- Email: [email protected]
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reported on the uptake of small unilamellar liposomes (mean diameter of about 80 nm) by human umbilical vein endothelial cells (HUVECs) or human promyelocytic leukemia cells (HL60). These authors found that internalization was clathrin independent, because it was not inhibited by sodium azide and deoxyglucose, but was affected by filipin. Consistent with these data, Kessner et al.20 concluded that liposomes depleted of specific ligands may be internalized into HUVECs through caveolae. In the previous section we reviewed results that indicated that widely used inhibitors of endosomal acidification do not interfere with internalization of liposomes via coated pits. Mineo and Anderson37 reported that bafilomycin A1 inhibited the uptake of 5-methyltetrahydrofolate into MA104 cells. These authors deduced that acidification can occur in plasmalemmal vesicles. Consequently, a measurement of intracellular pH by means of HPTS preencapsulated in liposomes may not necessarily constitute a proof for the residence of the liposomes in endosomes. Acknowledgments J.L.N. was supported by DGCYT (Grant PB96-0171), the Basque Government (EX-1998-28; PI-1998-32), and the University of the Basque Country (UPV 042.310-EA085/ 97; UPV 042.310-G03/98). 37
E. Mineo and R. G. W. Anderson, Exp. Cell Res. 224, 237 (1996).
[14] Assembly of Endocytosis-Associated Proteins on Liposomes By Markus R. Wenk and Pietro De Camilli Introduction
Synapses are intercellular junctions of a neuron with its target cells, and they are the sites of neurotransmission. At chemical synapses, depolarization of the synaptic plasma membrane triggers Ca2þ entry into the presynaptic nerve terminal, which leads to fusion of synaptic vesicles with the plasma membrane (exocytosis) and release of their contents into the synaptic cleft. After exocytosis, synaptic vesicle membranes are retrieved and recycled to generate new neurotransmitter-filled vesicles for a subsequent round of release. A major route of synaptic vesicle membrane retrieval involves clathrin-mediated endocytosis.1 The protein machinery underlying
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this endocytotic reaction has been characterized in detail. In addition to the major integral components of the clathrin coat, such as clathrin and the ‘‘classic’’ clathrin adaptor (AP-2), accessory factors (e.g., AP-180, amphiphysin, endophilin, eps-15, epsin, dynamin, Hip1R, intersectin, and synaptojanin) participate in coat assembly, pit formation, and vesicle fission.2–4 Cell-free reconstitution assays utilizing chemically defined liposomes (rather than biological membranes) as donor membranes, and cytosolic fractions or purified proteins as ligands, have been developed in order to identify and/or characterize the molecular requirements for vesicle biogenesis, including the biogenesis of clathrin-coated vesicles. Cell-free complete or partial reconstitutions have been reported for COPI-, COPII-, and clathrin-mediated vesicle budding,5–10 as well as for membrane tubulation reactions often observed in vivo at sites of vesicle budding.10–12 All these studies have emphasized the critical role of protein–lipid interfaces in the formation of tubulovesicular elements within the cell. Protein binding to lipids often involves both ionic interactions with the head groups of phospholipids, as well as hydrophobic interactions with the nonpolar domain of the membrane, and with specific lipids such as cholesterol and fatty acids.13 In addition, growing evidence indicates that lipids that can undergo rapid and reversible changes within the membrane play an important role in regulating the recruitment of cytosolic factors to 1
P. De Camilli, V. I. Slepnev, O. Shupliakov, and L. Brodin, in ‘‘Synapses’’ (M. W. Cowan, T. C. Sudhof, and C. F. Stevens, eds.), p. 217. Johns Hopkins University Press, Baltimore, MD, 2000. 2 M. Marsh and H. T. McMahon, Science 285, 215 (1999). 3 P. De Camilli, H. Chen, J. Hyman, E. Panepucci, A. Bateman, and A. T. Brunger, FEBS Lett. 513, 11 (2002). 4 V. I. Slepnev and P. De Camilli, Nat. Rev. 1, 161 (2000). 5 A. Spang, K. Matsuoka, S. Hamamoto, R. Schekman, and L. Orci, Proc. Natl. Acad. Sci. USA 95, 11199 (1998). 6 K. Matsuoka, L. Orci, M. Amherdt, S. Y. Bednarek, S. Hamamoto, R. Schekman, and T. Yeung, Cell 93, 263 (1998). 7 M. Bremser, W. Nickel, M. Schweikert, M. Ravazzola, M. Amherdt, C. A. Hughes, T. H. Sollner, J. E. Rothman, and F. T. Wieland, Cell 96, 495 (1999). 8 M. Kinuta, H. Yamada, T. Abe, M. Watanabe, S. A. Li, A. Kamitani, T. Yasuda, T. Matsukawa, H. Kumon, and K. Takei, Proc. Natl. Acad. Sci. USA 99, 2842 (2002). 9 M. G. Ford, B. M. Pearse, M. K. Higgins, Y. Vallis, D. J. Owen, A. Gibson, C. R. Hopkins, P. R. Evans, and H. T. McMahon, Science 291, 1051 (2001). 10 K. Takei, V. Haucke, V. Slepnev, K. Farsad, M. Salazar, H. Chen, and P. De Camilli, Cell 94, 131 (1998). 11 S. M. Sweitzer and J. E. Hinshaw, Cell 93, 1021 (1998). 12 K. Takei, V. I. Slepnev, V. Haucke, and P. De Camilli, Nat. Cell Biol. 1, 33 (1999). 13 W. B. Huttner and A. Schmidt, Curr. Opin. Neurobiol. 10, 543 (2000).
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the bilayer.14 Among such lipids, phosphoinositides (phosphorylated derivatives of phosphatidylinositol) are of critical importance and are best characterized. Rapid phosphorylation–dephosphorylation at different positions of the inositol ring by a multiplicity of enzymes generates a variety of stereoisomers, and phosphoinositides are now believed to be key players in the regulation of clathrin coat recruitment and dynamics.15,16 Although cytosolic proteins alone can generate from liposomes, membrane buds, and tubules that are similar to the different types of coated vesicle buds and tubules observed in the cell, integral membrane proteins often act as receptors for coat proteins and their accessory factors. In doing so, they contribute to the regulation of coating in time and space in the living cytoplasm. Thus, a synergistic effect of both membrane components, lipids and proteins, is central to the regulation of assembly reactions on membrane surfaces. Here we describe the methodology of a cell-free system utilizing liposomes and cytosol to study assembly reactions of proteins involved in clathrin-mediated endocytosis. Liposomes, when incubated with brain cytosol and nucleotides, undergo morphological changes and fragmentation into smaller structures that resemble closely intermediates of clathrin-mediated endocytosis8,10 (Fig. 1). Biochemical and biophysical analysis of these reactions show that elevated levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2, or PIP2] correlate with increased recruitment of clathrin and clathrin adaptors to the liposomal surface17,18 (Fig. 2). Furthermore, inclusion into the liposomes of a surface-exposed recombinant fragment of synaptotagmin enhances the recruitment of clathrin coat proteins, demonstrating the synergistic actions of proteins and lipids19 (Fig. 3). Three modes of analysis are applied: (1) observation of liposomal structure by electron microscopy, (2) protein biochemistry to assess amounts of endocytotic proteins recruited to the liposomal surface, and (3) lipid biochemistry to measure the metabolism of phosphoinositides during the incubation reaction.
14
J. H. Hurley and T. Meyer, Curr. Opin. Cell Biol. 13, 146 (2001). O. Cremona and P. De Camilli, J. Cell Sci. 114, 1041 (2001). 16 T. F. Martin, Curr. Opin. Cell Biol. 13, 493 (2001). 17 O. Cremona, G. Di Paolo, M. R. Wenk, A. Luthi, W. T. Kim, K. Takei, L. Daniell, Y. Nemoto, S. B. Shears, R. A. Flavell, D. A. McCormick, and P. De Camilli, Cell 99, 179 (1999). 18 M. R. Wenk, L. Pellegrini, V. A. Klenchin, G. Di Paolo, S. Chang, L. Daniell, M. Arioka, T. F. Martin, and P. De Camilli, Neuron 32, 79 (2001). 19 V. Haucke, M. R. Wenk, E. R. Chapman, K. Farsad, and P. De Camilli, EMBO J. 19, 6011 (2000). 15
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Fig. 1. Electron micrographs demonstrating the effect of brain cytosol on liposomes composed of brain lipids. Preparations were analyzed after plastic embedding and thin sectioning. (a and b) Control liposomes not incubated with cytosol. (c–f) Liposomes incubated with rat brain cytosol, ATP, and GTP S. High-power observation reveals the presence of dynamin-like rings and clathrin-coated profiles (d–f). Fields (e)–(h) demonstrate the similarity of dynamin-coated tubules and clathrin-coated pits observed on liposomes (e and f) and synaptic membranes (g and h) incubated with brain cytosol, ATP, and GTP S. Calibration bar: 500 nm in (a) and (c); 150 nm in (b) and (d); and 50 nm in (e)–(h). Reprinted from Cell, Vol. 94: K. Takei, V. Haucke, V. Slepnev, K. Farsad, M. Salazar, H. Chen, and P. De Camilli, Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes, pp. 131–141. Copyright (1998), with permission from Elsevier Inc.10
Reconstitution assays such as this one are a powerful tool with which to study the molecular events that are required for the formation as well as the dynamics of protein coats on membrane surfaces. We have used this integrated approach to detect alterations in phosphoinositide metabolism and clathrin coat dynamics in cytosolic fractions prepared from knockout mice.17,18,20 Furthermore, purified proteins, such as amphiphysin and dynamin,12,21 endophilin,22 or phosphatidylinositol-4-phosphate 5-kinase (PIP 20
G. Di Paolo, S. Sankaranarayanan, M. R. Wenk, L. Daniell, E. Perucco, B. J. Caldarone, R. Flavell, M. Picciotto, T. A. Ryan, O. Cremona, and P. De Camilli, Neuron 33, 789 (2002). 21 K. Takei, V. I. Slepnev, and P. De Camilli, Methods Enzymol. 329, 478 (2001). 22 K. Farsad, N. Ringstad, K. Takei, S. R. Floyd, K. Rose, and P. De Camilli, J. Cell Biol. 155, 193 (2001).
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Fig. 2. Phosphoinositide metabolism and clathrin coat recruitment to liposomes. Liposomes composed of brain lipids are incubated with brain cytosol, [ -32P]ATP, and GTP and analyzed for incorporation of phosphate into phosphoinositides (A and B) and for binding of clathrin coat proteins to the liposomal surface (C). After the reaction, phosphoinositides are extracted and deacylated, and glycerophosphoinositols are separated by HPLC. A typical chromatogram is shown in (A). (B) Quantification of PI(4,5)P2 levels in an experiment utilizing different sources of cytosol as indicated in (C) [knockout (KO) and wild-type (WT) denote cytosolic fractions from synaptojanin 1 knockout mice and control litter mates, respectively]. These cytosolic fractions were used in lanes 5 and 7 and were supplemented (lane 6) or depleted (lane 8) of PIP kinase I , the major PIP2-synthesizing enzyme in brain cytosol. Levels of PIP2 generated during the incubation of liposomes with cytosol (B) correlate nicely with the amount of clathrin coat that is recruited to the liposomes [elevated levels of clathrin heavy chain (HC) and -adaptin in lanes 6 and 7 compared with control incubations (lanes 5 and 8)]. A control protein (tubulin) does not show any significant variations between the different incubation conditions. Reprinted from Neuron, Vol. 32: M. R. Wenk, L. Pellegrini, V. A. Klenchin, G. Di Paolo, S. Chang, L. Daniell, M. Arioka, T. F. Martin, and P. De Camilli, PIP kinase I is the major PI(4,5)P2 synthesizing enzyme at the synapse, pp. 79–88. Copyright (2001), with permission from Elsevier Inc.18
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Fig. 3. Liposomes containing glutathione-linked GST fusion proteins. (A) Chemical structure of glutathione (GSH)-linked N-MPB-PE. (B) High-affinity binding of GST fusion proteins to liposomes containing GSH-PE (+) compared with control liposomes devoid of GSH-PE (–). (C) Liposomes with GSH-PE in their membranes were incubated with GST or GST-synaptotagmin I C2B (GST-C2B) proteins to allow coupling of GST fusion proteins to the liposomal membrane via GSH-PE. An aliquot (50 g of total lipid) was then analyzed by SDS–PAGE and Coomassie blue staining. (D) Liposomes containing glutathione-PE-linked GST or GST-C2B (C) were incubated with rat brain cytosol, reisolated, and washed, and bound proteins were analyzed by SDS–PAGE and immunoblotting. The presence of GSTC2B on the liposomal surface greatly facilitates clathrin/AP-2 recruitment, but has no effect on the binding of tubulin or dynamin I. Reprinted from EMBO J., Vol. 19: V. Haucke, M. R. Wenk, E. R. Chapman, K. Farsad, and P. De Camilli, Dual interaction of synaptotagmin with 2- and -adaptin facilitates clathrin-coated pit nucleation, pp. 6011–6019. By permission of Oxford University Press.19
kinase)18 can be used in this system in addition to, or instead of, cytosolic fractions. Functionalized liposomes, such as the proteoliposomes described here, are ideally suited to investigate the synergistic effect of membrane lipids and membrane proteins on coat structure and function. They are also a useful tool for future applications in the field of liposome research, such as targeting for drug delivery or the development of liposome-based affinity probes for proteins and lipids.
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Methods
Preparation of Liposomes Pure Lipid Vesicles. Large unilamellar vesicles are prepared as described23 with some modifications. A defined amount of lipid (4 mg) [bovine brain extract, type I Folch fraction I; Sigma, St. Louis, MO; stored as a 20-mg/ml stock solution in chloroform–methanol (1:2) at 20 ] is transferred to a 10-ml borosilicate glass tube and spiked with 0.05% (w/w) NBD-PC {1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]dodecanoyl]-sn-glycero-3-phosphocholine; Avanti Polar Lipids, Alabaster, AL} or, alternatively, [3H]PC (l- -dipalmitoyl-[2-palmitoyl9,10-3H(N)]-phosphatidylcholine) (PerkinElmer Life Sciences, Boston, MA) at 0.05 Ci/mg lipid. The low amounts of fluorescent or radioactive lipids are used for quantification of recovery after reisolation of the liposomes from the incubation reaction (see below). The lipid solution is then dried under a gentle stream of nitrogen. A thin and even film of lipid is formed on the glass wall by slowly (manually) rotating the glass tube during evaporation of the organic solvent. The lipid is further dried in a desiccator over phosphorous pentoxide (Sigma) and high vacuum for 2 h. The dried lipid film is then hydrated with a gentle stream of water-saturated nitrogen until it loses some of its opalescent appearance. A defined amount of 0.3 M sucrose (in water) (2 ml) is gently added to the test tube, which is flushed with nitrogen and sealed. Liposomes are allowed to form spontaneously for 2 h at 37 . The suspension is then agitated gently to resuspend the liposomes, and large aggregates and debris are removed by brief centrifugation. Thus obtained liposomes (final lipid concentration, 2 mg/ml) are mostly unilamellar spheres with diameters of >500 nm (Fig. 1a and b), which can be stored at 4 for several days. Instead of a lipid extract from a biological tissue, such as the total brain lipids described above, chemically defined components (i.e., synthetic lipids) can be used to generate these liposomes. For that aim, individual lipids are mixed in organic solvent at a defined ratio and then dried and processed as described above. The effect of the individual lipid species on protein recruitment can hence be studied.10 Lipoprotein-Containing Liposomes. Recombinant glutathione S-transferase (GST) fusion proteins can be chemically linked to a glutathione (GSH)-derivatized phosphatidylethanolamine (GSH-PE)7,19 (Fig. 3A). The latter is synthesized by mixing equal volumes (2 ml each) of [4-(p-maleimidophenyl)butyryl] phosphatidylethanolamine (MPB-PE, 5 mg/ml in 23
J. P. Reeves and R. M. Dowben, J. Cell. Physiol. 73, 49 (1969).
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CHCl3; Avanti; Polar Lipids) and glutathione [5 mM in dimethylformamide (DMF); Sigma] supplemented with 10 mM morpholinepropanesul Fonic acid (MOPS), pH 7.5. After incubation for 2 h at room temperature, free maleimido residues are blocked with 10 mM 2-mercaptoethanol, and the mixture is further incubated for 30 min at room temperature. The GSH-PE lipid is extracted by adding 4 ml of chloroform and 2 ml of methanol to the mixture, followed by vortexing and brief centrifugation to separate phases. The lower organic phase is then carefully removed, the aqueous phase is reextracted twice with 2 ml of chloroform, and the organic phases are pooled, dried, and resuspended in chloroform–methanol (2:1, v/v). Lipids (bovine brain extract; see above) and GSH-PE are mixed in chloroform at a ratio of 8:2 (w/w) and 0.05% (w/w) NBD-PE {1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphatidylethanolamine; Avanti Polar Lipids} is added as a fluorescent marker for quantification. The lipid film (5 mg) is dried under a stream of nitrogen and resuspended in 0.5 ml of 0.6 M sucrose [in phosphate-buffered saline (PBS)] by vigorously vortexing the suspension. The resulting multilamellar liposomes are then filtered five times through polycarbonate filters (pore size, 400 nm), using an extruder system (Liposofast; Avestin, Ottawa, ON, Canada) to form unilamellar liposomes. The nonencapsulated sucrose is removed by washing the liposomes in 3 ml of PBS followed by centrifugation for 20 min at 43,600 g [35,000 rpm in a Beckman Coulter (Fusterton, CA) TLA 100.2 rotor] and resuspension in 0.5 ml of PBS (final lipid concentration, 10 mg/ml). GSH-PE-containing liposomes are then coupled to GST or a GST fusion protein (GST-C2B domain of synaptotagmin) by incubation (200 g/ml of lipid) with protein (40 g/ml) in PBS for 4 h at 4 . An aliquot (50 g of total lipid) is analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fig. 3B). As expected, the presence of glutathione greatly enhances the binding of GST or GST-C2B to liposomes (Fig. 3B). Liposomes containing similar amounts of either GST or GSTC2B (Fig. 3C) are then incubated (0.5 mg/ml; 50 g of total lipid) with brain cytosol (2.5 mg/ml) for 10 min at 37 to assess the ability of the synaptotagmin C2B domain to recruit clathrin and AP-2. After the reaction, liposomes are reisolated by centrifugation (20 min at 35,000 rpm in a Beckman Coulter TLA 100.2 rotor) and washed (once in 1 ml of PBS), and bound proteins are analyzed by SDS–PAGE and Western blotting (Fig. 3D). Liposomes carrying the C2B domain are much more efficient in the recruitment of AP-2 and clathrin from cytosol to the liposome surface (Fig. 3D).
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Preparation of Cytosol Cytosol is prepared from fresh or frozen rat brains by high-speed centrifugation followed by a desalting step and ammonium sulfate precipitation to concentrate the sample.10 The brainstem is removed from 40 rat brains, and the brains are rinsed in breaking buffer (BB) [500 mM KCl, 10 mM MgCl2, 250 mM sucrose, 25 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol (DTT), 2 mM EGTA, protease inhibitors (complete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany)] and minced coarsely into small pieces with a razor blade. The tissue is then homogenized in 100 ml of breaking buffer with a Polytron blender (Ultra Turrax; Janke & Kunkel, Staufen, Germany) at medium speed for 1 min, and centrifuged for 60 min at 9000 g (8700 rpm in a Beckman Coulter SS34 rotor). The supernatant is recovered and centrifuged at 184,000 g (50,000 rpm in a Beckman Coulter 70Ti rotor) for 2 h. The supernatant is then dialyzed two times for 2 h at 4 against 4 liters of dialysis buffer (DB) [50 mM KCl, 25 mM Tris-HCl (pH 8.0), 1 mM DTT], using Spectra/Por 3 membranes (Spectrum, Gardena, CA). Ammonium sulfate [(NH4)2SO4] is added slowly to 60% saturation (over a duration of approximately 30 min), and the solution is stirred for an additional 30 min. The precipitate is recovered by centrifugation at 9000 g for 30 min, resuspended in 13 ml of buffer from the second dialysis step, and dialyzed for another 2 h against 4 liters of the same buffer, followed by 2 h against DB (4 liters) without DTT. Finally, the cytosol is centrifuged at 100,000 g (47,000 rpm in a 70Ti rotor) for 2 h and recovered in 15 ml of DB (final protein concentration is approximately 20 mg/ml), aliquoted (1.5-ml Eppendorf tubes), and snap-frozen by dropping the tubes into liquid nitrogen. Samples can be stored for several months at 70 . Incubation of Liposomes with Cytosol and Nucleotides A common incubation protocol that allows for morphological and biochemical analysis is presented here: liposomes, brain cytosol, nucleotides, and a stock (10) of cytosolic buffer (CB) {final concentrations after dilution: 25 mM HEPES-KOH (pH 7.4), 25 mM KCl, 2.5 mM magnesium acetate [Mg(CH3COO)2], 150 mM potassium glutamate, 10 M Ca2þ} are mixed in 1.5-ml Eppendorf tubes to obtain the following final concentrations: liposomes (0.1 mg/ml), cytosol (4 mg/ml), GTP (200 M), ATP-regenerating system [ATP (2 mM), phosphocreatine (17 mM; Sigma), creatine phosphokinase (17 units/ml; Sigma)]. For lipid and protein biochemistry (Figs. 2 and 3), total reaction volumes of 100 and 400 l, respectively, are sufficient, whereas larger amounts (1.5 ml) are needed for plastic embedding and thin sectioning (Fig. 1). The samples are incubated for 15 min at 37 and processed as described below.
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Analysis of Bound Protein by SDS–PAGE and Western Blotting At the end of incubation, liposomes are loaded onto a step gradient (600 l of 0.5 M sucrose over a cushion of 180 l of 2 M sucrose) and centrifuged for 30 min at 150,000 g (in a Beckman Coulter TLA 100.2 rotor) at 4 to separate bound from unbound protein. Liposomes are recovered from the interface of the step gradient, washed once with 400 l of CB, and resuspended in 200 l of CB. The recovery of liposomes is assessed by measuring NBD fluorescence (ex 360 nm, em 430 nm) of an aliquot (10 l), using a spectrofluorometer. If a radiolabeled lipid tracer ([3H]PC) is used, a small aliquot (10 l) is measured by liquid scintillation counting. On the basis of these measurements, equal amounts of lipid are then loaded for SDS–PAGE and Western blot analysis. Endocytosis-associated proteins are detected with the following primary antibodies: clathrin HC [American Type Culture Collection (ATCC), Manassas, VA], -adaptin (AP-2) (Affinity BioReagents, Golden, CO), dynamin 1 (Transduction Laboratories, Lexington, KY), and tubulin 1 (Sigma), which serves as a control (Figs. 2 and 3). For semiquantitative purposes, secondary antibodies conjugated to horseradish peroxidase in combination with a chemiluminescence kit (SuperSignal West Pico peroxide; Pierce, Rockford, IL) can be used (Fig. 3D). A more reliable and more quantitative analysis, however, requires detection by secondary antibodies conjugated to 125I, followed by quantitative autoradiography with a Storm PhosphoImager (Molecular Dynamics-Amersham Biosciences, Piscataway, NJ).24 Electron Microscopy Incubation mixtures are fixed in suspension by addition of an equal volume of 2 fixative [final concentration, 3% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) (freshly prepared from powder), 2% glutaraldehyde in 50 mM HEPES-KOH (pH 7.4)] for 30 min, and liposomes are pelleted by centrifugation in a Beckman Coulter TLA 100.3 rotor at 50,000 rpm for 10 min. The pellets are washed in 0.1 M sodium cacodylate (Electron Microscopy Sciences) buffer, pH 7.3, postfixed with 1% OsO4 in the same buffer for 1 h, and washed three times in buffer. They are then en bloc stained with 2% aqueous uranyl acetate (Electron Microscopy Sciences) for 1 h in the dark and dehydrated with increasing concentrations of ethanol (once each with 10, 50, 70, and 95%, and three times with 100%, for 10 min each), followed by substitution with 24
H. Gad, N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M. H. Ellisman, P. De Camilli, O. Shupliakov, and L. Brodin, Neuron 27, 301 (2000).
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propylene oxide (three times for 10 min each). The samples are embedded in Epon (Electron Microscopy Sciences) by incubation in a mixture of Epon–propylene oxide (1:1, v/v) in a rotating mixer at approximately 8 rpm (Pelco R2; Ted Palla, Redding, CA), uncapped and overnight. This Epon is then replaced with fresh Epon, and incubation is continued for several hours. The pellets are embedded in fresh Epon and baked for 48 h at 60 in Eppendorf tubes. Blocks are then trimmed and thinsectioned at 40 nm, using a microtome (Leica Ultracut; Leica, Wetzlar, Germany). Sections are stained on a grid with 2% aqueous uranyl acetate followed by lead citrate (0.4% in water) and observed at 80 kV in a transmission electron microscope. For some samples, OsO4 postfixation is followed by impregnation with 1% tannic acid in distilled water to enhance visualization of coat proteins.25 Lipid Biochemistry: Measurement of Phosphoinositide Levels For lipid analysis, incubations are performed in the presence of 5 Ci of [ -32P]ATP, at a final ATP concentration of 50 M, and typically in a reaction volume of 50–100 l in 1.5-ml screw-cap plastic Eppendorf tubes. Reactions are stopped by addition of 400 l of crude brain phosphoinositides (20 g/ml; Sigma) in chloroform–methanol (2:1, v/v), followed by 400 l of chloroform and 300 l of 1 M HCl. After vigorous vortexing, phases are separated by brief centrifugation (2 min at high speed in a table-top centrifuge) and the lower organic phase is removed with a Pasteur pipette or a transfer pipette. Care should be taken not to remove any of the upper aqueous phase and protein–aqueous interphase. The aqueous phases are reextracted with an additional 400 l of chloroform, and the organic phases are pooled and dried under a stream of nitrogen. A sample concentrator, which can hold and dry multiple samples in parallel (Dri-Block; Techne, Princeton, NJ), is used for this purpose. Work should be carried out under a fume hood. Phosphoinositides can be separated without further processing by thin-layer chromatography (TLC). Lipids are resuspended in 40 l of chloroform–methanol (2:1 v/v), and a 10-l aliquot is spotted onto a TLC plate [Merck Silica gel 60 TLC plate that has been preimpregnated with potassium oxalate: TLC plates are dipped into a solution of 1% potassium oxalate (Sigma) in methanol–water (1:1, v/v), air dried, and activated for 30 min at 120 before use]. Plates are developed in a solvent system of chloroform–acetone–methanol–acetic acid–water (64:30:24:32:14, by volume) and autoradiographed at 70 for several hours or overnight. Major radioactive spots [PIP2, phosphatidylinositol 25
L. Orci, B. S. Glick, and J. E. Rothman, Cell 46, 171 (1986).
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4-phosphate (PIP), and phosphatidic acid (PA)] are identified by comigration with purified lipid standards (Sigma and Avanti Polar Lipids) and visualized by placing the TLC plate into a tank saturated with iodine vapor [crystals of iodine (Sigma) are added to a TLC tank, the tank is covered with the lid, and iodine vapors are allowed to form by sublimation for 2 h]. Associated radioactivity is measured by quantitative autoradiography with a Phospholmager (Molecular Dynamics-Amersham Biosciences) or by scraping the area of silica gel on the TLC plate that harbors the spot into a scintillation vial, followed by liquid scintillation counting. Alternatively, lipids are deacylated for analysis by strong anionexchange (SAX) high-performance liquid chromatography (HPLC).26 Lipids are deacylated with methylamine as described.27 A mixture of 40% aqueous methylamine (Fluka, Milwaukee, WI)–water–n-butanol– methanol (36:8:9:47, by volume) (2 ml) is added to the dried lipid extract and incubated at 50 for 45 min in a tightly stoppered tube. The mixture is then cooled on ice and evaporated to dryness under vacuum in a Savant SpeedVac (Thermo Savant, Holbrook, NY). A mixture of n-butanol– petroleum ether–ethyl formate (20:40:1, by volume) (2 ml) is added, followed by water (2 ml). After thorough mixing and gentle centrifugation to separate phases, the lower aqueous phase, containing the deacylated lipids, is carefully removed. The organic phase is reextracted with 2 ml of water, and the combined aqueous phases are dried in a SpeedVac. Samples are taken up in water, loaded on a Partisphere SAX ion-exchange column (4.6 125 mm; Whatman, Clifton, NJ), and eluted at a flow rate of 1 ml/ min, using the following gradient profile28: 0–10 min, 0% B; 10–55 min, 0–35% B; and 55–70 min, 35–100% B [buffer B is 1.4 M (NH4)2HPO4, pH 3.7]. Fractions (1 ml) are collected and counted for radioactivity by scintillation counting. Alternatively, radioactivity is measured with an inline liquid scintillation counter (Packard, Meriden, CT).29 Peaks are identified by coelution with standards, most of which must be generated by enzymatic reactions using phosphoinositide kinases and phosphatases.26,30 Phophatidylinositol-[inositol-2-3H(N)] ([3H]PI) and phosphatidylinositol[inositol-2-3H(N)]-4,5-bisphosphate ([3H]PI(4,5)P2) are available from Perkin Elmer Life Sciences.
26
L. A. Serunian, K. R. Auger, and L. C. Cantley, Methods Enzymol. 198, 78 (1991). C. J. Kirk, A. J. Morris, and S. B. Shears, in ‘‘Peptide Hormone Action: A Practical Approach’’ (K. Siddle and J. C. Hutton, eds.), p. 151. IRL Press, Oxford, 1990. 28 I. M. Bird, Methods Mol. Biol. 105, 25 (1998). 29 L. Zhang and I. L. Buxton, Methods Mol. Biol. 105, 47 (1998). 30 X. Zhang, J. C. Loijens, I. V. Boronenkov, G. J. Parker, F. A. Norris, J. Chen, O. Thum, G. D. Prestwich, P. W. Majerus, and R. A. Anderson, J. Biol. Chem. 272, 17756 (1997). 27
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Concluding Remarks
The methodology described above allows for the investigation of key aspects of lipids and their metabolism in coat dynamics. Among the advantages offered is the possibility to study the role of individual proteins or of cytosol and cytosolic fractions under similar assay conditions. The power of this methodology is further enhanced by the use of proteoliposomes, rather than pure lipid vesicles, to study the cooperativity of proteins and lipids in membrane dynamics. Some limitations of in vitro systems such as this one include the relatively low efficiency, slow kinetics, and lack of physiological ‘‘compartmentalization’’ of the components (e.g., the lack of segregation of proteins and lipids into distinct subcompartments). Clearly, the validity of results obtained by these techniques must be ultimately assessed by studies in situ and by observations from genetic approaches.
[15] Fluorescence Assays for Liposome Fusion By Nejat Du¨zgu¨nes, Introduction
Membrane fusion is a biophysical reaction that is of fundamental importance in biological systems and takes place in such diverse processess as fertilization, viral infection, exocytosis, and intracellular membrane traffic.1 Because their membrane composition can be manipulated readily, liposomes have provided a convenient model system to study the molecular determinants and mechanisms of membrane fusion.2,3 The roles of membrane composition, the ionic environment, and membrane phase state have been elucidated by the use of liposome fusion assays. The role of cytosolic or viral membrane proteins in fusion, as well as the role of fusion activity of peptides derived from viral proteins, have been studied in detail with liposomes.2,3 Liposomes containing fluorescent lipids have been used to study the fusion characteristics of intracellular membranes
1
N. Du¨ zgu¨ nes, and F. Bronner (eds.), ‘‘Membrane Fusion in Fertilization, Cellular Transport and Viral Infection,’’ p. xviii and 384. Academic Press, New York, 1988. 2 N. Du¨ zgu¨ nes, and S. Nir, in ‘‘Liposomes as Tools in Basic Research and Industry’’ (J. R. Philippot and F. Schuber, eds.), p. 103. CRC Press, Boca Raton, FL, 1995. 3 N. Du¨ zgu¨ nes,, in ‘‘Trafficking of Intracellular Membranes: From Molecular Sorting to Membrane Fusion’’ (M. C. Pedroso de Lima, N. Du¨ zgu¨ nes,, and D. Hoekstra, eds.), p. 97. Springer-Verlag, Berlin, 1995.
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reported on the uptake of small unilamellar liposomes (mean diameter of about 80 nm) by human umbilical vein endothelial cells (HUVECs) or human promyelocytic leukemia cells (HL60). These authors found that internalization was clathrin independent, because it was not inhibited by sodium azide and deoxyglucose, but was affected by filipin. Consistent with these data, Kessner et al.20 concluded that liposomes depleted of specific ligands may be internalized into HUVECs through caveolae. In the previous section we reviewed results that indicated that widely used inhibitors of endosomal acidification do not interfere with internalization of liposomes via coated pits. Mineo and Anderson37 reported that bafilomycin A1 inhibited the uptake of 5-methyltetrahydrofolate into MA104 cells. These authors deduced that acidification can occur in plasmalemmal vesicles. Consequently, a measurement of intracellular pH by means of HPTS preencapsulated in liposomes may not necessarily constitute a proof for the residence of the liposomes in endosomes. Acknowledgments J.L.N. was supported by DGCYT (Grant PB96-0171), the Basque Government (EX-1998-28; PI-1998-32), and the University of the Basque Country (UPV 042.310-EA085/ 97; UPV 042.310-G03/98). 37
E. Mineo and R. G. W. Anderson, Exp. Cell Res. 224, 237 (1996).
[14] Assembly of Endocytosis-Associated Proteins on Liposomes By Markus R. Wenk and Pietro De Camilli Introduction
Synapses are intercellular junctions of a neuron with its target cells, and they are the sites of neurotransmission. At chemical synapses, depolarization of the synaptic plasma membrane triggers Ca2þ entry into the presynaptic nerve terminal, which leads to fusion of synaptic vesicles with the plasma membrane (exocytosis) and release of their contents into the synaptic cleft. After exocytosis, synaptic vesicle membranes are retrieved and recycled to generate new neurotransmitter-filled vesicles for a subsequent round of release. A major route of synaptic vesicle membrane retrieval involves clathrin-mediated endocytosis.1 The protein machinery underlying
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this endocytotic reaction has been characterized in detail. In addition to the major integral components of the clathrin coat, such as clathrin and the ‘‘classic’’ clathrin adaptor (AP-2), accessory factors (e.g., AP-180, amphiphysin, endophilin, eps-15, epsin, dynamin, Hip1R, intersectin, and synaptojanin) participate in coat assembly, pit formation, and vesicle fission.2–4 Cell-free reconstitution assays utilizing chemically defined liposomes (rather than biological membranes) as donor membranes, and cytosolic fractions or purified proteins as ligands, have been developed in order to identify and/or characterize the molecular requirements for vesicle biogenesis, including the biogenesis of clathrin-coated vesicles. Cell-free complete or partial reconstitutions have been reported for COPI-, COPII-, and clathrin-mediated vesicle budding,5–10 as well as for membrane tubulation reactions often observed in vivo at sites of vesicle budding.10–12 All these studies have emphasized the critical role of protein–lipid interfaces in the formation of tubulovesicular elements within the cell. Protein binding to lipids often involves both ionic interactions with the head groups of phospholipids, as well as hydrophobic interactions with the nonpolar domain of the membrane, and with specific lipids such as cholesterol and fatty acids.13 In addition, growing evidence indicates that lipids that can undergo rapid and reversible changes within the membrane play an important role in regulating the recruitment of cytosolic factors to 1
P. De Camilli, V. I. Slepnev, O. Shupliakov, and L. Brodin, in ‘‘Synapses’’ (M. W. Cowan, T. C. Sudhof, and C. F. Stevens, eds.), p. 217. Johns Hopkins University Press, Baltimore, MD, 2000. 2 M. Marsh and H. T. McMahon, Science 285, 215 (1999). 3 P. De Camilli, H. Chen, J. Hyman, E. Panepucci, A. Bateman, and A. T. Brunger, FEBS Lett. 513, 11 (2002). 4 V. I. Slepnev and P. De Camilli, Nat. Rev. 1, 161 (2000). 5 A. Spang, K. Matsuoka, S. Hamamoto, R. Schekman, and L. Orci, Proc. Natl. Acad. Sci. USA 95, 11199 (1998). 6 K. Matsuoka, L. Orci, M. Amherdt, S. Y. Bednarek, S. Hamamoto, R. Schekman, and T. Yeung, Cell 93, 263 (1998). 7 M. Bremser, W. Nickel, M. Schweikert, M. Ravazzola, M. Amherdt, C. A. Hughes, T. H. Sollner, J. E. Rothman, and F. T. Wieland, Cell 96, 495 (1999). 8 M. Kinuta, H. Yamada, T. Abe, M. Watanabe, S. A. Li, A. Kamitani, T. Yasuda, T. Matsukawa, H. Kumon, and K. Takei, Proc. Natl. Acad. Sci. USA 99, 2842 (2002). 9 M. G. Ford, B. M. Pearse, M. K. Higgins, Y. Vallis, D. J. Owen, A. Gibson, C. R. Hopkins, P. R. Evans, and H. T. McMahon, Science 291, 1051 (2001). 10 K. Takei, V. Haucke, V. Slepnev, K. Farsad, M. Salazar, H. Chen, and P. De Camilli, Cell 94, 131 (1998). 11 S. M. Sweitzer and J. E. Hinshaw, Cell 93, 1021 (1998). 12 K. Takei, V. I. Slepnev, V. Haucke, and P. De Camilli, Nat. Cell Biol. 1, 33 (1999). 13 W. B. Huttner and A. Schmidt, Curr. Opin. Neurobiol. 10, 543 (2000).
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the bilayer.14 Among such lipids, phosphoinositides (phosphorylated derivatives of phosphatidylinositol) are of critical importance and are best characterized. Rapid phosphorylation–dephosphorylation at different positions of the inositol ring by a multiplicity of enzymes generates a variety of stereoisomers, and phosphoinositides are now believed to be key players in the regulation of clathrin coat recruitment and dynamics.15,16 Although cytosolic proteins alone can generate from liposomes, membrane buds, and tubules that are similar to the different types of coated vesicle buds and tubules observed in the cell, integral membrane proteins often act as receptors for coat proteins and their accessory factors. In doing so, they contribute to the regulation of coating in time and space in the living cytoplasm. Thus, a synergistic effect of both membrane components, lipids and proteins, is central to the regulation of assembly reactions on membrane surfaces. Here we describe the methodology of a cell-free system utilizing liposomes and cytosol to study assembly reactions of proteins involved in clathrin-mediated endocytosis. Liposomes, when incubated with brain cytosol and nucleotides, undergo morphological changes and fragmentation into smaller structures that resemble closely intermediates of clathrin-mediated endocytosis8,10 (Fig. 1). Biochemical and biophysical analysis of these reactions show that elevated levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2, or PIP2] correlate with increased recruitment of clathrin and clathrin adaptors to the liposomal surface17,18 (Fig. 2). Furthermore, inclusion into the liposomes of a surface-exposed recombinant fragment of synaptotagmin enhances the recruitment of clathrin coat proteins, demonstrating the synergistic actions of proteins and lipids19 (Fig. 3). Three modes of analysis are applied: (1) observation of liposomal structure by electron microscopy, (2) protein biochemistry to assess amounts of endocytotic proteins recruited to the liposomal surface, and (3) lipid biochemistry to measure the metabolism of phosphoinositides during the incubation reaction.
14
J. H. Hurley and T. Meyer, Curr. Opin. Cell Biol. 13, 146 (2001). O. Cremona and P. De Camilli, J. Cell Sci. 114, 1041 (2001). 16 T. F. Martin, Curr. Opin. Cell Biol. 13, 493 (2001). 17 O. Cremona, G. Di Paolo, M. R. Wenk, A. Luthi, W. T. Kim, K. Takei, L. Daniell, Y. Nemoto, S. B. Shears, R. A. Flavell, D. A. McCormick, and P. De Camilli, Cell 99, 179 (1999). 18 M. R. Wenk, L. Pellegrini, V. A. Klenchin, G. Di Paolo, S. Chang, L. Daniell, M. Arioka, T. F. Martin, and P. De Camilli, Neuron 32, 79 (2001). 19 V. Haucke, M. R. Wenk, E. R. Chapman, K. Farsad, and P. De Camilli, EMBO J. 19, 6011 (2000). 15
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Fig. 1. Electron micrographs demonstrating the effect of brain cytosol on liposomes composed of brain lipids. Preparations were analyzed after plastic embedding and thin sectioning. (a and b) Control liposomes not incubated with cytosol. (c–f) Liposomes incubated with rat brain cytosol, ATP, and GTP S. High-power observation reveals the presence of dynamin-like rings and clathrin-coated profiles (d–f). Fields (e)–(h) demonstrate the similarity of dynamin-coated tubules and clathrin-coated pits observed on liposomes (e and f) and synaptic membranes (g and h) incubated with brain cytosol, ATP, and GTP S. Calibration bar: 500 nm in (a) and (c); 150 nm in (b) and (d); and 50 nm in (e)–(h). Reprinted from Cell, Vol. 94: K. Takei, V. Haucke, V. Slepnev, K. Farsad, M. Salazar, H. Chen, and P. De Camilli, Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes, pp. 131–141. Copyright (1998), with permission from Elsevier Inc.10
Reconstitution assays such as this one are a powerful tool with which to study the molecular events that are required for the formation as well as the dynamics of protein coats on membrane surfaces. We have used this integrated approach to detect alterations in phosphoinositide metabolism and clathrin coat dynamics in cytosolic fractions prepared from knockout mice.17,18,20 Furthermore, purified proteins, such as amphiphysin and dynamin,12,21 endophilin,22 or phosphatidylinositol-4-phosphate 5-kinase (PIP 20
G. Di Paolo, S. Sankaranarayanan, M. R. Wenk, L. Daniell, E. Perucco, B. J. Caldarone, R. Flavell, M. Picciotto, T. A. Ryan, O. Cremona, and P. De Camilli, Neuron 33, 789 (2002). 21 K. Takei, V. I. Slepnev, and P. De Camilli, Methods Enzymol. 329, 478 (2001). 22 K. Farsad, N. Ringstad, K. Takei, S. R. Floyd, K. Rose, and P. De Camilli, J. Cell Biol. 155, 193 (2001).
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Fig. 2. Phosphoinositide metabolism and clathrin coat recruitment to liposomes. Liposomes composed of brain lipids are incubated with brain cytosol, [ -32P]ATP, and GTP and analyzed for incorporation of phosphate into phosphoinositides (A and B) and for binding of clathrin coat proteins to the liposomal surface (C). After the reaction, phosphoinositides are extracted and deacylated, and glycerophosphoinositols are separated by HPLC. A typical chromatogram is shown in (A). (B) Quantification of PI(4,5)P2 levels in an experiment utilizing different sources of cytosol as indicated in (C) [knockout (KO) and wild-type (WT) denote cytosolic fractions from synaptojanin 1 knockout mice and control litter mates, respectively]. These cytosolic fractions were used in lanes 5 and 7 and were supplemented (lane 6) or depleted (lane 8) of PIP kinase I , the major PIP2-synthesizing enzyme in brain cytosol. Levels of PIP2 generated during the incubation of liposomes with cytosol (B) correlate nicely with the amount of clathrin coat that is recruited to the liposomes [elevated levels of clathrin heavy chain (HC) and -adaptin in lanes 6 and 7 compared with control incubations (lanes 5 and 8)]. A control protein (tubulin) does not show any significant variations between the different incubation conditions. Reprinted from Neuron, Vol. 32: M. R. Wenk, L. Pellegrini, V. A. Klenchin, G. Di Paolo, S. Chang, L. Daniell, M. Arioka, T. F. Martin, and P. De Camilli, PIP kinase I is the major PI(4,5)P2 synthesizing enzyme at the synapse, pp. 79–88. Copyright (2001), with permission from Elsevier Inc.18
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Fig. 3. Liposomes containing glutathione-linked GST fusion proteins. (A) Chemical structure of glutathione (GSH)-linked N-MPB-PE. (B) High-affinity binding of GST fusion proteins to liposomes containing GSH-PE (+) compared with control liposomes devoid of GSH-PE (–). (C) Liposomes with GSH-PE in their membranes were incubated with GST or GST-synaptotagmin I C2B (GST-C2B) proteins to allow coupling of GST fusion proteins to the liposomal membrane via GSH-PE. An aliquot (50 g of total lipid) was then analyzed by SDS–PAGE and Coomassie blue staining. (D) Liposomes containing glutathione-PE-linked GST or GST-C2B (C) were incubated with rat brain cytosol, reisolated, and washed, and bound proteins were analyzed by SDS–PAGE and immunoblotting. The presence of GSTC2B on the liposomal surface greatly facilitates clathrin/AP-2 recruitment, but has no effect on the binding of tubulin or dynamin I. Reprinted from EMBO J., Vol. 19: V. Haucke, M. R. Wenk, E. R. Chapman, K. Farsad, and P. De Camilli, Dual interaction of synaptotagmin with 2- and -adaptin facilitates clathrin-coated pit nucleation, pp. 6011–6019. By permission of Oxford University Press.19
kinase)18 can be used in this system in addition to, or instead of, cytosolic fractions. Functionalized liposomes, such as the proteoliposomes described here, are ideally suited to investigate the synergistic effect of membrane lipids and membrane proteins on coat structure and function. They are also a useful tool for future applications in the field of liposome research, such as targeting for drug delivery or the development of liposome-based affinity probes for proteins and lipids.
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Methods
Preparation of Liposomes Pure Lipid Vesicles. Large unilamellar vesicles are prepared as described23 with some modifications. A defined amount of lipid (4 mg) [bovine brain extract, type I Folch fraction I; Sigma, St. Louis, MO; stored as a 20-mg/ml stock solution in chloroform–methanol (1:2) at 20 ] is transferred to a 10-ml borosilicate glass tube and spiked with 0.05% (w/w) NBD-PC {1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]dodecanoyl]-sn-glycero-3-phosphocholine; Avanti Polar Lipids, Alabaster, AL} or, alternatively, [3H]PC (l- -dipalmitoyl-[2-palmitoyl9,10-3H(N)]-phosphatidylcholine) (PerkinElmer Life Sciences, Boston, MA) at 0.05 Ci/mg lipid. The low amounts of fluorescent or radioactive lipids are used for quantification of recovery after reisolation of the liposomes from the incubation reaction (see below). The lipid solution is then dried under a gentle stream of nitrogen. A thin and even film of lipid is formed on the glass wall by slowly (manually) rotating the glass tube during evaporation of the organic solvent. The lipid is further dried in a desiccator over phosphorous pentoxide (Sigma) and high vacuum for 2 h. The dried lipid film is then hydrated with a gentle stream of water-saturated nitrogen until it loses some of its opalescent appearance. A defined amount of 0.3 M sucrose (in water) (2 ml) is gently added to the test tube, which is flushed with nitrogen and sealed. Liposomes are allowed to form spontaneously for 2 h at 37 . The suspension is then agitated gently to resuspend the liposomes, and large aggregates and debris are removed by brief centrifugation. Thus obtained liposomes (final lipid concentration, 2 mg/ml) are mostly unilamellar spheres with diameters of >500 nm (Fig. 1a and b), which can be stored at 4 for several days. Instead of a lipid extract from a biological tissue, such as the total brain lipids described above, chemically defined components (i.e., synthetic lipids) can be used to generate these liposomes. For that aim, individual lipids are mixed in organic solvent at a defined ratio and then dried and processed as described above. The effect of the individual lipid species on protein recruitment can hence be studied.10 Lipoprotein-Containing Liposomes. Recombinant glutathione S-transferase (GST) fusion proteins can be chemically linked to a glutathione (GSH)-derivatized phosphatidylethanolamine (GSH-PE)7,19 (Fig. 3A). The latter is synthesized by mixing equal volumes (2 ml each) of [4-(p-maleimidophenyl)butyryl] phosphatidylethanolamine (MPB-PE, 5 mg/ml in 23
J. P. Reeves and R. M. Dowben, J. Cell. Physiol. 73, 49 (1969).
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CHCl3; Avanti; Polar Lipids) and glutathione [5 mM in dimethylformamide (DMF); Sigma] supplemented with 10 mM morpholinepropanesul Fonic acid (MOPS), pH 7.5. After incubation for 2 h at room temperature, free maleimido residues are blocked with 10 mM 2-mercaptoethanol, and the mixture is further incubated for 30 min at room temperature. The GSH-PE lipid is extracted by adding 4 ml of chloroform and 2 ml of methanol to the mixture, followed by vortexing and brief centrifugation to separate phases. The lower organic phase is then carefully removed, the aqueous phase is reextracted twice with 2 ml of chloroform, and the organic phases are pooled, dried, and resuspended in chloroform–methanol (2:1, v/v). Lipids (bovine brain extract; see above) and GSH-PE are mixed in chloroform at a ratio of 8:2 (w/w) and 0.05% (w/w) NBD-PE {1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphatidylethanolamine; Avanti Polar Lipids} is added as a fluorescent marker for quantification. The lipid film (5 mg) is dried under a stream of nitrogen and resuspended in 0.5 ml of 0.6 M sucrose [in phosphate-buffered saline (PBS)] by vigorously vortexing the suspension. The resulting multilamellar liposomes are then filtered five times through polycarbonate filters (pore size, 400 nm), using an extruder system (Liposofast; Avestin, Ottawa, ON, Canada) to form unilamellar liposomes. The nonencapsulated sucrose is removed by washing the liposomes in 3 ml of PBS followed by centrifugation for 20 min at 43,600 g [35,000 rpm in a Beckman Coulter (Fusterton, CA) TLA 100.2 rotor] and resuspension in 0.5 ml of PBS (final lipid concentration, 10 mg/ml). GSH-PE-containing liposomes are then coupled to GST or a GST fusion protein (GST-C2B domain of synaptotagmin) by incubation (200 g/ml of lipid) with protein (40 g/ml) in PBS for 4 h at 4 . An aliquot (50 g of total lipid) is analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fig. 3B). As expected, the presence of glutathione greatly enhances the binding of GST or GST-C2B to liposomes (Fig. 3B). Liposomes containing similar amounts of either GST or GSTC2B (Fig. 3C) are then incubated (0.5 mg/ml; 50 g of total lipid) with brain cytosol (2.5 mg/ml) for 10 min at 37 to assess the ability of the synaptotagmin C2B domain to recruit clathrin and AP-2. After the reaction, liposomes are reisolated by centrifugation (20 min at 35,000 rpm in a Beckman Coulter TLA 100.2 rotor) and washed (once in 1 ml of PBS), and bound proteins are analyzed by SDS–PAGE and Western blotting (Fig. 3D). Liposomes carrying the C2B domain are much more efficient in the recruitment of AP-2 and clathrin from cytosol to the liposome surface (Fig. 3D).
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Preparation of Cytosol Cytosol is prepared from fresh or frozen rat brains by high-speed centrifugation followed by a desalting step and ammonium sulfate precipitation to concentrate the sample.10 The brainstem is removed from 40 rat brains, and the brains are rinsed in breaking buffer (BB) [500 mM KCl, 10 mM MgCl2, 250 mM sucrose, 25 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol (DTT), 2 mM EGTA, protease inhibitors (complete EDTA-free tablets; Roche Diagnostics, Mannheim, Germany)] and minced coarsely into small pieces with a razor blade. The tissue is then homogenized in 100 ml of breaking buffer with a Polytron blender (Ultra Turrax; Janke & Kunkel, Staufen, Germany) at medium speed for 1 min, and centrifuged for 60 min at 9000 g (8700 rpm in a Beckman Coulter SS34 rotor). The supernatant is recovered and centrifuged at 184,000 g (50,000 rpm in a Beckman Coulter 70Ti rotor) for 2 h. The supernatant is then dialyzed two times for 2 h at 4 against 4 liters of dialysis buffer (DB) [50 mM KCl, 25 mM Tris-HCl (pH 8.0), 1 mM DTT], using Spectra/Por 3 membranes (Spectrum, Gardena, CA). Ammonium sulfate [(NH4)2SO4] is added slowly to 60% saturation (over a duration of approximately 30 min), and the solution is stirred for an additional 30 min. The precipitate is recovered by centrifugation at 9000 g for 30 min, resuspended in 13 ml of buffer from the second dialysis step, and dialyzed for another 2 h against 4 liters of the same buffer, followed by 2 h against DB (4 liters) without DTT. Finally, the cytosol is centrifuged at 100,000 g (47,000 rpm in a 70Ti rotor) for 2 h and recovered in 15 ml of DB (final protein concentration is approximately 20 mg/ml), aliquoted (1.5-ml Eppendorf tubes), and snap-frozen by dropping the tubes into liquid nitrogen. Samples can be stored for several months at 70 . Incubation of Liposomes with Cytosol and Nucleotides A common incubation protocol that allows for morphological and biochemical analysis is presented here: liposomes, brain cytosol, nucleotides, and a stock (10) of cytosolic buffer (CB) {final concentrations after dilution: 25 mM HEPES-KOH (pH 7.4), 25 mM KCl, 2.5 mM magnesium acetate [Mg(CH3COO)2], 150 mM potassium glutamate, 10 M Ca2þ} are mixed in 1.5-ml Eppendorf tubes to obtain the following final concentrations: liposomes (0.1 mg/ml), cytosol (4 mg/ml), GTP (200 M), ATP-regenerating system [ATP (2 mM), phosphocreatine (17 mM; Sigma), creatine phosphokinase (17 units/ml; Sigma)]. For lipid and protein biochemistry (Figs. 2 and 3), total reaction volumes of 100 and 400 l, respectively, are sufficient, whereas larger amounts (1.5 ml) are needed for plastic embedding and thin sectioning (Fig. 1). The samples are incubated for 15 min at 37 and processed as described below.
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Analysis of Bound Protein by SDS–PAGE and Western Blotting At the end of incubation, liposomes are loaded onto a step gradient (600 l of 0.5 M sucrose over a cushion of 180 l of 2 M sucrose) and centrifuged for 30 min at 150,000 g (in a Beckman Coulter TLA 100.2 rotor) at 4 to separate bound from unbound protein. Liposomes are recovered from the interface of the step gradient, washed once with 400 l of CB, and resuspended in 200 l of CB. The recovery of liposomes is assessed by measuring NBD fluorescence (ex 360 nm, em 430 nm) of an aliquot (10 l), using a spectrofluorometer. If a radiolabeled lipid tracer ([3H]PC) is used, a small aliquot (10 l) is measured by liquid scintillation counting. On the basis of these measurements, equal amounts of lipid are then loaded for SDS–PAGE and Western blot analysis. Endocytosis-associated proteins are detected with the following primary antibodies: clathrin HC [American Type Culture Collection (ATCC), Manassas, VA], -adaptin (AP-2) (Affinity BioReagents, Golden, CO), dynamin 1 (Transduction Laboratories, Lexington, KY), and tubulin 1 (Sigma), which serves as a control (Figs. 2 and 3). For semiquantitative purposes, secondary antibodies conjugated to horseradish peroxidase in combination with a chemiluminescence kit (SuperSignal West Pico peroxide; Pierce, Rockford, IL) can be used (Fig. 3D). A more reliable and more quantitative analysis, however, requires detection by secondary antibodies conjugated to 125I, followed by quantitative autoradiography with a Storm PhosphoImager (Molecular Dynamics-Amersham Biosciences, Piscataway, NJ).24 Electron Microscopy Incubation mixtures are fixed in suspension by addition of an equal volume of 2 fixative [final concentration, 3% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) (freshly prepared from powder), 2% glutaraldehyde in 50 mM HEPES-KOH (pH 7.4)] for 30 min, and liposomes are pelleted by centrifugation in a Beckman Coulter TLA 100.3 rotor at 50,000 rpm for 10 min. The pellets are washed in 0.1 M sodium cacodylate (Electron Microscopy Sciences) buffer, pH 7.3, postfixed with 1% OsO4 in the same buffer for 1 h, and washed three times in buffer. They are then en bloc stained with 2% aqueous uranyl acetate (Electron Microscopy Sciences) for 1 h in the dark and dehydrated with increasing concentrations of ethanol (once each with 10, 50, 70, and 95%, and three times with 100%, for 10 min each), followed by substitution with 24
H. Gad, N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M. H. Ellisman, P. De Camilli, O. Shupliakov, and L. Brodin, Neuron 27, 301 (2000).
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propylene oxide (three times for 10 min each). The samples are embedded in Epon (Electron Microscopy Sciences) by incubation in a mixture of Epon–propylene oxide (1:1, v/v) in a rotating mixer at approximately 8 rpm (Pelco R2; Ted Palla, Redding, CA), uncapped and overnight. This Epon is then replaced with fresh Epon, and incubation is continued for several hours. The pellets are embedded in fresh Epon and baked for 48 h at 60 in Eppendorf tubes. Blocks are then trimmed and thinsectioned at 40 nm, using a microtome (Leica Ultracut; Leica, Wetzlar, Germany). Sections are stained on a grid with 2% aqueous uranyl acetate followed by lead citrate (0.4% in water) and observed at 80 kV in a transmission electron microscope. For some samples, OsO4 postfixation is followed by impregnation with 1% tannic acid in distilled water to enhance visualization of coat proteins.25 Lipid Biochemistry: Measurement of Phosphoinositide Levels For lipid analysis, incubations are performed in the presence of 5 Ci of [ -32P]ATP, at a final ATP concentration of 50 M, and typically in a reaction volume of 50–100 l in 1.5-ml screw-cap plastic Eppendorf tubes. Reactions are stopped by addition of 400 l of crude brain phosphoinositides (20 g/ml; Sigma) in chloroform–methanol (2:1, v/v), followed by 400 l of chloroform and 300 l of 1 M HCl. After vigorous vortexing, phases are separated by brief centrifugation (2 min at high speed in a table-top centrifuge) and the lower organic phase is removed with a Pasteur pipette or a transfer pipette. Care should be taken not to remove any of the upper aqueous phase and protein–aqueous interphase. The aqueous phases are reextracted with an additional 400 l of chloroform, and the organic phases are pooled and dried under a stream of nitrogen. A sample concentrator, which can hold and dry multiple samples in parallel (Dri-Block; Techne, Princeton, NJ), is used for this purpose. Work should be carried out under a fume hood. Phosphoinositides can be separated without further processing by thin-layer chromatography (TLC). Lipids are resuspended in 40 l of chloroform–methanol (2:1 v/v), and a 10-l aliquot is spotted onto a TLC plate [Merck Silica gel 60 TLC plate that has been preimpregnated with potassium oxalate: TLC plates are dipped into a solution of 1% potassium oxalate (Sigma) in methanol–water (1:1, v/v), air dried, and activated for 30 min at 120 before use]. Plates are developed in a solvent system of chloroform–acetone–methanol–acetic acid–water (64:30:24:32:14, by volume) and autoradiographed at 70 for several hours or overnight. Major radioactive spots [PIP2, phosphatidylinositol 25
L. Orci, B. S. Glick, and J. E. Rothman, Cell 46, 171 (1986).
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4-phosphate (PIP), and phosphatidic acid (PA)] are identified by comigration with purified lipid standards (Sigma and Avanti Polar Lipids) and visualized by placing the TLC plate into a tank saturated with iodine vapor [crystals of iodine (Sigma) are added to a TLC tank, the tank is covered with the lid, and iodine vapors are allowed to form by sublimation for 2 h]. Associated radioactivity is measured by quantitative autoradiography with a Phospholmager (Molecular Dynamics-Amersham Biosciences) or by scraping the area of silica gel on the TLC plate that harbors the spot into a scintillation vial, followed by liquid scintillation counting. Alternatively, lipids are deacylated for analysis by strong anionexchange (SAX) high-performance liquid chromatography (HPLC).26 Lipids are deacylated with methylamine as described.27 A mixture of 40% aqueous methylamine (Fluka, Milwaukee, WI)–water–n-butanol– methanol (36:8:9:47, by volume) (2 ml) is added to the dried lipid extract and incubated at 50 for 45 min in a tightly stoppered tube. The mixture is then cooled on ice and evaporated to dryness under vacuum in a Savant SpeedVac (Thermo Savant, Holbrook, NY). A mixture of n-butanol– petroleum ether–ethyl formate (20:40:1, by volume) (2 ml) is added, followed by water (2 ml). After thorough mixing and gentle centrifugation to separate phases, the lower aqueous phase, containing the deacylated lipids, is carefully removed. The organic phase is reextracted with 2 ml of water, and the combined aqueous phases are dried in a SpeedVac. Samples are taken up in water, loaded on a Partisphere SAX ion-exchange column (4.6 125 mm; Whatman, Clifton, NJ), and eluted at a flow rate of 1 ml/ min, using the following gradient profile28: 0–10 min, 0% B; 10–55 min, 0–35% B; and 55–70 min, 35–100% B [buffer B is 1.4 M (NH4)2HPO4, pH 3.7]. Fractions (1 ml) are collected and counted for radioactivity by scintillation counting. Alternatively, radioactivity is measured with an inline liquid scintillation counter (Packard, Meriden, CT).29 Peaks are identified by coelution with standards, most of which must be generated by enzymatic reactions using phosphoinositide kinases and phosphatases.26,30 Phophatidylinositol-[inositol-2-3H(N)] ([3H]PI) and phosphatidylinositol[inositol-2-3H(N)]-4,5-bisphosphate ([3H]PI(4,5)P2) are available from Perkin Elmer Life Sciences.
26
L. A. Serunian, K. R. Auger, and L. C. Cantley, Methods Enzymol. 198, 78 (1991). C. J. Kirk, A. J. Morris, and S. B. Shears, in ‘‘Peptide Hormone Action: A Practical Approach’’ (K. Siddle and J. C. Hutton, eds.), p. 151. IRL Press, Oxford, 1990. 28 I. M. Bird, Methods Mol. Biol. 105, 25 (1998). 29 L. Zhang and I. L. Buxton, Methods Mol. Biol. 105, 47 (1998). 30 X. Zhang, J. C. Loijens, I. V. Boronenkov, G. J. Parker, F. A. Norris, J. Chen, O. Thum, G. D. Prestwich, P. W. Majerus, and R. A. Anderson, J. Biol. Chem. 272, 17756 (1997). 27
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liposomes in molecular cell biology
Concluding Remarks
The methodology described above allows for the investigation of key aspects of lipids and their metabolism in coat dynamics. Among the advantages offered is the possibility to study the role of individual proteins or of cytosol and cytosolic fractions under similar assay conditions. The power of this methodology is further enhanced by the use of proteoliposomes, rather than pure lipid vesicles, to study the cooperativity of proteins and lipids in membrane dynamics. Some limitations of in vitro systems such as this one include the relatively low efficiency, slow kinetics, and lack of physiological ‘‘compartmentalization’’ of the components (e.g., the lack of segregation of proteins and lipids into distinct subcompartments). Clearly, the validity of results obtained by these techniques must be ultimately assessed by studies in situ and by observations from genetic approaches.
[15] Fluorescence Assays for Liposome Fusion By Nejat Du¨zgu¨nes, Introduction
Membrane fusion is a biophysical reaction that is of fundamental importance in biological systems and takes place in such diverse processess as fertilization, viral infection, exocytosis, and intracellular membrane traffic.1 Because their membrane composition can be manipulated readily, liposomes have provided a convenient model system to study the molecular determinants and mechanisms of membrane fusion.2,3 The roles of membrane composition, the ionic environment, and membrane phase state have been elucidated by the use of liposome fusion assays. The role of cytosolic or viral membrane proteins in fusion, as well as the role of fusion activity of peptides derived from viral proteins, have been studied in detail with liposomes.2,3 Liposomes containing fluorescent lipids have been used to study the fusion characteristics of intracellular membranes
1
N. Du¨ zgu¨ nes, and F. Bronner (eds.), ‘‘Membrane Fusion in Fertilization, Cellular Transport and Viral Infection,’’ p. xviii and 384. Academic Press, New York, 1988. 2 N. Du¨ zgu¨ nes, and S. Nir, in ‘‘Liposomes as Tools in Basic Research and Industry’’ (J. R. Philippot and F. Schuber, eds.), p. 103. CRC Press, Boca Raton, FL, 1995. 3 N. Du¨ zgu¨ nes,, in ‘‘Trafficking of Intracellular Membranes: From Molecular Sorting to Membrane Fusion’’ (M. C. Pedroso de Lima, N. Du¨ zgu¨ nes,, and D. Hoekstra, eds.), p. 97. Springer-Verlag, Berlin, 1995.
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