使用Annexin V染色可视化质膜脂质不对称性的丧失

Julia F. Baum; Huriye D. Uzun; Thomas  Günther Pomorski

doi:10.21769/BioProtoc.4754

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Peer-reviewed

Visualizing Loss of Plasma Membrane Lipid Asymmetry Using Annexin V Staining

使用Annexin V染色可视化质膜脂质不对称性的丧失

JB Julia F. Baum ^*

HU Huriye D. Uzun ^*

TP Thomas Günther Pomorski email

(*contributed equally to this work) 发布: 2023年07月20日第13卷第14期 DOI: 10.21769/BioProtoc.4754 浏览次数: 2721

评审: Alexandros AlexandratosVishal NehruEVANGELOS THEODOROUTakashi Nishina

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Cover of Journal of Cell Science, featuring study using the protocol.

Mar 2022

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Abstract

Loss of plasma membrane lipid asymmetry contributes to many cellular functions and responses, including apoptosis, blood coagulation, and cell fusion. In this protocol, we describe the use of fluorescently labeled annexin V to detect loss of lipid asymmetry in the plasma membrane of adherent living cells by fluorescence microscopy. The approach provides a simple, sensitive, and reproducible method to detect changes in lipid asymmetry but is limited by low sample throughput. The protocol can also be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. To validate the lipid binding properties of such probes, we additionally describe here the preparation and use of giant unilamellar vesicles as simple model membrane systems that have a size comparable to cells.

Key features

• Monitoring loss of lipid asymmetry in the plasma membrane via confocal microscopy.

• Protocol can be applied to any type of cell that is adherent in culture, including primary cells.

• Assay can be adapted to other fluorescently labeled lipid-binding proteins or peptide probes.

• Giant unilamellar vesicles serve as a tool to validate the lipid binding properties of such probes.

Graphical overview

Imaging the binding of fluorescent annexin V to adherent mammalian cells and giant vesicles by confocal microscopy. Annexin V labeling is a useful method for detecting a loss of plasma membrane lipid asymmetry in cells (top image, red); DAPI can be used to identify nuclei (top image, blue). Giant vesicles are used as a tool to validate the lipid binding properties of annexin V to anionic lipids (lower image, red).

Keywords: Confocal microscopy (共聚焦显微镜)

Giant vesicle (巨大囊泡)

Lipid asymmetry (脂质不对称)

Lipid-binding protein (脂质结合蛋白)

Mammalian cells (哺乳动物细胞)

Plasma membrane (质膜)

Background

A characteristic feature of many biological membranes is that their phospholipids are asymmetrically distributed across the lipid bilayer, a phenomenon known as transbilayer lipid asymmetry. A prominent example is the plasma membrane of animal cells, in which the phospholipids phosphatidylcholine and sphingomyelin are concentrated in the exoplasmic leaflet, whereas the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) are restricted to the cytosolic leaflet (van Meer et al., 2008). Transbilayer lipid asymmetry is essential for several vital cellular functions, including the regulation of membrane protein activity, signaling, and vesicle formation in the secretory and endocytic pathways (Sprong et al., 2001; Ewers and Helenius, 2011; van Meer, 2011; Sebastian et al., 2012). In animals, loss of transbilayer lipid asymmetry has been linked to processes such as blood coagulation (Lentz, 2003; Jackson, 2011), cell adhesion (Schlegel et al., 1985; Malhotra et al., 1996; Wautier et al., 2011), macrophage recognition (Krahling et al., 1999), apoptosis (Bevers and Williamson, 2016), and myotube formation (van den Eijnde et al., 2001). The establishment and regulation of lipid asymmetry are therefore crucial for cells, and several membrane proteins have evolved to fulfill the function of cross-bilayer phospholipid transporters, comprising lipid flippases, floppases, and scramblases (Hankins et al., 2015; Ristovski et al., 2021).

Several methods have been developed to analyze the loss of phospholipid asymmetry in the plasma membrane of eukaryotic cells. These include chemical approaches using e.g., trinitrobenzene sulfonic acid or fluorescamine, which covalently react with amino groups of lipids and proteins (Marinetti et al., 1976; Pomorski et al., 2003). As the probes are membrane impermeant, only aminophospholipids exposed to the cell surface are modified and can then be detected by thin-layer chromatography or mass spectrometry. However, this approach is not suitable for live-cell imaging. More recent methods are based on fluorescently labeled lipid-binding proteins that can be added to the cells. One example is the PS-specific probe lactadherin, which binds to PS with a nanomolar affinity and without the need for cofactors (Waehrens et al., 2009). Another example is annexin V, a member of the annexin family of Ca²⁺-dependent, non-covalent lipid-binding proteins. Annexin V binds negatively charged lipids with relatively high affinity and is used extensively for the detection of exofacial PS by flow cytometry or microscopy (Koopman et al., 1994; Vermes et al., 1995; Tait et al., 2004). A new generation of fluorescent probes is based on cyclic peptides that successfully mimic the function of lipid-binding proteins and benefit from their small size, ease of labeling, and cofactor-free PS recognition (Hanshaw et al., 2005; DiVittorio et al., 2006; Zheng et al., 2011).

In this protocol, we describe the use of fluorescently labeled lipid-binding protein sensors to detect the loss of lipid asymmetry in living cells by fluorescence microscopy, exemplified on mouse C2C12 wild-type myoblasts and corresponding knockout cells lacking the P4-ATPase flippase subunit CDC50A (also known as TMEM30A). Deletion of CDC50A results in loss of the aminophospholipid flippase activity and constitutive loss of plasma membrane lipid asymmetry (Grifell-Junyent et al., 2022). The approach is illustrated using commercially available annexin V conjugated to Alexa Fluor 568, but other fluorescently labeled lipid-binding proteins or peptide probes can also be used.

To validate the specificity and sensitivity of such lipid binding probes, we also describe here the use of giant unilamellar vesicles (GUVs) as simple model membrane systems. One of the major advantages of using GUVs as model membrane systems is their similarity in size to cells. This allows GUVs to be observed directly under the microscope, making them a convenient and accessible tool for lipid-binding studies. By preparing GUVs with defined lipid compositions, the specificity and sensitivity of lipid-binding probes can be evaluated and their accuracy and reproducibility in live cell experiments can be ensured (Weingärtner et al., 2012; Chandra and Datta, 2022). GUVs with defined lipid compositions can be prepared by various methods, including swelling, PVA or agarose swelling, and electroformation using indium tin oxide glass slides and droplet transfer methods (Angelova and Dimitrov, 1986; Weinberger et al., 2013; Bhatia et al., 2015; Shimane and Kuruma, 2022). In this study, we describe the swelling method due to its simplicity. For alternative preparation methods, the reader is referred to other bio-protocols (Parigoris et al., 2020; Mathiassen and Pomorski, 2022). Our protocol provides a reliable and efficient method for detecting loss of lipid asymmetry in living cells and can be adapted for use with a variety of lipid-binding proteins.

Materials and reagents

Mammalian cell culture
In this study, we used mouse myoblast cells (C2C12; cell number: ACC 565, DSMZ Braunschweig, Germany) and the corresponding knockout cells lacking CDC50A (Grifell-Junyent et al., 2022) that were cultured in growth medium (see Recipe 1). Optimal culture media and conditions may differ for other cell lines.
1. Basal cell culture medium for growth (e.g., high glucose DMEM, without pyruvate; Sigma-Aldrich, catalog number: D5796), store at 4 °C
2. Ethanol absolute ≥ 99.8% (VWR, catalog number: 20821.321)
3. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (e.g., Sigma-Aldrich, catalog number: E4378)
4. Fetal bovine serum (FBS), heat inactivated before use (e.g., Capricorn Scientific, catalog number: FBS-11A), store at -20 °C
5. Hanks’ balanced salt solution, Ca²⁺ and Mg²⁺ free (HBSS) (e.g., Sigma-Aldrich, catalog number: H6648), store at 4 °C
6. 35 mm polymer bottom dishes (e.g., Ibidi, catalog number: 81156)
7. 1.5 mL microcentrifuge tubes (Sarstedt, catalog number: 72.690.001)
8. Penicillin-streptomycin, 100× solution (e.g., Sigma-Aldrich, catalog number: P4333), store at -20 °C
9. Pipette controller (e.g., accu-jet pro, Brand, catalog number: 263 00)
10. Polypropylene tubes, 15 mL capacity (e.g., Falcon tubes, Sarstedt, catalog numbers: 62.554.502 and 62.547.254)
11. Sterile serological pipettes (e.g., Serological pipettes of 5, 10, and 25 mL; Sarstedt, catalog numbers: 86.1253.001, 86.1254.001, and 86.1685.001)
12. Sterile culture vessels T-75 flasks (e.g., Sarstedt, catalog number: 83.3911)
13. Trypsin-EDTA solution (e.g., Sigma-Aldrich, catalog number: T3924), store at -20 °C
14. Trypan Blue solution, 0.4% (e.g., Thermo Fischer Scientific, catalog number: 15250061)
15. Growth medium (see Recipe 1)
For annexin V labeling
1. Annexin V conjugated to Alexa Fluor 568 (e.g., Roche, catalog number: A13202), store at 4 °C
2. 4’,6-Diamidino-2-phenyl-indol-dihydrochlorid (DAPI) (e.g., Sigma-Aldrich, catalog number: D9542)
3. Dead cell staining reagents, e.g., SYTOX Blue (Thermo Scientific, catalog number: S34857)
4. Ice
5. Tyrode’s balanced salt solution with Ca²⁺ (TBSS + Ca²⁺; see Recipe 2), store at 4 °C
6. Tyrode’s balanced salt solution without Ca²⁺ (TBSS - Ca²⁺; see Recipe 3), store at 4 °C
7. DAPI stock solution (1 mg/mL) (see Recipe 4)
  Note: This procedure has also been successfully performed using FITC-labeled lactadherin (e.g., Haematologic Technologies, catalog number: BLAC-FITC).
For the preparation of giant unilamellar vesicles (GUVs)
1. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti^® Polar Lipids, catalog number: 850375)
2. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti^® Polar Lipids, catalog number: 850725)
3. 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) (Avanti^® Polar Lipids, catalog number: 840035)
4. Calcium chloride (CaCl₂) (Grüssing, catalog number: 10043-52-4)
5. Chloroform, ethanol-stabilized and certified for absence of HCl (Sigma-Aldrich, catalog number: 32211-M)
6. Detergent/soap
7. Ethanol, 70% (Sigma-Aldrich, catalog number: 64-17-5)
8. Glucose (Duchefa Biochemie, catalog number: G0802.5000)
9. HEPES (Carl Roth, catalog number: 7365-45-9)
10. Ice
11. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: 7791-18-6)
12. Methanol ≥ 99.8% (VWR, catalog number: 67-56-1)
13. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 1310-58-3)
14. Potassium chloride (KCl) (Merck, catalog number: 7447-40-7)
15. Sodium chloride (NaCl) (Carl Roth, catalog number: 7647-14-5)
16. Sucrose (Duchefa Biochemie, catalog number: S0809.5000)
17. Lipid stock in chloroform (see Recipe 5)
18. Swelling buffer (320 mM sucrose) (see Recipe 6)
19. Binding buffer (see Recipe 7)
Note: Store or keep all reagents at room temperature, except indicated items. All buffers are prepared the day before and stored at 4 °C.

Equipment

General
1. Analytical balance (e.g., Sartorius Entris-I II, 220 g/0.1 mg; Buch Holm, catalog number: 4669128)
2. Computer with monitor (e.g., DELL U2415)
3. Confocal laser scanning microscope (e.g., Leica TCS SP8 confocal laser scanning microscope, Leitz, Wetzlar, Germany, equipped with a 63×/1.20 water objective)
4. Eppendorf Research^® plus pipettes P20, P200, P1000 (Eppendorf, catalog numbers: 3123000039, 3123000055, 3123000063)
5. Eppendorf tube, 2 mL (Merck, catalog number: EP0030120094)
6. Freezers -20 °C and -80 °C
7. Magnetic stirrer (e.g., IKAMAG^®, DREHZAHL ELECTRONIC, IKA, Staufen im Breisgau, Germany)
8. Magnets
9. pH meter (pH-Meter 761 Calimatic, Knick, Berlin, Germany)
10. Pipette tips 10, 200, 1,000 μL (Sarstedt, catalog numbers: 70.760.002, 70.3030.020, 70.3050.020)
11. Refrigerator (5 °C)
12. Water distillation system
For cell culture
1. Autoclave sterilizer (e.g., Systec VX-65, Systec, Linden, Germany)
2. Biological safety cabinet certified for handling biological materials (e.g., Herasafe KSP Class II Biological Safety Cabinets, Thermo Fisher Scientific)
3. Centrifuge with rotor for 15 and 50 mL polypropylene tubes (e.g., Eppendorf 5810 R; Wesseling, Germany)
4. Incubator with humidity and gas control to maintain 37 °C and 95% humidity in an atmosphere of 5% CO₂ in air (e.g., Binder, Tuttlingen, Germany)
5. Inverted phase contrast microscope equipped with a 10× objective (HI PLAN I 10×/0.22 PH1; Leica DMi1, Mannheim, Germany)
6. Neubauer counting chamber (improved dark lines, 0.1 mm) and cover glasses (20 mm × 26 mm × 0.4 mm)
7. Water bath (e.g., WPE45 Memmert, Schwabach, Germany) for mammalian cells and for NBD-lipid labeling (Julabo CORIO C-BT5, catalog number: 9011305)
For preparation of GUVs
1. Cover glass slides (26 mm × 76 mm, #1.5, Thermo Fisher Scientific, Life Technologies Corporation Eugene)
2. Flow cabinet to work with organic solvents
3. Glass beads, 3 mm (Merck, catalog number: 104015)
4. Glass desiccator Boro 3.3 with a socket in the lid, 20 cm, including stopcock (Brand, catalog number: 65238)
5. Glass pipettes (e.g., graduated pipettes BLAUBRAND^® Type 3 Class AS, 10 mL, graduation: 10 mL; Carl Roth, catalog number: HXT8.1)
6. Glass slide (Thermo Scientific, microscope slides 76 mm × 26 mm, catalog number: MEZ 101026)
7. Glass vials (Rotilabo^® screw neck ND8 vials, brown/white glass, 1.5 mL; Carl Roth, catalog number: KE30.1) with screw caps (without a borehole, without septum, PP, black, ND8; Carl Roth, catalog number: KE39.1)
8. Glass tubes (Carl Roth, catalog number: DURAN C208.1)
9. Hamilton 700 Series syringes 25, 100, 1,000 μL (Hamilton Company, Nevada, USA)
10. High vacuum grease (DOW CORNING, 65201 Wiesbaden, made in USA, Artwork Nr. 0315)
11. Ice bucket (e.g., Magic Touch 2^TM ice bucket with lid; Sigma-Aldrich, catalog number: BAM168072002)
12. O-ring (28 mm × 1 mm, Nanion Technologies, München)
13. Parafilm (PARAFILM^® M; Sigma-Aldrich, catalog number: P7793-1EA)
14. Rotavapor^® R-100 Evaporator with I-100 Controller and V-100 vacuum pump (Flawil, Switzerland)
15. Scissors
16. Ultra-violet/ozone probe and surface decontamination unit (e.g., Novascan Technologies Inc., Boone, IA, USA)
17. Vortex mixer (e.g., Vortex Genie 2 Scientific Industries Inc., catalog number: SI-0236)
18. Vacuum Pump V-100 with Interface I-100 (Buchi, catalog numbers: 11593636 and 11593655D)
19. Wipes (Precision Wipes, KIMTECH Science, Kimberly-Clark^® Professional, catalog number: 7552)

Software

ImageJ (Wayne, Rasband, S., U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/index.html , version v.153q)
Leica Application Suite AF (LAS AF, Leitz, Wetzlar, Germany)
Microsoft Excel (Microsoft Corporation, 2018)
PowerPoint (Microsoft Corporation, 2018)
OriginPro (OriginLab, 2023)

Procedure

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如何引用

Baum, J. F., Uzun, H. D. and Pomorski, T. G. (2023). Visualizing Loss of Plasma Membrane Lipid Asymmetry Using Annexin V Staining. Bio-protocol 13(14): e4754. DOI: 10.21769/BioProtoc.4754.