- Email: [email protected]
Dynamics of sphingomyelin- and cholesterol-enriched lipid domains during cytokinesis
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Dynamics of sphingomyelin- and cholesterol-enriched lipid domains during cytokinesis M. Abe*, T. Kobayashi*, x, 1 *RIKEN, Wako, Saitama, Japan x CNRS, Illkirch, France 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE Introduction ................................................................................................................ 2 1. Methods ................................................................................................................ 3 1.1 Preparation of Lipid Probes ...................................................................... 3 1.2 Synchronizing HeLa Cells......................................................................... 5 1.3 Staining Cells With Lipid Probes............................................................... 5 Conclusion ................................................................................................................. 7 Acknowledgment......................................................................................................... 8 References ................................................................................................................. 8
Abstract Sphingomyelin (SM) and cholesterol (Chol) are the major lipids in the mammalian cells, which are mainly localized to the plasma membrane. Multiple lines of evidence suggest that these lipids form local lipid domains in the plasma membrane, playing functional roles in the cell. Several observations have suggested that these lipid domains are required for cytokinesis. In this chapter, we show the methods for visualizing SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins. Lysenin, equinatoxin II, perfringolysin O, and pleurotolysin A2 bind specifically to clustered SM-rich domain, dispersed SM-rich domain, Chol-rich domain, and SM/ Chol mixtures, respectively. Nontoxic forms of these lipid-binding proteins fused to fluorescent proteins are used for imaging lipid domains in biological membranes at cytokinesis. The image analysis reveals the structures and functions of SM-rich and/or Chol-rich domains at the time of cytokinesis. Methods in Cell Biology, Volume 137, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2016.03.030 © 2017 Elsevier Inc. All rights reserved.
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INTRODUCTION Sphingomyelin (SM) is one of the major lipids in the mammalian plasma membrane, accounting for 2e15% of the total phospholipids (Koval & Pagano, 1991). SM has been shown to form local clusters under particular conditions in model membranes (Pandit, Jakobsson, & Scott, 2004; Pandit et al., 2004). One attractive model of the presence of SM-rich domains in biological membranes is the “lipid raft” concept (Simons & Ikonen, 1997). Lipid rafts have been suggested to contain certain characteristic lipids, including SM, glycosphingolipids, and cholesterol (Chol), playing important roles in signal transduction, membrane trafficking, cytoskeleton, and pathogen entry (Pike, 2006). Since most of the evidence for lipid raft relied on indirect methods such as detergent extraction, structure, function, and even existence of raft have been under debate. Imaging the lipid components of the lipid raft may provide conclusive evidence showing the structures and functions. Several observations have suggested that lipid rafts are involved in cytokinesis (Atilla-Gokcumen et al., 2014; Gudejko, Alford, & Burgess, 2012; Ng, Chang, & Burgess, 2005). Depletion of cholesterol or inhibition of its synthesis impairs cytokinesis (Feng, Schwarz, & Jesuthasan, 2002; Fernandez, Lobo Md Mdel, Gomez-Coronado, & Lasuncion, 2004). Depletion of SM disperses PIP2 and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis (Abe et al., 2012). Together with these observations, imaging the lipid components of lipid raft supports the idea that lipid raft is required for cytokinesis in mammalian cells (Abe et al., 2012; Makino et al., 2015; Ng et al., 2005). In this chapter, we focus on SM-rich and/or Chol-rich binding proteins to observe these lipid domains in biological membrane at cytokinesis. As SM-rich and/or Chol-rich binding proteins, we show here lysenin (Lys), equinatoxin II (EqtII), perfringolysin O (PFO), and pleurotolysin A2 (PlyA2). Lys is a 297 amino acid protein, which was originally isolated from the coelomic fluid of the earthworm Eisenia foetida (Sekizawa, Kubo, Kobayashi, Nakajima, & Natori, 1997). Lys specifically binds to SM (Bruhn, Winkelmann, Andersen, Andra, & Leippe, 2006; Yamaji et al., 1998), especially clustered SM composed of fewer than 10 SM molecules (Makino et al., 2015). Lys has a cytolytic activity to vertebrate spermatozoa, amphibian larvae, and cultured mammalian cells (Hanada et al., 1998; Kobayashi, Sekizawa, Aizu, & Umeda, 2000; Kobayashi, Suzuki, & Ohta, 2006; Yamaji et al., 1998). Due to the cytolytic activity, full-length Lys is not suitable for use in living cells as an SM-specific probe. Study with several truncated Lys indicates that the fragment 161e297 amino acid (NT-Lys) has no pore-forming activity and retains its binding specificity to SM (Kiyokawa et al., 2005). The EqtII is a member of the actinoporins and was isolated from the sea anemone, Actinia equina. The EqtII belongs to a family of pore-forming toxins showing hemolytic activity to mammalian cells. Biochemical assays showed that EqtII specifically binds SM (Bakrac et al., 2008), especially dispersed SM rather than clustered SM (Makino et al., 2015). Pore formation by EqtII is multiple
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1. Methods
processes, which include binding to SM, intramolecular conformational change, oligomerization on the membrane, and formation of the pore. Inhibition of intramolecular conformational change by disulfide bridge formation in the mutant EqtII(8e69) prevents the hemolytic activity without perturbing SM-binding activity (Hong et al., 2002; Kristan et al., 2004; Rojko et al., 2013). PFO, also referred as q toxin, secreted from Clostridium perfringens binds to Chol-rich domains both in model and biological membranes (Verherstraeten et al., 2015). PFO exhibits a pore-forming activity in a Chol-dependent manner and causes cytolytic activity to mammalian membranes (Heuck, Savva, Holzenburg, & Johnson, 2007; Hotze & Tweten, 2012). Study with several truncated fragments of PFO indicates C-terminal domain (D4) of PFO is the smallest functional unit that retains the Chol-binding ability without cytolytic activity (Shimada, Maruya, Iwashita, & Ohno-Iwashita, 2002). Recently, we identified a novel protein, which binds to SM and Chol mixtures (Bhat et al., 2013). We screened for proteins binding specifically to SM/Chol liposomes from edible mushroom Pleurotus eryngii. Biochemical analyses indicate that PlyA2, a pleurotolysin ortholog from P. eryngii, binds to SM/Chol mixtures but not to phosphatidylcholine/Chol mixtures in model membranes. PlyA2 also binds ceramide phosphoethanolamine, a quite minor sphingolipid in mammalian cells (Bhat et al., 2015). PlyA2 does not have cytolytic activity to mammalian cells. Using these lipid-binding proteins, SM-rich and/or Chol-rich domains in the outer leaflet of the plasma membrane have been visualized under a confocal microscope. First, the lipid-binding proteins fused to fluorescent proteins are expressed in and purified from Escherichia coli. Subsequently, lipid domains in the outer leaflet of the plasma membrane in living mammalian cells are stained by exogenously added nontoxic recombinant proteins. Here we show the methods for visualizing SM-enriched and/or Chol-enriched domains in cell membrane.
1. METHODS 1.1 PREPARATION OF LIPID PROBES To prepare the lipid probes, the lipid-binding proteins fused to fluorescent proteins are produced in E. coli. Since all fusion proteins are tagged with poly histidine, the recombinant proteins can be purified with Ni2þ or Co2þ columns. In the case of EqtII(8e69)-EGFP, the recombinant protein is oxidized after the purification to prevent its cytolytic activity. 1. Transform the plasmids expressing lipid probes into E. coli strain BL-21 (DE3). 2. Grow the bacterial culture in 250 mL lysogeny broth (LB) medium with 50 mg/mL kanamycin until an optical density (600 nm) reaches 0.2 at 30 C. 3. Add 250 mL of LB containing 2M sorbitol, 50 mg/mL kanamycin, 5 mM betaine hydrochloride, and 1 mM isopropyl-D-thiogalactopyranoside, and further incubate overnight at 30 C. Osmotic stress in the presence of sorbitol
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and betaine has been shown to increase the yield of recombinant proteins (Blackwell & Horgan, 1991). 4. Harvest the bacterial cells by centrifugation at 5000 g for 10 min at 4 C. The pellet can be stored at 80 C. 5. Suspend the pellet in binding buffer consisting of 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl, 20 mM imidazole, and protease inhibitors. 6. Disrupt the cells by sonication on ice. We use a Misonix XL2020 sonicator with a 1/800 probe (30 5-s pulses, a 10-s incubation on ice in between, 40% full power). 7. Remove insoluble protein and cell debris by centrifugation at 9000 g at 4 C for 20 min. Transfer the supernatant carefully, to avoid disturbing the pellet, to a fresh tube. 8. For purification of recombinant proteins, use Ni2þ (GE Healthcare, UK, No. 29048631) or Co2þ columns (GE Healthcare, UK, No. 29048565). Elute recombinant proteins from columns by elution buffer consisting of 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl and 500 mM imidazole. 9. Suspend the obtained fractions in 15 mL of PBS and concentrate them by using an Amicon Ultra-15 Ultracel-30K centrifugal unit. 10. In the case of EqtII(8e69), the fusion protein should be oxidized to form a disulfide bridge between Cys8 and Cys69, preventing the formation of pores (Hong et al., 2002; Kristan et al., 2004). Incubate the EqtII(8e69) fusion protein in 0.5 mM 1,10-phenantroline and 0.1 mM CuSO4 for 1 h at 30 C. Suspend the resulting fractions in 15 mL of PBS and concentrate them by using an Amicon Ultra-15 Ultracel-30K centrifugal unit. 11. Add 30% of glycerol, and store at 20 C. Notes • For step 9, stepwise gradient elution can highly purify the recombinant proteins with low yield. However, we give priority to yield over purity since extracts from E. coli hardly influence the binding ability of the lipidbinding proteins. • Here we describe lipid-binding proteins fused to fluorescent proteins. Lipidbinding proteins can also be labeled with a fluorescent dye such as Alexa Fluor 647 using labeling kit (Thermo Fisher Scientific, MA, No. A20173) (Abe et al., 2012). Materials Plasmids for expressing lipid-binding proteins fused to EGFP (pET28/HisEGFP-NT-Lys, pET28/EqtII(8e69)-EGFP-His, pET28/His-EGFP-D4 and pET28/PlyA2-EGFP-His) can be obtained from RIKEN BRC (http://dna. brc.riken.jp/). Ni2þ or Co2þ columns. We use HisTrap FF crude (GE Healthcare, UK, No. 29048631), HiTrap TALON crude (GE Healthcare, UK, No. 29048565), or Ni-NTA superflow columns (Qiagen, Germany, No. 30622).
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Amicon Ultra-15 Ultracel-30K centrifugal unit (Merck Millipore, Germany, No. UFC903008). Sonicator. Any sonicators for disrupting cells can be used. We use a Misonix XL2020 sonicator with a 1/800 probe (Misonix, NY).
1.2 SYNCHRONIZING HeLa CELLS To enrich HeLa cells at the time of cytokinesis, mitotic cells are collected and released from G2/M arrest. 1. Grow HeLa cells in a T-25 flask at 37 C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 2. For synchronizing HeLa cells, incubate HeLa cells in the presence of 40 ng/mL nocodazole for 3 h, and harvest mitotic cells by mechanical shake-off. 3. Plate the harvested cells in a poly-D-lysine-coated dish, and incubate in the presence of 40 ng/mL nocodazole for additional 30 min. 4. Wash nocodazole out, and the cells are incubated in DMEM supplemented with 10% FBS for 1 h to release the cells from the mitotic arrest. Notes • During the mechanical shake-off, the efficiency should be monitored under a light microscope. • Longer incubation in the presence of nocodazole may take longer to release the cells from mitosis in the fresh medium. Materials T-25 flask (Thermo Fisher Scientific, MA or Sigma-Aldrich, MO). Poly-D-lysine-coated dish (Corning, NY, No. 354077).
1.3 STAINING CELLS WITH LIPID PROBES 1. Replace the medium to DMEM supplemented with 10% lipoprotein deficient serum (LPDS). 2. Stain the cells with 10 mg/mL of the recombinant proteins. 3. Incubate the cells at 37 C for 10 min. 4. Observe the living cells under a confocal microscope (typical images are shown in Fig. 1) without washing. Notes • Since lipid-binding proteins bind lipoproteins, it is better to use LPDS instead of FBS. Due to this, when FBS is used, a high concentration of lipid-binding protein is needed. Cells do not divide in the absence of serum. Either LPDS or FBS is required for progression of cytokinesis. • If you need to fix cells, cells are incubated with 4% paraformaldehyde and 0.2% glutaraldehyde for 30 min after step 3, because chemical fixation does not immobilize lipid molecules (Tanaka et al., 2010). Sometimes fixation before labeling gives different signals compared with living cell staining.
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FIGURE 1 Typical images of sphingomyelin (SM)-rich and/or cholesterol (Chol)-rich domains in the cleavage furrow. (A) Selective accumulation of SM clusters to the midbody. HeLa cells were synchronized, and cells were doubly labeled with EGFP-NT-Lys (green (light gray in print versions)) and EqtII(8e69)-mKate (red (gray in print versions)). The arrow indicates the midbody. (B) Chol-rich domains and SM/Chol domains in the outer leaflet of the plasma membrane at the time of cytokinesis. Synchronized HeLa cells were doubly labeled with PlyA2-EGFP (green (light gray in print versions)) and mKate-D4 (red (gray in print versions)). (C) Chol-rich domains in the outer but not the inner leaflet of the plasma membrane accumulated in the cleavage furrow. HeLa cells transiently expressing mCherry-D4 (red (gray in print versions)) were stained with exogenous recombinant EGFP-D4 (green (light gray in print versions)). Bars, 10 mm.
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Conclusion
FIGURE 2 Fluorescence intensity of EGFP-NT-Lys and a nonspecific membrane probe. (A) Clustered sphingomyelin labeled with NT-Lys. HeLa cells were stained with EGFP-NT-Lys (green (light gray in print versions)) and DiIC18 (red (gray in print versions)). Bar, 5 mm. (B) Quantitative analysis of fluorescence intensity. Fluorescence intensity of EGFP-NT-Lys (green (light gray in print versions)) and DiIC18 (red (gray in print versions)) was measured along the white line shown in (A).
•
For staining of the Chol-rich domains at the inner leaflet of the plasma membrane, the labeled D4 is transiently expressed in the cells after plasmid transfection (Fig. 1C). It should be noted that artificial aggregates of the labeled D4 are seen in the cells when the labeled D4 is expressed. • To exclude the possibility that the increased fluorescence intensity is due to the closely apposed membranes in the furrow region, it is important to compare the lipid-binding protein staining with that of a nonspecific membrane probe, such as 1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiIC18) as shown in Fig. 2. Materials LPDS (Sigma-Aldrich, MO, No. S5394). A confocal microscope equipped with an environmental chamber maintained with humidity, 37 C, and 5% CO2. We use an FV 1000 confocal microscope with a 60 1.1 NA PlanApo objective lens (Olympus, Japan).
CONCLUSION Here we presented methods for visualizing endogenous SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins. Imaging
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SM-rich and/or Chol-rich membrane domains has provided evidence showing their structures and functions at the time of cytokinesis. The combined use of lipid-binding proteins and several fluorescent proteins and/or small molecules coupled with advanced microscopy will ultimately reveal accurate dynamics and organization of SM-rich and/or Chol-rich domains in cell membranes.
ACKNOWLEDGMENT The work of authors’ laboratory was supported by the Integrated Lipidology Program of RIKEN, and the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25293015 and 15K07957) and Mizutani Foundation.
REFERENCES Abe, M., Makino, A., Hullin-Matsuda, F., Kamijo, K., Ohno-Iwashita, Y., Hanada, K., … Kobayashi, T. (2012). A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol-4,5-bisphosphate to the cleavage furrow during cytokinesis. Molecular and Cellular Biology, 32, 1396e1407. Atilla-Gokcumen, G. E., Muro, E., Relat-Goberna, J., Sasse, S., Bedigian, A., Coughlin, M. L., … Eggert, U. S. (2014). Dividing cells regulate their lipid composition and localization. Cell, 156, 428e439. Bakrac, B., Gutierrez-Aguirre, I., Podlesek, Z., Sonnen, A. F., Gilbert, R. J., Macek, P., … Anderluh, G. (2008). Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin. The Journal of Biological Chemistry, 283, 18665e18677. Bhat, H. B., Ishitsuka, R., Inaba, T., Murate, M., Abe, M., Makino, A., … Kobayashi, T. (2015). Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 29, 3920e3934. Bhat, H. B., Kishimoto, T., Abe, M., Makino, A., Inaba, T., Murate, M., … Kobayashi, T. (2013). Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains. Journal of Lipid Research, 54, 2933e2943. Blackwell, J. R., & Horgan, R. (1991). A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Letters, 295, 10e12. Bruhn, H., Winkelmann, J., Andersen, C., Andra, J., & Leippe, M. (2006). Dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defence protein of the annelid Eisenia fetida. Developmental and Comparative Immunology, 30, 597e606. Feng, B., Schwarz, H., & Jesuthasan, S. (2002). Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres. Experimental Cell Research, 279, 14e20. Fernandez, C., Lobo Md Mdel, V., Gomez-Coronado, D., & Lasuncion, M. A. (2004). Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation. Experimental Cell Research, 300, 109e120. Gudejko, H. F., Alford, L. M., & Burgess, D. R. (2012). Polar expansion during cytokinesis. Cytoskeleton (Hoboken, NJ), 69, 1000e1009.
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Hanada, K., Hara, T., Fukasawa, M., Yamaji, A., Umeda, M., & Nishijima, M. (1998). Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase. The Journal of Biological Chemistry, 273, 33787e33794. Heuck, A. P., Savva, C. G., Holzenburg, A., & Johnson, A. E. (2007). Conformational changes that effect oligomerization and initiate pore formation are triggered throughout perfringolysin O upon binding to cholesterol. The Journal of Biological Chemistry, 282, 22629e22637. Hong, Q., Gutierrez-Aguirre, I., Barlic, A., Malovrh, P., Kristan, K., Podlesek, Z., … Anderluh, G. (2002). Two-step membrane binding by Equinatoxin II, a pore-forming toxin from the sea anemone, involves an exposed aromatic cluster and a flexible helix. The Journal of Biological Chemistry, 277, 41916e41924. Hotze, E. M., & Tweten, R. K. (2012). Membrane assembly of the cholesterol-dependent cytolysin pore complex. Biochimica et Biophysica Acta, 1818, 1028e1038. Kiyokawa, E., Baba, T., Otsuka, N., Makino, A., Ohno, S., & Kobayashi, T. (2005). Spatial and functional heterogeneity of sphingolipid-rich membrane domains. The Journal of Biological Chemistry, 280, 24072e24084. Kobayashi, H., Sekizawa, Y., Aizu, M., & Umeda, M. (2000). Lethal and non-lethal responses of spermatozoa from a wide variety of vertebrates and invertebrates to lysenin, a protein from the coelomic fluid of the earthworm Eisenia foetida. Journal of Experimental Zoology, 286, 538e549. Kobayashi, H., Suzuki, H., & Ohta, N. (2006). Exfoliation of the epidermal cells and defecation by amphibian larvae in response to coelomic fluid and lysenin from the earthworm Eisenia foetida. Biomedical Research, 27, 169e181. Koval, M., & Pagano, R. E. (1991). Intracellular transport and metabolism of sphingomyelin. Biochimica et Biophysica Acta, 1082, 113e125. Kristan, K., Podlesek, Z., Hojnik, V., Gutierrez-Aguirre, I., Guncar, G., Turk, D., … Anderluh, G. (2004). Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich. The Journal of Biological Chemistry, 279, 46509e46517. Makino, A., Abe, M., Murate, M., Inaba, T., Yilmaz, N., Hullin-Matsuda, F., … Kobayashi, T. (2015). Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 29, 477e493. Ng, M. M., Chang, F., & Burgess, D. R. (2005). Movement of membrane domains and requirement of membrane signaling molecules for cytokinesis. Developmental Cell, 9, 781e790. Pandit, S. A., Jakobsson, E., & Scott, H. L. (2004). Simulation of the early stages of nano-domain formation in mixed bilayers of sphingomyelin, cholesterol, and dioleoylphosphatidylcholine. Biophysical Journal, 87, 3312e3322. Pandit, S. A., Vasudevan, S., Chiu, S. W., Mashl, R. J., Jakobsson, E., & Scott, H. L. (2004). Sphingomyelin-cholesterol domains in phospholipid membranes: atomistic simulation. Biophysical Journal, 87, 1092e1100. Pike, L. J. (2006). Rafts defined: a report on the keystone symposium on lipid rafts and cell function. Journal of Lipid Research, 47, 1597e1598.
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Rojko, N., Kristan, K. C., Viero, G., Zerovnik, E., Macek, P., Dalla Serra, M., & Anderluh, G. (2013). Membrane damage by an alpha-helical pore forming protein equinatoxin II proceeds through succesion of ordered steps. The Journal of Biological Chemistry, 288(33). Sekizawa, Y., Kubo, T., Kobayashi, H., Nakajima, T., & Natori, S. (1997). Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle. Gene, 191, 97e102. Shimada, Y., Maruya, M., Iwashita, S., & Ohno-Iwashita, Y. (2002). The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains. European Journal of Biochemistry, 269, 6195e6203. Simons, K., & Ikonen, E. (1997). Functional rafts in cell membranes. Nature, 387, 569e572. Tanaka, K. A., Suzuki, K. G., Shirai, Y. M., Shibutani, S. T., Miyahara, M. S., Tsuboi, H., … Kusumi, A. (2010). Membrane molecules mobile even after chemical fixation. Nature Methods, 7, 865e866. Verherstraeten, S., Goossens, E., Valgaeren, B., Pardon, B., Timbermont, L., Haesebrouck, F., … Van Immerseel, F. (2015). Perfringolysin O: the underrated Clostridium perfringens toxin? Toxins (Basel), 7, 1702e1721. Yamaji, A., Sekizawa, Y., Emoto, K., Sakuraba, H., Inoue, K., Kobayashi, H., & Umeda, M. (1998). Lysenin, a novel sphingomyelin-specific binding protein. The Journal of Biological Chemistry, 273, 5300e5306.
Dynamics of sphingomyelin- and cholesterol-enriched lipid domains during cytokinesis M. Abe*, T. Kobayashi*, x, 1 *RIKEN, Wako, Saitama, Japan x CNRS, Illkirch, France 1
Corresponding author: E-mail: [email protected]
CHAPTER OUTLINE Introduction ................................................................................................................ 2 1. Methods ................................................................................................................ 3 1.1 Preparation of Lipid Probes ...................................................................... 3 1.2 Synchronizing HeLa Cells......................................................................... 5 1.3 Staining Cells With Lipid Probes............................................................... 5 Conclusion ................................................................................................................. 7 Acknowledgment......................................................................................................... 8 References ................................................................................................................. 8
Abstract Sphingomyelin (SM) and cholesterol (Chol) are the major lipids in the mammalian cells, which are mainly localized to the plasma membrane. Multiple lines of evidence suggest that these lipids form local lipid domains in the plasma membrane, playing functional roles in the cell. Several observations have suggested that these lipid domains are required for cytokinesis. In this chapter, we show the methods for visualizing SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins. Lysenin, equinatoxin II, perfringolysin O, and pleurotolysin A2 bind specifically to clustered SM-rich domain, dispersed SM-rich domain, Chol-rich domain, and SM/ Chol mixtures, respectively. Nontoxic forms of these lipid-binding proteins fused to fluorescent proteins are used for imaging lipid domains in biological membranes at cytokinesis. The image analysis reveals the structures and functions of SM-rich and/or Chol-rich domains at the time of cytokinesis. Methods in Cell Biology, Volume 137, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2016.03.030 © 2017 Elsevier Inc. All rights reserved.
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Dynamics of sphingomyelin- and cholesterol-enriched lipid domains
INTRODUCTION Sphingomyelin (SM) is one of the major lipids in the mammalian plasma membrane, accounting for 2e15% of the total phospholipids (Koval & Pagano, 1991). SM has been shown to form local clusters under particular conditions in model membranes (Pandit, Jakobsson, & Scott, 2004; Pandit et al., 2004). One attractive model of the presence of SM-rich domains in biological membranes is the “lipid raft” concept (Simons & Ikonen, 1997). Lipid rafts have been suggested to contain certain characteristic lipids, including SM, glycosphingolipids, and cholesterol (Chol), playing important roles in signal transduction, membrane trafficking, cytoskeleton, and pathogen entry (Pike, 2006). Since most of the evidence for lipid raft relied on indirect methods such as detergent extraction, structure, function, and even existence of raft have been under debate. Imaging the lipid components of the lipid raft may provide conclusive evidence showing the structures and functions. Several observations have suggested that lipid rafts are involved in cytokinesis (Atilla-Gokcumen et al., 2014; Gudejko, Alford, & Burgess, 2012; Ng, Chang, & Burgess, 2005). Depletion of cholesterol or inhibition of its synthesis impairs cytokinesis (Feng, Schwarz, & Jesuthasan, 2002; Fernandez, Lobo Md Mdel, Gomez-Coronado, & Lasuncion, 2004). Depletion of SM disperses PIP2 and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis (Abe et al., 2012). Together with these observations, imaging the lipid components of lipid raft supports the idea that lipid raft is required for cytokinesis in mammalian cells (Abe et al., 2012; Makino et al., 2015; Ng et al., 2005). In this chapter, we focus on SM-rich and/or Chol-rich binding proteins to observe these lipid domains in biological membrane at cytokinesis. As SM-rich and/or Chol-rich binding proteins, we show here lysenin (Lys), equinatoxin II (EqtII), perfringolysin O (PFO), and pleurotolysin A2 (PlyA2). Lys is a 297 amino acid protein, which was originally isolated from the coelomic fluid of the earthworm Eisenia foetida (Sekizawa, Kubo, Kobayashi, Nakajima, & Natori, 1997). Lys specifically binds to SM (Bruhn, Winkelmann, Andersen, Andra, & Leippe, 2006; Yamaji et al., 1998), especially clustered SM composed of fewer than 10 SM molecules (Makino et al., 2015). Lys has a cytolytic activity to vertebrate spermatozoa, amphibian larvae, and cultured mammalian cells (Hanada et al., 1998; Kobayashi, Sekizawa, Aizu, & Umeda, 2000; Kobayashi, Suzuki, & Ohta, 2006; Yamaji et al., 1998). Due to the cytolytic activity, full-length Lys is not suitable for use in living cells as an SM-specific probe. Study with several truncated Lys indicates that the fragment 161e297 amino acid (NT-Lys) has no pore-forming activity and retains its binding specificity to SM (Kiyokawa et al., 2005). The EqtII is a member of the actinoporins and was isolated from the sea anemone, Actinia equina. The EqtII belongs to a family of pore-forming toxins showing hemolytic activity to mammalian cells. Biochemical assays showed that EqtII specifically binds SM (Bakrac et al., 2008), especially dispersed SM rather than clustered SM (Makino et al., 2015). Pore formation by EqtII is multiple
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1. Methods
processes, which include binding to SM, intramolecular conformational change, oligomerization on the membrane, and formation of the pore. Inhibition of intramolecular conformational change by disulfide bridge formation in the mutant EqtII(8e69) prevents the hemolytic activity without perturbing SM-binding activity (Hong et al., 2002; Kristan et al., 2004; Rojko et al., 2013). PFO, also referred as q toxin, secreted from Clostridium perfringens binds to Chol-rich domains both in model and biological membranes (Verherstraeten et al., 2015). PFO exhibits a pore-forming activity in a Chol-dependent manner and causes cytolytic activity to mammalian membranes (Heuck, Savva, Holzenburg, & Johnson, 2007; Hotze & Tweten, 2012). Study with several truncated fragments of PFO indicates C-terminal domain (D4) of PFO is the smallest functional unit that retains the Chol-binding ability without cytolytic activity (Shimada, Maruya, Iwashita, & Ohno-Iwashita, 2002). Recently, we identified a novel protein, which binds to SM and Chol mixtures (Bhat et al., 2013). We screened for proteins binding specifically to SM/Chol liposomes from edible mushroom Pleurotus eryngii. Biochemical analyses indicate that PlyA2, a pleurotolysin ortholog from P. eryngii, binds to SM/Chol mixtures but not to phosphatidylcholine/Chol mixtures in model membranes. PlyA2 also binds ceramide phosphoethanolamine, a quite minor sphingolipid in mammalian cells (Bhat et al., 2015). PlyA2 does not have cytolytic activity to mammalian cells. Using these lipid-binding proteins, SM-rich and/or Chol-rich domains in the outer leaflet of the plasma membrane have been visualized under a confocal microscope. First, the lipid-binding proteins fused to fluorescent proteins are expressed in and purified from Escherichia coli. Subsequently, lipid domains in the outer leaflet of the plasma membrane in living mammalian cells are stained by exogenously added nontoxic recombinant proteins. Here we show the methods for visualizing SM-enriched and/or Chol-enriched domains in cell membrane.
1. METHODS 1.1 PREPARATION OF LIPID PROBES To prepare the lipid probes, the lipid-binding proteins fused to fluorescent proteins are produced in E. coli. Since all fusion proteins are tagged with poly histidine, the recombinant proteins can be purified with Ni2þ or Co2þ columns. In the case of EqtII(8e69)-EGFP, the recombinant protein is oxidized after the purification to prevent its cytolytic activity. 1. Transform the plasmids expressing lipid probes into E. coli strain BL-21 (DE3). 2. Grow the bacterial culture in 250 mL lysogeny broth (LB) medium with 50 mg/mL kanamycin until an optical density (600 nm) reaches 0.2 at 30 C. 3. Add 250 mL of LB containing 2M sorbitol, 50 mg/mL kanamycin, 5 mM betaine hydrochloride, and 1 mM isopropyl-D-thiogalactopyranoside, and further incubate overnight at 30 C. Osmotic stress in the presence of sorbitol
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and betaine has been shown to increase the yield of recombinant proteins (Blackwell & Horgan, 1991). 4. Harvest the bacterial cells by centrifugation at 5000 g for 10 min at 4 C. The pellet can be stored at 80 C. 5. Suspend the pellet in binding buffer consisting of 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl, 20 mM imidazole, and protease inhibitors. 6. Disrupt the cells by sonication on ice. We use a Misonix XL2020 sonicator with a 1/800 probe (30 5-s pulses, a 10-s incubation on ice in between, 40% full power). 7. Remove insoluble protein and cell debris by centrifugation at 9000 g at 4 C for 20 min. Transfer the supernatant carefully, to avoid disturbing the pellet, to a fresh tube. 8. For purification of recombinant proteins, use Ni2þ (GE Healthcare, UK, No. 29048631) or Co2þ columns (GE Healthcare, UK, No. 29048565). Elute recombinant proteins from columns by elution buffer consisting of 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl and 500 mM imidazole. 9. Suspend the obtained fractions in 15 mL of PBS and concentrate them by using an Amicon Ultra-15 Ultracel-30K centrifugal unit. 10. In the case of EqtII(8e69), the fusion protein should be oxidized to form a disulfide bridge between Cys8 and Cys69, preventing the formation of pores (Hong et al., 2002; Kristan et al., 2004). Incubate the EqtII(8e69) fusion protein in 0.5 mM 1,10-phenantroline and 0.1 mM CuSO4 for 1 h at 30 C. Suspend the resulting fractions in 15 mL of PBS and concentrate them by using an Amicon Ultra-15 Ultracel-30K centrifugal unit. 11. Add 30% of glycerol, and store at 20 C. Notes • For step 9, stepwise gradient elution can highly purify the recombinant proteins with low yield. However, we give priority to yield over purity since extracts from E. coli hardly influence the binding ability of the lipidbinding proteins. • Here we describe lipid-binding proteins fused to fluorescent proteins. Lipidbinding proteins can also be labeled with a fluorescent dye such as Alexa Fluor 647 using labeling kit (Thermo Fisher Scientific, MA, No. A20173) (Abe et al., 2012). Materials Plasmids for expressing lipid-binding proteins fused to EGFP (pET28/HisEGFP-NT-Lys, pET28/EqtII(8e69)-EGFP-His, pET28/His-EGFP-D4 and pET28/PlyA2-EGFP-His) can be obtained from RIKEN BRC (http://dna. brc.riken.jp/). Ni2þ or Co2þ columns. We use HisTrap FF crude (GE Healthcare, UK, No. 29048631), HiTrap TALON crude (GE Healthcare, UK, No. 29048565), or Ni-NTA superflow columns (Qiagen, Germany, No. 30622).
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1. Methods
Amicon Ultra-15 Ultracel-30K centrifugal unit (Merck Millipore, Germany, No. UFC903008). Sonicator. Any sonicators for disrupting cells can be used. We use a Misonix XL2020 sonicator with a 1/800 probe (Misonix, NY).
1.2 SYNCHRONIZING HeLa CELLS To enrich HeLa cells at the time of cytokinesis, mitotic cells are collected and released from G2/M arrest. 1. Grow HeLa cells in a T-25 flask at 37 C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 2. For synchronizing HeLa cells, incubate HeLa cells in the presence of 40 ng/mL nocodazole for 3 h, and harvest mitotic cells by mechanical shake-off. 3. Plate the harvested cells in a poly-D-lysine-coated dish, and incubate in the presence of 40 ng/mL nocodazole for additional 30 min. 4. Wash nocodazole out, and the cells are incubated in DMEM supplemented with 10% FBS for 1 h to release the cells from the mitotic arrest. Notes • During the mechanical shake-off, the efficiency should be monitored under a light microscope. • Longer incubation in the presence of nocodazole may take longer to release the cells from mitosis in the fresh medium. Materials T-25 flask (Thermo Fisher Scientific, MA or Sigma-Aldrich, MO). Poly-D-lysine-coated dish (Corning, NY, No. 354077).
1.3 STAINING CELLS WITH LIPID PROBES 1. Replace the medium to DMEM supplemented with 10% lipoprotein deficient serum (LPDS). 2. Stain the cells with 10 mg/mL of the recombinant proteins. 3. Incubate the cells at 37 C for 10 min. 4. Observe the living cells under a confocal microscope (typical images are shown in Fig. 1) without washing. Notes • Since lipid-binding proteins bind lipoproteins, it is better to use LPDS instead of FBS. Due to this, when FBS is used, a high concentration of lipid-binding protein is needed. Cells do not divide in the absence of serum. Either LPDS or FBS is required for progression of cytokinesis. • If you need to fix cells, cells are incubated with 4% paraformaldehyde and 0.2% glutaraldehyde for 30 min after step 3, because chemical fixation does not immobilize lipid molecules (Tanaka et al., 2010). Sometimes fixation before labeling gives different signals compared with living cell staining.
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FIGURE 1 Typical images of sphingomyelin (SM)-rich and/or cholesterol (Chol)-rich domains in the cleavage furrow. (A) Selective accumulation of SM clusters to the midbody. HeLa cells were synchronized, and cells were doubly labeled with EGFP-NT-Lys (green (light gray in print versions)) and EqtII(8e69)-mKate (red (gray in print versions)). The arrow indicates the midbody. (B) Chol-rich domains and SM/Chol domains in the outer leaflet of the plasma membrane at the time of cytokinesis. Synchronized HeLa cells were doubly labeled with PlyA2-EGFP (green (light gray in print versions)) and mKate-D4 (red (gray in print versions)). (C) Chol-rich domains in the outer but not the inner leaflet of the plasma membrane accumulated in the cleavage furrow. HeLa cells transiently expressing mCherry-D4 (red (gray in print versions)) were stained with exogenous recombinant EGFP-D4 (green (light gray in print versions)). Bars, 10 mm.
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Conclusion
FIGURE 2 Fluorescence intensity of EGFP-NT-Lys and a nonspecific membrane probe. (A) Clustered sphingomyelin labeled with NT-Lys. HeLa cells were stained with EGFP-NT-Lys (green (light gray in print versions)) and DiIC18 (red (gray in print versions)). Bar, 5 mm. (B) Quantitative analysis of fluorescence intensity. Fluorescence intensity of EGFP-NT-Lys (green (light gray in print versions)) and DiIC18 (red (gray in print versions)) was measured along the white line shown in (A).
•
For staining of the Chol-rich domains at the inner leaflet of the plasma membrane, the labeled D4 is transiently expressed in the cells after plasmid transfection (Fig. 1C). It should be noted that artificial aggregates of the labeled D4 are seen in the cells when the labeled D4 is expressed. • To exclude the possibility that the increased fluorescence intensity is due to the closely apposed membranes in the furrow region, it is important to compare the lipid-binding protein staining with that of a nonspecific membrane probe, such as 1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiIC18) as shown in Fig. 2. Materials LPDS (Sigma-Aldrich, MO, No. S5394). A confocal microscope equipped with an environmental chamber maintained with humidity, 37 C, and 5% CO2. We use an FV 1000 confocal microscope with a 60 1.1 NA PlanApo objective lens (Olympus, Japan).
CONCLUSION Here we presented methods for visualizing endogenous SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins. Imaging
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SM-rich and/or Chol-rich membrane domains has provided evidence showing their structures and functions at the time of cytokinesis. The combined use of lipid-binding proteins and several fluorescent proteins and/or small molecules coupled with advanced microscopy will ultimately reveal accurate dynamics and organization of SM-rich and/or Chol-rich domains in cell membranes.
ACKNOWLEDGMENT The work of authors’ laboratory was supported by the Integrated Lipidology Program of RIKEN, and the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25293015 and 15K07957) and Mizutani Foundation.
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