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Tenascins are associated with lipid rafts isolated from mouse brain
BBRC Biochemical and Biophysical Research Communications 294 (2002) 742–747 www.academicpress.com
Tenascins are associated with lipid rafts isolated from mouse brainq Joachim Kappler,a,* Stephan L. Baader,b Sebastian Franken,a Penka Pesheva,c Karl Schilling,b Uwe Rauch,d and Volkmar Gieselmanna a
c
Physiologisch-Chemisches Institut, Nussallee 11, 53115 Bonn, Germany b Anatomisches Institut, Nussallee 10, 53115 Bonn, Germany Klinik f€ ur Nuklearmedizin, Sigmund-Freud-Straße 25, Bonn, Rheinische Friedrich-Wilhelms-Universit€at Bonn, Bonn, Germany d Department of Experimental Pathology, Universitet Lund, Lund, Sweden Received 8 May 2002
Abstract Lipid rafts are microdomains of the plasma membrane which are enriched in glycosphingolipids and specific proteins. The reported interactions of several raft-associated proteins (such as, e.g., F3) with tenascin C and tenascin R prompted us to consider that these oligomeric multidomain glycoproteins of the extracellular matrix (ECM) could associate with rafts. Here, we show punctate immunocytochemical distributions of tenascin C (TN-C) and tenascin R (TN-R) at the membrane surface of neural cells resembling the pattern reported for raft-associated proteins. Moreover, cholesterol depletion with methyl-b-cyclodextrin reduced the punctate surface staining of TN-C. Consistently, TN-C was associated with lipid rafts of neonatal mouse brain according to sucrose density gradient centrifugation experiments. Furthermore, TN-R was also found in rafts prepared from myelin of adult mice. Thus, brainderived tenascins are able to associate with lipid rafts. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Tenascin; Lipid rafts; Cerebellum
The tenascin glycoproteins of the extracellular matrix (ECM) share a characteristic multidomain structure [1]. Tenascin C (TN-C) and tenascin R (TN-R) are found in the brain where their expression is developmentally regulated [2]. Oligomerization gives rise to the characteristic six-armed hexabrachion of TN-C [3] and to dimers or trimers of TN-R. TN-C and TN-R interact with a broad and largely overlapping spectrum of molecules. TN-C, for example, binds multiple ECM glycoproteins [4] and presumably organizes ECM molecules into
q Abbreviations: ECM, extracellular matrix; TN-C, tenascin C; TN-R, tenascin R; GPI, glycosylphosphatidylinositol; RPTP, receptor-type protein tyrosine phosphatase; CALEB, chicken acidic leucinerich EGF-like domain containing brain protein; HEPES, N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid]; EDTA, ethylenediaminetetraacetic acid, PBS, phosphate-buffered saline; N-CAM, neural cell adhesion molecule; HS-PG, heparan sulfate proteoglycan; PrP, prion protein; MAP-2, microtubuli-associated protein 2; GFAP, glial fibrillary acidic protein. * Corresponding author. Fax: +49-228-732-416. E-mail address: [email protected] (J. Kappler).
networks [3,5]. Furthermore, TN-C can link the ECM to the cell surface because it interacts with several membrane-bound proteins. These include F3/F11/contactin, heparan sulfate proteoglycans, annexin-2, integrins, chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), and receptor-type phosphotyrosine phosphatase b=f (RPTP b=f) (reviewed in [4,6]). Similar binding properties have also been described for TN-R (for summary, see [2]). In addition, TN-C and TN-R bind sulfatide and disialogangliosides [7–10]. Thus, the affinity of tenascins towards glycosphingolipids may localize them to specialized cholesterol-containing, glycosphingolipid-rich microdomains of the membrane called lipid rafts [11–13]. Here, we examine the possibility that brain-expressed tenascins, TN-C and TN-R, could associate with lipid rafts. We used immunocytochemistry to visualize their distributions at the surface of various neural cell types and to assess the influence of cholesterol. Moreover, we employed sucrose density gradient centrifugation of membranes from neonatal and adult mouse brain extracted at 4 °C with Triton X-100 to determine the
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subcellular localization of TN-C and TN-R. Based on these experiments we propose that tenascins may aid the organization of rafts and their relations to the ECM.
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each fraction were loaded with exception of the bottom fraction which was diluted tenfold because it contained much more protein than the other fractions. Raft preparation from myelin. Myelin was isolated from adult mice and lipid rafts were prepared following an established standard protocol exactly as described [22].
Materials and methods Antibodies. Rat anti-mouse TN-C [Sigma, clone MTn-12, used for Western blotting (WB) at 1:2500, immunofluorescence staining (IF) 1:400], rabbit anti-TN-R [14] (WB: 1:2500, IF: 1:200), anti-MAP-2 and anti-GFAP [15], anti-N-CAM 5B8 (Developmental Hybridoma Bank, WB 1:100), rat anti-BSP-2/N-CAM (WB 1:2500) [16], and rabbit antimouse F3 [17] (WB 1:2,000) and monoclonal O4 [18] (IF 1:20). Secondary Alexa-conjugated antibodies were from Molecular Probes (anti-mouse Alexa 488 and anti-rabbit Alexa 546, IF 1:500). Anti-rat Cy3 (IF 1:500) and horseradish peroxidase-conjugated anti-rabbit (WB 1:10,000), anti-mouse (WB 1:10,000), and anti-rat (WB 1:2500) secondary antibodies were from Jackson Immunoresearch. Primary dissociated cerebellar cultures. Cultures were prepared from cerebellar tissue of 17-day-old fetal B6C3F1 mice or of mice at postnatal day 8 as described [19]. Immunofluorescence. Cultured cells were fixed in 0.5% paraformaldehyde and 0.5% glutaraldehyde diluted in culture medium for 30 min, treated with 0.5% sodium borohydride/PBS for 5 min and incubated in 0.1 M glycine dissolved in PBS at 4 °C overnight. After blocking in PBS containing 4% goat serum, cells were incubated with the primary antibody diluted in 2% goat serum/PBS for 1 h at room temperature. After intensive washing, cells were incubated with the secondary antibodies for 1 h. Fluorescent labeling of oligodendrocytes was done by adding the O4 monoclonal antibody in a dilution of 1:20 to the culture medium of unfixed cells. Cholesterol extraction was performed as described [20]. Briefly, cerebellar cultures were extracted with 10 mM methyl-b-cyclodextrin (Sigma) in MEM, 50 mM HEPES, pH 7.3, 0.35 g/liter carbonate at 37 °C for 1 h on a rocking platform prior to fixation. Differential centrifugation of brain homogenates. Per gram of wet brain tissue 2.5 ml buffer (either 25 mM HEPES, pH 7.2, 2.5 mM CaCl2 , 1 mM MgCl2 , 4 mM KCl, 300 mM sucrose or 25 mM HEPES, pH 7.2, 5 mM EDTA, 300 mM sucrose, each supplemented with 1 mM phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) was added and the brains were homogenized in a Teflon-glass douncer. After removal of the nuclei (at 1000g for 10 min) the membrane fraction was pelleted at 20,000g for 30 min. After removal of the microsomes at 120,000g for 60 min a supernatant containing the soluble proteins was obtained. Raft preparation from newborn mouse brain. Lipid rafts were prepared as described [21] with modifications. Briefly, the membrane fraction was resuspended in 25 mM HEPES, pH 7.2, 2.5 mM CaCl2 , 1 mM MgCl2 , 4 mM KCl, 300 mM sucrose or in 25 mM HEPES, pH 7.2, 5 mM EDTA, 300 mM sucrose supplemented with protease inhibitors and mixed with an equal volume of 2 detergent (2% Triton X-100) for 60 min at 4 °C; the solution was mixed with an equal volume of 80% sucrose and 1 ml of this sample was overlaid with a 11-ml gradient of 5–30% sucrose in a Beckman SW41 ultracentrifuge tube. All sucrose solutions contained 1% Triton X-100. Following centrifugation at 218,000g for at least 16 h at 4 °C, 12 1 ml fractions of the gradient were precipitated with aceton and analyzed by SDS–PAGE and Western blotting. Proteins were electrophoresed on 7.5 or 10% SDS–polyacrylamide gels in Mini Protean Cell (Bio-Rad) and silver stained or blotted onto nitrocellulose (Schleicher & Schuell) in a transblot cell (Bio-Rad) according to standard protocols. Blots were blocked overnight (at 4 °C) with nonfat dry milk powder in Tris-buffered saline containing 0.05% Tween (TBS-T), incubated with appropriate dilutions of the primary and secondary antibodies, and developed using chemiluminescence (Supersignal West Pico, Pierce). For electrophoresis, equal aliquots of
Results The distributions of TN-C and TN-R and several marker proteins were examined in primary cultures from mouse cerebellum [15,19]. Glutaraldehyde-containing fixatives were used to prevent antibody-induced redistribution of raft-associated proteins [23]. Punctate deposits of TN-C were found at the cell surface of most cells (Fig. 1A, arrowhead). Double stainings of cultures with antibodies against TN-C followed by permeabilization and staining for GFAP or MAP-2, respectively, revealed that astrocytes and neurons were stained for TN-C (data not shown). The average size of the punctate TN-C deposits was about 500 nm. Less frequently, patchy aggregates were observed which reached a diameter of 1 lm and more. Extraction of cholesterol with methyl-b-cyclodextrin prior to immunocytochemistry reduced the number of punctate TN-C deposits and caused a diffuse appearance of the staining (Fig. 1B). Cerebellar cultures from 8-day-old animals contained a large number of typically structured oligodendrocytes characterized by multipolar, heavily branched processes. These cells were positive for the oligodendroglial marker O4 (Fig. 1C). TN-R was expressed at the surface of oligodendrocytes (Fig. 1D) which is in agreement with previous observations [24]. Some of the TN-R immunoreactivity was present in fine spots (Fig. 1D, arrowheads) and patches similar to the TN-C staining pattern (Fig. 1D). Furthermore, we observed more fibrillar TN-R aggregates on the proximal part of oligodendrocyte processes (Fig. 1D). To further examine the possibility that the discontinuous immunocytochemical staining patterns of TN-C and TN-R at the cell surface could reflect association with rafts we analyzed the subcellular localization of tenascins using differential centrifugation and sucrose density gradient centrifugation. After homogenization of brain tissue in the presence of 5 mM EDTA, about 20% of TN-C was found at the membrane fraction (Fig. 2). When the homogenization was carried out in buffers containing Ca and Mg ions, however, approximately 80% of TN-C was associated with the membrane fraction (Fig. 2). In contrast, the membrane association of polysialylated N-CAM was not affected by divalent cations or EDTA (Fig. 2). These data are consistent with the observation that interactions of tenascins with membrane components such as sulfatide depend on divalent cations [7,25].
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Fig. 1. Immunofluorescent labeling of dissociated cerebellar cultures using tenascin C-, tenascin R-, and O4-specific antibodies. (A) High-power micrographs showing staining for TN-C. TN-C immunoreactivity is distributed not only on glia, but also on neurons in small puncta (arrowheads) and larger aggregates. Bar, 5 lm. (B) Staining for TN-C after extraction of cholesterol with methyl-b-cyclodextrin. (C) Mature oligodendrocyte positively stained for O4, bar, 10 lm. (D) High-power micrograph of an oligodendrocyte stained for tenascin R without prior treatment with Triton X-100, bar, 5 lm.
Fig. 2. Western blot analysis of subcellular fractions from neonatal mouse brain. Brain tissue was homogenized in buffers containing CaCl2 and MgCl2 or EDTA, respectively, as indicated at the top. Equal aliquots of the crude membrane fractions (M) and the soluble fractions (S) were analyzed with anti-TN-C and mouse anti-N-CAM (5B8) antibodies, as indicated above the lanes. Positions of molecular weight marker bands are indicated at the left (in kDa).
To assess the membrane association of TN-C in more detail, we isolated lipid rafts from neonatal mouse brain in the presence of divalent cations to stabilize the interaction of TN-C with membrane components. Western blot analysis of fractions obtained after sucrose
density gradient centrifugation of membranes extracted with Triton X-100 in the cold detected transmembrane forms of N-CAM (using antibody 5B8) only in the bottom fractions while most of the raft marker F3 floated in the gradient (Figs. 3C and D). Consistently, silver staining revealed specific patterns of protein bands in the bottom fractions and in the fractions containing floating proteins (Fig. 3E). Under these conditions, a considerable amount of TN-C was in the gradient fractions containing floating proteins (Fig. 3B) which also contained the bulk of the raft marker F3 (Fig. 3D). However, most of TN-C partitioned with the bottom fractions of the gradient containing solubilized proteins. The membrane association of TN-R was analyzed in adult mouse brain tissue. Initial attempts to fractionate this material by differential centrifugation revealed that most TN-R partitioned with the initial 1000g pellets (data not shown). Thus, we isolated myelin to obtain a defined membrane fraction enriched in TN-R [25]. Myelin was extracted with Triton X-100 and fractionated with a two-step sucrose density gradient centrifugation. TN-R immunoreactivity was found only in the floating fraction (Fig. 4B) containing the marker proteins 20 ,30 -cyclic nucleotide 30 -phosphodiesterase (CNPase, Fig. 4C) and the GPI-linked adult N-CAM isoform of 120 kDa detected with the BSP-2 antibody
J. Kappler et al. / Biochemical and Biophysical Research Communications 294 (2002) 742–747
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Fig. 4. Sucrose density gradient centrifugation of Triton X-100 extracted membranes from adult mouse myelin. (A) Schematic drawing of the density gradient; (B, C, D) Western blot analyses with antiTN-R (B), anti-CNPase (C), and rat anti-N-CAM (D) antibodies; (E) silver staining. Positions of molecular weight marker bands are indicated at the right (in kDa). Fig. 3. Sucrose density gradient centrifugation of Triton X-100 extracted membranes from neonatal mouse brain. (A) Schematic drawing of the density gradient; (B, C, D) Western blot analyses with antiTN-C (B), anti-N-CAM (C), and anti-F3 (D) antibodies; (E) silver staining. Positions of molecular weight marker bands are indicated at the right (in kDa).
(Fig. 4D). A similar distribution in raft-containing fractions was also reported for F3 [22].
Discussion TN-C and TN-R bind to glycosphingolipids [7,9,10] and many receptor proteins for TN-C or TN-R have properties common for raft-associated molecules because they are glypiated (F3 and glypicans) or were reported to be detergent-resistant (syndecan [26], annexin-2 [27], and b 1-integrins [28]). Thus, these interactions may tie tenascins to rafts. For raft-associated proteins like PrP or Thy-1, characteristic spotty or patchy distributions at the cell surface were described following fixation with glutaraldehyde [21]. Consistently, we observed a spotted pattern of TN-C at the surface of astrocytes and neurons (Fig. 1A). The size of most TN-
C-positive spots was approximately 500 nm (Fig. 1A). Interestingly, this size is only slightly above the dimensions reported for rafts (25–100 nm) [29,30]. Moreover, the reduced number of punctate TN-C deposits at the cell surface after extraction of cholesterol with methyl-bcyclodextrin (Fig. 1B) indicates that this discontinuous distribution depends on an intact scaffold of membrane lipids. Consistently, sucrose density gradient centrifugation experiments with Triton X-100 extracts from neonatal rat brain membranes revealed the presence of TN-C in the floating fractions containing rafts and raftassociated proteins (Fig. 3). Thus, both morphological and biochemical criteria indicate an association of TN-C with lipid rafts. The fact that a substantial amount of TN-C partitioned with the solubilized proteins (Fig. 3), points to a dynamic TN-C/raft interaction, because rafts prepared with sucrose density gradient centrifugation are depleted in proteins which dissociate quickly from rafts [13]. The high local concentrations of TN-C at certain areas of the plasma membrane at defined stages of development according to immunocytochemical data (Fig. 1, [31]), however, imply that in situ probably a much higher percentage of TN-C is associated with rafts than anticipated from the sucrose density gradient data.
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Our data indicate that TN-R is also associated with lipid rafts since immunocytochemistry demonstrated a discontinuous distribution of TN-R at the surface of oligodendrocytes (Fig. 1D) and in sucrose density gradient centrifugation experiments myelin-derived TN-R was mostly associated with rafts (Fig. 4). The fact that TN-R was virtually absent from the bottom fractions of the gradient indicates a stable association of TN-R with rafts from adult myelin. Thus, in spite of differences with respect to their developmental expression, association with rafts seems to be a property of both brain-derived tenascins. As a consequence of their oligomeric structure, TN-C and TN-R can cluster multiple receptor molecules [32,33]. It is noteworthy that clustering of rafts, e.g., after exposure to antibodies against molecules with raftaffinity, can recruit signal-transducing proteins like src-family tyrosine kinases to rafts and thereby can activate them [13]. Interestingly, binding of TN-R to F3-expressing HeLa cells recruits and activates the src kinase fyn [34]. Since the average length of one fully extended TN-C subunit is approximately 70 nm according to rotatory shadowing experiments [3] the size of TN-C hexamers is very close to the dimensions reported for rafts (see above). TN-R dimers and trimers are only slightly smaller [2]. Therefore, single tenascin molecules could cover individual rafts. Accordingly, after their recruitment to the plasma membrane tenascin molecules could aid the co-ordinated spatial assembly of raft components.
Acknowledgments We thank Norbert R€ osel for expert technical assistance, Prof. Jacqueline Trotter for the generous gift of BSP-2-, F3-, and CNPasespecific antibodies, Prof. Andreas Faissner for helpful discussions, and Prof. Hans Kresse and Dr. Andreas Schwarz for critically reading the manuscript. This work is supported by DFG Grant Ka 762/2-1 and BONFOR O161.0008 to J.K. and BONFOR O167.0004 to S.L.B.
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Tenascins are associated with lipid rafts isolated from mouse brainq Joachim Kappler,a,* Stephan L. Baader,b Sebastian Franken,a Penka Pesheva,c Karl Schilling,b Uwe Rauch,d and Volkmar Gieselmanna a
c
Physiologisch-Chemisches Institut, Nussallee 11, 53115 Bonn, Germany b Anatomisches Institut, Nussallee 10, 53115 Bonn, Germany Klinik f€ ur Nuklearmedizin, Sigmund-Freud-Straße 25, Bonn, Rheinische Friedrich-Wilhelms-Universit€at Bonn, Bonn, Germany d Department of Experimental Pathology, Universitet Lund, Lund, Sweden Received 8 May 2002
Abstract Lipid rafts are microdomains of the plasma membrane which are enriched in glycosphingolipids and specific proteins. The reported interactions of several raft-associated proteins (such as, e.g., F3) with tenascin C and tenascin R prompted us to consider that these oligomeric multidomain glycoproteins of the extracellular matrix (ECM) could associate with rafts. Here, we show punctate immunocytochemical distributions of tenascin C (TN-C) and tenascin R (TN-R) at the membrane surface of neural cells resembling the pattern reported for raft-associated proteins. Moreover, cholesterol depletion with methyl-b-cyclodextrin reduced the punctate surface staining of TN-C. Consistently, TN-C was associated with lipid rafts of neonatal mouse brain according to sucrose density gradient centrifugation experiments. Furthermore, TN-R was also found in rafts prepared from myelin of adult mice. Thus, brainderived tenascins are able to associate with lipid rafts. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Tenascin; Lipid rafts; Cerebellum
The tenascin glycoproteins of the extracellular matrix (ECM) share a characteristic multidomain structure [1]. Tenascin C (TN-C) and tenascin R (TN-R) are found in the brain where their expression is developmentally regulated [2]. Oligomerization gives rise to the characteristic six-armed hexabrachion of TN-C [3] and to dimers or trimers of TN-R. TN-C and TN-R interact with a broad and largely overlapping spectrum of molecules. TN-C, for example, binds multiple ECM glycoproteins [4] and presumably organizes ECM molecules into
q Abbreviations: ECM, extracellular matrix; TN-C, tenascin C; TN-R, tenascin R; GPI, glycosylphosphatidylinositol; RPTP, receptor-type protein tyrosine phosphatase; CALEB, chicken acidic leucinerich EGF-like domain containing brain protein; HEPES, N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid]; EDTA, ethylenediaminetetraacetic acid, PBS, phosphate-buffered saline; N-CAM, neural cell adhesion molecule; HS-PG, heparan sulfate proteoglycan; PrP, prion protein; MAP-2, microtubuli-associated protein 2; GFAP, glial fibrillary acidic protein. * Corresponding author. Fax: +49-228-732-416. E-mail address: [email protected] (J. Kappler).
networks [3,5]. Furthermore, TN-C can link the ECM to the cell surface because it interacts with several membrane-bound proteins. These include F3/F11/contactin, heparan sulfate proteoglycans, annexin-2, integrins, chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), and receptor-type phosphotyrosine phosphatase b=f (RPTP b=f) (reviewed in [4,6]). Similar binding properties have also been described for TN-R (for summary, see [2]). In addition, TN-C and TN-R bind sulfatide and disialogangliosides [7–10]. Thus, the affinity of tenascins towards glycosphingolipids may localize them to specialized cholesterol-containing, glycosphingolipid-rich microdomains of the membrane called lipid rafts [11–13]. Here, we examine the possibility that brain-expressed tenascins, TN-C and TN-R, could associate with lipid rafts. We used immunocytochemistry to visualize their distributions at the surface of various neural cell types and to assess the influence of cholesterol. Moreover, we employed sucrose density gradient centrifugation of membranes from neonatal and adult mouse brain extracted at 4 °C with Triton X-100 to determine the
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 5 2 0 - X
J. Kappler et al. / Biochemical and Biophysical Research Communications 294 (2002) 742–747
subcellular localization of TN-C and TN-R. Based on these experiments we propose that tenascins may aid the organization of rafts and their relations to the ECM.
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each fraction were loaded with exception of the bottom fraction which was diluted tenfold because it contained much more protein than the other fractions. Raft preparation from myelin. Myelin was isolated from adult mice and lipid rafts were prepared following an established standard protocol exactly as described [22].
Materials and methods Antibodies. Rat anti-mouse TN-C [Sigma, clone MTn-12, used for Western blotting (WB) at 1:2500, immunofluorescence staining (IF) 1:400], rabbit anti-TN-R [14] (WB: 1:2500, IF: 1:200), anti-MAP-2 and anti-GFAP [15], anti-N-CAM 5B8 (Developmental Hybridoma Bank, WB 1:100), rat anti-BSP-2/N-CAM (WB 1:2500) [16], and rabbit antimouse F3 [17] (WB 1:2,000) and monoclonal O4 [18] (IF 1:20). Secondary Alexa-conjugated antibodies were from Molecular Probes (anti-mouse Alexa 488 and anti-rabbit Alexa 546, IF 1:500). Anti-rat Cy3 (IF 1:500) and horseradish peroxidase-conjugated anti-rabbit (WB 1:10,000), anti-mouse (WB 1:10,000), and anti-rat (WB 1:2500) secondary antibodies were from Jackson Immunoresearch. Primary dissociated cerebellar cultures. Cultures were prepared from cerebellar tissue of 17-day-old fetal B6C3F1 mice or of mice at postnatal day 8 as described [19]. Immunofluorescence. Cultured cells were fixed in 0.5% paraformaldehyde and 0.5% glutaraldehyde diluted in culture medium for 30 min, treated with 0.5% sodium borohydride/PBS for 5 min and incubated in 0.1 M glycine dissolved in PBS at 4 °C overnight. After blocking in PBS containing 4% goat serum, cells were incubated with the primary antibody diluted in 2% goat serum/PBS for 1 h at room temperature. After intensive washing, cells were incubated with the secondary antibodies for 1 h. Fluorescent labeling of oligodendrocytes was done by adding the O4 monoclonal antibody in a dilution of 1:20 to the culture medium of unfixed cells. Cholesterol extraction was performed as described [20]. Briefly, cerebellar cultures were extracted with 10 mM methyl-b-cyclodextrin (Sigma) in MEM, 50 mM HEPES, pH 7.3, 0.35 g/liter carbonate at 37 °C for 1 h on a rocking platform prior to fixation. Differential centrifugation of brain homogenates. Per gram of wet brain tissue 2.5 ml buffer (either 25 mM HEPES, pH 7.2, 2.5 mM CaCl2 , 1 mM MgCl2 , 4 mM KCl, 300 mM sucrose or 25 mM HEPES, pH 7.2, 5 mM EDTA, 300 mM sucrose, each supplemented with 1 mM phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) was added and the brains were homogenized in a Teflon-glass douncer. After removal of the nuclei (at 1000g for 10 min) the membrane fraction was pelleted at 20,000g for 30 min. After removal of the microsomes at 120,000g for 60 min a supernatant containing the soluble proteins was obtained. Raft preparation from newborn mouse brain. Lipid rafts were prepared as described [21] with modifications. Briefly, the membrane fraction was resuspended in 25 mM HEPES, pH 7.2, 2.5 mM CaCl2 , 1 mM MgCl2 , 4 mM KCl, 300 mM sucrose or in 25 mM HEPES, pH 7.2, 5 mM EDTA, 300 mM sucrose supplemented with protease inhibitors and mixed with an equal volume of 2 detergent (2% Triton X-100) for 60 min at 4 °C; the solution was mixed with an equal volume of 80% sucrose and 1 ml of this sample was overlaid with a 11-ml gradient of 5–30% sucrose in a Beckman SW41 ultracentrifuge tube. All sucrose solutions contained 1% Triton X-100. Following centrifugation at 218,000g for at least 16 h at 4 °C, 12 1 ml fractions of the gradient were precipitated with aceton and analyzed by SDS–PAGE and Western blotting. Proteins were electrophoresed on 7.5 or 10% SDS–polyacrylamide gels in Mini Protean Cell (Bio-Rad) and silver stained or blotted onto nitrocellulose (Schleicher & Schuell) in a transblot cell (Bio-Rad) according to standard protocols. Blots were blocked overnight (at 4 °C) with nonfat dry milk powder in Tris-buffered saline containing 0.05% Tween (TBS-T), incubated with appropriate dilutions of the primary and secondary antibodies, and developed using chemiluminescence (Supersignal West Pico, Pierce). For electrophoresis, equal aliquots of
Results The distributions of TN-C and TN-R and several marker proteins were examined in primary cultures from mouse cerebellum [15,19]. Glutaraldehyde-containing fixatives were used to prevent antibody-induced redistribution of raft-associated proteins [23]. Punctate deposits of TN-C were found at the cell surface of most cells (Fig. 1A, arrowhead). Double stainings of cultures with antibodies against TN-C followed by permeabilization and staining for GFAP or MAP-2, respectively, revealed that astrocytes and neurons were stained for TN-C (data not shown). The average size of the punctate TN-C deposits was about 500 nm. Less frequently, patchy aggregates were observed which reached a diameter of 1 lm and more. Extraction of cholesterol with methyl-b-cyclodextrin prior to immunocytochemistry reduced the number of punctate TN-C deposits and caused a diffuse appearance of the staining (Fig. 1B). Cerebellar cultures from 8-day-old animals contained a large number of typically structured oligodendrocytes characterized by multipolar, heavily branched processes. These cells were positive for the oligodendroglial marker O4 (Fig. 1C). TN-R was expressed at the surface of oligodendrocytes (Fig. 1D) which is in agreement with previous observations [24]. Some of the TN-R immunoreactivity was present in fine spots (Fig. 1D, arrowheads) and patches similar to the TN-C staining pattern (Fig. 1D). Furthermore, we observed more fibrillar TN-R aggregates on the proximal part of oligodendrocyte processes (Fig. 1D). To further examine the possibility that the discontinuous immunocytochemical staining patterns of TN-C and TN-R at the cell surface could reflect association with rafts we analyzed the subcellular localization of tenascins using differential centrifugation and sucrose density gradient centrifugation. After homogenization of brain tissue in the presence of 5 mM EDTA, about 20% of TN-C was found at the membrane fraction (Fig. 2). When the homogenization was carried out in buffers containing Ca and Mg ions, however, approximately 80% of TN-C was associated with the membrane fraction (Fig. 2). In contrast, the membrane association of polysialylated N-CAM was not affected by divalent cations or EDTA (Fig. 2). These data are consistent with the observation that interactions of tenascins with membrane components such as sulfatide depend on divalent cations [7,25].
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Fig. 1. Immunofluorescent labeling of dissociated cerebellar cultures using tenascin C-, tenascin R-, and O4-specific antibodies. (A) High-power micrographs showing staining for TN-C. TN-C immunoreactivity is distributed not only on glia, but also on neurons in small puncta (arrowheads) and larger aggregates. Bar, 5 lm. (B) Staining for TN-C after extraction of cholesterol with methyl-b-cyclodextrin. (C) Mature oligodendrocyte positively stained for O4, bar, 10 lm. (D) High-power micrograph of an oligodendrocyte stained for tenascin R without prior treatment with Triton X-100, bar, 5 lm.
Fig. 2. Western blot analysis of subcellular fractions from neonatal mouse brain. Brain tissue was homogenized in buffers containing CaCl2 and MgCl2 or EDTA, respectively, as indicated at the top. Equal aliquots of the crude membrane fractions (M) and the soluble fractions (S) were analyzed with anti-TN-C and mouse anti-N-CAM (5B8) antibodies, as indicated above the lanes. Positions of molecular weight marker bands are indicated at the left (in kDa).
To assess the membrane association of TN-C in more detail, we isolated lipid rafts from neonatal mouse brain in the presence of divalent cations to stabilize the interaction of TN-C with membrane components. Western blot analysis of fractions obtained after sucrose
density gradient centrifugation of membranes extracted with Triton X-100 in the cold detected transmembrane forms of N-CAM (using antibody 5B8) only in the bottom fractions while most of the raft marker F3 floated in the gradient (Figs. 3C and D). Consistently, silver staining revealed specific patterns of protein bands in the bottom fractions and in the fractions containing floating proteins (Fig. 3E). Under these conditions, a considerable amount of TN-C was in the gradient fractions containing floating proteins (Fig. 3B) which also contained the bulk of the raft marker F3 (Fig. 3D). However, most of TN-C partitioned with the bottom fractions of the gradient containing solubilized proteins. The membrane association of TN-R was analyzed in adult mouse brain tissue. Initial attempts to fractionate this material by differential centrifugation revealed that most TN-R partitioned with the initial 1000g pellets (data not shown). Thus, we isolated myelin to obtain a defined membrane fraction enriched in TN-R [25]. Myelin was extracted with Triton X-100 and fractionated with a two-step sucrose density gradient centrifugation. TN-R immunoreactivity was found only in the floating fraction (Fig. 4B) containing the marker proteins 20 ,30 -cyclic nucleotide 30 -phosphodiesterase (CNPase, Fig. 4C) and the GPI-linked adult N-CAM isoform of 120 kDa detected with the BSP-2 antibody
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Fig. 4. Sucrose density gradient centrifugation of Triton X-100 extracted membranes from adult mouse myelin. (A) Schematic drawing of the density gradient; (B, C, D) Western blot analyses with antiTN-R (B), anti-CNPase (C), and rat anti-N-CAM (D) antibodies; (E) silver staining. Positions of molecular weight marker bands are indicated at the right (in kDa). Fig. 3. Sucrose density gradient centrifugation of Triton X-100 extracted membranes from neonatal mouse brain. (A) Schematic drawing of the density gradient; (B, C, D) Western blot analyses with antiTN-C (B), anti-N-CAM (C), and anti-F3 (D) antibodies; (E) silver staining. Positions of molecular weight marker bands are indicated at the right (in kDa).
(Fig. 4D). A similar distribution in raft-containing fractions was also reported for F3 [22].
Discussion TN-C and TN-R bind to glycosphingolipids [7,9,10] and many receptor proteins for TN-C or TN-R have properties common for raft-associated molecules because they are glypiated (F3 and glypicans) or were reported to be detergent-resistant (syndecan [26], annexin-2 [27], and b 1-integrins [28]). Thus, these interactions may tie tenascins to rafts. For raft-associated proteins like PrP or Thy-1, characteristic spotty or patchy distributions at the cell surface were described following fixation with glutaraldehyde [21]. Consistently, we observed a spotted pattern of TN-C at the surface of astrocytes and neurons (Fig. 1A). The size of most TN-
C-positive spots was approximately 500 nm (Fig. 1A). Interestingly, this size is only slightly above the dimensions reported for rafts (25–100 nm) [29,30]. Moreover, the reduced number of punctate TN-C deposits at the cell surface after extraction of cholesterol with methyl-bcyclodextrin (Fig. 1B) indicates that this discontinuous distribution depends on an intact scaffold of membrane lipids. Consistently, sucrose density gradient centrifugation experiments with Triton X-100 extracts from neonatal rat brain membranes revealed the presence of TN-C in the floating fractions containing rafts and raftassociated proteins (Fig. 3). Thus, both morphological and biochemical criteria indicate an association of TN-C with lipid rafts. The fact that a substantial amount of TN-C partitioned with the solubilized proteins (Fig. 3), points to a dynamic TN-C/raft interaction, because rafts prepared with sucrose density gradient centrifugation are depleted in proteins which dissociate quickly from rafts [13]. The high local concentrations of TN-C at certain areas of the plasma membrane at defined stages of development according to immunocytochemical data (Fig. 1, [31]), however, imply that in situ probably a much higher percentage of TN-C is associated with rafts than anticipated from the sucrose density gradient data.
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Our data indicate that TN-R is also associated with lipid rafts since immunocytochemistry demonstrated a discontinuous distribution of TN-R at the surface of oligodendrocytes (Fig. 1D) and in sucrose density gradient centrifugation experiments myelin-derived TN-R was mostly associated with rafts (Fig. 4). The fact that TN-R was virtually absent from the bottom fractions of the gradient indicates a stable association of TN-R with rafts from adult myelin. Thus, in spite of differences with respect to their developmental expression, association with rafts seems to be a property of both brain-derived tenascins. As a consequence of their oligomeric structure, TN-C and TN-R can cluster multiple receptor molecules [32,33]. It is noteworthy that clustering of rafts, e.g., after exposure to antibodies against molecules with raftaffinity, can recruit signal-transducing proteins like src-family tyrosine kinases to rafts and thereby can activate them [13]. Interestingly, binding of TN-R to F3-expressing HeLa cells recruits and activates the src kinase fyn [34]. Since the average length of one fully extended TN-C subunit is approximately 70 nm according to rotatory shadowing experiments [3] the size of TN-C hexamers is very close to the dimensions reported for rafts (see above). TN-R dimers and trimers are only slightly smaller [2]. Therefore, single tenascin molecules could cover individual rafts. Accordingly, after their recruitment to the plasma membrane tenascin molecules could aid the co-ordinated spatial assembly of raft components.
Acknowledgments We thank Norbert R€ osel for expert technical assistance, Prof. Jacqueline Trotter for the generous gift of BSP-2-, F3-, and CNPasespecific antibodies, Prof. Andreas Faissner for helpful discussions, and Prof. Hans Kresse and Dr. Andreas Schwarz for critically reading the manuscript. This work is supported by DFG Grant Ka 762/2-1 and BONFOR O161.0008 to J.K. and BONFOR O167.0004 to S.L.B.
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