- Email: [email protected]
[21] Analysis of sphingomyelin hydrolysis in caveolar membranes
184
SPHINGOLIPIDMETABOLISM
[211
Automated Assays Multiple simultaneous liquid handlers can be utilized to greatly increase the efficiency of assaying large numbers of samples. Dispensing test compounds, enzyme, assay mixture, BSA, TCA, scintillation cocktail, and stopped reaction supernatants to multiwell plates via automated liquid handlers greatly reduces time, effort, and errors when assaying many samples. Automation of all aspects of the assay may be possible but is not necessarily practical or efficient. Sealing and mixing plates must be performed carefully to minimize sample loss before quantitation and thus are best performed manually. In our laboratory, an assay automated at all but the sealing and mixing steps allowed screening of several hundred thousand compounds in the search for inhibitors of the N-SMase. Acknowledgment The authors thank Dr. Robert M. Bell for valuable suggestions in the development of the assay and for his support of this work.
[21] A n a l y s i s o f S p h i n g o m y e l i n H y d r o l y s i s i n Caveolar Membranes
By
R I C K T . DOBROWSKY a n d VALESWARA R A O G A Z U L A
Introduction Caveolae and caveolae-related domains (CRDs) are distinct, specialized regions of the plasma membrane that share a somewhat similar lipid constitution enriched in cholesterol, sphingomyelin (SM), phosphatidylinositols, and glycosphingolipids.1 4 CRDs are likely to be ubiquitous entities in most if not all cells, 5'6 whereas caveolae are not necessarily present in all cell types. 7 Caveolae are nonclathrin-coated invaginations of the plasma membrane that possess a distinct flask-like morphology that arises from the i D. A. Brown and J. K. Rose, Cell 68, 533 (1992). 2 j. Liu, P. Oh, T. H o m e r , R. A. Rogers, and J. E. Schnitzer, J. Biol. Chem. 272, 7211 (1997). 3 L. J. Pike and L. Casey, J. Biol. Chem. 271, 26453 (1996). 4 M. P. Lisanti, P. E. Scherer, Z. Tang, and M. Sargiacomo, Trends" Cell BioL 4, 213 (1994). s D. A. Brown and E. London, Biochem. Biophys. Res. Commun. 240, 1 (1997). 6 R. J. Schroeder, S. N. Ahmed, Y. Zhu, E. London, and D. A. Brown, J. BioL Chem. 273, 1150 (1998). 7 A. Gorodinsky and D. A. Harris, J. Cell Biol. 129, 619 (1995).
METHODS IN ENZYMOLOGY,VOL. 311
Copyright © 1999by Academic Press All rights of reproduction in any form reserved. 0076-6879/99 $3(/.00
[21]
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association of lipids within these domains with the structural protein of caveolae, caveolin,s Indeed, expression of caveolin in cells lacking caveolae, but possessing CRDs, leads to the formation of morphologic caveolae. These results suggest that CRDs may be viewed as precaveolae. 9 Many experimental approaches to the isolation of caveolae and CRDs have exploited the detergent insolubility of these domains, which is in large part due to the sphingolipid composition of these membranes. Relative to phospholipids, sphingolipids are more hydrophobic, undergo more hydrogen bonding, and tend to cluster within cell membranes, l° Consequently, these regions and their associated proteins tend to be insoluble in nonionic detergent at low temperature and display a low buoyant density in sucrose gradients. It is the lipid composition of these regions and not the presence of caveolin or other proteins that is primarily responsible for their detergent insolubility.5 This is exemplified by the detergent solubility of caveolin prior to its interaction with lipids within CRDs, which then renders caveolin detergent insoluble, l Caveolae and CRDs have emerged as potential sites for the sequestering and integration of signal transduction pathways.9 Numerous signaling molecules such as G-proteins, ras, protein kinase C isoforms, nitric oxide synthase, and src-related tyrosine kinases have been demonstrated to localize to caveolae or CRDs. 9 Further, several receptor systems have also been demonstrated to signal from caveolae, including the epidermal growth factor receptor, 12platelet-derived growth factor receptor,13 and the low-affinity neurotrophin receptor, p75NTR]4 Evidence has implicated caveolae and CRDs as sites for ligand-induced SM hydrolysis. ~4"ls Interestingly, although the Mg2*-dependent neutral sphingomyelinase (SMase) is localized primarily to the plasma membrane] 6 an acid SMase activity has been reported to localize to caveolae. ~5 Thus, caveolae and CRDs are emerging as potential sites for organizing and sequestering molecular components involved in regulating receptor-linked SM metabolism. Further, it is likely that caveolae may be critical sites for
s S. Li, K. S. Song, S. S. Koh, A. Kikuchi, and M. P. Lisanti, Z BioL Chem. 271, 28647 (1996). 9 T. Okamoto, A. Schlegel, P. E. Scherer, and M. P. Lisanti, J. Biol. Chem. 273, 5419 (1998). 10 T. Harder and K. Simons, Curr. Opin. Cell Biol. 9, 534 (1997). 1~ M. Murata, J. Peranen, R. Schreiner, F. Wieland, T. V. Kurzchalia, and K. Simons, Proc. Natl. Acad. Sci. U.S.A 92, 10339 (1995). ~2 p. Liu, Y. Ying, Y. G. Ko, and R. G. W. A n d e r s o n , J. Biol. Chem. 271, 10299 (1996). 13 C. Mineo, G. L. James, E. J. Smart, and R. G. W. A n d e r s o n , J. Biol. Chem. 271,11930 (1996). t~ T. R. Bilderback, R. J. Grigsby, and R. T. Dobrowsky, J. Biol. Chem. 272, 10922 (1997). t5 p. Liu and R. G. W. A n d e r s o n , Z Biol. Chem. 270, 27179 (1995). 16 M. W. Spence, J. Wakkary, and H. W. Cook, Biochem. Biophys. Acta 719, 162 (1982).
186
SPHINGOLIPIDMETABOLISM
[211
interactions between tyrosine kinase and sphingolipid-signaling pathways. 17 Understanding the biochemical mechanisms affecting interactions between sphingolipid and tyrosine kinase signaling pathways in caveolae represents a novel and emerging area of cell regulation. This article describes some of the commonly used procedures for isolating caveolar m e m b r a n e s and examining ligand-dependent SM turnover. Additionally, we provide some cautionary notes on the use of these procedures in the identification of c a v e o l a e / C R D s as localized sites for signal transduction and for the enrichment of various signaling molecules.
M e a s u r e m e n t of S p h i n g o m y e l i n H y d r o l y s i s in C a v e o l i n - E n r i c h e d M e m b r a n e s from C u l t u r e d Cells Detergent Extraction
This procedure exploits the detergent insolubility of caveolae in Triton X-100 at low t e m p e r a t u r e and the low buoyant density of this m e m b r a n e fraction in sucrose gradients, is The purification of caveolae is monitored by assaying for the presence of caveolin, the m a r k e r protein for caveolae. Although this procedure readily separates caveolin-enriched m e m b r a n e s (CEMs) from noncaveolar m e m b r a n e s (NCMs), it does not necessarily produce pure caveolar vesicles. Therefore, it is p r e m a t u r e to call this m e m brane fraction " c a v e o l a e " without morphologic documentation of the homogeneity of the m e m b r a n e vesicles. Similarly, this procedure will also purify C R D s in cells lacking caveolae. It is important to note that C R D s and caveolae may coexist in some cells and that these domains will likely copurify in the following procedure. Although these domains may be separated using cationic silica beads, 19 the current lack of availability of these beads renders this separation problematic.
Buffers Phosphate-buffered saline (PBS) MES-buffered saline (MBS): 25 m M MES, p H 6.5, 5 m M E D T A , and 150 m M NaC1
17C. Wu, S. Butz, Y. Ying, and R. G. W. Anderson, J. Biol. Chem. 272, 3554 (1997). ~8M. P. Lisanti, P. E. Scherer, J. Vidugririene, Z. Tang, A. Hermanowski-Vosatka, Y.-H. Tu, R. F. Cook, and M. Sargiacomo, J. Cell Biol. 126, 111 (1994). 19j. E. Schnitzer, D. P. Mclntosh, A. M. Dvorak, J. Liu, and P. Oh, Science 269, 1435 (1995).
[21 ]
CAVEOLARSPHINGOMYEL1NHYDROLYSIS
187
MBST: MBS plus 1% Triton X-100, 1 mM Pefabloc or phenylmethylsulfonyl fluoride (PMSF), 10 /xg/ml leupeptin, aprotinin, and bestatin each 80, 35, and 5% (w/v) sucrose in MBS Procedure
Metabolic Labeling Prepare two 15-cm dishes of cells and allow them to reach at least 70% confluency. Four or more 10-cm dishes may also be substituted. If liganddependent SM hydrolysis is to be measured, label the cells for 3 days in medium containing 0.5/zCi/ml [3H]choline chloride. Other isotopes such as [3H]palmitate or [14C]serine (with serine-deficient medium) may also be used if desired. Because previous work has suggested that the ligand-sensitive pool of SM is the last to incorporate the radiolabel, 2° it is important to incubate the cells for at least 48 to 72 hr with the desired radioisotope to reach metabolic equilibrium. Although these times are influenced by the growth rate of particular cell lines, 48 hr is usually sufficient to reach metabolic equilibrium for cells with a rapid doubling time, whereas longer incubation times may be required for cells with prolonged population doublings. After the end of the incubation, dispose of the radioactive medium, wash the monolayers twice with fresh serum-free medium or PBS, and place the cells in serum-free medium for at least 4 hr. This resting period is important as simple medium changes can dramatically affect sphingolipid levels in cultured cells. 2~
Isolation of CEMs Treat the cells with the ligand of choice and, following treatment, aspirate the medium rapidly and wash the cells twice with ice-cold PBS. After aspirating the PBS, add 2 ml of ice-cold MBST and quickly scrape the cells from the plate with a cell scraper. Care should be exercised that the pH of the MBST is adjusted to 6.5 because neutral pH buffers give lower yields of caveolar membranes. 2~ Transfer the lysate to a 10-ml homogenization tube and set on ice for at least 15-20 rain. Homogenize the samples using a tight-fitting Teflon pestle (20 strokes), maintaining the tube on ice at all times. Transfer 2 ml of the sample to a 12-ml ultracentrifuge tube that can be accommodated within the buckets of a SW41 or comparable rotor. 2~ C. M. Linardic and Y. A. H a n n u n , J. Biol. Chem. 269, 23530 (1994). ~-i E. R. Smith, P. L. Jones, J. M. Boss, and A. H., Merrill, Jr., J. Biol. Chem. 272, 5640 (1997). 22 M. Sargiacomo, M. Sudol, Z. Tang, and M. P. Lisanti, J. Cell Biol. 122, 789 (1993).
]88
SPHINGOLIPID METABOLISM
[21]
Adjust the extract to 40% sucrose by the addition of 2 ml of 80% sucrose in MBS lacking Triton X-100. Vortex the tube to mix the solutions thoroughly. Form a discontinuous sucrose gradient by carefully overlaying this solution with 4 ml of the 35% sucrose in MBS. A clear interface should be evident between the 35 and 40% sucrose layers. Next, carefully overlay the 35% sucrose layer with 4 ml of the 5% sucrose in MBS. The difference in density results in a readily visible interface. A linear 5-35% sucrose gradient may also be substituted for the discontinuous gradient. Prepare a balance tube if necessary using the same solutions (substitute 2 ml of MBS for the lysate) and centrifuge the samples at 39,000 rpm in a SW41 rotor for 16-18 hr at 4°. Alternatively, a minimal centrifugation time of 3 hr may be used because it also gives satisfactory recovery of CEMs. s We typically collect 15 x 0.8ml fractions beginning from the top of the gradient. An aliquot of each fraction may be used to determine the distribution of total radiolabel over the gradient (Fig. 1A). Additional aliquots of each fraction are used for the determination of protein content. Because total cellular protein distributes primarily to high density regions of the gradient (Fig. 2, closed circles), reliable protein measurements can be obtained using 0.1-0.2 ml of fractions 3-8 and 0.02-0.05 ml of fractions 9-15. CEMs are typically recovered as a visible band of material located at the interface of the 5 and 35% sucrose layers, fractions 4-6.~4 Alternatively, CEMs may be located by light scattering (600 nm). The presence of CEMs is indicated by comigration of caveolin with these gradient fractions. This is performed easily by S D S - P A G E and immunoblot analysis using any of several commercially available caveolin antibodies. If desired, the CEMs can be concentrated by pooling together the fractions, diluting in MBS, and concentrating the membranes by centrifugation at 100,000g for 30 min. However, some investigators concentrate the membranes by centrifugation in a microfuge. 22 Caveolin is well resolved from the bulk of cellular protein as over 95% of the total cellular protein is typically recovered in NCMs (fractions 10-15) (Fig. 2, closed circles). However, depending on the cell type, about 50-70% of the cellular SM is recovered in the CEMs, 14 whereas the remainder distributes primarily to fractions 12 15 representing bulk plasma membrane (Fig. 1A). Thus, relative to cellular protein, SM is highly enriched in CEMs (Fig. 2, closed squares).
Advantages and Disadvantages A major advantage of preparing CEMs using detergent insolubility as a criterion is the relative ease of the procedure. However, a cautionary note is that slight alterations in the protein-to-detergent ratio may alter
[9,1 ]
CAVEOLAR SPHINGOMYELIN HYDROLYSIS 40
A 0
35
189
Total Choline cpm SM cpm
~ o
30 5© 25
E 2o ~ 15 10
/•0
5
0 0
0
2
4
6
8
10
12
E) ;)
14
16
Fraction # 40: 35
B i
-" :'
Total Choline cpm SM cpm
30 O
25
10 i
t 0 ~H() 0 2
c~ ,-- ~?C .... 4 6 8 10
, ~ , 12 14
16
Fraction # FIG. 1. Distribution of [3H]SM following detergent (A) and nondetergent extraction (B) and centrifugation through discontinuous sucrose gradients. Cells were fractionated as described in the text and the amount of [3H]SM in each fraction was plotted as a percentage of the total [3HJSM recovered in the gradient. The distribution of [3H]SM relative to the tolal amount of radioactivity present is also shown.
the solubility of caveolar components. 23 Because the mechanisms whereby receptors couple to both neutral or acid SMases are poorly understood, loss of critical c o m p o n e n t s due to changes in this ratio may cause inconsistent results. In this regard, detergent extraction results in the selective loss 2~ A. Uinenbogaard, Y. Ying, and E. J. Smart, J. Biol. Chem. 273, 6525 (1998).
190
SPHINGOLIPID
[21]
METABOLISM
• 25
70 O @
i
i i
20
60 50
~D
.¢..a
o
~
o
-4,,,,o
=
15
© ¢z,
40 30
10
d
-4...a
20
5 10
m
~D
.+.a
0
0 0
2
4
6
8
10
12
14
~D ~D
6
Fraction # FIG. 2. Enrichment of [3H]SM in CEMs relative to cellular protein following detergent (Q, II) or nondetergent extraction (O, [3) and centrifugation through discontinuous sucrose gradients. Cells were fractionated as described in the text and the amount of protein (O, O) in each fraction was plotted as a percentage of the total recovered in the gradient. The amount of SM in the gradient fractions was determined in the same manner and the ratio of SM/ protein plotted (ll, [3).
of lipid-modified proteins from caveolae, z4 An additional concern is that this procedure does not result in a homogeneous preparation of caveolar vesicles. Whereas isolated caveolae are composed primarily of vesicles ranging in diameter from about 50 to 80 nm, detergent extraction likely results in a very heterogeneous population of vesicles, many of which may be derived from the plasma membrane proper. 25 Thus, although useful for indicating whether ligand-induced SM hydrolysis may localize to detergentinsoluble membrane domains, it is not a definitive demonstration of a caveolar compartmentalization of this response. 24 K. S. Song, S. Li, T. Okamoto, L. A. Quilliam, M. Sargiacomo, and M. P. Lisanti, J2 Biol. Chem. 271, 9690 (1996). 25 R. V. Stan, W. G. Roberts, D. Predescu, K. Ihida, L. Saucan, L. Ghitescu, and G. E. Palade, Mol. Biol. Cell 8, 595 (1997).
[21 ]
CAVEOLARSPHINGOMYELINHYDROLYSIS
191
Detergent-Free Extraction Because detergent can preferentially solubilize lipid-modified caveolaeassociated proteins, several detergent-free methods have been developed to avoid this problem. The sodium carbonate procedure is a variation to that described earlier. 24 Buffers MBS containing 0.5 M sodium carbonate, pH 11 90% sucrose in MBS 5 and 35% sucrose in MBS containing 250 mM sodium carbonate Procedure
Isolation of CEMs Cells are scraped into 2 ml of ice-cold MBS containing 0.5 M sodium carbonate, pH 11, producing a very viscous cell lysate. The cells are then homogenized using 20 strokes in a Dounce homogenizer, three 10-sec pulses with a Polytron tissue grinder (half-maximal speed), and three 20-sec bursts with a microtip sonicator to break up the plasma membrane and detach the caveolae. The lysate is then brought to 45% sucrose containing 250 mM sodium carbonate by the addition of 2 ml of 90% sucrose in MBS (prepare the 90% sucrose by dissolving the sugar with mild heating). The 45% sucrose is overlaid with 4 ml of 35% sucrose in MBS followed by 4 ml of 5% sucrose in MBS (both containing 250 mM sodium carbonate). The tubes are then centrifuged as described earlier. Under this protocol, CEMs also migrate in fractions 4-6 and noncaveolar membranes (NCMs) comprise the bulk of fractions 10-15.
Advantages and Disadvantages Similar to detergent extraction, the sodium carbonate extraction procedure is easy and reliably separates CEMs from NCMs and results in a similar protein profile (Fig. 2, open circles). Additionally, SM is similarly distributed over the gradient (Fig. 1B) and enriched in CEMs relative to protein (Fig. 2, open squares). Importantly, the recovery of CEMs is somewhat improved over the detergent extraction method and does not remove lipid-modified proteins from these membrane domains. However, sodium carbonate has been used extensively to determine if proteins strongly attach to membranes. Thus, this procedure may remove loosely attached protein components that may impair the development of in vitro approaches to
192
SPHINGOLIPIDMETABOLISM Control
4
5
6
100 ng/ml NGF 7
4
5
6
7
10 ng/ml NT-3 4
5
6
10 ng/ml IL- l I3 7
,
' QO, i flO,,
• 44-
- 4 - .
SM, cpm
24294
Percent Control
- 6 . .
[2 1]
4
5
6
7 Frac.#
- a.
16977
13146
11048
69.9
54.1
45.4
F~G. 3. Hydrolysis of [3H]SM in CEMs. Cells were labeled with [3H]choline and treated with the indicated ligands. CEMs were isolated and [3H]SM content determined by TLC. After normalization to total protein in each fraction the percentage SM hydrolysis was determined. PC, phosphatidylcholine. determine h o w receptors m a y couple to e n z y m e s regulating SM lism in CEMs. M o r e o v e r , because the extraction is p e r f o r m e d in the p H range of 9-11, the direct m e a s u r e m e n t of e n z y m e s in sphingolipid metabolism is problematic. T h e m e m b r a n e s dialyzed against buffer to r e m o v e the sodium c a r b o n a t e and activity assessed.
metabotypically involved m a y be enzyme
A s s a y of S p h i n g o m y e l i n C o n t e n t of C a v e o l i n - E n r i c h e d M e m b r a n e s O n c e C E M s have been isolated, we m e a s u r e SM levels in the m e m b r a n e s by thin-layer c h r o m a t o g r a p h y ( T L C ) or by using the bacterial SMase assay. 14 Bacterial SMase Assay
Transfer a 0.4-ml aliquot of each fraction to a screw-cap glass tube and p e r f o r m a Bligh and D y e r lipid extraction. 26 Quantitation of SM is d e t e r m i n e d as described. 27 E v a p o r a t e one-half of the organic layer and 26E. G, Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 2vS. Jayadev, C. M. Linardic, and Y. A. Hannum, J. Biol. Chem. 269, 5757 (1994).
[21]
CAVEOLAR SPH1NGOMYELINHYDROLYSIS
193
solubilize the residue in 0.05 ml of 200 m M Tris-HC1, pH 7.5, 20 m M MgC1 containing 1% Triton X-100. Add 0.05 ml of a 2 U/ml solution of Streptomyces sp. SMase (diluted in 10 m M Tris-HCl, p H 7.5) and incubate the tubes for 2 hr at 37 °. Terminate the reaction by adding 1.5 ml of c h l o r o f o r m : m e t h a n o l (2:1). Add 0.2 ml of water and recover the released [3H]choline in the aqueous layer after centrifugation. Normalize the amount of SM hydrolyzed to the protein content of each fraction. Because the bacterial SMase quantitatively hydrolyzes any SM present in the s a m p l e s ligand-induced SM hydrolysis is revealed by a decrease in the amount of SM present in CEMs from treated cells versus control cells.
Thin-Layer Chromatography of SM Alternatively, SM hydrolysis may be measured by TLC. Numerous solvent systems exist for the separation of SM from other lipids, but two common systems used in our laboratory are chloroform : methanol : glacial acetic acid : water (65 : 35 : 8 : 4) or chloroform : methanol : 2 N NH4OH (65 : 35 : 8). Fit a T L C chamber with a paper wick (Whatman filter paper) and place enough solvent into the chamber to cover about the bottom 1-2 cm of the T L C plate. Do not individually add the solvent components to the chamber, mix them in a separate flask and add the solution to the chamber. Next, allow the chamber to equilibrate for at least 3-4 hr prior to placing the TLC plate in the chamber. If two T L C plates are to be developed, a single chamber may be used. If this is the case, place both plates into the chamber together and ensure that they do not touch one another and are adequately immersed in the solvent. Following the Bligh and Dyer lipid extraction, evaporate 1.5 ml of the chloroform extract and resuspend the residue in 0.03 ml of chloroform. Apply 0.02 ml to the silica gel and dry the spot thoroughly. Develop the plate to about 2 cm from the top and allow the solvent to evaporate in a fume hood. Spray the silica gel with an autoradiography enhancer such as En3Hance (spray liberally but do not create runs), wrap the plate in plastic wrap, and expose to film for 2-3 days at - 8 0 °. Figure 3 shows a typical result for the analysis of SM hydrolysis from CEMs by this method from p75NrR-NIH 3T3 cells labeled with [3H]choline and treated with several agonists.
SPHINGOLIPIDMETABOLISM
[211
Automated Assays Multiple simultaneous liquid handlers can be utilized to greatly increase the efficiency of assaying large numbers of samples. Dispensing test compounds, enzyme, assay mixture, BSA, TCA, scintillation cocktail, and stopped reaction supernatants to multiwell plates via automated liquid handlers greatly reduces time, effort, and errors when assaying many samples. Automation of all aspects of the assay may be possible but is not necessarily practical or efficient. Sealing and mixing plates must be performed carefully to minimize sample loss before quantitation and thus are best performed manually. In our laboratory, an assay automated at all but the sealing and mixing steps allowed screening of several hundred thousand compounds in the search for inhibitors of the N-SMase. Acknowledgment The authors thank Dr. Robert M. Bell for valuable suggestions in the development of the assay and for his support of this work.
[21] A n a l y s i s o f S p h i n g o m y e l i n H y d r o l y s i s i n Caveolar Membranes
By
R I C K T . DOBROWSKY a n d VALESWARA R A O G A Z U L A
Introduction Caveolae and caveolae-related domains (CRDs) are distinct, specialized regions of the plasma membrane that share a somewhat similar lipid constitution enriched in cholesterol, sphingomyelin (SM), phosphatidylinositols, and glycosphingolipids.1 4 CRDs are likely to be ubiquitous entities in most if not all cells, 5'6 whereas caveolae are not necessarily present in all cell types. 7 Caveolae are nonclathrin-coated invaginations of the plasma membrane that possess a distinct flask-like morphology that arises from the i D. A. Brown and J. K. Rose, Cell 68, 533 (1992). 2 j. Liu, P. Oh, T. H o m e r , R. A. Rogers, and J. E. Schnitzer, J. Biol. Chem. 272, 7211 (1997). 3 L. J. Pike and L. Casey, J. Biol. Chem. 271, 26453 (1996). 4 M. P. Lisanti, P. E. Scherer, Z. Tang, and M. Sargiacomo, Trends" Cell BioL 4, 213 (1994). s D. A. Brown and E. London, Biochem. Biophys. Res. Commun. 240, 1 (1997). 6 R. J. Schroeder, S. N. Ahmed, Y. Zhu, E. London, and D. A. Brown, J. BioL Chem. 273, 1150 (1998). 7 A. Gorodinsky and D. A. Harris, J. Cell Biol. 129, 619 (1995).
METHODS IN ENZYMOLOGY,VOL. 311
Copyright © 1999by Academic Press All rights of reproduction in any form reserved. 0076-6879/99 $3(/.00
[21]
CAVEOLARSPHINGOMYELINHYDROLYSIS
185
association of lipids within these domains with the structural protein of caveolae, caveolin,s Indeed, expression of caveolin in cells lacking caveolae, but possessing CRDs, leads to the formation of morphologic caveolae. These results suggest that CRDs may be viewed as precaveolae. 9 Many experimental approaches to the isolation of caveolae and CRDs have exploited the detergent insolubility of these domains, which is in large part due to the sphingolipid composition of these membranes. Relative to phospholipids, sphingolipids are more hydrophobic, undergo more hydrogen bonding, and tend to cluster within cell membranes, l° Consequently, these regions and their associated proteins tend to be insoluble in nonionic detergent at low temperature and display a low buoyant density in sucrose gradients. It is the lipid composition of these regions and not the presence of caveolin or other proteins that is primarily responsible for their detergent insolubility.5 This is exemplified by the detergent solubility of caveolin prior to its interaction with lipids within CRDs, which then renders caveolin detergent insoluble, l Caveolae and CRDs have emerged as potential sites for the sequestering and integration of signal transduction pathways.9 Numerous signaling molecules such as G-proteins, ras, protein kinase C isoforms, nitric oxide synthase, and src-related tyrosine kinases have been demonstrated to localize to caveolae or CRDs. 9 Further, several receptor systems have also been demonstrated to signal from caveolae, including the epidermal growth factor receptor, 12platelet-derived growth factor receptor,13 and the low-affinity neurotrophin receptor, p75NTR]4 Evidence has implicated caveolae and CRDs as sites for ligand-induced SM hydrolysis. ~4"ls Interestingly, although the Mg2*-dependent neutral sphingomyelinase (SMase) is localized primarily to the plasma membrane] 6 an acid SMase activity has been reported to localize to caveolae. ~5 Thus, caveolae and CRDs are emerging as potential sites for organizing and sequestering molecular components involved in regulating receptor-linked SM metabolism. Further, it is likely that caveolae may be critical sites for
s S. Li, K. S. Song, S. S. Koh, A. Kikuchi, and M. P. Lisanti, Z BioL Chem. 271, 28647 (1996). 9 T. Okamoto, A. Schlegel, P. E. Scherer, and M. P. Lisanti, J. Biol. Chem. 273, 5419 (1998). 10 T. Harder and K. Simons, Curr. Opin. Cell Biol. 9, 534 (1997). 1~ M. Murata, J. Peranen, R. Schreiner, F. Wieland, T. V. Kurzchalia, and K. Simons, Proc. Natl. Acad. Sci. U.S.A 92, 10339 (1995). ~2 p. Liu, Y. Ying, Y. G. Ko, and R. G. W. A n d e r s o n , J. Biol. Chem. 271, 10299 (1996). 13 C. Mineo, G. L. James, E. J. Smart, and R. G. W. A n d e r s o n , J. Biol. Chem. 271,11930 (1996). t~ T. R. Bilderback, R. J. Grigsby, and R. T. Dobrowsky, J. Biol. Chem. 272, 10922 (1997). t5 p. Liu and R. G. W. A n d e r s o n , Z Biol. Chem. 270, 27179 (1995). 16 M. W. Spence, J. Wakkary, and H. W. Cook, Biochem. Biophys. Acta 719, 162 (1982).
186
SPHINGOLIPIDMETABOLISM
[211
interactions between tyrosine kinase and sphingolipid-signaling pathways. 17 Understanding the biochemical mechanisms affecting interactions between sphingolipid and tyrosine kinase signaling pathways in caveolae represents a novel and emerging area of cell regulation. This article describes some of the commonly used procedures for isolating caveolar m e m b r a n e s and examining ligand-dependent SM turnover. Additionally, we provide some cautionary notes on the use of these procedures in the identification of c a v e o l a e / C R D s as localized sites for signal transduction and for the enrichment of various signaling molecules.
M e a s u r e m e n t of S p h i n g o m y e l i n H y d r o l y s i s in C a v e o l i n - E n r i c h e d M e m b r a n e s from C u l t u r e d Cells Detergent Extraction
This procedure exploits the detergent insolubility of caveolae in Triton X-100 at low t e m p e r a t u r e and the low buoyant density of this m e m b r a n e fraction in sucrose gradients, is The purification of caveolae is monitored by assaying for the presence of caveolin, the m a r k e r protein for caveolae. Although this procedure readily separates caveolin-enriched m e m b r a n e s (CEMs) from noncaveolar m e m b r a n e s (NCMs), it does not necessarily produce pure caveolar vesicles. Therefore, it is p r e m a t u r e to call this m e m brane fraction " c a v e o l a e " without morphologic documentation of the homogeneity of the m e m b r a n e vesicles. Similarly, this procedure will also purify C R D s in cells lacking caveolae. It is important to note that C R D s and caveolae may coexist in some cells and that these domains will likely copurify in the following procedure. Although these domains may be separated using cationic silica beads, 19 the current lack of availability of these beads renders this separation problematic.
Buffers Phosphate-buffered saline (PBS) MES-buffered saline (MBS): 25 m M MES, p H 6.5, 5 m M E D T A , and 150 m M NaC1
17C. Wu, S. Butz, Y. Ying, and R. G. W. Anderson, J. Biol. Chem. 272, 3554 (1997). ~8M. P. Lisanti, P. E. Scherer, J. Vidugririene, Z. Tang, A. Hermanowski-Vosatka, Y.-H. Tu, R. F. Cook, and M. Sargiacomo, J. Cell Biol. 126, 111 (1994). 19j. E. Schnitzer, D. P. Mclntosh, A. M. Dvorak, J. Liu, and P. Oh, Science 269, 1435 (1995).
[21 ]
CAVEOLARSPHINGOMYEL1NHYDROLYSIS
187
MBST: MBS plus 1% Triton X-100, 1 mM Pefabloc or phenylmethylsulfonyl fluoride (PMSF), 10 /xg/ml leupeptin, aprotinin, and bestatin each 80, 35, and 5% (w/v) sucrose in MBS Procedure
Metabolic Labeling Prepare two 15-cm dishes of cells and allow them to reach at least 70% confluency. Four or more 10-cm dishes may also be substituted. If liganddependent SM hydrolysis is to be measured, label the cells for 3 days in medium containing 0.5/zCi/ml [3H]choline chloride. Other isotopes such as [3H]palmitate or [14C]serine (with serine-deficient medium) may also be used if desired. Because previous work has suggested that the ligand-sensitive pool of SM is the last to incorporate the radiolabel, 2° it is important to incubate the cells for at least 48 to 72 hr with the desired radioisotope to reach metabolic equilibrium. Although these times are influenced by the growth rate of particular cell lines, 48 hr is usually sufficient to reach metabolic equilibrium for cells with a rapid doubling time, whereas longer incubation times may be required for cells with prolonged population doublings. After the end of the incubation, dispose of the radioactive medium, wash the monolayers twice with fresh serum-free medium or PBS, and place the cells in serum-free medium for at least 4 hr. This resting period is important as simple medium changes can dramatically affect sphingolipid levels in cultured cells. 2~
Isolation of CEMs Treat the cells with the ligand of choice and, following treatment, aspirate the medium rapidly and wash the cells twice with ice-cold PBS. After aspirating the PBS, add 2 ml of ice-cold MBST and quickly scrape the cells from the plate with a cell scraper. Care should be exercised that the pH of the MBST is adjusted to 6.5 because neutral pH buffers give lower yields of caveolar membranes. 2~ Transfer the lysate to a 10-ml homogenization tube and set on ice for at least 15-20 rain. Homogenize the samples using a tight-fitting Teflon pestle (20 strokes), maintaining the tube on ice at all times. Transfer 2 ml of the sample to a 12-ml ultracentrifuge tube that can be accommodated within the buckets of a SW41 or comparable rotor. 2~ C. M. Linardic and Y. A. H a n n u n , J. Biol. Chem. 269, 23530 (1994). ~-i E. R. Smith, P. L. Jones, J. M. Boss, and A. H., Merrill, Jr., J. Biol. Chem. 272, 5640 (1997). 22 M. Sargiacomo, M. Sudol, Z. Tang, and M. P. Lisanti, J. Cell Biol. 122, 789 (1993).
]88
SPHINGOLIPID METABOLISM
[21]
Adjust the extract to 40% sucrose by the addition of 2 ml of 80% sucrose in MBS lacking Triton X-100. Vortex the tube to mix the solutions thoroughly. Form a discontinuous sucrose gradient by carefully overlaying this solution with 4 ml of the 35% sucrose in MBS. A clear interface should be evident between the 35 and 40% sucrose layers. Next, carefully overlay the 35% sucrose layer with 4 ml of the 5% sucrose in MBS. The difference in density results in a readily visible interface. A linear 5-35% sucrose gradient may also be substituted for the discontinuous gradient. Prepare a balance tube if necessary using the same solutions (substitute 2 ml of MBS for the lysate) and centrifuge the samples at 39,000 rpm in a SW41 rotor for 16-18 hr at 4°. Alternatively, a minimal centrifugation time of 3 hr may be used because it also gives satisfactory recovery of CEMs. s We typically collect 15 x 0.8ml fractions beginning from the top of the gradient. An aliquot of each fraction may be used to determine the distribution of total radiolabel over the gradient (Fig. 1A). Additional aliquots of each fraction are used for the determination of protein content. Because total cellular protein distributes primarily to high density regions of the gradient (Fig. 2, closed circles), reliable protein measurements can be obtained using 0.1-0.2 ml of fractions 3-8 and 0.02-0.05 ml of fractions 9-15. CEMs are typically recovered as a visible band of material located at the interface of the 5 and 35% sucrose layers, fractions 4-6.~4 Alternatively, CEMs may be located by light scattering (600 nm). The presence of CEMs is indicated by comigration of caveolin with these gradient fractions. This is performed easily by S D S - P A G E and immunoblot analysis using any of several commercially available caveolin antibodies. If desired, the CEMs can be concentrated by pooling together the fractions, diluting in MBS, and concentrating the membranes by centrifugation at 100,000g for 30 min. However, some investigators concentrate the membranes by centrifugation in a microfuge. 22 Caveolin is well resolved from the bulk of cellular protein as over 95% of the total cellular protein is typically recovered in NCMs (fractions 10-15) (Fig. 2, closed circles). However, depending on the cell type, about 50-70% of the cellular SM is recovered in the CEMs, 14 whereas the remainder distributes primarily to fractions 12 15 representing bulk plasma membrane (Fig. 1A). Thus, relative to cellular protein, SM is highly enriched in CEMs (Fig. 2, closed squares).
Advantages and Disadvantages A major advantage of preparing CEMs using detergent insolubility as a criterion is the relative ease of the procedure. However, a cautionary note is that slight alterations in the protein-to-detergent ratio may alter
[9,1 ]
CAVEOLAR SPHINGOMYELIN HYDROLYSIS 40
A 0
35
189
Total Choline cpm SM cpm
~ o
30 5© 25
E 2o ~ 15 10
/•0
5
0 0
0
2
4
6
8
10
12
E) ;)
14
16
Fraction # 40: 35
B i
-" :'
Total Choline cpm SM cpm
30 O
25
10 i
t 0 ~H() 0 2
c~ ,-- ~?C .... 4 6 8 10
, ~ , 12 14
16
Fraction # FIG. 1. Distribution of [3H]SM following detergent (A) and nondetergent extraction (B) and centrifugation through discontinuous sucrose gradients. Cells were fractionated as described in the text and the amount of [3H]SM in each fraction was plotted as a percentage of the total [3HJSM recovered in the gradient. The distribution of [3H]SM relative to the tolal amount of radioactivity present is also shown.
the solubility of caveolar components. 23 Because the mechanisms whereby receptors couple to both neutral or acid SMases are poorly understood, loss of critical c o m p o n e n t s due to changes in this ratio may cause inconsistent results. In this regard, detergent extraction results in the selective loss 2~ A. Uinenbogaard, Y. Ying, and E. J. Smart, J. Biol. Chem. 273, 6525 (1998).
190
SPHINGOLIPID
[21]
METABOLISM
• 25
70 O @
i
i i
20
60 50
~D
.¢..a
o
~
o
-4,,,,o
=
15
© ¢z,
40 30
10
d
-4...a
20
5 10
m
~D
.+.a
0
0 0
2
4
6
8
10
12
14
~D ~D
6
Fraction # FIG. 2. Enrichment of [3H]SM in CEMs relative to cellular protein following detergent (Q, II) or nondetergent extraction (O, [3) and centrifugation through discontinuous sucrose gradients. Cells were fractionated as described in the text and the amount of protein (O, O) in each fraction was plotted as a percentage of the total recovered in the gradient. The amount of SM in the gradient fractions was determined in the same manner and the ratio of SM/ protein plotted (ll, [3).
of lipid-modified proteins from caveolae, z4 An additional concern is that this procedure does not result in a homogeneous preparation of caveolar vesicles. Whereas isolated caveolae are composed primarily of vesicles ranging in diameter from about 50 to 80 nm, detergent extraction likely results in a very heterogeneous population of vesicles, many of which may be derived from the plasma membrane proper. 25 Thus, although useful for indicating whether ligand-induced SM hydrolysis may localize to detergentinsoluble membrane domains, it is not a definitive demonstration of a caveolar compartmentalization of this response. 24 K. S. Song, S. Li, T. Okamoto, L. A. Quilliam, M. Sargiacomo, and M. P. Lisanti, J2 Biol. Chem. 271, 9690 (1996). 25 R. V. Stan, W. G. Roberts, D. Predescu, K. Ihida, L. Saucan, L. Ghitescu, and G. E. Palade, Mol. Biol. Cell 8, 595 (1997).
[21 ]
CAVEOLARSPHINGOMYELINHYDROLYSIS
191
Detergent-Free Extraction Because detergent can preferentially solubilize lipid-modified caveolaeassociated proteins, several detergent-free methods have been developed to avoid this problem. The sodium carbonate procedure is a variation to that described earlier. 24 Buffers MBS containing 0.5 M sodium carbonate, pH 11 90% sucrose in MBS 5 and 35% sucrose in MBS containing 250 mM sodium carbonate Procedure
Isolation of CEMs Cells are scraped into 2 ml of ice-cold MBS containing 0.5 M sodium carbonate, pH 11, producing a very viscous cell lysate. The cells are then homogenized using 20 strokes in a Dounce homogenizer, three 10-sec pulses with a Polytron tissue grinder (half-maximal speed), and three 20-sec bursts with a microtip sonicator to break up the plasma membrane and detach the caveolae. The lysate is then brought to 45% sucrose containing 250 mM sodium carbonate by the addition of 2 ml of 90% sucrose in MBS (prepare the 90% sucrose by dissolving the sugar with mild heating). The 45% sucrose is overlaid with 4 ml of 35% sucrose in MBS followed by 4 ml of 5% sucrose in MBS (both containing 250 mM sodium carbonate). The tubes are then centrifuged as described earlier. Under this protocol, CEMs also migrate in fractions 4-6 and noncaveolar membranes (NCMs) comprise the bulk of fractions 10-15.
Advantages and Disadvantages Similar to detergent extraction, the sodium carbonate extraction procedure is easy and reliably separates CEMs from NCMs and results in a similar protein profile (Fig. 2, open circles). Additionally, SM is similarly distributed over the gradient (Fig. 1B) and enriched in CEMs relative to protein (Fig. 2, open squares). Importantly, the recovery of CEMs is somewhat improved over the detergent extraction method and does not remove lipid-modified proteins from these membrane domains. However, sodium carbonate has been used extensively to determine if proteins strongly attach to membranes. Thus, this procedure may remove loosely attached protein components that may impair the development of in vitro approaches to
192
SPHINGOLIPIDMETABOLISM Control
4
5
6
100 ng/ml NGF 7
4
5
6
7
10 ng/ml NT-3 4
5
6
10 ng/ml IL- l I3 7
,
' QO, i flO,,
• 44-
- 4 - .
SM, cpm
24294
Percent Control
- 6 . .
[2 1]
4
5
6
7 Frac.#
- a.
16977
13146
11048
69.9
54.1
45.4
F~G. 3. Hydrolysis of [3H]SM in CEMs. Cells were labeled with [3H]choline and treated with the indicated ligands. CEMs were isolated and [3H]SM content determined by TLC. After normalization to total protein in each fraction the percentage SM hydrolysis was determined. PC, phosphatidylcholine. determine h o w receptors m a y couple to e n z y m e s regulating SM lism in CEMs. M o r e o v e r , because the extraction is p e r f o r m e d in the p H range of 9-11, the direct m e a s u r e m e n t of e n z y m e s in sphingolipid metabolism is problematic. T h e m e m b r a n e s dialyzed against buffer to r e m o v e the sodium c a r b o n a t e and activity assessed.
metabotypically involved m a y be enzyme
A s s a y of S p h i n g o m y e l i n C o n t e n t of C a v e o l i n - E n r i c h e d M e m b r a n e s O n c e C E M s have been isolated, we m e a s u r e SM levels in the m e m b r a n e s by thin-layer c h r o m a t o g r a p h y ( T L C ) or by using the bacterial SMase assay. 14 Bacterial SMase Assay
Transfer a 0.4-ml aliquot of each fraction to a screw-cap glass tube and p e r f o r m a Bligh and D y e r lipid extraction. 26 Quantitation of SM is d e t e r m i n e d as described. 27 E v a p o r a t e one-half of the organic layer and 26E. G, Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 2vS. Jayadev, C. M. Linardic, and Y. A. Hannum, J. Biol. Chem. 269, 5757 (1994).
[21]
CAVEOLAR SPH1NGOMYELINHYDROLYSIS
193
solubilize the residue in 0.05 ml of 200 m M Tris-HC1, pH 7.5, 20 m M MgC1 containing 1% Triton X-100. Add 0.05 ml of a 2 U/ml solution of Streptomyces sp. SMase (diluted in 10 m M Tris-HCl, p H 7.5) and incubate the tubes for 2 hr at 37 °. Terminate the reaction by adding 1.5 ml of c h l o r o f o r m : m e t h a n o l (2:1). Add 0.2 ml of water and recover the released [3H]choline in the aqueous layer after centrifugation. Normalize the amount of SM hydrolyzed to the protein content of each fraction. Because the bacterial SMase quantitatively hydrolyzes any SM present in the s a m p l e s ligand-induced SM hydrolysis is revealed by a decrease in the amount of SM present in CEMs from treated cells versus control cells.
Thin-Layer Chromatography of SM Alternatively, SM hydrolysis may be measured by TLC. Numerous solvent systems exist for the separation of SM from other lipids, but two common systems used in our laboratory are chloroform : methanol : glacial acetic acid : water (65 : 35 : 8 : 4) or chloroform : methanol : 2 N NH4OH (65 : 35 : 8). Fit a T L C chamber with a paper wick (Whatman filter paper) and place enough solvent into the chamber to cover about the bottom 1-2 cm of the T L C plate. Do not individually add the solvent components to the chamber, mix them in a separate flask and add the solution to the chamber. Next, allow the chamber to equilibrate for at least 3-4 hr prior to placing the TLC plate in the chamber. If two T L C plates are to be developed, a single chamber may be used. If this is the case, place both plates into the chamber together and ensure that they do not touch one another and are adequately immersed in the solvent. Following the Bligh and Dyer lipid extraction, evaporate 1.5 ml of the chloroform extract and resuspend the residue in 0.03 ml of chloroform. Apply 0.02 ml to the silica gel and dry the spot thoroughly. Develop the plate to about 2 cm from the top and allow the solvent to evaporate in a fume hood. Spray the silica gel with an autoradiography enhancer such as En3Hance (spray liberally but do not create runs), wrap the plate in plastic wrap, and expose to film for 2-3 days at - 8 0 °. Figure 3 shows a typical result for the analysis of SM hydrolysis from CEMs by this method from p75NrR-NIH 3T3 cells labeled with [3H]choline and treated with several agonists.