were treated again with 0.04% Digitonin in 2 mL uptake buffer (UB) containing 120 mM KCl, 10 mM NaCl, 25 mM HEPES, 2 mM MgCl2, 5 mM Na2HPO4x2H2O, pH 7.2 for 10 min at RT, then washed twice with 2 mL UB and kept on ice until used in uptake assays. Between thawing DVs and assay performance completion, not more than 2.5 h should elapse.Fluo-4 uptake assay: One assay setup was composed of one Ca2+ concentration (three concentrations were implemented) and two parasite strains with 24 samples each. Here, the assay is described for 1.2 µM free Ca2+. For each strain, 10 samples with IB/1.2Ca2+/pH 7.2/+Fluo-4/-ATP, 10 samples with IB/1.2Ca2+/pH 7.2/+Fluo-4/+ATP and four samples only with IB/1.2Ca2+/pH 7.2 as a blank control were prepared. 20 × 106 DVs for each sample were washed in 100 µL IB/0.1Ca2+/pH 7.2 and centrifuged at 8000× g for 3 min at 4°C. These centrifuge settings were used for all subsequent steps. The SN was discarded, and for Fluo-4 uptake, the pellet was resuspended in 75 µL IB/1.2Ca2+/pH 7.2/+Fluo-4/-ATP, or IB/1.2Ca2+/pH 7.2/+Fluo-4/+ATP, or solely in IB/1.2Ca2+/pH 7.2 and incubated for 30 min in a dark 37 °C water bath. All following steps were protected from light. Subsequently, 200 µL IB/1.2Ca2+/pH 7.2/+TG was added to the sample and then centrifuged. The supernatant was discarded and DVs were washed once in 100 µL IB/1.2Ca2+/pH 7.2/+TG and once in IB/1.2Ca2+/pH 5.2/+TG. Each sample was then taken up in 100 µL IB/1.2Ca2+/pH 5.2/+TG and transferred to an ELISA plate well. All samples were cooled down for 30 min to 4 °C, and Fluo-4 intensity was measured immediately in a plate reader with an excitation of 485 nm and emission of 535 nm with 0.1 s exposure time. For control setups, samples were incubated with 3 µM of the PfMDR1 specific inhibitor Tariquidar = XR-9576 and for perforation of DV membranes with 0.2 mM Triton X-100 [on X-100 .For the entire study, one large batch of isolated DVs for each parasite strain was produced to ensure comparability between all data sets.Viable DVs from parasite trophozoite stages of RBCs infected with multi-drug resistant and sensitive strains can be obtained with an optimized bio-separation strategy described here.2+ imaging’ to monitor PfMDR1 transport rates in the isolated compartment, free of cytoplasmic influences of the parasite.DVs isolated in this manner contain functional PfMDR1 transporters that are responsive to both ATP and drugs and can be used for ‘reverse Fluo-4 Capfmdr1 gene might lead to higher Fluo-4 transport rates of the PfMDR1 transport proteinAmino acid exchanges due to mutations in the Isolated DVs can be subjected to a high-content assay that, alongside with morphometric analyses of DV surface area and quantitative ELISA for PfMDR1 binding studies, provide the unique opportunity to relate global transport rates from whole DVs to molecular PfMDR1 rates.The high content transport rate assay described here will be of high value for upcoming in vitro drug screening assays, as DVs can be stably stored following the bio-separation process and allow a constant sample quality.


Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent. Listeria monocytogenes (L. monocytogenes) and is its major virulence factor. LLO induces the internalization of L. monocytogenes into host cells. This internalization pathway requires the formation of LLO pores in the plasma membrane of the host cells, and subsequent calcium flux controls L. monocytogenes internalization [L. monocytogenes infection.Bladder cancer is the fourth most common cancer in men and the eighth most common in women ,2,3. Whilization . Inside lization ,6. LLO blization ,8,9. Cholization ,11,12. Alization ,14. Whillization ,15,16. Tlization ,18. MembMany LLO mutants with different pH-dependent pore-forming, membrane-binding, and cholesterol-binding activities have been constructed ,20,21,22In the present study, RT4 cells, a model of low-grade tumor derived from papillary superficial human bladder cancer , and porWe used NPU and RT4 cells to assess cell-dependent susceptibility to LLO Y406A in comparison to the wt LLO. The viability was determined using the CellTiter-Glo assay by measuring cellular ATP levels. Treatment of cells with final 0.1 µM LLO Y406A did not affect the viability of NPU and RT4 cells A. HoweveTo compare the short- and long-term effects of LLO Y406A, apoptosis was analyzed 1 and 24 h after 0.5 µM LLO Y406A incubation. Apoptosis in RT4 cells was monitored by immunofluorescence of the active form of caspase 3 (Cas3) G. IncubaTransmission electron microscopy revealed that in untreated RT4 cells, no vesicles were observed at the cell surface A, while Evaluation of viability after treatments with LLO Y406A and wt LLO revealed LLO Y406A as the preferred toxin variant for selective elimination of >50% RT4 cells while maintaining high viability of NPU cells (~95%). Although 2.5 µM LLO Y406A eliminated RT4 cells even more rigorously, 0.5 µM LLO Y406A outperformed the effect because fewer NPU cells were sacrificed. wt LLO was excluded as a potential agent in cancer cell treatment since concentrations of the wt LLO that were toxic to >40% of RT4 cells were considerably toxic also to NPU cells. Therefore, we chose the final concentration of 0.5 µM LLO Y406A, which was markedly toxic to cancer urothelial cells and non-toxic to normal urothelial cells and used it for the treatment of cancer urothelial cells in the following experiments.Fluorescent dextrans are commonly used as macropinocytotic probes to study endocytosis. We co-incubated LLO Y406A and FITC-dextran to identify dispersion of endosomal content as an indication of the pore-forming activity of LLO Y406A in endosomal membranes. The pattern of FITC-dextran distribution was dotted in the control untreated RT4 cells A, reflecAt the fluorescence-microscopy level, neither dextran nor LysoTracker revealed dispersion of endosomal content into the cytosol after LLO Y406A treatment. Plasma membrane labeling with mCherry-LLO Y406A implies that LLO Y406A perforates the plasma membrane of RT4 cells, thereby allowing passage of dextran into the cytosol.To reveal the escape of endosomal content after LLO Y406A treatment, which could not be resolved by light microscopy, we utilized transmission electron microscopy. As a proof of concept, we used gold nanoparticles (gold NPs) as candidate markers to be co-delivered with LLO Y406A. We prepared gold NPs with an average size of 10 ± 2 nm  Supplem. In the Listeria infection [Our results confirmed that LLO Y406A did not perforate endosomal membranes, because gold NPs were not released from endosomes into the cytosol. We speculate that the prevention of endosomal escape could be due to the absence of necessary virulence factors, membrane-active phospholipase C enzymes that contribute to pore formation in endosomes during nfection  or by thnfection . HoweverIt has been shown before that the pore-forming activity of LLO is not necessarily restricted to intracellular targets but can also be directed to the plasma membrane of cells ,32,33,34This examination revealed that LLO Y406A activity is targeted specifically to the plasma membrane of RT4 cells, resulting in calcium influx, immediate membrane blebbing, and shedding of extracellular vesicles.The novelty of this study is the selective pore-forming activity of LLO Y406A for urothelial cells in vitro where cancer cells respond with high sensitivity, while the sensitivity of normal cells is only negligible. To our knowledge, this is the first study in which wt LLO or its mutants have been used on urothelial cells. To date, wt LLO activity has been studied in a variety of other cell types, including colon epithelial cells Caco-2 and HT-29/B6, embryonic kidney cells HEK 293, human brain endothelial cells, and Jurkat cells, each without comparing the response of wt LLO in cancer vs. normal cells ,36,37,38In addition to cholesterol content, another possible reason for the cell-type specific action of LLO Y406A is the acidic microenvironment that likely surrounds RT4 cells. The acidity is provided by lactate as a metabolic product of cancer cell-specific glycolysis known as the Warburg effect ,40. ThisL. monocytogenes [In addition to the evident binding of LLO Y406A to the plasma membrane, its pore-forming activity has been proven with the calcium influx into RT4 cells. It happened within the first 20 s after LLO Y406A application. However, the exact size of pores has yet to be determined. According to the hydrodynamic diameter of 2.3 nm of 3 kDa dextran that entered the cytosol and ~10 nm of gold NPs that could not enter, we can roughly estimate the pore size to be between 2 and 10 nm . We founytogenes .2. The porcine normal urothelial (NPU) cells from the fourth to twelfth passage were prepared from at least three porcine normal urinary bladders  as described previously [Monocultures: Human cancer urothelial RT4 and T24 cells  were cultured in advanced Dulbecco’s modified Eagle’s medium (A-DMEM)/F12 (1:1), 5% heat-inactivated fetal bovine serum (FBS), 10,000 U/mL penicillin, and 10,000 µg/mL streptomycin solution (determined as control medium) at 37 °C in a humidified atmosphere with 5% COeviously . NPU cel5 cells/cm2) and RT4 cells (1 × 105 cells/cm2) were cultured for 2 days in Petri dishes or in 96-well plates. To discriminate RT4 cells from NPU cells, RT4 cells were labeled with the orange—red-fluorescent lipophilic dye DiI  before co-culturing. Briefly, 4 µl DiI was added to 1 × 106 RT4 cells for 30 min at 37 °C, cells were rinsed five times with fresh control medium and seeded with unlabeled NPU cells. Co-cultures were left untreated or were treated with 0.5 µM LLO Y406A, grown for an hour in a control medium. The medium was collected and detached cells were pelleted  and fixed in 4% formaldehyde in phosphate-buffered saline . The attached cells were either fixed in 4% formaldehyde in phosphate-buffered saline  or DiI fluorescence was measured with a microplate reader . Fixed cells were rinsed with PBS and embedded in Vectashield with DAPI , and analyzed with 10×/NA 0.3 objective on a fluorescence microscope AxioImager Z.1. .Co-cultures: The NPU cells  for 2 h in control medium, washed and left in fresh control medium for 1 h, or were untreated. Cell viability was tested with CellTiter-Glo Luminescent Cell Viability Assay  according to the manufacturer’s instructions. The luminescence was measured using a microplate reader (Safire2). Data from viability assays are expressed as percentages of luminescence of treated to untreated cells, as an average ± standard error of the mean (SEM) of 3 independent experiments, each performed in triplicate.The RT4 cells and NPU cells were cultured for 2 days in 96-well plates , each at a seeding density of 1 × 10Escherichia coli BL21(DE3) pLysS cells in Terrific Broth medium. Cells were grown at 37 °C with shaking until A600 reached ~1 and protein expression was induced with 0.5  mM isopropyl 1-thio-β-D-galactopyranoside. After shaking for 20  h at 20  °C, the cells were harvested by centrifugation at 4  °C and 4410× g for 5 min. Cells were resuspended in a minimal amount of lysis buffer  glycerol, pH 6.5) and 2-sulfanylethanol was added to a final concentration of 5  mM. Protease inhibitor phenylmethanesulfonyl fluoride was added to a final concentration of 2  mM. The cell suspension was sonicated and debris was removed by centrifugation at 50,000× g, 4 °C for 1 h. The supernatant was filtered through 0.22  μm filter, aliquoted, and frozen at −80  °C until use. For protein purification, the cell lysate was loaded to Ni-NTA,  column, washed extensively with the wash buffer , and the bound fraction eluted with the same wash buffer except the imidazole concentration was 300  mM. Tobacco etch protease (TEV) was added to a final concentration of ~30  μg/mL and the solution was dialyzed 1:60 overnight at 4 °C in a dialysis buffer . The dialyzed sample was loaded again on Ni-NTA column and the unbound fraction, containing purified protein was concentrated with Amicon Ultra 30  kDa MWCO  for the final step, size exclusion chromatography. The Superdex 200 column  was equilibrated with GF buffer (20  mM 2-(N-morpholino)ethanesulfonic acid (MES), 150  mM NaCl, pH 5.7). Fractions of pure protein (determined with SDS-PAGE) were pooled, concentrated, aliquoted, and flash frozen in liquid nitrogen and stored at −80  °C.The wt LLO and LLO Y406A were prepared as previously described . ProteinE. coli BL21(DE3) cells as described above for wt LLO and LLO Y406A. The cell lysate was loaded onto Ni-NTA  column for protein purification. The column was washed with 50 mM NaH2PO4/Na2HPO4 pH 7.5, 250 mM NaCl, 5% v/v glycerol and the bound recombinant protein was eluted with 50 mM NaH2PO4/Na2HPO4 pH 7.5, 250 mM NaCl, 300 mM NaCl, 5% v/v glycerol. 6xHis-Tag was cleaved with TEV protease in dialysis buffer  overnight at 4 °C. After dialysis, the protein solution was loaded to the Ni-NTA column and the unbound fraction with the purified protein was concentrated in Amicon Ultra 30 kDa MWCO  and the buffer exchanged so the final protein solution was in 20 mM NaH2PO4/Na2HPO4 pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT) and stored at −80 °C until used.The gene for mCherry-LLO Y406A recombinant protein was inserted into pET17b plasmid. The mCherry was linked to LLO Y406A at the N-terminus using a linker with amino acid sequence GGGSGGGSPR. C-terminally to the LLO Y406A sequence, the TEV protease cleavage site, and a hexa-histidine tag were added. mCherry-LLO Y406A protein was expressed in Stock solutions of LLO-Y406A (69.1 µM), mCherry-LLO Y406A (22.8 µM) and wt LLO (101.0 µM) were mixed in control medium alone or combination with FITC-dextran or gold nanoparticles (see preparation below) at 22 °C in a laminar flow hood. To reveal endosomal escape, RT4 cells were co-incubated with 0.5 µM LLO Y406A and 0.5 mg/mL FITC-dextran  for 2 h, washed in control medium, and cultured in control medium for 1 h. Alternatively, RT4 cells were co-incubated with 5.0 µM mCherry-LLO Y406A and FITC-dextran following the same protocol. Then cells were then fixed in 4% formaldehyde in PBS (pH 7.4) at 22 °C for 15 min and mounted in Vectashield with DAPI  for nuclear staining (Vector Laboratories). Additionally, LysoTracker Red DND-99  was used. In brief, RT4 cells were incubated with 50 nM LysoTracker at 37 °C for 45 min. Cells were then washed and LLO Y406A was added to a final concentration of 0.5 µM for 2 h and washed with control medium. One h later, cells were fixed in 4% formaldehyde in PBS at 22 °C for 15 min and mounted in Vectashield with DAPI. Cells were analyzed using a 20×/NA 0.75 and 63×/NA 1.4 objectives on a fluorescence microscope AxioImager Z.1 with an Apotome device for optical section generation (Zeiss).4Cl, 0.02% NaN3) at 37 °C for 30 min. Then cells were incubated with rabbit anti-cleaved Cas3 antibodies  overnight at 4 °C, washed with PBS, and incubated with Alexa Fluor 555-conjugated anti-rabbit secondary antibodies  at 37 °C for 30 min. In parallel, untreated RT4 cells were processed for Cas3 immunolabeling using the same protocol as for LLO Y406A-treated cells. The cells were mounted in Vectashield with DAPI for nuclear staining and observed under fluorescence microscope AxioImager Z1. Cas3 positive cells and nuclei were counted using the Cell Counter Plugin .For caspase 3 (Cas3) labeling, RT4 cells were incubated with 0.5 µM LLO Y406A for 2 h, washed in control medium, and cultured for 1 and 24 h in control medium, then fixed in 4% formaldehyde at 22 °C for 15 min, and incubated in a blocking-permeabilization buffer  were seeded on glass-bottom dishes  and grown for 2 days. Cells were washed with HBSS+  buffer and incubated with 2 μM Fluo-4 AM  at 37 °C for 45 min in the dark. Cells were then washed with HBSS+ buffer and FluoroBright DMEM microscopy medium without FBS (Gibco) was added to the cells. Imaging was made with Zeiss Axio Imager Z.1 fluorescence microscope at 37 °C  and images were acquired using the AxioVision program. Then LLO Y406A was added at the final concentration 0.5 µM and 20 s later images were taken at 10-s intervals with the same settings as in pre-treatment. Gray values of the fluorescence intensity (a.u.) were measured with AxioVision 4.8 software (Zeiss) in three independent experiments, each time in 10 cells.The RT4 and NPU cells  sodium citrate was brought to a boil and 1 mL of 24.3 mM HAuCl4  was added with rapid mixing. The reaction was completed within 1–2 min and the solution was further boiled for 15 min. Gold NPs in solution were centrifuged at 6500× g for 30 min at 4 °C. Nine tenths of the supernatant was discarded to increase the concentration of gold NPs in the solution. Next, 5 μL of the gold NPs solution were deposited onto copper grids and air-dried. Images of gold NPs were taken at 20,000–100,000× magnifications with a Philips CM100 transmission electron microscope  at 80 kV using an AMT camera .The procedure for gold nanoparticles (gold NPs) preparation was adopted from Hayat (Hayat 1989). Gold NPs were prepared by reduction of HAuCl4 for 1 h at 22 °C. The cells were then dehydrated and embedded in Epon . Ultrathin sections were stained with uranyl acetate and lead citrate . The sections were examined with a Philips CM100 transmission electron microscope at 80 kV using an AMT camera.The RT4 and NPU cells treated with 0.5 µM LLO Y406A, with 0.5 µM LLO Y406A and gold NPs, and the untreated cells were fixed with 4% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at 4 °C. After overnight washing in 0.33 M sucrose in cacodylate buffer, the cells were incubated with 1% OsOp < 0.05; ** p < 0.005; and *** p < 0.001.The data are presented as average ± SEM of three independent experiments, each performed in triplicates. Data were analyzed with Student’s two-tailed t-test or one-way Anova test . Statistically significant results were represented by * LLO Y406A has great selectivity for the urothelial cancer cells, which is the most important feature of an effective anticancer drug. We envision the use of LLO Y406A in combination with other conventional drugs such as Bacillus Calmette–Guérin (BCG) or mitomycin C that are used after bladder tumor resection to destroy remnant tumor cells ,55. LLO


Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers  PI4Kα1 is targeted to plasma membrane nanodomains by a lipid-anchored heterotetrameric complex essential for plant cell survival, including gametophytic, embryonic, and post-embryonic development. Eukaryotic cells are composed of several membrane-surrounded compartments. Each compartment has a unique physicochemical environment delimited by a membrane with a specific biochemical and biophysical identity . The mem2, and PIP2—depending on the number and position of phosphates around the inositol ring Ptol ring . They actol ring . Recent tol ring , 2020. Itol ring . The platol ring . These ttol ring . In animtol ring . Similar mammals . In PI4K mammals .In plants, PI4P massively accumulates at the plasma membrane and is less abundant at the TGN , 2016. TArabidopsis thaliana genome codes four PI4-kinases: PI4Kα1, PI4Kα2, PI4Kβ1, and PI4Kβ2 , HYCCIN-CONTAINING (HYC), and EFR3 OF PLANTS (EFOP) protein families. Using fluorescent protein tagging, immunolocalization, and subcellular fractionation, we confirmed the presence of the PI4Kα1 complex at the plasma membrane. Furthermore, we show that 2  domain was previously reported in its C-terminal, upstream from the catalytic domain . This PH2 . HoweverAt2g43040), NPG-Related 1 (NPGR1–At1g27460), and NPGR2  motifs that are protein–protein interaction motifs. In the yeast-two-hybrid screen, the selected interaction domain identified for NPG1, NPGR1, and NPGR2 correspond to the C-terminal part of the proteins . This is also the part of the sequence that contains the highest density of predicted TPR motifs suggesting that the interaction between PI4Kα1 and NPGs could be mediated by the C-terminal TPR motifs.https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). We chose NPGR2, as it has a stronger and wider expression level than the other members of the NPG family. Indeed, NPG1 was predicted to be specifically expressed in the pollen, while NPGR1 expression matched that of NPGR2 but was predicted to be much weaker.Because all three members of the NPG family interacted with PI4Kα1 in yeast-two hybrids and given the high degree of identity and similar architecture of the three proteins, we decided to focus on one member of the family to confirm the interaction in planta. We guided this choice based on the RNAseq expression data compiled in the eFP browser (pro:NPGR2-mCITRINEUBQ10. We raised antibodies against the native PI4Kα1 (residues 1–344 of PI4Kα1). In immnoblot, the antibody recognized PI4Kα1 around the expected size (225 kDa) and the tagged version of PI4Kα1 with mCITRINE and 2xmCHERRY slightly higher  and HYCCIN-CONTAINING2 .Next, we asked whether the PI4Kα1 and NPG proteins could interact with additional protein partners. To this end, we used the lines expressing mCITRINE–PI4Kα1 and NPGR2–mCITRINE to perform immunoprecipitation (IP) followed by mass spectrometry analyses. Two lines expressing membrane-associated (myristoylation–2xmCITRINE) and nuclear-excluded (mCITRINE–NES–mCITRINE) proteins were also used as generic controls for plasma membrane and cytosolic proteins, respectively. In the NPGR2 IP, we found PI4Kα1, further confirming that these two proteins are present in the same complex in plants. Only one common protein was found in both NPGR2 and PI4Kα1 IPs but excluded from the two controls . This prHYC2 is broadly expressed, while the HYC1 expression is restricted to pollen according to the eFP browser data set (https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Hence, we chose to test whether HYC2 interacted with PI4Kα1 and NPGR2 in sporophytic tissues. To this end, we raised UBQ10::HYC2-mCITRINE expressing lines and successfully isolated antibodies raised against NPGR2 (residues 1–273 of NPGR2). The expected size of NPGR2 is 82 kDa. The antibody recognized a band at ∼80 kDa that is not present in npgr2-1 or npgr2-3 knockout mutants or npgr1 npgr2-1 double mutant  has a HYCCIN domain and is part of a complex containing PI4KIIIα . In thisThe structure of the human trimeric complex formed by PI4KIIIα–TTC7–FAM126 has been determined by cryo-electron microscopy . We thusThe modeling approach together with interaction data suggest that PI4Kα1, HYC, and NPG proteins form a stable heterotrimeric complex in planta, which is likely equivalent to its metazoan counterpart.At5g21080), EFOP2 (At2g41830), EFOP3 (At1g05960), and EFOP4 (At5g26850).In human cells and yeast, the PI4-kinase complex contains an additional subunit called EFR3/Efr3p . We thusMarchantia polymorpha, mutants for MpPI4Kα1 (the homolog of PI4Kα1) and MpSRI4, the homolog of EFOP2, display short rhizoids, suggesting that they could act in the same pathway and/or protein complex  and HEAT-like repeats. ARM and HEAT repeats are difficult to be distinguished bioinformatically, but all four EFOP proteins belong to the ARM-repeat super-family, which includes both ARM and HEAT repeat containing proteins . In Marc complex . In addimCITRINE , suggestThe summary of these interactions suggests that PI4Kα1 is part of an heterotetrameric complex in which NPG proteins may act as a scaffold that bridges EFOP, HYC, and PI4Kα1 proteins together .PI4Kα1 because it is the catalytic subunit and it is present as a single-copy gene in the Arabidopsis genome for which we isolated two T-DNA insertion alleles. The first allele  had an insertion in the first exon, while the second insertion  was in the 20th intron  to test whether they showed morphological defects . In addition, self-fertilized −/−; PI4Kα1pro:PI4Kα1pi4kα1-1 plants showed a low number of shriveled pollen grains, comparable to control plants, indicating that a wild-type copy of PI4Kα1 is required for transmission through the male gametophyte and normal pollen morphology  pollen grains . Thus, depending on the alleles, these double mutant combinations presented from 70% to 90% of abnormal pollens  or partially  mimic the pi4kα1 phenotype. Thus, these proteins likely act together in plants, potentially as a single protein complex.None of the s family . We thuss family ; Table 3 pollens . In addie lethal . SEM shoe lethal . FinallyHYC1 is specifically expressed in pollen and is male sterile, HYC2 is predicted to be expressed in the sporophyte, which suggests that HYC2 loss-of-function could lead to sporophytic phenotypes. We characterized two T-DNA alleles corresponding to two putative hyc2 loss-of-function mutants. The segregation rate of hyc2-2 heterozygous plants was 60%  while the rest of the embryos pursued their development normally , but it can impact the localization of coincident detectors such as 1xPHFAPP1 (which interacts both with PI4P and ARF1). Consistently, the amount of Phosphorus-32 labelled phosphatidylinositol monophosphate (32P-PIP) in lipid extracts of 32Pi-prelabeled seedlings grown on β-estradiol was unchanged compared to the nontreated control  also failed to complement pi4kα1-1  fluorescence recovered in less than a minute after bleaching. Accordingly, the mobile fraction calculated of NPGR2–mCITRINE and EFOP2–mCITRINE was low (around 20%), while the mobile fraction of the sensor reached 100% . After bleaching, the signal of NPGR2–mCITRINE, HYC2–mCITRINE, and EFOP2–mCITRINE did not recover after 2 min of acquisition . In comphed 100% . This inefop3-1 efop4-4 and efop3-2 efop4-4 mutants showed the same pollen lethality phenotype as the efop3-1 efop4-2 and efop3-2 efop4-2 mutant , the efop4-4 allele led to a near full-length protein with only a small in frame deletion of 19 residues close to the EFOP4 N-terminus. This suggested that this N-terminal region is crucial for EFOP4 function  and the positively charged surface visualized in the modeled EFOP structure , a PI4K inhibitor  and 26 roots out of 44 for the +/− npgr1−/− npgr2-2−/−npg1-2 allelic combination . This result indicates that PI4Kα1 plasma membrane targeting requires NPG proteins.Because NPG proteins likely bridge PI4Kα1 to the membrane-binding EFOP subunits, we reasoned that they should contribute to the targeting of PI4Kα1 at the plasma membrane. To test this hypothesis, we monitored the localization of PI4Kα1 in the  of NPG1 . Such cypro:PI4Kα1-mCPI4Kα1ITRINE-Lti6b construct was able to rescue the npg1-2 as we were able to recover homozygous npg1-2 mutants upon expression of this particular transgene  the functional link between NPG proteins and PI4Kα1 and (2) that the function of NPG proteins is to target PI4Kα1 to the plasma membrane. As discussed above, tagged versions of PI4Kα1, including PI4Kα1–mCITRINE, were not functional as they did not complement the pi4kα1 mutant P2 P2 . Like npn lethal . sec3a mHYC2 induces embryo lethality while npg triple sesquimutants and PI4Kα1 knockdown display severe growth phenotypes at rosette and seedling stages. This is concordant with the idea that PI4Kα1 has a critical role for the cell function, not only during gametophytic but also sporophytic development. PI4P is crucial for the plasma membrane surface charge  the tal PI4P  and (2) tal PI4P . Togethepi4kα1, npg triple sesquimutants, hyc1 and efop3 efop4 pollens reveal that they present a thicker an irregular intine layer. Thus, it is likely that the secretion of cell wall materials by the micropores is affected. The intine layer has a composition similar to a primary cell wall with cellulose, hemicellulose and pectin. Loss-of-function mutants for subunit of the cellulose synthase complex—cesa1 and cesa3—are pollen sterile with shrivelled pollen grains similar to the pi4kα1 complex mutants P2 clusters and facilitating membrane fusion -binding protein. Indeed, NPG1 can interact with several Cam isoforms in the presence of Camination . Ca2+ isfficking . Ca2+ cae fusion . As phosOTAGMINs . For insositides . The PI4hyc1 and hyc2 mutations are lethal as the corresponding wild-type genes are required for male gametophyte and embryo development, respectively. Our result thus suggests that HYC is an essential subunit for the function of the PI4Kα1 complex in plants. In humans, there are additional HYCCIN-containing proteins, including FAM126B. Our results thus open the possibility that FAM126A is not the only HYCCIN-containing protein contributing to PI4KIIIα function in metazoa.If EFOPs anchor the complex at the membrane and NPGs bridge PI4Kα1 and EFOPs, the role of HYCCIN-CONTAING proteins in the complex is less clear. In mammals, FAM126A, which has a HYCCIN domain, is involved in the stability of the complex and TTC7 folding . In humaThe PI4Kα1 complex localizes at the plasma membrane in nanodomains. In yeast, Stt4 is found in large clusters called PIK patches . HoweverDaucus carota) and associates with polymerized F-actin in vitro Pen tubes . These nArabidopsis thaliana, ecotype Columbia (Col-0) was used in this study, except for pi4kα1-2, which is in the Ws background. Plants expressing pro:myrimCITRINE-mCITRINE2xp35S were obtained from pro::mCITRINE-1xPHFAPP1UBQ10 and pro:mCITRINE-P4MSidMUBQ10, were obtained from  for 7–10 days. Resistant and sensitive seedlings were counted for segregations.Seeds were surface-sterilized by the addition of 4 mL of HCl 37% in 100 mL of bleach for 4 h before plating on Murashige and Skoog  media supplemented with 0.8% plant agar and containing the appropriate antibiotic or herbicide. Glufosinate, kanamycin, hygromycin, and sulfadyacin were used at 10 mg·L−2·s−1).Seeds were directly sown in soil or 7-day-old seedlings were transferred from in vitro plates to soil. Plants were grown at 21°C under long day conditions  or Gibson Assembly mix  containing kanamycin at 50 mg·L−1. Plasmids were purified using the Nucleospin Plasmid kit (Macherey-Nagel) and inserts sequenced. The expression vectors containing the promoter-gene-fluorescent tag cassette or 3′-UTR were obtained using the LR clonase-based three-fragment recombination system (Invitrogen), the pB7m34GW/pH7m34GW/pK7m34GW/pS7m43GW  containing 100 mg·L−1 spectinomycin. Plasmids were purified using the Nucleospin Plasmid kit (Macherey-Nagel) and inserts sequenced.The following vectors were published before: Lti6b/pDONR207 , UBQ10pr Biolab; . ThermocpS7m43GW  and pLOKpS7m43GW . Only EFpS7m43GW . Thermoc). PCR products were digested using Dpn1 and cutsmart buffer (Biolab). Thermocompetent DH5α E. coli were transformed with the digested PCR product and plated on LB  containing the appropriate antibiotic. Plasmids were purified using the Nucleospin Plasmid kit (Macherey-Nagel) and mutations were sequenced.Plasmids were amplified by PCR using the high-fidelity Phusion Hot Start II (Thermo Fisher Scientific) taq polymerase and the indicated overlapping primers carrying mutations . PCR prohttp://wmd3.weigelworld.org/cgi-bin/webapp.cgi). DNA synthesis containing AttB1-AttB2 sites and the miR319a modified with the sequence of the artificial microRNA  were transformed by electroporation. One microliter of DNA plasmid at a concentration of 0.25–1 μg·μL−1 was added into 50 µL of electrocompetent agrobacterium on ice. The agrobacteria were transferred into a cold 1mm wide electroporation chamber . A pulse of 2 kV, 335 Ω, 15 µF, for 5 ms was performed on the electroporation chamber using the MicropulserTM . One microliter of liquid LB media was added and the bacteria were placed into a new tube and incubated at 29°C for 2–3 h. The agrobacteria were selected on LB , or YEB  plates containing the appropriate antibiotics to select the agrobacterium strain (50 mg·L−1 of rifampicin and 20 mg·L−1 of gentamycin) and the target construct (250 mg·L−1 of spectinomycin). Plates were incubated at 29°C for 48 h.Electrocompetent Agrobacteria were spun and resuspended in 5% sucrose and 0.02% Silwet L-77 detergent. Arabidopsis were transformed by floral dipping.−1, 50 mg·L−1, 30 mg·L−1, and 75 mg·L−1, respectively. Selection of the pLOK180_pR7m34g plasmid was done using the FastRed method (i.e. red fluorescent seeds).Transformed seedlings were selected on MS (Duchefa Biochemie) media supplemented with 0.8% plant agar containing the appropriate antibiotic or herbicide. Basta, kanamycin, hygromycin, and sulfadiazin have been used at 10 mg·LLeaf tissue from wild-type plants and/or transgenic lines was collected, frozen in liquid nitrogen and ground to powder. The powder was resuspended in −20°C MetOH; 1% Protease inhibitor  and incubated at −20°C for 5 min. Proteins were pelleted, resuspended in −20°C acetone and incubated at −20°C for 5min. Proteins were pelleted. The pellet was dried and resuspended in Protein Extraction Reagent  supplemented with 1% protease inhibitor . Protein extraction was then kept at −20°C.E. coli and purified a PI4Kα1 fragment corresponding to AA1-344, fused with 6xHis at its N-terminus and the antibodies were produced in Rat. For the anti-NPGR2 antibody, Proteogenix cloned, expressed in E. coli and purified a NPGR2 fragment corresponding to AA1-269, fused with 6xHis at its N-terminus, and the antibodies were produced in Rabbit. Secondary antibodies were used at 1/5,000  in 1X TBS, 0.1% Tween-20 for 1 h. Protein revelation was done using the Clarity or Clarity Max ECL Western Blotting Substrates (Biorad) and the Chemidoc MP imaging system (Biorad).Protein samples in 1 × SDG  supplemented with bromophenol blue were denaturated at 95°C for 5 min. Samples migrated on 7.5% (for proteins over 200 kDa) or 10% SDS–polyacrylamide gel electrophoresis polyacrylamide gel at 140 V with 1×Tris-Glycine SDS running buffer (Euromedex). Proteins were transferred to a nitrocellulose membrane (0.45 µm) in 1×Tris-Glycine (Euromedex), 20%EtOH transfer buffer at 100 V for 1 h. Membrane was blocked with 5% milk, 1X phosphate buffered saline-T (Dominique Dutscher), 2% Tween-20 for 1 h. Primary antibodies were used at 1/1,000  or at 1/2,000 . Crude extracts were put in 1XSDG supplemented with bromophenol blue and denatured at 95°C for 5 min, while IP samples were directly ready for migration on polyacrylamide gels.Each co-IP experiment was repeated at least three times independently with similar outcomes.Mass spectrometry analysis was performed by the Protein Science Facility of SFR (Structure Fédérative de Recherche) Bioscience Lyon. Trypsine-digested samples were analyzed by liquid chromatography-tandem mass spectrometry . The peptides identified were compared to those in the UniProtKb database.https://www.hybrigenics-services.com/contents/our-services/interaction-discovery/ultimate-y2h-2), using the ULTImate yeast two hybrid (Y2H) screen against their Universal Arabidopsis Normalized library obtained using oligo_dT. The residues 2–1479 of PI4Kα1 were used. The screen was performed on 0.5-mM 3AT, 58.6 million interactions were analyzed, and 313 positive clones were sequenced.The initial screen was performed by hybrigenics services (AD vectors (prey) and DB vectors (bait) were transformed in the Y8800 and Y8930 yeast strains, respectively. Transformed yeasts were plated on a minimal synthetic defined (SD) medium  with all amino acids except tryptophan (SD-T) for AD vectors and SD-L for DB vectors for selection.−1 final) to determine the autoactivated DB clones; SD-LH+CHX + 3AT to determine which concentration of 3AT erased the autoactivation of DB clones. The experiment was repeated three times independently and similar results were obtained.Yeast colony transformed with AD or DB vectors were grown in SD-T and SD-L liquid media, 30°C, 200 rpm for 48 h. Mating was performed at 30°C, 200 rpm, overnight using 10 µL of AD and 10-µL DB clones in 180-µL YEPD  liquid media. Diploid yeasts were selected by addition of 100 µL of SD-LT liquid media. Yeast was grown for 48 h at 30°C, 200 rpm. Diploid yeasts were plated on different selective media: SD-LT to verify the mating; SD-LTH to select the positive interactions; SD-LTH + 3AT  to select strong interactions only: SD-LH+CHX (cycloheximide at 10 µg·mLwww.rgenome.net/). Primers were designed following the methods developed in Supplemental Table S7).A 20-bp target sequence upstream of a 5′-NGG-3′ sequence in an exon of the gene of interest was found using the CrisPr RGEN tool . DNA was precipitated with isopropanol and the DNA pellet was washed with 75% ethanol before resuspension in water.Plants were genotyped by PCR using the GoTaq polymerase (Promega) and the indicated primers . PCR proPollen grains from mutant or WT flowers were placed on tape and observed using the mini SEM Hirox 3000 at −10°C, 10 kV.2HPO4 0.1 M, NaH2PO4 0.1 M overnight and postfixed in 1% OsO4, Na2HPO4 0.1 M, NaH2PO4 0.1 M. Anthers were dehydrated through a graded ethanol series from 30% to 100% and embedded in SPURR resin. Sections were made using an ultramicrotome Leica UC7 at 70–80 nm and poststained with acetate uranyle 5% (in ethanol), lead citrate (in NaOH). Pollen was observed using a transmission electron microscope Jeol 1400 Flash. For the genotypes +/− npgr1−/− npgr2-2−/−pi4kα1-1, npg1-2, hyc1, and −/− efop4-2−/−efop3-1, shriveled pollen grains were selected for imaging and compared with the wild-type pollen.For transmission electron microscopy, anthers were placed in a fixative solution of 3.7% paraformaldehyde and 2.5% glutaraldehyde, NaTo perform Alexander staining, flowers that were about to open were dissected and anthers were put between slide and coverslip in Alexander staining solution  for 7 h at 50°C before observation under a stereomicroscope.http://www.semrock.com/).For DAPI staining, flowers that were about to open were dissected and their anthers were put between slide and coverslip in DAPI solution  for 5 min at RT before observation with the confocal microscope. DAPI was excited with a 405 nm laser (80 mW) and fluorescence emission was filtered by a 447/60 nm BrightLine single-band bandpass filter  for 1 h before observation at the microscope with differential interference contrast.−1) containing 5-μM β-estradiol or the equivalent volume of DMSO. Plates were vernalized 3 days before being placed under continuous white light conditions for 11 days. Plates were scanned. Root length was measured using ImageJ software. Similar results were obtained for five independent experiments.Surface-sterilized seeds were plated on MS (Duchefa BiochemieR) media supplemented with sucrose (10 g·Lhttp://www.zeiss.com/) with a spinning disk module  and a Prime 95B camera  using a 63x Plan-Apochromat objective  and the appropriate laser and bandpath filter.The 7- to 10-day-old seedlings were observed with a Zeiss microscope . mCITRINE was excited with a 515 nm laser (60 mW) and fluorescence emission was filtered by a 578/105 nm BrightLine single-band bandpass filter . mCHERRY was excited with a 561 nm laser (80 mW) and fluorescence emission was filtered by a 609/54 nm BrightLine single-band bandpass filter . In the case of seedling expressing mCHERRY and mCITRINE markers, mCITRINE was excited and emission was filtered using the GFP settings.GFP was excited with a 488 nm laser (150 mW) and fluorescence emission was filtered by a 525/50 nm BrightLine single-band bandpass filter  with 100× Apo NA 1.46 Oil objective. The exposure time used was 1s. HYC2-mCITRINE and NPGR2-mCITRINE were excited and emission was filtered using the GFP settings, while EFOP2-mCITRINE was excited and emission was filtered using the YFP settings.4, pH 7) for 2 h. Roots were placed on Superfrost Plus slides , dried at RT for 1 h, and rehydrated using MTBS + 0.1% tritonx100. Permeabilization was done using enzymatic digestion  for 40 min  in labeling buffer. Incubations were stopped by adding 50 µL of 50% (w/v) perchloric acid and the lipids extracted (32P-labeled phospholipids. Experiments were performed in quadruplicate.Nine-day-old seedlings grown on 1/2 MS plates with 1% (w/v) sucrose, 1% (w/v) agar, and 5-μM β-estradiol or DMSO (control) were transferred to 2-mL Safe-lock Eppendorf tubes containing 190-µL labelling buffer  supplemented with either 5-μM β-estradiol or DMSO (control), with each tube containing three seedlings. Seedlings were metabolically labeled with radioactive phosphate by incubating them overnight for ∼16–20 h with 10 µL (5–10 µCi) carrier-free xtracted . PIP wasxtracted . Each laMicrosomes were purified as described in  and resuFor PI4Kα1, proteins were precipitated using five volumes of −20°C acetone for one volume of protein extraction and incubated for 10 min at −20°C. Proteins were pelleted. This process was repeated two more times. Pellet was dried and resuspended in 1×SDG supplemented with bromophenol blue. All steps were performed at 4°C.The 7 to 10-day-old seedlings were placed between side and coverslip in MES 10 mM at pH 5.8 with or without 0.75-M sorbitol and observed using a confocal microscope.http://www.biovis.com/ilas.htm). Fluorescence recovery was measured in the ROIs and in controlled ROIs (rectangle with the same dimension in unbleached area). FRAP was recorded continuously during 120 s with a delay of 1s between frames. Fluorescence intensity data were normalized as previously described (t = 120s with the following formula: I(t)−Min(I)/Ictrl(t)/Min(I), where I(t) and Ictrl(t) are the intensity of the bleached and control region at time t, respectively. Data were obtained in several independent experiments.Fluorescence in a rectangle region of interest (ROI) (5× 1.7 μm) at the plasma membrane was bleached in the root by successive scans at full laser power (150 W) using the iLas2 FRAP module  by pipetting. The Optical Density at 600 nm (OD600) was measured using a spectrophotometer  and adjusted to 1 by adding infiltration media.Transformed agrobacterium were directly taken from plate with a tip and resuspended into 2 mL of infiltration media , the infiltration solution with the agrobacterium was pressed onto the abaxial side of the chosen tobacco leaf. The plants were returned to the growth chamber for 2–3 days under long day conditions.2 regions of the leaf surrounding the place where the infiltration had been made were cut out. The pieces of leaf were mounted in water between slide and coverslip with the abaxial side of the leaf facing the coverslip. Using the appropriate wavelength, an epifluorescent microscope and the smallest objective (10×), the surface of the leaf was screened to find the transformed cells. Then, the subcellular localization of the fluorescent protein was observed using a spinning confocal microscope and 63× objective.The 5 mmwww.sigmaaldrich.com, PAO stock solution at 60 mM in DMSO) for 30 min before observation using a confocal microscope.Seedlings (7- to 10-day old) were incubated in liquid MS with 30-µM PAO ) and the R studio interface .PI4Kα1, At1g46340; NPG1, At2g43040; NPGR1, At1g27460; NPGR2, At4g28600; HYC1, At5g21050; HYC2, At5g64090; EFOP1, At5g21080; EFOP2, At2g41830; EFOP3, At1g05960; EFOP4, At5g26850The following materials are available in the online version of this article. Immunoblot validation of the custom-made anti-PI4Kα1 and anti-NPGR2 antibodies. Template-based modeling of PI4Kα1, NPG1, and HYC1. Characterization of pi4kα1 pollen phenotype. Characterization of the pollen phenotype of single and multiple npg, hyc, and efop mutants. Sporophytic phenotypes and complementation of npg multiple mutants and hyc2 single mutant. PI4Kα1, NPG, HYC, and EFOP protein localization in pollen grains. FRAP analysis of NPGR2/EFOP2/HYC2 fused with mCITRINE in Arabidopsis root. Position of the putative PH domain of PI4Kα1. Antibodies used in this study. Description of the single and multiple mutants analyzed in this study. Transgenic lines. Published vectors used in this study. Primers used for cloning into gateway entry vectors. Primers used for site directed mutagenesis. Primers used for Crispr constructs. Genotyping primers. microRNA sequences. Statistical analysis.koab135_Supplementary_DataClick here for additional data file.