Cell Biology Protocols

PROTOCOL 6.19

Measuring cytochrome c release in isolated mitochondria by Western blot analysis [2, 4]

Equipment

SDS-PAGE apparatus

96-well U-bottom tissue culture plate

Pipette and tips

5% CO2/37 ◦C humidified incubator

Reagents

Freshly isolated mitochondria and S100 (see Protocols 4.7–4.10)

Assay buffer: 220 mM sucrose, 68 mM mannitol, 10 mM Hepes-KOH, 10 mM KCl, 5 mM KH2PO4, 2 mM MgCl2, 0.5 mM EGTA, 5 mM succinate, 2 µM rotenone, pH 7.2

Cyclosporin A (10 mM stock in ethanol)

PT inducers

Cytochrome c antibody (Pharmingen)

Procedure

1.Add 1 mg/ml freshly isolated mitochondria in assay buffer to a 96-well

U-bottom plate with or without S100, in a final volume of 60 µl.

2. Pretreat with any PT inhibitors (i.e. 10 µM cyclosporin A, 30 min) as necessary before incubating the mitochondria with the PT inducers at 37 ◦C for the desired time period, usually within 0–4 h.

3.Centrifuge the plate at 760g, 5 min.

Add 30 µl of supernatant to 7.5 µl of 5× SDS loading buffer. Boil for 5 min then add 10 µl to a standard 15% SDSPAGE gel for Western blotting. Use antibodies recognizing cytochrome c to verify cytochrome c release into the

supernatant and mitochondrial dysfunction. 1

Note

1 Cytochrome c release is a measure of mitochondrial dysfunction but not necessarily PT. Pre-incubating with PT pore inhibitors can confirm that the release of cytochrome c may be due to PT.

PROTOCOL 6.20

Protein import into isolated mitochondria [3, 4]

The influence of anti-apoptotic proteins such as Bcl-2 on the inhibition of PT

can be examined with mitochondria overexpressing Bcl-2. Bcl-2high mitochondria

can be obtained by isolating them from cells over-expressing Bcl-2, or by loading the isolated mitochondria with Bcl-2 using a protein import protocol.

Reagents

Freshly isolated mitochondria and S100 (see Protocols 4.7–4.10 )

Buffer 1: 250 mM sucrose, 10 mM HepesKOH, 2 mM K2HPO4, 5 mM Na succinate, 1 mM ATP, 0.08 mM ADP, 1 mM DTT, pH 7.5

Buffer 2: 20 mM Hepes-KOH, 10 mM KCl, 2.5 mM Mg Cl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH 7.5

Buffer 3: 250 mM sucrose, 10 mM HepesKOH, 1 mM DTT, pH 7.5

Assay buffer: 220 mM sucrose, 68 mM mannitol, 10 mM Hepes-KOH, 10 mM KCl, 5 mM KH2PO4, 2 mM MgCl2, 0.5 mM EGTA, 5 mM succinate, 2 µM rotenone, pH 7.2

Bcl-2 protein

Equipment

37 ◦C water bath

Eppendorfs

Pipettes and tips

Microcentrifuge

Procedure

1.50 µl 1 mg/ml freshly isolated mitochondria in buffer 1 is added to 50 µl

buffer 2 containing 0.125–0.5 µg/ml Bcl-2 protein (higher concentrations may be toxic). Incubate 30 min at 37 ◦C.

2. Layer 100 µl onto a 500	µl cushion
of buffer 3 and centrifuge	at 14 500g,

5 min, 4 ◦C.

3.Resuspend pellet in 10 µl of assay buffer. The mitochondria can then be used in the confocal, fluorometer, cellfree (Western), or FACS protocols.

References

1.Susin, S. A., Larochette, N., Gueskens, M. and Kroemer, G. (2000) Quantitation of the

mitochondrial transmembrane potential in cells and isolated mitochondria. Meth. Enzymol., 322, 205–208.

2. Alimonti, J. B., Shi, L., Baijal, P. K. and Greenberg, A. H. (2001) Granzyme B induces BID-mediated cytochrome c release and mitochondrial permeability transition. J. Biol. Chem., 276, 6974–6982.

3.Goping, I. S., Gross, A., Lavoie, J. N., Nguyen, M., Jemmerson, R., Roth, K., Korsmeyer, S. J. and Shore, G. C. (1998) Regulated targeting of BAX to mitochondria. J. Cell Biol., 143, 207–215.

4.Vande Velde, C., Cizeau, J., Dubik, D., Alimonti, J., Brown, T., Israels, S., Hakem, R. and Greenberg, A. H. (2000) BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.

Molec. Cell. Biol., 20, 5454–5468.

PROTOCOL 6.20

273

5.Castedo, M., Ferri, K., Roumier, T., Metivier, D., Zamzami, N. and Kroemer, G. (2002) Quantitation of mitochondrial alterations associated with apoptosis. J. Immunol. Meth., 265, 39–47.

PROTOCOL 6.21

Formation of ternary SNARE complexes in vitro

Jinnan Xiao, Anuradha Pradhan and Yuechueng Liu

Introduction

SNARE (soluble NSF attachment protein receptor) proteins have been shown to play essential roles in vesicular transport [1]. In nervous systems, v (vesicle)-SNARE and t (target)-SNAREs interact with each other to form a tight ternary complex. The assembly of such a ternary complex allows the opposing membranes to be pulled into close proximity, permitting synaptic vesicle fusion to proceed and release neurotransmitters [2]. The neuronal SNARE core complex includes v-SNARE synaptobrevin (also known as VAMP), t- SNAREs syntaxin and SNAP-25. While both synaptobrevin and syntaxin are membrane proteins with a single C-terminal transmembrane domain, SNAP-25 attaches to membranes via post-translational palmitoylation of its cysteine residues.

Many studies involving vesicular trafficking require reconstitution of the SNARE core complex in vitro. For instance, GST-fusion protein pull-down assay is often used to investigate proteins implicated in regulation of membrane fusion via their interactions with the SNAREs. This short protocol will focus on the isolation of recombinant SNAREs and the reconstitution of the SNARE core complex in vitro (see Figure 6.13).

Equipment

Tabletop microcentrifuge and 1.5 ml microcentrifuge tubes

Figure 6.13 An example of reconstituted SNARE ternary complexes analysed by SDSPAGE and stained with Coomassie Blue. The apparent molecular weights for the SNAREs are: 60 kDa (EST-Syntaxin TM), 32 kDa (6 × His- SNAP-25), and 20 kDa (Synaptobrevin). Prestained molecular weight marker is shown on the left. The molecular weights for the markers are (in kDa): 106, 78, 50, 32, 26, 20

37 ◦C Incubator, with shaker

Sonicator (e.g. Virtis Virsonic 50)

Rotating shaker (e.g. Labquake Shaker)

Reagents

LB bacterial culture media

IPTG (e.g. Sigma, GIBCO)

Glutathione-agarose (e.g. Pierce Chemi-

cals)

Phosphate-buffered saline (PBS)

Thrombin cleavage and capture kit (e.g. Novagen)

6 × His-tagged SNAP-25, in a bacterial expression plasmid vector (e.g. Invitrogen’s pTrcHis, Qiagen’s pQE)

Syntaxin with deleted transmembrane domain (syntaxin TM) in pGEX-2T

Synaptobrevin in pGEX-2T

Procedure

1. Prepare 6 × His-SNAP-25,	GST-
syntaxin TM and GST-synaptobrevin.
The procedure for GST fusion	pro-

tein purification has been described in detail by Smith and Johnson [3]. For affinity purification of 6 × His-tagged proteins using Ni-NTA agarose, follow the manufacturers’ instructions (e.g. Qiagen’s The QIAexpressionist). Dialyse the purified His-SNAP-25 against PBS overnight at 4 ◦C with at least three buffer changes. The purified proteins should be kept at 4 ◦C for shortterm (<5 days) storage. For long-term storage, the proteins can be kept in −20 ◦C freezer (avoid repeated freezing/thawing). The GST-syntaxin TM agarose is kept at 4 ◦C and it is stable for at least 2 weeks.

2.Add 5 units of biotinated thrombin to1 ml slurry of GST-synaptobrevin in PBS containing 0.1% Triton X-100. Mix the sample well by gently reversing the microcentrifuge tube several times

and put the tube onto a rotating shaker. Incubate overnight at 4 ◦C. 1

3.Centrifuge for 2 min in a microcentrifuge at maximum speed at 4 ◦C. Collect the supernatant containing synap-

tobrevin. Wash the agarose beads once with PBS by centrifugation at 4 ◦C. Use 0.4–0.5 ml PBS which is about the same volume as the agarose beads.

PROTOCOL 6.21

275

Combine the supernatants. However, if one wants a more concentrated synaptobrevin preparation, it will be better to keep them separated, since the first supernatant usually has more synaptobrevin.

4. Add at least 30 µl streptoavidin-agarose to the supernatant and incubate for 20–30 min at room temperature. Again, use a rotating shaker for continuous mixing of the sample.

5.Centrifuge the sample for 5 min at room temperature and at maximum speed. Collect the supernatant and dis-

card the pellet. Dialyse the sample against PBS overnight at 4 ◦C with three buffer changes.

6.Aliquot 100 µl of His-SNAP-25 (0.2 mg/ml), 100 µl of synaptobrevin (0.15

mg/ml) and 100 µl agarose slurry of GST-syntaxin TM ( 0.5 mg/ml) and mix in a 1.5 ml microcentrifuge tube

by gently reversing the tube several times. 2

7.Place the mixture on a rotator and incubate overnight at 4 ◦C. 3

8.Centrifuge the sample in a microcen-

trifuge at maximum speed for 2 min at 4 ◦C. Discard the supernatant (or keep it for further experiments). Wash the agarose resin with 1 ml PBS three times at 4 ◦C by centrifugation.

9.The agarose beads containing ternary complexes can be boiled in SDS sample buffer and directly analysed by SDSPAGE, or immunoblot analysis using specific antibodies against the SNAREs. Alternatively, the complexes can be eluted with 5 mM reduced glutathione for further experiments.

Notes

This procedure will take approximately 48 h (not including the preparation of

	276	IN VITRO TECHNIQUES
	His-SNAP-25, GST-syntaxin TM, and			overnight incubation at 4 ◦C seems to
	GST-synaptobrevin).			produce more complexes.
	1	Triton X-100 is helpful in releas-	References
		ing the synaptobrevin from the GST
		agarose.	1.	Jahn R. and Sudhof¨ T. C. (1999) Ann. Rev.
	2	The molar ratio for the ternary com-
				Biochem., 66, 863–911.
		plex in vivo is 1 : 1 : 1.	2.	Brunger A. T. (2001) Curr. Opin. Struct. Biol.,
	3	Incubation time can be shorter, e.g.		11, 163–173.
			3.	Smith, D. B. and Johnson, K. (1988) Gene,
		1 h at room temperature. However,		67, 31–40.

PROTOCOL 6.22

In vitro reconstitution of liver endoplasmic reticulum

Jacques Paiement and Robin Young

Introduction

In vitro reconstitution is among the most important and widely used strategies in studying membrane traffic [1]. We describe here an in vitro assay which allows study of the reconstitution of rat liver endoplasmic reticulum. Membrane derivatives of liver endoplasmic reticulum (ER) are incubated under specific conditions leading to membrane fusion and the formation of large ER membrane complexes. Following incubation the reconstituted ER membranes are studied by electron microscopy. Depending on the type of ER derivative (i.e. rough or smooth?) used in the reconstitution assay the assembly products will exhibit different structural properties and can be distinguished based on morphological criteria. Examples are described below.

Reagents

ATP (100 mM) to be stored in aliquots in powder form in a dessicator at −80 ◦C (High grade. Sigma Chemicals cat. no. A-7699)

Complete Protease Inhibitor Cocktail (1 tablet/ml = 50 × concentrated) stored at −20 ◦C (Roche Applied Science cat. no. 1697498)

Creatine Phosphokinase (1 U/µl) to be stored in aliquots in powder form in a dessicator at 4 ◦C (Roche Applied Science cat. no. 127566)

DTT (250 mM) to be stored in aliquots in powder form in a dessicator at 4 ◦C (Sigma Chemicals cat. no. D-0632)

GTP (25 mM) to be stored in aliquots in powder form in a dessicator at −80 ◦C (High grade. Sigma Chemicals cat. no. G-8877)

MgCl2 (125 mM), filtered on Whatman no. 42 ashless filter to remove any particulate matter in solution

Phosphocreatine (50 mM) to be stored in aliquots in powder form in a dessicator at 4 ◦C (Roche Applied Science cat. no. 621714)

Sucrose-Imidazole (0.25 mM sucrose, 3 mM imidazole, pH 7.4), filtered on Whatman no. 42 ashless filter to remove any particulate matter in solution

tER derived membranes (see ‘Fractionation of rough and smooth microsomes from rat liver homogenates’, Protocol 6.22 for preparation)

Tris-HCl buffer (200 mM, pH 7.4), filtered on Whatman no. 42 ashless filter to remove any particulate matter in solution

278 IN VITRO TECHNIQUES

Equipment

Polypropylene conical microcentrifuge tubes (1 ml)

Incubation bath, with shaker (37 ◦C)

Procedure 1

1. Prepare test samples and all stock solutions on ice (ensure all solutions are at least 4 ◦C prior to preparation of medium).

2. Prepare stock solutions of DTT, creatine phosphokinase and phosphocreatine. Remove protease inhibitor stock from −20 ◦C and put on ice to thaw.

3.The following reagents are added to each tube to produce a final incubation mixture of 250 µl:

•125 µl Tris buffer to produce final concentration of 100 mM,

•10 µl MgCl2 to produce final concentration of 5 mM,

•1.8 µl creatine phosphokinase to produce final concentration of 7.3 U/ml,

•10 µl phosphocreatine to produce final concentration of 2 mM,

•10 µl DTT to produce final concentration of 0.1 mM,

•5 µl protease inhibitor cocktail to produce final concentration of 1×.

4.Take membranes out of −80 ◦C and place on ice.

5.Add sucrose-imidazole (amount added is calculated to bring final incubation volume to 250 µl, after addition of all reagents).

6.Take GTP and ATP out of −80 ◦C and keep on ice.

7.Prepare stock ATP and add 5 µl to each tube to produce a concentration of 2 mM.

8.Prepare stock GTP and add 10 µl to each tube to produce a concentration of 1 mM.

9.Verify membranes are thawed and add 150 µg of membrane protein to each tube.

10.Incubate at 37 ◦C for 90 min with constant oscillation, with additional manual agitation every half-hour.

11.At end of 90 min add 5 and 10 µl of ATP and GTP respectively. 2

12.Continue incubation for an additional 90 min at 37 ◦C with constant oscillation, with additional manual agitation of individual tubes every half-hour.

Comments

The incubation medium described here contains factors that will stimulate fusion of membranes of classical rough microsomes [2, 3], fusion of membranes of lowdensity rough microsomes [4–7], fusion of membranes of smooth microsomes [4–7] and fusion of membranes of the nuclear envelope [8]. The reconstituted elements formed as a consequence of membrane fusion have been characterized by a variety of biochemical, histochemical and immunocytochemical procedures [2–8]. The factors involved in the transformation of various ER membrane subdomains are summarized in a review [9].

Figure 6.14 shows in vitro reconstituted transitional endoplasmic reticulum (tER). Derivatives of liver tER were incubated using the specific incubation conditions described above. Following incubation the membrane sample was fixed in suspension and processed as described previously [7]. The two domains of the tER are recognizable based on morphological criteria. The

PROTOCOL 6.22

279

Figure 6.14 Micrograph of reconstituted hepatocyte ER showing a membrane complex consisting of branching and anastomosing smooth tubules in continuity with peripheral rough ER cisternae. The arrow points to closely apposed membranes of two rough ER cisternae. One cisterna is dilated and contains the arrow and the arrowheads, the other cisterna is flattened and tightly bound at the periphery of the dilated cisterna. Ribosomes are mainly observed between the two cisternae. Arrowheads point to ribosomes. f, fenestrations; t, smooth tubes.

rER domain consists of parallel cisternae limited by ribosome-studded membranes and the sER domain consists of a network of interconnecting tubules limited by smooth membranes devoid of attached ribosomes.

Notes

This procedure will take approximately 4 h.

1 Similar cell-free incubation conditions for reconstitution of tER have previously been summarized and include variations in concentration of certain reagents and other parameters of incubation [4–6].

2 This will compensate for potential nucleotide hydrolysis during incubation by membrane nucleotidases.

2.Paiement, J. and Bergeron, J. J. M. (1983) J. Cell Biol., 96, 1791–1796.

3.Paiement, J., Beaufay, H. and Godelaine, D. (1980) J. Cell Biol., 86, 29–37.

4.Roy, L., Bergeron, J. J. M., Lavoie, C., Hendriks, R., Gushue, J., Fazel, A., Pelletier, A., Morre,´ D. J., Subramaniam, V. N., Hong, W. and Paiement, J. (2000) Mol. Biol. Cell., 11, 2529–2542.

5.Lavoie, C. Chevet, E., Roy, L., Tonks, N. K., Fazel, A., Posner, B. I., Paiement, J. and Bergeron, J. J. M. (2000) Proc. Nat. Acad. Sci., 25, 13 637–13 642.

6.Lavoie, C., Paiement, J., Dominguez, M., Roy,

L., Dahan, S., Gushue,	J. N.	and	Berg-
eron, J. J. M. (1999) J.	Cell	Biol.,	146,
285–299.

7.Lavoie, C., Lanoix, J., Kan, F. W. K. and Paiement, J. (1996) J. Cell Sci., 109, 1415– 1425.

8.Paiement, J. (1984) Exp. Cell Res., 151, 354–366.

9.Paiement, J. and Bergeron, J. (2001) Biochem. Cell Biol., 79, 587–592.

References

1.Mellman, I. and Warren, G. (2000) Cell, 100, 99–112.

PROTOCOL 6.23

Asymmetric incorporation of glycolipids into membranes and detection of lipid flip-flop movement

Felix´ M. Goni*,˜ Ana-Victoria Villar, F.-Xabier Contreras

and Alicia Alonso

Introduction

Assemblies of glycosphingolipids and cholesterol are believed to form microdomains (‘rafts’) with specific functions in membrane traffic and signal transduction. Flask-shaped 60 nm invaginations of the cell plasma membrane, termed caveolae, may be the sites at which these domains cluster and selfstabilize [1, 2]. Rafts and caveolae recruit specific membrane proteins which are implicated in cell signalling [3, 4].

These domains have a particular asymmetric disposition as glycosphingolipids and glycosylphosphatidylinositol (GPI)- linked proteins locate preferentially in the outer leaflet [1, 2, 5, 6]. While artificial lipidic vesicles (liposomes) have been extremely useful in the understanding of multiple aspects of membrane structure and function, no simple technology was available for the reliable preparation of liposomes with asymmetrically distributed lipids. In view of the structural and functional importance of rafts and caveolae, we have recently described the preparation of liposomes with GPI and/or glycosphingolipids located preferentially in the outer monolayer [7].

Cell membrane asymmetry, however, is not a static phenomenon. Instead, it arises from a series of concerted transmembrane

movements, leading to a time-invariant distribution of bilayer components. Lipid asymmetry in particular is known to be altered in physiological or pathological events such as recognition by phagocytes, blood coagulation, or apoptosis [8]. The collapse of lipid asymmetry is often known as lipid ‘scrambling’.

Sphingomyelinases cleave the sphingophospholipid sphingomyelin yielding phosphorylcholine and ceramide [9, 10]. In turn, ceramide may alter a number of membrane properties: it increases lipid order, gives rise to ceramide-rich separate domains, and destabilizes the lamellar structures, inducing membrane permeabilization and membrane fusion [4, 11–14].

In this context, we have developed an assay to test transmembrane (‘flip-flop’) lipid movement induced by sphingomyelinase via ceramide formation [15]. Both the preparation of asymmetric vesicles and the flip-flop assay are described below.

A. Asymmetric incorporation of glycolipids into liposomal membranes

Materials

GM3 monosialoganglioside (e.g. Matreya Inc. or other supplier) 1