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Jul 2015

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Imaging of Lipid Uptake in Arabidopsis Seedlings Utilizing Fluorescent Lipids and Confocal Microscopy
利用荧光脂质和共聚焦显微镜对拟南芥幼苗的脂质摄取进行成像   

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Abstract

Eukaryotic cells use a diverse set of transporters to control the movement of lipids across their plasma membrane, which drastically affects membrane properties. Various tools and techniques to analyze the activity of these transporters have been developed. Among them, assays based on fluorescent phospholipid probes are particularly suitable, allowing for imaging and quantification of lipid internalization in living cells. Classically, these assays have been applied to yeast and animal cells. Here, we describe the adaptation of this powerful approach to characterize lipid internalization in plant roots and aerial tissues using confocal imaging.


Graphic abstract:


Fluorescent lipid uptake in Arabidopsis seedlings. Scale bars: seedling, 25 mm; leaf, 10 μm; root, 25 μm.


Keywords: Arabidopsis thaliana (拟南芥), Flippases (翻转酶), P4 ATPases (P4 ATP酶), Lipid transport (脂质运输), NBD-lipids (NBD-脂质), Roots (根部), Leaves (叶子), Guard cells (保卫细胞)

Background

In eukaryotic cells, movement of lipids across biological membranes (known as lipid flip-flop) is regulated by a diverse set of membrane transporters that can be classified into two categories: (i) ATP-independent transporters, also called scramblases, that facilitate a rapid bi-directional movement of lipids without metabolic energy input, and (ii) ATP-driven vectorial transporters that actively translocate lipids from one membrane leaflet to the other, often with high specificity. The latter group comprises ATP-dependent flippases and floppases, which catalyze the inward movement of lipids to the cytoplasmic membrane leaflet, and the outward movement to the extracellular/luminal side, respectively. A subgroup of P-type ATPases, the P4 ATPases, has emerged as a major group of lipid flippases that form heterodimeric complexes with members of the Cdc50 (cell division control 50) protein family (reviewed in Lopez-Marques et al., 2014; Andersen et al., 2016). While initially characterized as aminophospholipid flipases, recent studies of individual family members from fungi, plants, and animals show that P4 ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates, including lysophospholipids, synthetic alkylphospholipids, and sugar-modified ceramides (Roland et al., 2019; Shin and Takatsu, 2019).


Quantitative assessment of P4 ATPase lipid transport activity is essential for the determination of substrate specificities and to establish whether and how this activity is regulated in the living cell. As P4-ATPases are trapped in an environment (cellular membranes) formed by their own substrate (lipids), analyzing their activity is not a trivial task, and most assays are based on the use of fluorescent lipid analogs, typically nitrobenzoxadiazol (NBD)-labeled lipids. These analogs have a fluorescent reporter group attached to a short-chain fatty acid (C6) and maintain most of the properties of endogenous phospholipids, except that they are more water-soluble, which facilitates incorporation from the medium into the outer monolayer of the plasma membrane.


Traditionally, lipid uptake assays employing NBD-lipids are carried out after heterologous expression of the plant P4 ATPases in yeast strains devoid of their endogenous lipid transporters (for a review, see Nintemann et al., 2019). However, many plant P4 ATPases express poorly, fail to fold, and/or traffic improperly when produced in heterologous systems. In addition, successful expression does not always result in active lipid translocation, probably due to the absence of plant-specific accessory proteins and/or cofactors required for the functioning of plant lipid transporters (McDowell et al., 2015).


The method presented here utilizes C6-NBD-lipids to study lipid internalization in intact plants, exemplified on Arabidopsis seedlings as a model. Small 5-day old seedlings are grown on plates under sterile conditions and then transferred to liquid growth medium supplemented with C6-NBD-lipids. After incubation for the desired time, the seedlings are washed with medium containing small amounts of a specific detergent to remove excess lipids attached to the cell walls. Finally, plants are visualized using confocal microscopy, and the data quantified using imaging software. This protocol can be used to characterize lipid internalization both in roots and aerial tissues and can be easily adapted to other plant species. For experiments in roots, growth of seedlings on agar plates is preferred to regular cultivation on soil, as removal from the soil causes damage to the root surface.

Materials and Reagents

Materials

  1. Arabidopsis seeds (ecotype Col-0 or as desired)

  2. Pipette tips PIPETMAN DIAMOND D10, D200, D1000 (Gilson, catalog numbers: F161630, F161930, F161670)

  3. 2-ml microcentrifuge snap-cap tubes with round bottom (e.g., BRAND, microcentrifuge tube, 2 ml with lid, PP; Merck, catalog number: BR780546-500EA)

  4. Circular holder for 2-ml microcentrifuge tubes (e.g., PrepSafeTM microcentrifuge tube mini floating rack, clear; Merck, catalog number: Z756385)

  5. 50-ml glass beaker (e.g., BRAND, catalog number: 91217)

  6. Square Petri dishes 120 × 120 × 17 mm, Greiner Bio-One (Fisher Scientific, catalog number: 07-000-330)

  7. Micropore tape (3M, catalog number: 1530-1)

  8. Aluminum foil (e.g., Sigma, catalog number: Z185140-1EA)

  9. Centrifuge glasses DURAN® with conical bottom, 12 ml (Carl Roth, catalog number: K211.1)

  10. 25-μl calibrated glass syringe (model 702 N; Hamilton, catalog number: CAL80400)

  11. 1.5 ml screw amber glass vials with 8-mmTeflon-lined screw caps (e.g., VWR, catalog numbers: VWRI548-0019 and 548-0360)

  12. Corning® Pasteur pipettes, non-sterile, 228 mm (Merck, catalog number: CLS7095B9)

  13. PARAFILM® M (Merck, catalog number: P7793)

  14. Clear glass jars with snap-cap, 11 ml, 22 × 45 mm (VWR, catalog number: 548-0625)

  15. For aerial tissues, microscope slides 76 × 26 × 1 mm with cut edges (Histolab, catalog number: 06300)

  16. For root tissues, diagnostic microscope slides 25 × 75 mm, 8 Wells of 6 mm, with black epoxy field around cavities (Histolab, catalog number: 06260)

  17. Cover glass No. 1, 18 × 24 mm and 24 × 24 mm (Histolab, catalog numbers: 06602 and 06608)


Reagents

All reagents can be stored at room temperature, except NBD-phospholipid solutions, which should be kept at -20°C for long-term storage.

  1. Sodium hypochlorite (14% Cl2) in aqueous solution, GPR RECTAPUR® (VWR Chemicals, catalog number: 27900.296)

  2. 37% hydrochloric acid solution (Sigma, catalog number: 320331)

  3. Phytoagar (Duchefa Biochemie, catalog number: P1003)

  4. 2-(N-morpholino)ethanesulfonic acid (MES) (Merck-Millipore, catalog number: 1061261000; CAS number: 4432-31-9)

  5. Potassium hydroxide (Sigma, catalog number: 484016-1KG; CAS number: 1310-58-3)

  6. Dimethyl Sulfoxide (DMSO), sterile-filtered, BioPerformance Certified (Sigma, catalog number: D2438)

  7. Methanol (Sigma, catalog number: 179337; CAS number: 67-56-1)

  8. Chloroform, ethanol-stabilized and certified for absence of phosgene and HCl (Sigma, catalog number: 650471)

  9. Fluorescent C6-NBD-phospholipids in chloroform

    C6-NBD-phosphatidylethanolamine (Avanti Polar Lipids, catalog number: 810153)

    C6-NBD-phosphatidylserine (Avanti Polar Lipids, catalog number: 810192)

    C6-NBD-phosphatidylcholine (Avanti Polar Lipids, catalog number: 810130)

    NBD-lysophosphatidylcholine (Avanti Polar Lipids, catalog number: 810128)

    C6-NBD sphingomyelin (Avanti Polar Lipids, catalog number: 810218)

  10. Tergitol solution type NP-40 (Sigma, catalog number: NP40S)

  11. Low-melting-point agarose, analytical grade (Promega Corporation, catalog number: V2111)

  12. Murashige and Skoog (MS) salts with vitamins (Phyto Technology Laboratories, catalog number: M519)

  13. Half strength MS liquid medium (see Recipes)

  14. Half strength MS plates (see Recipes)

  15. Agarose solution (see Recipes)

  16. C6-NBD-lipid stocks (see Recipes)

Equipment

  1. Pipettes PIPETMAN Classic P2, P20, P200, P1000 (Gilson, models: F144801, F123600, F123601, F123602)

  2. Precision tweezers Style #5, fine needle-sharp, anti-magnetic stainless steel (Merck, catalog number: T4537)

  3. Scalpel (e.g., Sigma, catalog numbers: S2646 and S2896)

  4. Glass dessicator (e.g., Boro 3.3 dessicator 20 cm with knob lid; BRAND, catalog number: 65038)

  5. Shallow water bath (e.g., Precision GP 2S, 2L shallow water bath; ThermoFisher, catalog number: TSGP2S)

  6. Incubator or heating block at 60°C (e.g., VWR, catalog number: 75838-270)

  7. Analytical balance (e.g., Sartorius Entris-i II, 220 g/0.1 mg, Buch Holm, catalog number: 4669128)

  8. Autoclave sterilizer (e.g., Presoclave III, 80 liters, Ø: 40 × 62 cm, Buch Holm, catalog number: 5083042)

  9. Freezer (e.g., GRAM Bioline, model: BioCompact 210RF)

  10. Refrigerator (e.g., GRAM Bioline, model: BioCompact 210RR)

  11. Rotary evaporator equipped with vacuum pump or nitrogen gas supply [e.g., Büchi® Rotavapor® RII evaporator with jack and water bath (Sigma, catalog number: Z564036), equipped with a Vacuubrand diaphragm vacuum pump model MD1C (Sigma, catalog number: Z656194)]

  12. Water purification systems (e.g., Milli-Q® Direct water purification system, Merck-Millipore, catalog number: ZR0Q008WW)

  13. Fume hood (e.g., ErlabTM Captair 391 Smart Fume Hood, Fisher Scientific, catalog number: 15514360)

  14. Microwave oven (e.g., H2100 Microwave Oven 220 Volt, Merck, catalog number: A9209)

  15. Laminar flow cabinet (e.g., Fortuna Clean Bench, ScanLaf, Labogene)

  16. Plant growth chamber (e.g., Sanyo Versatile Environmental Test Chamber, model: MLR-351H)

  17. Confocal microscope, e.g., Leica TCS SP5 Confocal Laser Scanning Microscope equipped with an argon laser (Leica Microsystems A/S) and a C-Apochromat 63×/1.2 W autocorr M27 (CG=0.14-0.19 mm) (FWD=0.28 mm at CG=0.17 mm) objective (Carl Zeiss Microscopy, catalog number: 421787-9971-790)

Software

  1. Leica LAS X software, version 3.5.7.23225 (Leica Microsystems A/S)

  2. ImageJ 1.52n (National Institutes of Health, USA, http://imagej.nih.gov/ij) (Schneider et al., 2012)

Procedure

  1. Prepare plant growth medium

    Prepare ½ MS media plates (see Recipes) considering that one plate will be needed for every 20-25 seeds. For each lipid, uptake should be quantified for at least five seedlings per experiment. Allow some extra seeds to account for possible damaged seedlings along the procedure.

    Note: Plates can be made in advance and kept at 4°C for about a month in a sterile bag.


  2. Seed sterilization and plant growth conditions

    We sterilize our seeds using chlorine vapor inside a desiccator, but the protocol can be used with any other sterilization procedure.

    1. Transfer seeds to 2-ml microcentrifuge tubes (maximum of 50 seeds/tube) marked with a pencil or chlorine-resistant pen and place in a circular rack.

    2. Place the rack with the seed-containing tubes (with lids open) and a 50-ml beaker with 25 ml of 14% hypochlorite solution into a desiccator jar placed in a fume hood (Figure 1A and 1B).



      Figure 1. Processing of plant samples in lipid uptake assays.

      Seeds are placed into open 2-ml round bottom tubes (A) and taken to a desiccator jar together with 25 ml of sodium hypochlorite solution in a 50-ml glass beaker (B). Sterilization by chlorine gas is triggered by addition of 37% hydrochloric acid. (C) Sterile seeds are plated on square ½ MS plates and sealed with micropore 3M tape. (D) After stratification, plates are placed vertically in a growth chamber under a long-day regime. (E) Seedlings are ready for lipid uptake assays after five days of growth. (F) Seedlings are transferred to small glass jars containing ½ MS media and placed at 25°C. Lipid uptake assays start with addition of the desired NBD-lipid. (G-H) After washing, roots (G) or aerial parts (H) are transferred to microscope slides for visualization. Scale bars: A, F, and H, 1 cm; B and D, 5 cm; C and E, 2.5 cm.


    3. Carefully add 1 ml of 37% hydrochloric acid solution to the hypochlorite solution and immediately close the desiccator jar.

    4. Allow sterilization by chlorine fumes to proceed for a period of approximately 3 to 4 h before opening the container and immediately closing the tube lids as fast as possible to preserve sterility.

      Note: Sterilization periods longer than 4 h reduce seed viability.

    5. Take the tubes with seeds to a sterile bench and plate on square ½ MS plates (see Recipes), trying to cover the whole surface so that seedlings do not touch each other as they grow (Figure 1C).

    6. Close the plates with micropore tape and stratify for 2 to 4 days at 4°C before placing them vertically in a plant growth chamber at 22°C under a 16 h light/8 h dark light regime for 5 days (Figure 1D-1E).

      Note: Lipid uptake seems to be poor in plants coming directly from the dark period. Allow the seedlings to stand in the light for at least 90-120 min before starting the experiment.


  3. NBD-lipid uptake assay

    1. For each lipid and plant line, prepare a glass jar with 250 µl of liquid ½ MS medium and incubate at 25°C for 5-15 min in a shallow water bath (Figure 2).

    2. Using precision tweezers, transfer 2-4 seedlings to each glass jar and shake gently. Avoid touching the part of the seedling that will be visualized later (Figure 1F and Figure 2).

      Start labeling by adding 1 µl of 10 mM C6-NBD-lipid (see Recipes) to each jar (40 µM final concentration in the medium) without touching the seedlings. Shake gently and incubate at 25°C for the desired time.

      Note: Incubation time depends on the permeability of the cell wall to the specific lipid, which depends on both fatty acid tail length and head group charge. For C6-NBD-sphingomyelin, 15 min are sufficient, while lipids like C6-NBD-phosphatidylethanolamine and C6-NBD-phosphatidylserine are first detectable after 3 h.



      Figure 2. Lipid uptake assays in plants.

      Using precision tweezers, 5-day old seedlings are transferred to small glass jars containing pre-warmed ½ MS liquid medium without touching the part of the seedling that will be visualized later. After addition of the desired NBD-lipid, seedlings are incubated for different time periods. Subsequently, seedlings are washed once with detergent-containing ½ MS medium and twice with medium without detergent, before transfer to microscopic slides. Roots are immobilized using a low-concentration agarose solution, while aerial parts are simply placed in water. Plant material that will not be visualized is removed with the help of a scalpel before the sample is covered with an objective glass and visualized using confocal microscopy.


    3. Remove the supernatant, being careful not to touch the seedlings, and add 400 µl of ½ MS liquid medium with 1% tergitol solution at 25°C. Shake gently and incubate for 2-3 min at 25°C.

    4. Repeat the wash step twice with 400 µl of ½ MS liquid medium without detergent at 25 °C, and keep the plants in ½ MS liquid medium at room temperature until visualization (not longer than 1 h).

      Note: At this point, plants can be tested for metabolic conversion of the fluorescent lipid analogs using lipid extraction and thin layer chromatography analysis, as previously described (Poulsen et al., 2015).


  4. Microscopic visualization

  1. Visualization of roots

    1. Taking care not to touch the areas to be visualized, place a seedling with the root inside a 6-mm well on a microscopic slide and add a 3-µl drop of 0.5% agarose solution on top (Figures 1G and Figure 2).

    2. Use a scalpel to remove the aerial parts, and cover with an objective glass.

    3. Mount the slide on the microscope and adjust the focus on the area of interest using bright field mode with a 63× objective.

      Note: Exposure to laser light will cause bleaching. Therefore, it is recommended to adjust the focus in bright field mode before switching to fluorescent mode and immediately acquire an image.

    1. Switch to confocal acquisition with excitation at 488 nm and emission recording at 490-508 nm. Pinhole diameter should be below two airy discs (about 100 μm).

      Note: These settings are for lipids carrying an NBD group and will need to be adapted when using other fluorophores.

    2. Set imaging parameters (laser power, detector gain, or exposure time) to get a bright signal without overexposure. We typically use 20-40% laser power and a 4-time line average scanning to prevent sample bleaching and compensate for the low signal intensity by increasing the detector gain and lowering the offset. Under these settings, the signal is stable for 25-45 s before significant bleaching occurs.

    3. Acquire images without changing any of the set parameters and in a similar area and confocal plane for all plants (for an example, see Figure 3).



      Figure 3. Examples of lipid uptake imaging in roots and aerial plant tissues.

      Seedlings (5-day old) were incubated with NBD-lipids for the indicated times and visualized by confocal microscopy after removal of excess lipid. (A) Time-course of a lipid uptake assay in roots and guard cells. Scale bars: roots, 25 μm; guard cells, 10 μm. (B) Examples of damaged roots. White polygons mark damaged areas. Scale bars: 20 μm.


  2. Visualization of leaves

    1. Drop 40-50 µl of water on a plain microscopy slide and use tweezers to place a seedling with the aerial parts inside the water (adaxial side up). Take care not to touch the areas to be visualized (Figure 1H and Figure 2).

    2. Remove the root using a scalpel and cover with an objective glass.

    3. Mount the slide on the microscope and locate cells using bright field mode with a 63× objective.

      Note: Exposure to laser light will cause bleaching. Therefore, we recommend adjusting the focus in bright field mode before switching to fluorescent mode and acquiring an image immediately.

    4. Switch to confocal acquisition with excitation at 488 nm and emission recording at 490-508 nm. Pinhole diameter should be below two airy discs (about 100 μm).

      Note: These settings are for lipids carrying an NBD group and will need to be adapted when using other fluorophores.

    5. Set imaging parameters (laser power, detector gain, or exposure time) to get a bright signal without overexposure. We typically use 20-40% laser power and 4-time line average scanning to prevent sample bleaching, and compensate for the low signal intensity by increasing the detector gain and lowering the offset. Under these settings, the signal is stable for 25-45 s before significant bleaching occurs.

    6. Acquire images without changing any of the set parameters and in a similar area and confocal plane for all plants (for an example, see Figure 3).

Data analysis

  1. Export the raw image data in a format compatible with ImageJ (e.g., tif) from the imaging system and import into ImageJ.

  2. Go to Analyze > Set measurements and select mean gray value.

  3. Draw a region of interest (ROI) in an area of the image where no fluorescence is present, then use Crtl + m to get the mean gray value of your background (Figure 4).

  4. Deselect the ROI, go to Process > Math > Substract, type the value of the background in, and press OK to adjust the image.

  5. Draw a new ROI covering an area that can be defined with clear parameters applicable to all images (e.g., the confocal image area covering from the root tip to the 8th epidermal cell above the tip, a region including five epidermal cells, a whole stoma, etc.) (Figure 4). Measure the mean gray value using Ctrl + m. Values appear sequentially in a table that can be exported to Excel.

    Note: Damaged tissue will show an abnormal fluorescence compared to the rest of the sample (see Figure 3B). Be sure not to include this in the quantification.

  6. For each plant line and lipid, use the quantification of at least three independent experiments with five seedlings each to calculate the average values for the whole population, and carry out a statistical analysis using an appropriate t-test.



    Figure 4. Image analysis using ImageJ.

    After setting the system to measure mean gray value (top right), define a convenient region of interest (ROI) for each image to quantify the background (top left). Apply a background correction by using the Process > Math > Substract command (bottom left). Finally, define a new ROI and measure the mean gray values of the samples. Scale bars: 25 μm.

Recipes

Note: Prepare all media using ultrapure water with purification sensitivity of 18 MΩ.cm at 25°C.

  1. Half strength MS liquid medium

    1. Mix 2.21 g L-1 Murashige and Skoog (MS) salts with vitamins and 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES) in the desired water volume.

    2. Adjust the pH to 5.7 with 2 M KOH.

    3. Autoclave at 121°C for 20 min and store at 4°C.

  2. Half strength MS plates

    1. Mix MS salts and MES and adjust the pH, as above.

    2. Add 0.7% phytoagar.

    3. Autoclave at 121°C for 20 min.

    4. Let the media cool down to approximately 60°C and pour onto square Petri dishes inside a laminar flow bench.

    5. Let the plates cool down and solidify, place them in a sterile bag, and store at 4°C.

  3. Agarose solution

    1. Mix low-melting-point agarose with the desired amount of water to obtain a 0.5% (w/v) solution.

    2. Heat up in the microwave until the solution starts boiling, then take out of the microwave and mix well.

    3. If necessary, repeat the boiling step until all the agarose is fully melted.

    4. Keep in a heating block or an incubator at 60°C to prevent solidification.

  4. C6-NBD-lipid stocks

    All steps must be performed in glass tubes in order to prevent nonspecific binding of lipids.

    Note: Chloroform is a chemical hazard. Do not breathe gas/fumes/vapor/spray. Wear suitable protective clothing. Work in a fume hood.

    1. Use a glass syringe to transfer the desired amount of C6-NBD-lipid into a 12-ml glass tube.

    2. Dry the lipids under a gentle stream of nitrogen gas so that a dried lipid film is formed at the bottom of the tube.

    3. Resuspend the C6-NBD-lipids in DMSO to a final concentration of 10 mM.

      Note: DMSO lipid suspensions can be stored at -20°C in a well-covered glass container and used for up to 2 weeks. However, they are prone to precipitation owing their hygroscopic nature.

Acknowledgments

This protocol was adapted from our previous work (Poulsen et al., 2015). This work was supported by the Danish National Research Foundation (DNRF85) and the Danish Council for Independent Research|Natural Sciences (FNU, project number 10-083406). Current work in RLLM's group is supported by the Novo Nordisk Foundation (NovoCrops; Project Number NNF19OC0056580) and the Independent Research Fund Denmark | Nature and Universe (Project Number 1026-00024B). Imaging data were collected at the Center for Advanced Bioimaging Denmark (CAB), University of Copenhagen.

Competing interests

The authors declare no competing interests.

Ethics

No human or animal subjects are used in this protocol.

References

  1. Andersen, J. P., Vestergaard, A. L., Mikkelsen, S. A., Mogensen, L. S., Chalat, M. and Molday, R. S. (2016). P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas. Front Physiol 7275.
  2. Lopez-Marques, R. L., Theorin, L., Palmgren, M. G. and Pomorski, T. G. (2014). P4-ATPases: lipid flippases in cell membranes. Pflugers Arch 466(7): 1227-1240.
  3. Poulsen, L. R., Lopez-Marques, R. L., Pedas, P. R., McDowell, S. C., Brown, E., Kunze, R., Harper, J. F., Pomorski, T. G. and Palmgren, M. (2015). A phospholipid uptake system in the model plant Arabidopsis thaliana. Nat Commun 67649.
  4. Roland, B. P., Naito, T., Best, J. T., Arnaiz-Yépez, C., Takatsu, H., Yu, R. J., Shin, H.-W. W. and Graham, T. R. (2019). Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs. J Biol Chem 2941794-1806.
  5. Nintemann, S. J., Palmgren, M. and López-Marqués, R. L. (2019). Catch you on the flip side: A critical review of flippase mutant phenotypes. Trends Plant Sci 24468-478.
  6. McDowell, S. C., López-Marqués, R. L., Cohen, T., Brown, E., Rosenberg, A., Palmgren, M. G. and Harper, J. F(2015). Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane. Front Plant Sci 6197.
  7. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9671-675.
  8. Shin, H. W. and Takatsu, H. (2019). Substrates of P4-ATPases: beyond aminophospholipids(phosphatidylserine and phosphatidylethanolamine). FASEB J 333087-3096.

简介

[摘要]真核细胞使用多种转运蛋白来控制脂质跨质膜的运动,这会极大地影响膜特性。已经开发了各种工具和技术来分析这些转运蛋白的活动。其中,基于荧光磷脂探针的检测特别适合,可以对活细胞中的脂质内化进行成像和量化。传统上,这些测定已应用于酵母和动物细胞。在这里,我们描述了这种强大的方法的适应性,以使用共聚焦成像来表征植物根部和地上组织中的脂质内化。

[背景]在真核细胞中,脂质跨生物膜的运动(称为脂质触发器)受多种膜转运蛋白的调节,膜转运蛋白可分为两类:(i)不依赖 ATP 的转运蛋白,也称为乱序,在没有代谢能量输入的情况下促进脂质的快速双向运动,以及 (ii) ATP 驱动的载体转运蛋白,可将脂质从一个膜小叶主动转移到另一个膜小叶,通常具有高度特异性。后一组包括 ATP 依赖性翻转酶和翻转酶,它们分别催化脂质向内运动到细胞质膜小叶,以及向外运动到细胞外/管腔侧。P 型 ATP 酶的一个亚组,即 P4 ATP 酶,已成为主要的脂质翻转酶组,它们与 Cdc50(细胞分裂控制 50)蛋白家族的成员形成异二聚体复合物(Lopez-Marques等人综述,2014 年;安徒生等人,2016 年)。虽然最初被定性为氨基磷脂翻转酶,但最近对真菌、植物和动物的个体家庭成员的研究表明,P4 ATPases 的底物特异性不同,并介导更广泛的脂质底物的转运,包括溶血磷脂、合成烷基磷脂和糖修饰的神经酰胺(Roland等人,2019 年;Shin 和 Takatsu,2019 年)。

P4 ATPase 脂质转运活性的定量评估对于确定底物特异性以及确定该活性是否以及如何在活细胞中受到调节至关重要。由于 P4-ATPases 被困在由它们自己的底物(脂质)形成的环境(细胞膜)中,因此分析它们的活性并不是一项简单的任务,大多数分析都是基于使用荧光脂质类似物,通常是硝基苯并恶二唑 (NBD)-标记的脂质。这些类似物具有连接到短链脂肪酸 (C 6 )的荧光报告基团,并保留了内源性磷脂的大部分特性,但它们更易溶于水,这有助于从培养基中掺入到外层单层中。质膜。

传统上,在植物 P4 ATP 酶在缺乏内源性脂质转运蛋白的酵母菌株中异源表达后,使用 NBD 脂质进行脂质吸收测定(综述参见 Nintemann等,2019)。然而,当在异源系统中产生时,许多植物 P4 ATPase 表达不佳、无法折叠和/或运输不当。此外,成功表达并不总是导致活性脂质易位,这可能是由于缺乏植物脂质转运蛋白功能所需的植物特异性辅助蛋白和/或辅因子(McDowell等,2015)。

这里介绍的方法利用 C 6 -NBD-脂质来研究完整植物中的脂质内化,以拟南芥幼苗为例。5 天龄的小幼苗在无菌条件下在平板上生长,然后转移到补充有 C 6 -NBD-脂质的液体生长培养基中。孵育所需时间后,用含有少量特定洗涤剂的培养基清洗幼苗,以去除附着在细胞壁上的多余脂质。最后,使用共聚焦显微镜对植物进行可视化,并使用成像软件对数据进行量化。该协议可用于表征根和地上组织中的脂质内化,并可轻松适应其他植物物种。对于根部实验,在琼脂平板上生长的幼苗优于在土壤上常规培养,因为从土壤中移除会损坏根表面。

关键字:拟南芥, 翻转酶, P4 ATP酶, 脂质运输, NBD-脂质, 根部, 叶子, 保卫细胞

材料和试剂

 

材料

1.     拟南芥种子(生态型 Col-0 或根据需要)

2.     移液器吸头 PIPETMAN DIAMOND D10D200D1000Gilson,目录号:F161630F161930F161670

3.     2 ml 圆底微量离心管卡扣管(例如BRAND,微量离心管,2 ml 带盖,PP;默克,目录号:BR780546-500EA

4.     用于 2 毫升微量离心管的圆形支架(例如PrepSafe TM微量离心管迷你浮动架,透明;默克,目录号:Z756385

5.     50 毫升玻璃烧杯(例如BRAND,目录号:91217

6.     方形培养皿 120 × 120 × 17 mmGreiner Bio-OneFisher Scientific,目录号:07-000-330

7.     微孔胶带(3M,目录号:1530-1

8.     铝箔(例如Sigma,目录号:Z185140-1EA

9.     带有锥形底部的离心玻璃 DURAN ® 12 mlCarl Roth,目录号:K211.1

10.  25-μl 校准玻璃注射器(型号 702 NHamilton,目录号:CAL80400

11.  1.5 毫升带 8 毫米特氟龙内衬螺旋盖的螺旋琥珀色玻璃小瓶(例如VWR,目录号:VWRI548-0019 548-0360

12.  Corning ® Pasteur 移液器,非无菌,228 mmMerck,目录号:CLS7095B9

13.  PARAFILM ® M(默克,目录号:P7793

14.  带卡扣盖的透明玻璃罐,11 毫升,22 × 45 毫米(VWR,目录号:548-0625

15.  对于气生组织,显微镜载玻片 76 × 26 × 1 mm,带切边(Histolab,目录号:06300

16.  对于根组织,诊断显微镜载玻片 25 × 75 mm8 6 mm 孔,腔周围有黑色环氧树脂场(Histolab,目录号:06260

17.  盖玻片 118 × 24 毫米和 24 × 24 毫米(Histolab,目录号:06602 06608

 

试剂

所有试剂都可以在室温下储存,除了 NBD-磷脂溶液,它应保持在 -20°C 以长期储存。

1.     次氯酸钠(14% Cl )水溶液,GPR RECTAPUR ® VWR Chemicals,目录号:27900.296

2.     37%盐酸溶液(Sigma,目录号:320331

3.     PhytoagarDuchefa Biochemie,目录号:P1003

4.     2-N-吗啉代)乙磺酸(MES)(Merck-Millipore,目录号:1061261000CAS号:4432-31-9

5.     氢氧化钾(Sigma,目录号:484016-1KGCAS 号:1310-58-3

6.     二甲基亚砜(DMSO),无菌过滤,生物性能认证(Sigma,目录号:D2438

7.     甲醇(Sigma,目录号:179337CAS 号:67-56-1

8.     氯仿,乙醇稳定并经认证不含光气和 HClSigma,目录号:650471

9.     氯仿中的荧光-NBD-磷脂

-NBD-磷脂酰乙醇胺(Avanti Polar Lipids,目录号:810153

-NBD-磷脂酰丝氨酸(Avanti Polar Lipids,目录号:810192

-NBD-磷脂酰胆碱(Avanti Polar Lipids,目录号:810130

NBD-溶血磷脂酰胆碱(Avanti Polar Lipids,目录号:810128

-NBD 鞘磷脂(Avanti Polar Lipids,目录号:810218

10.  Tergitol NP-40型溶液(Sigma,目录号:NP40S

11.  低熔点琼脂糖,分析级(Promega Corporation,目录号:V2111

12.  含维生素的 Murashige SkoogMS)盐(Phyto Technology Laboratories,目录号:M519

13.  半强度 MS 液体培养基(见配方)

14.  半强度 MS 板(见配方)

15.  琼脂糖溶液(见食谱)

16.  -NBD-脂质储备(见配方)

 

设备

 

1.     移液器 PIPETMAN Classic P2P20P200P1000Gilson,型号:F144801F123600F123601F123602

2.     5 型精密镊子,细针锋利,防磁不锈钢(默克,目录号:T4537

3.     手术刀(例如Sigma,目录号:S2646 S2896

4.     玻璃干燥器(例如Boro 3.3 干燥器 20 cm 带旋钮盖;BRAND,目录号:65038

5.     浅水浴(例如Precision GP 2S2L 浅水浴;ThermoFisher,目录号:TSGP2S

6.     60°C 的培养箱或加热块(例如VWR,目录号:75838-270

7.     分析天平(例如Sartorius Entris-i II220 g/0.1 mgBuch Holm,目录号:4669128

8.     高压灭菌器(例如Presoclave III80 升,Ø40 × 62 cmBuch Holm,目录号:5083042

9.     冷冻机(例如GRAM Bioline,型号:BioCompact 210RF

  1. 冰箱(例如GRAM Bioline,型号:BioCompact 210RR
  2. 旋转蒸发仪配备有真空泵或氮气供给[例如,步琪®旋转蒸发仪® RII蒸发器与插座和水浴(Sigma,目录号:Z564036),配备有VACUUBRAND隔膜真空泵模型MD1CSigma,目录号:Z656194 ]
  3. 水净化系统(例如,的Milli-Q ®直接水净化系统,默克-Millipore公司,目录号:ZR0Q008WW
  4. 通风柜(例如Erlab TM Captair 391 Smart Fume HoodFisher Scientific,目录号:15514360
  5. 微波炉(例如H2100 Microwave Oven 220 VoltMerck,目录号:A9209
  6. 层流柜(例如Fortuna Clean BenchScanLafLabogene
  7. 植物生长箱(三洋多功能环境试验箱,型号:MLR-351H
  8. 共聚焦显微镜,例如配备氩激光器(Leica Microsystems A/S)和 C-Apochromat 63×/1.2 W autocorr M27CG=0.14-0.19 mm)(FWD=0.28 mm)的 Leica TCS SP5 共聚焦激光扫描显微镜在 CG=0.17 mm)物镜(Carl Zeiss Microscopy,目录号:421787-9971-790

 

软件

 

1.     Leica LAS X 软件,版本 3.5.7.23225 (Leica Microsystems A/S)

2.     ImageJ 1.52n(美国国立卫生研究院,http: //imagej.nih.gov/ij )(Schneider2012

 

程序

 

A.    准备植物生长培养基

考虑到每 20-25 个种子需要一个板,准备 ½ MS 培养基板(参见配方)。对于每种脂质,每个实验至少应量化五株幼苗的吸收。允许一些额外的种子来解释过程中可能损坏的幼苗。

注意:平板可以提前制作并在无菌袋中在 4°C 下保存约一个月。

 

B.    种子杀菌和植物生长条件

我们使用干燥器内的氯蒸气对种子进行消毒,但该协议可用于任何其他消毒程序。

1.     将种子转移到用铅笔或耐氯笔标记的 2 毫升微量离心管(最多 50 粒种子/管)中,然后放在圆形架子上。

2.     将装有种子的管子(打开盖子)和 50 毫升烧杯和 25 毫升 14% 次氯酸盐溶液放入放置在通风橱中的干燥器罐中(图 1A 1B)。

 

 

1. 在脂质吸收测定中处理植物样品。

将种子放入开口的 2 毫升圆底试管 (A) 中,并与 50 毫升玻璃烧杯 (B) 中的 25 毫升次氯酸钠溶液一起放入干燥器罐中。通过添加 37% 的盐酸触发氯气灭菌。(C) 无菌种子镀在方形 ½ MS 板上,并用微孔 3M 胶带密封。(D) 分层后,将板垂直放置在长日制下的生长室中。(E) 幼苗在生长五天后即可进行脂质吸收测定。(F) 将幼苗转移到装有 ½ MS 培养基的小玻璃罐中,并置于 25°C。脂质摄取测定从添加所需的 NBD 脂质开始。(GH) 洗涤后,根 (G) 或地上部分 (H) 被转移到显微镜载玻片上进行可视化。比例尺:AF H1 厘米;BD5厘米;C E2.5 厘米。

 

3.     小心地将 1 ml 37% 的盐酸溶液加入次氯酸盐溶液中,并立即关闭干燥器罐。

4.     在打开容器并立即尽快关闭管盖以保持无菌之前,允许通过氯气进行大约 3 4 小时的灭菌。

注意:灭菌时间超过 4 小时会降低种子活力。

5.     将装有种子的管子放到无菌工作台上,然后放在 ½ MS 方形板上(参见食谱),尝试覆盖整个表面,以便幼苗在生长时不会相互接触(图 1C)。

6.     用微孔胶带封闭板并在 4°C 下分层 2 4 天,然后将它们垂直放置在 22°C 的植物生长室中,在 16 小时光照/8 小时暗光制度下持续 5 天(图 1D-1E .

注意:直接来自黑暗时期的植物的脂质吸收似乎很差。在开始实验之前,让幼苗在光照下至少站立 90-120 分钟。

 

C.    NBD-脂质摄取测定

1.     对于每个脂质和植物系,准备一个装有 250 µl 液体 ½ MS 培养基的玻璃罐,并在 25°C 的浅水浴中孵育 5-15 分钟(图 2)。

2.     使用精密镊子,将 2-4 株幼苗转移到每个玻璃罐中并轻轻摇晃。避免接触稍后将被可视化的幼苗部分(图 1F 和图 2)。

在不接触幼苗的情况下向每个罐子(培养基中的最终浓度为 40 µM)添加 1 µl 10 mM C -NBD-脂质(参见配方),开始标记。轻轻摇动并在 25°C 下孵育所需的时间。

注意:孵育时间取决于细胞壁对特定脂质的渗透性,这取决于脂肪酸尾长和头基电荷。对于-NBD-鞘磷脂,15 分钟就足够了,而像-NBD-磷脂酰乙醇胺和-NBD-磷脂酰丝氨酸这样的脂质在 3 小时后首先可检测到。

 

 

2. 植物中的脂质吸收测定。

使用精密镊子,将 5 天大的幼苗转移到装有预热的 ½ MS 液体培养基的小玻璃罐中,而不会接触稍后将看到的幼苗部分。添加所需的 NBD 脂质后,将幼苗培养不同的时间段。随后,幼苗用含有洗涤剂的 ½ MS 培养基洗涤一次,用不含洗涤剂的培养基洗涤两次,然后转移到显微载玻片上。使用低浓度琼脂糖溶液固定根部,而地上部分则简单地置于水中。在样品用物镜覆盖并使用共聚焦显微镜进行可视化之前,在手术刀的帮助下去除不会被可视化的植物材料。

 

3.     去除上清液,小心不要接触幼苗,并在 25°C 下加入 400 µl ½ MS 液体培养基和 1% tergitol 溶液。轻轻摇晃并在 25°C 下孵育 2-3 分钟。

4.     25 °C 下用 400 µl 不含洗涤剂的 ½ MS 液体培养基重复洗涤步骤两次,并将植物保持在室温下的 ½ MS 液体培养基中,直至可视化(不超过 1 小时)。

注意:此时,可以使用脂质提取和薄层色谱分析来测试植物的荧光脂质类似物的代谢转化,如前所述Poulsen2015)。

 

D.    显微可视化

1.     根的可视化

a.     注意不要触摸要可视化的区域,将根部置于 6 毫米孔内的幼苗放在显微载玻片上,并在顶部添加 3 微升 0.5% 琼脂糖溶液(图 1G 和图 2)。

b.     用手术刀去除地上部分,并用物镜盖住。

c.     将载玻片安装在显微镜上,并使用带有 63 倍物镜的明场模式调整感兴趣区域的焦点。

注意:暴露在激光下会导致漂白。因此,建议在切换到荧光模式之前在明场模式下调整焦点并立即获取图像。

d.     切换到 488 nm 激发和 490-508 nm 发射记录的共聚焦采集。针孔直径应低于两个艾里斑(约 100 μm)。

注意:这些设置适用于携带 NBD 组的脂质,在使用其他荧光团时需要进行调整。

e.     设置成像参数(激光功率、探测器增益或曝光时间)以获得明亮的信号而不会过度曝光。我们通常使用 20-40% 的激光功率和 4 次线平均扫描来防止样品漂白并通过增加检测器增益和降低偏移来补偿低信号强度。在这些设置下,信号在发生显着漂白之前稳定 25-45 秒。

f. 在不更改任何设置参数的情况下获取图像,并在所有植物的相似区域和共焦平面中获取图像(例如,参见图 3)。

 

 

3. 根和气生植物组织中脂质吸收成像的示例。

将幼苗(5 天大)与 NBD 脂质孵育指定的时间,并在去除多余的脂质后通过共聚焦显微镜观察。(A) 根和保卫细胞中脂质吸收测定的时间过程。比例尺:根,25 μm;保卫细胞,10 μm(B) 受损根的例子。白色多边形标记损坏区域。比例尺:20 μm

 

2.     叶子的可视化

a.     40-50 µl 水滴在普通显微镜载玻片上,并使用镊子将地上部分的幼苗放入水中(正面朝上)。注意不要触摸要可视化的区域(图 1H 和图 2)。

b.     使用手术刀去除根部并用物镜盖住。

c.     将载玻片安装在显微镜上,并使用明场模式和 63 倍物镜定位细胞。

注意:暴露在激光下会导致漂白。因此,我们建议在切换到荧光模式并立即获取图像之前,先在明场模式下调整焦点。

d.     切换到 488 nm 激发和 490-508 nm 发射记录的共聚焦采集。针孔直径应低于两个艾里斑(约 100 μm)。

注意:这些设置适用于携带 NBD 组的脂质,在使用其他荧光团时需要进行调整。

e.     设置成像参数(激光功率、探测器增益或曝光时间)以获得明亮的信号而不会过度曝光。我们通常使用 20-40% 的激光功率和 4 次线平均扫描来防止样品漂白,并通过增加检测器增益和降低偏移来补偿低信号强度。在这些设置下,信号在发生显着漂白之前稳定 25-45 秒。

f.      在不更改任何设置参数的情况下获取图像,并在所有植物的相似区域和共焦平面中获取图像(例如,参见图 3)。

 

数据分析

 

1.     以与 ImageJ 兼容的格式(例如tif)从成像系统导出原始图像数据并导入 ImageJ

2.     转到分析 > 设置测量并选择平均灰度值。

3.     在不存在荧光的图像区域中绘制感兴趣区域 (ROI),然后使用 Crtl + m 获得背景的平均灰度值(图 4)。

4.     取消选择 ROI,转到 Process > Math > Substract,输入背景值,然后按 OK 调整图像。

5.     绘制一个新的ROI覆盖一个区域,该区域可以用适用于所有图像的清晰参数定义(例如,覆盖从根尖到尖端上方8表皮细胞的共聚焦图像区域,一个包括五个表皮细胞的区域,一个整体造口)(图 4)。使用 Ctrl + m 测量平均灰度值。值按顺序出现在可以导出到 Excel 的表格中。

注意:与样品的其余部分相比,受损组织将显示异常荧光(见图 3B)。确保不要将其包含在量化中。

6.     对于每个植物系和脂质,使用至少三个独立实验的量化,每个实验有五个幼苗来计算整个种群的平均值,并使用适当的t检验进行统计分析。

 

 

4. 使用 ImageJ 进行图像分析。

将系统设置为测量平均灰度值(右上)后,为每个图像定义一个方便的感兴趣区域 (ROI) 以量化背景(左上)。使用 Process > Math > Substract 命令(左下角)应用背景校正。最后,定义一个新的 ROI 并测量样本的平均灰度值。比例尺:25 μm

 

食谱

 

注意:在 25°C 下使用纯化灵敏度为 18 MΩ.cm 的超纯水制备所有培养基。

1.     半强度 MS 液体培养基

a.     2.21 g L -1 Murashige Skoog (MS) 盐与维生素和 0.5 g/L 2-(N-morpholino) 乙磺酸 (MES) 混合在所需的水量中。

b.     2 M KOH pH 值调节到 5.7

c.     121°C 下高压灭菌 20 分钟并在 4°C 下储存。

2.     半强度 MS

a.     混合 MS 盐和 MES 并调整 pH 值,如上。

b.     添加 0.7% 植物琼脂。

c.     121°C 下高压灭菌 20 分钟。

d.     让培养基冷却至大约 60°C,然后倒入层流工作台内的方形培养皿中。

e.     让板冷却并凝固,将它们放入无菌袋中,并在 4°C 下储存。

3.     琼脂糖溶液

a.     将低熔点琼脂糖与所需量的水混合,以获得 0.5% (w/v) 的溶液。

b.     在微波炉中加热直到溶液开始沸腾,然后从微波炉中取出并混合均匀。

c.     如有必要,重复煮沸步骤,直到所有琼脂糖完全融化。

d.     保存在 60°C 的加热块或培养箱中以防止凝固。

4.     -NBD-脂质储备

所有步骤都必须在玻璃管中进行,以防止脂质的非特异性结合。

注意:氯仿是一种化学危害。不要吸入气体/烟雾/蒸气/喷雾。穿戴合适的防护服。在通风橱中工作。

a.     使用玻璃注射器将所需量的-NBD-脂质转移到 12 毫升玻璃管中。

b.     在温和的氮气流下干燥脂质,使管底部形成干燥的脂质膜。

c.     -NBD-脂质在 DMSO 中重悬至 10 mM 的最终浓度。

注意:DMSO 脂质悬浮液可在 -20°C 下储存在盖好玻璃容器中,最多可使用 2 周。然而,由于它们的吸湿性,它们易于沉淀。


致谢

 

该协议改编自我们之前的工作(Poulsen2015)。这项工作得到了丹麦国家研究基金会 (DNRF85) 和丹麦独立研究委员会|自然科学 (FNU,项目编号 10-083406) 的支持。RLLM 小组目前的工作得到了诺和诺德基金会(NovoCrops;项目编号 NNF19OC0056580)和丹麦独立研究基金的支持自然与宇宙(项目编号 1026-00024B)。成像数据是在哥本哈根大学丹麦高级生物成像中心 (CAB) 收集的。

 

利益争夺

 

作者声明没有竞争利益。

 

伦理

 

本协议中不使用人类或动物受试者。

 

参考

 

1.     Andersen, JP, Vestergaard, AL, Mikkelsen, SA, Mogensen, LS, Chalat, M. Molday, RS (2016)P4-ATPases 作为磷脂翻转酶 - 结构、功能和谜团。前生理学7275              

2.     Lopez-Marques, RL, Theorin, L., Palmgren, MG Pomorski, TG (2014)P4-ATPases:细胞膜中的脂质翻转酶。 Pflugers Arch 466(7): 1227-1240

3.     Poulsen, LR, Lopez-Marques, RL, Pedas, PR, McDowell, SC, Brown, E., Kunze, R., Harper, JF, Pomorski, TG Palmgren, M. (2015)模式植物拟南芥中的磷脂吸收系统。 国家通讯社67649

4.     Roland, BP, Naito, T., Best, JT, Arnaiz-Yépez, C., Takatsu, H., Yu, RJ, Shin, H.-WW Graham, TR (2019)酵母和人类 P4-ATP 酶使用保守的结构基序运输鞘糖脂。J Biol Chem 2941794-1806

5.     Nintemann, SJ, Palmgren, M. López-Marqués, RL (2019)抓住你的另一面:对翻转酶突变表型的批判性审查。趋势植物科学24468-478

6.     McDowell, SC, López-Marqués, RL, Cohen, T., Brown, E., Rosenberg, A., Palmgren, MG Harper, J. F (2015)的损失拟南芥P4-ATPALA6ALA7也妨碍花粉健身和改变花粉管质膜Front Plant Sci 6197

7.     Schneider, CA, Rasband, WS Eliceiri, KW (2012)NIH Image to ImageJ25 年的图像分析Nat 方法9671-675

8.     Shin, HW Takatsu, H.2019 年)。P4-ATPases 的底物:除了氨基磷脂(磷脂酰丝氨酸和磷脂酰乙醇胺)。FASEB J 333087-3096

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引用:López-Marqués, R. L. and Pomorski, T. G. (2021). Imaging of Lipid Uptake in Arabidopsis Seedlings Utilizing Fluorescent Lipids and Confocal Microscopy. Bio-protocol 11(22): e4228. DOI: 10.21769/BioProtoc.4228.
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