Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M411781200 on January 19, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11641-11647, March 25, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11641    most recent
M411781200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gajate, C.
Right arrow Articles by Mollinedo, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gajate, C.
Right arrow Articles by Mollinedo, F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cytoskeleton-mediated Death Receptor and Ligand Concentration in Lipid Rafts Forms Apoptosis-promoting Clusters in Cancer Chemotherapy*

Consuelo Gajate and Faustino Mollinedo{ddagger}

From the Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain

Received for publication, October 18, 2004 , and in revised form, January 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
While investigating the mechanism of action of the novel antitumor drug Aplidin, we have discovered a potent and novel cell-killing mechanism that involves the formation of Fas/CD95-driven scaffolds in membrane raft clusters housing death receptors and apoptosis-related molecules. Fas, tumor necrosis factor-receptor 1, and tumor necrosis factor-related apoptosis-inducing ligand receptor 2/death receptor 5 were clustered into lipid rafts in leukemic Jurkat cells following Aplidin treatment, the presence of Fas being essential for apoptosis. Preformed membrane-bound Fas ligand (FasL) as well as downstream signaling molecules, including Fas-associated death domain-containing protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid, were also translocated into lipid rafts, connecting death receptor extrinsic and mitochondrial intrinsic apoptotic pathways. Blocking Fas/FasL interaction partially inhibited Aplidin-induced apoptosis. Aplidin was rapidly incorporated into membrane rafts, and drug uptake was inhibited by lipid raft disruption. Actin-linking proteins ezrin, moesin, RhoA, and RhoGDI were conveyed into Fas-enriched rafts in drug-treated leukemic cells. Disruption of lipid rafts and interference with actin cytoskeleton prevented Fas clustering and apoptosis. Thus, Aplidin-induced apoptosis involves Fas activation in both a FasL-independent way and, following Fas/FasL interaction, an autocrine way through the concentration of Fas, membrane-bound FasL, and signaling molecules in membrane rafts. These data indicate a major role of actin cytoskeleton in the formation of Fas caps and highlight the crucial role of the clusters of apoptotic signaling molecule-enriched rafts in apoptosis, acting as concentrators of death receptors and downstream signaling molecules and as the linchpin from which a potent death signal is launched.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Drug-induced apoptosis in cancer chemotherapy has been postulated to be mediated by Fas ligand (FasL)1 expression followed by its subsequent interaction with death receptor Fas (a.k.a. CD95 or APO-1) (1). However, this hypothesis was questioned when FasL up-regulation, and even FasL itself, was found nonessential for Fas activation (2, 3). Fas is a major member of the death receptor family, a subgroup of the tumor necrosis factor receptor superfamily characterized by a cytoplasmic death domain that is responsible for the transmission of apoptotic signaling through interaction with death domain-bearing adaptor molecules (4). Stimulation of Fas results in receptor oligomerization and recruitment of the adaptor molecule Fas-associated death domain-containing protein (FADD) through interaction between its own death domain and the clustered receptor death domains. In turn, FADD recruits procaspase-8 via a death effector domain interaction forming the so-called death-inducing signaling complex (5). The close proximity of procaspase-8 molecules in the complex drives its activation by self-cleavage, triggering downstream effector caspases and leading to apoptosis (4). We have recently shown the translocation of Fas and downstream signaling molecules into clusters of lipid rafts in the killing of tumor cells, independently of FasL, by the antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; Edelfosine) (6, 7). Fas association with membrane rafts has also been reported following its engagement by the ligand (8, 9), and lipid rafts have been found to be essential for the initiation of Fas-mediated cell death signaling (6–10). These data have led to the notion that Fas-mediated apoptosis is triggered by its recruitment and clustering in membrane rafts. Lipid rafts are membrane microdomains highly enriched in cholesterol and sphingolipids with an estimated size between 50–70 nm. The proteins located in these microdomains are severely limited in their ability to freely diffuse over the plasma membrane (11). Thus, raft association tends to concentrate specific proteins within plasma membrane microdomains, and this could affect protein function (12).

Aplidin (Aplidine; dehydrodidemnin B) is a naturally occurring cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans that shows strong in vitro and in vivo antitumor activity (13). Aplidin behaves as the most potent and rapid apoptosis inducer ever described and as a promising drug in the treatment of leukemia (14). Its apoptotic action is mediated by Fas/FasL, c-Jun amino-terminal kinase (JNK), and mitochondria (14, 15). Here, we have found a potent and novel mechanism of cell killing mediated not only by the clustering of Fas but also by the concentration of additional death receptors and membrane-bound FasL, together with downstream signaling molecules, into aggregated lipid rafts through a cytoskeleton-mediated process.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and Cell Culture—Aplidin was obtained from PharmaMar (Colmenar Viejo, Madrid, Spain) and prepared as a 1-mM stock solution dissolved in Me2SO. Final solvent concentrations in cell culture were less than 0.1% (v/v), which had no effect in any of the parameters studied. [14C]Aplidin (87.2 mCi/mmol) was from PharmaMar. The human acute T-cell leukemia cell line Jurkat was grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 24 µg/ml gentamicin. Fas-deficient Jurkat cells were generated following protracted cultures (over 10 months) of parental Jurkat cells. Mouse fibroblasts L929 and Fas-transfected L929 cells (7) were grown in Dulbecco's modified Eagle's medium-10% fetal calf serum.

Immunofluorescence Flow Cytometry—Cell surface expression of death receptors was analyzed by immunofluorescence flow cytometry (16) in a BD Biosciences FACSCalibur flow cytometer, using anti-Fas SM1/1 monoclonal antibody (mAb) (Bender MedSystems, Vienna, Austria) and specific antibodies against TNF receptor 1 (TNF-R1) and TRAIL-receptor 2/death receptor 5 (DR5) (Alexis Biochemicals, Lausen, Switzerland; Santa Cruz Biotechnology, Santa Cruz, CA). P3X63 myeloma culture supernatant, provided by F. Sánchez-Madrid (Hospital de La Princesa, Madrid, Spain), was used as a negative control. Measurement of FasL levels was achieved by flow cytometry in permeabilized cells as previously described (16).

Aplidin Uptake—Drug uptake was measured after incubating 1 x 106/ml cells in fetal calf serum containing culture medium with 10–100 nM [14C]Aplidin for the indicated times and subsequent exhaustive washing (five times) with 2% bovine serum albumin-phosphate-buffered saline, followed by radioactivity counting in the cell pellet.

Apoptosis Assay—Quantitation of apoptotic cells was calculated by flow cytometry as the percentage of cells with a DNA content less than G1 (hypodiploidy) in cell cycle analysis (16). Death receptor-mediated apoptosis was induced with rhTNF-related apoptosis-inducing ligand (TRAIL) or rhFasL (Alexis), active in human and murine systems, rhTNF{alpha} (Alexis), murine TNF{alpha} (provided by M. Modolell, Max-Planck-Institut für Immunbiologie, Freiburg, Germany), and cytotoxic anti-Fas CH-11 mAb (Upstate Biotechnology, Lake Placid, NY) as previously described (7). The implication of Fas/FasL interaction in Aplidin-induced apoptosis was evaluated by using the blocking anti-Fas SM1/23 mAb (Bender MedSystems) as described previously (14).

Confocal Microscopy—Cells were settled onto poly-L-lysine-coated slides and analyzed with a Zeiss LSM 510 laser scan confocal microscope (Oberkochen, Germany) for membrane raft and protein visualization as described (6). Colocalization assays were analyzed by excitation of the corresponding fluorochromes in the same section. Negative controls, lacking the primary antibody or using an irrelevant antibody, showed no staining.

Lipid Raft Isolation and Western Blotting—Lipid rafts were isolated from 7 x 107 cells by lysis conditions for 30 min on ice in 1% Triton X-100-containing buffer and centrifugation on discontinuous sucrose gradients as previously reported (6). 1-ml fractions were collected from the top of the gradient, and 20 µl of the individual fractions were subjected to SDS-PAGE, immunoblotting, and an enhanced chemiluminescence detection (ECL) system (Amersham Biosciences). The location of ganglioside GM1-containing lipid rafts was determined using cholera toxin B subunit (CTx) conjugated to horseradish peroxidase (Sigma) as described previously (17). Proteins were identified using the following specific antibodies: anti-48-kDa Fas (C-20), anti-46-kDa JNK1 (C-17), and anti-30-kDa RhoGDI (K-21) rabbit polyclonal antibodies, anti-23-kDa Bid (N-19) and anti-45-kDa DR5 (N-19) goat polyclonal antibodies, and anti-21-kDa RhoA mAb (Santa Cruz Biotechnology); anti-29-kDa FADD (clone-1), anti-80-kDa ezrin (clone-18), and anti-78-kDa moesin mAbs (BD Transduction Laboratories, Lexington, KY); anti-60-kDa TNF-R1 (H398) mAb (Alexis Biochemicals); anti-45-kDa DR5 (Ab-1) rabbit polyclonal antibody, anti-55-kDa procaspase-8 (Ab-3) and anti-58-kDa procaspase-10 (Ab-2) mAbs (Oncogene Research Products). Anti-human FasL (NOK-1) mAb that recognized both 40-kDa membrane-bound FasL and 26-kDa soluble FasL was from Pharmingen (San Diego, CA). Anti-human CD45 (clone HI30) mAb that reacts with the 180-, 190-, 205-, and 220-kDa isoforms of the leukocyte common antigen present on all human leukocytes was from BD Transduction Laboratories. Prestained protein molecular mass standards (Bio-Rad) were run in parallel.

Biotinylation and Immunoprecipitation—Biotinylation of cell surface proteins from untreated and Aplidin-treated Jurkat cells was carried out by exhaustive cell washing with phosphate-buffered saline, followed by addition of 0.5 mg of the membrane-impermeable reagent sulfosuccinimidyl-6-(biotinamido) hexanoate (Pierce) to 2.5 x 107 cells in 1 ml of phosphate-buffered saline for 30 min at room temperature. Cells were then washed three times with ice-cold phosphate-buffered saline and submitted to lipid raft isolation as above. Lipid raft-enriched fractions (fractions 3–5) were pooled and precleared with protein A-Sepharose and then incubated for 2 h at 4 °C with anti-FasL (NOK-1) mAb or X63 myeloma supernatant, used as a negative control, coupled to protein A-Sepharose. Sepharose beads were pelleted, washed five times, and submitted to SDS-PAGE. Proteins were transferred to nitrocellulose filters, and visualization of biotinylated proteins was performed by ECL.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Fas Involvement in Aplidin-induced Apoptosis—We have previously found that L929 cells, lacking Fas, were resistant to Aplidin but became sensitive after being stably transfected with human Fas cDNA (14). Both L929 and Fas-transfected L929 cells possessed functional receptors for TRAIL and TNF{alpha} (>90 and 74% apoptosis after incubation for 24 h with 50 ng/ml TRAIL and 100 units/ml TNF{alpha}, respectively, in the presence of 500 ng/ml actinomycin D). We then asked for the reverse approach, that is whether a Fas-positive cancer cell sensitive to Aplidin could become drug-resistant upon Fas loss. Toward this goal we generated a Fas-deficient subline from human T-lymphoid leukemia Jurkat cells, which are over 85% Fas-positive and very sensitive to Aplidin (Fig. 1). The cell surface expression of additional death receptors, such as TNF-R1 and TRAIL-receptor 2/DR5, remained at similar levels in both parental and Fas-deficient Jurkat cells (~40 and 63% positive cells for TNF-R1 and DR5, respectively). Both cell types were equally sensitive to apoptosis after incubation with rhTNF{alpha} and rhTRAIL (>42% apoptosis after 24 h of incubation), indicating that TNF-R1 and DR5 receptors were functional. However, unlike parental Jurkat cells, Fas-deficient Jurkat cells (<1% Fas-positive cells) were resistant to Aplidin as well as to the agonistic cytotoxic CH-11 anti-Fas monoclonal antibody or rhFasL (Fig. 1). The antitumor drug ET-18-OCH3, which triggers Fas-mediated apoptosis (6, 7), also failed to induce cell death in Fas-deficient Jurkat cells (Fig. 1). These data indicate a major role of Fas in Aplidin-induced apoptosis that cannot be replaced by DR5 or TNF-R1.



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 1.
Lack of Fas prevents Aplidin-induced apoptosis. Induction of apoptosis in Jurkat (JK) and Fas-deficient Jurkat cells was determined by flow cytometry following a 24-h incubation with 50 ng/ml cytotoxic anti-Fas CH-11 mAb, 100 ng/ml rhFasL, 10 nM Aplidin, or 10 µM ET-18-OCH3. Untreated control cells were run in parallel. Data shown are means ± S.D. of three independent experiments.

 
Aplidin Induces Recruitment of Death Receptors and Downstream Signaling Molecules into Clusters of Membrane Rafts—We found Aplidin induced a rapid capping of lipid rafts (Fig. 2A) as assessed by the raft marker fluorescein isothiocyanate-labeled cholera toxin B subunit (FITC-CTx) that binds ganglioside GM1 (18), mainly found in rafts (19). In addition, Fas cocapped with membrane rafts in Aplidin-treated Jurkat cells (Fig. 2B). Translocation of Fas into lipid rafts was further confirmed by isolation of membrane rafts in both untreated and Aplidin-treated Jurkat cells in sucrose gradients (Fig. 3A). Lipid rafts were isolated based on their insolubility in Triton X-100 lysis buffer at 4 °C and fractionated by discontinuous sucrose gradient centrifugation (7, 17). GM1-containing lipid rafts, at the upper part (fractions 3–5) of the sucrose gradient (Fig. 3A), were located using CTx conjugated to horseradish peroxidase (17). Interestingly, we also found that additional death receptors, DR5 and TNF-R1, were translocated into lipid rafts in Aplidin-treated cells (Fig. 3A). We found that Aplidin induced colocalization of DR5 and TNF-R1 with clusters of lipid rafts as assessed by confocal microscopy (data not shown). These data show for the first time the clustering of three major death receptors in lipid rafts and could explain the outstanding proapoptotic effect of Aplidin in cancer cells (14).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 2.
Coclustering of membrane rafts and Fas in Aplidin-treated Jurkat cells. A, T-leukemic Jurkat cells were either untreated (Control) or treated with 10 nM Aplidin for 30 min, stained with FITC-CTx, and analyzed by confocal microscopy for raft visualization. Bar, 10 µm. B, colocalization of membrane rafts (Raft) and Fas in Aplidin-treated Jurkat cells. Cells were either untreated (Control) or treated with 10 nM Aplidin for 3 h and processed for confocal microscopy using FITC-CTx (green fluorescence for lipid rafts) and anti-Fas mAb, followed by CY3-conjugated anti-mouse antibody (red fluorescence for Fas). Areas of colocalization between membrane rafts and Fas in the merge panels are yellow. Bar, 10 µm. Images shown are representative of three independent experiments.

 



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 3.
Recruitment of death receptors, membrane-bound FasL, and downstream signaling molecules into membrane rafts following Aplidin treatment. A, untreated Jurkat cells (Control) and Jurkat cells treated with 10 nM Aplidin for 3 h were lysed in 1% Triton X-100 and fractionated by centrifugation on a discontinuous sucrose density gradient. An equal volume of each collected fraction was subjected to SDS-PAGE before analysis of the indicated proteins using specific antibodies. Procaspases 8 and 10 as well as active p18 caspase-8 and p23 caspase-10 cleavage forms are indicated. Location of GM1-containing rafts (fractions 3–5) was determined using CTx conjugated to horseradish peroxidase. Representative blots of three separate experiments are shown. B, the cell surface proteins of Jurkat cells were biotinylated, and membrane rafts were isolated as above from untreated control cells as well as from cells treated with 10 nM Aplidin for 3 h. Fractions 3–5 were pooled and used as the membrane raft fraction. Then, equal amounts of protein (45 µg) from the raft-enriched fraction were immunoprecipitated with X63 myeloma supernatant, used as a negative control, or anti-FasL mAb. The molecular masses (kDa) of protein markers are indicated on the left. C, Jurkat cells were treated with 10 nM Aplidin for 3 h and then analyzed for membrane raft and FasL localization using FITC-CTx (green fluorescence) and anti-FasL mAb, followed by CY3-conjugated anti-mouse antibody (red fluorescence), respectively. Areas of colocalization between membrane rafts and FasL in the merge panels are yellow. Images shown are representative of three independent experiments. Bar, 10 µm.

 
A number of additional apoptosis-related molecules were also translocated into membrane rafts, including FADD, procaspase-8, procaspase-10, JNK, and Bid (Fig. 3A). Caspase-10 was activated at a higher level than caspase-8, although only a minor proportion of caspase-10 and caspase-8 cleavage forms remained in lipid rafts (Fig. 3A). The recruitment in membrane rafts of JNK and Bid, the latter reported to act as a bridge between Fas signaling and mitochondria (20), may explain the dependence on both JNK and mitochondrial signaling of Aplidin-mediated apoptosis (14, 15).

On the other hand, we found that the protein tyrosine phosphatase CD45 was excluded from the membrane raft-containing fractions in both untreated and Aplidin-treated cells (Fig. 3A). This is in agreement with previous reports showing CD45 exclusion from membrane rafts under a wide array of experimental conditions (21–23). Taken together, our results indicate that Aplidin induces a reorganization of lipid raft protein content, translocating some proteins to these detergent-insoluble fractions, whereas others remain excluded from these fractions.

Membrane-bound FasL Is Concentrated in Membrane Rafts following Aplidin Treatment—Because Fas was required for an effective Aplidin-induced apoptotic response we investigated whether its cognate ligand FasL was affected following drug treatment. Aplidin did not induce FasL synthesis before triggering of the apoptotic response (data not shown) as assessed by flow cytometry analysis of FasL expression in permeabilized cells. In addition, Aplidin-induced apoptosis is independent of protein biosynthesis (14), further supporting that FasL synthesis is not required for the apoptotic response induced by Aplidin. However, we found translocation of a FasL-immunoreactive band into membrane rafts following Aplidin treatment (Fig. 3A). This band corresponded to ~40-kDa molecular mass, which is the expected size for membrane-bound FasL. We further confirmed the presence of membrane-bound FasL in membrane rafts isolated from Aplidin-treated Jurkat cells by biotinylation of cell surface proteins followed by treatment with Aplidin and subsequent immunoprecipitation of raft-enriched pooled fractions with a specific anti-FasL mAb (Fig. 3B). However, FasL was found to be virtually undetectable or in negligible amounts in membrane rafts isolated from biotinylated untreated control Jurkat cells (Fig. 3B). In addition, by confocal microscopy we found coclustering of membrane-bound FasL and lipid rafts in Aplidin-treated Jurkat cells (Fig. 3C). Untreated cells showed a very faint and diffuse staining for membrane-bound FasL (data not shown). FasL translocation to lipid rafts following treatment with 10 nM Aplidin showed a kinetics similar to that of Fas receptor (data not shown), being optimum after 3 h treatment. Prevention of Fas/FasL interaction by the blocking anti-Fas SM1/23 antibody partially inhibited Aplidininduced apoptosis (43.2% inhibition), confirming our previous findings (14) and suggesting that Aplidin induces cell death through both the Fas/FasL system and a FasL-independent process. This can be explained by our present data as Fas and membrane-bound FasL located in membrane rafts may interact with each other through adjacent cells exposing the same complementary set of Fas and FasL proteins concentrated in lipid rafts. In addition, recruitment of Fas and the subsequent downstream signaling molecules into lipid rafts can facilitate triggering of the apoptotic signal with no participation of FasL (3, 7).

Integrity of Lipid Rafts Is Required for Aplidin-induced Fas Capping and Apoptosis—Disruption of rafts following pretreatment with methyl-{beta}-cyclodextrin (MCD), which extracts cholesterol, prevented Aplidin-induced apoptosis (Fig. 4) and Fas capping (data not shown), indicating the critical role of lipid rafts in Aplidin-induced apoptosis.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4.
Effect of MCD on Aplidin-induced apoptosis. Jurkat cells were pretreated with MCD at the indicated concentrations for 30 min at 37 °C in serum-free medium and washed three times; 10 nM Aplidin was then added for 3 h. The percentage of apoptotic cells was assessed by flow cytometry. Control untreated cells and cells treated only with MCD were run in parallel. Data shown are means of three independent experiments ± S.D.

 
Aplidin Is Incorporated into Lipid Rafts—We next asked whether Aplidin could promote the above rearrangements in lipid raft composition and clustering through its direct interaction with membrane rafts. We tested this directly by incubating Jurkat cells with [14C]Aplidin and subsequent isolation of membrane rafts using the well established method of Triton X-100 solubilization and sucrose gradient centrifugation (Fig. 5A). When cells were incubated with radiolabeled Aplidin for 10 min, most of the drug was found accumulated in raft fractions 3–5 (Fig. 5A). Radiolabeled Aplidin codistributed with the typical raft marker ganglioside GM1 (Fig. 5A). This association of Aplidin with lipid rafts preceded Fas translocation to these membrane microdomains (data not shown). Aplidin was rapidly incorporated by Jurkat cells, and disruption of rafts by treatment of cells with MCD resulted in the inhibition of Aplidin uptake (Fig. 5B), further demonstrating the association of Aplidin with membrane rafts.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 5.
Aplidin is rapidly incorporated into lipid rafts. A, Jurkat cells treated with 10 nM [14C]Aplidin for 10 min were lysed in 1% Triton X-100 and fractionated by centrifugation on a discontinuous sucrose density gradient. An equal volume of each collected fraction was counted for radioactivity and subjected to SDS-PAGE, and the location of GM1-containing rafts (fractions 3–5) and the distribution of [14C]Aplidin over the gradient fractions are shown. Data are representative of three separate experiments. B, Jurkat cells (1 x 106) were untreated (Control) or pretreated with 2.5 mg/ml MCD for 30 min. Drug uptake was determined after incubation with 100 nM [14C]Aplidin in 1-ml final volume for the indicated times.

 
Fas Capping Requires Actin Cytoskeleton—We next asked how Fas is concentrated in lipid rafts. Actin cytoskeleton is a dynamic intracellular structure that because of its continuous assembly/disassembly could be perfectly equipped to translocate proteins and transmit signals. A link between raft-mediated signaling and the interaction of actin cytoskeleton with raft membrane domains has been suggested (24). Ezrin, a major protein of the so-called ERM proteins (ezrin, radixin, moesin) linking the actin cytoskeleton to the plasma membrane, has been reported to interact with Fas and mediate Fas cell membrane polarization during Fas-induced apoptosis in human T lymphocytes (25). We found that the actin-linking proteins ezrin, moesin, RhoA, and RhoGDI were accumulated in membrane rafts following treatment of leukemic cells with Aplidin (Fig. 6A). Pretreatment of Jurkat cells with the microfilament-disrupting agent cytochalasin B or with jasplakinolide, a cell-permeable inhibitor of microfilament-mediated events that induces microfilament polymerization and stabilization (26), inhibited Aplidin-induced apoptosis (Fig. 6B) as well as Fas clustering (Fig. 6C, and data not shown). When actin cytoskeleton was disorganized, the Aplidin-promoted Fas caps were no longer observed and Fas staining was weak and showed a rather scattered distribution (Fig. 6C). These data support the notion that actin cytoskeleton is involved in the generation of Fas-enriched lipid raft clusters.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 6.
Translocation of actin-linking proteins into membrane rafts and involvement of actin cytoskeleton on Fas capping and apoptosis in drug-treated Jurkat cells. A, untreated control cells and cells treated with 10 nM Aplidin for 3 h were lysed in 1% Triton X-100 and fractionated by centrifugation on a discontinuous sucrose density gradient. Individual fractions were subjected to SDS-PAGE and Western blotting to analyze the indicated proteins using specific antibodies and for the location of GM1-containing rafts (fractions 3–5). Representative blots of three separate experiments are shown. B, Jurkat cells were pretreated with 5 µg/ml cytochalasin B (CB) or 1 µM jasplakinolide (JASPL) for 30 min, incubated with 10 nM Aplidin for 3 h, and analyzed for apoptosis by flow cytometry. Data shown are means of three independent experiments ± S.D. C, Jurkat cells were pretreated with jasplakinolide and then incubated with 10 nM Aplidin for 3 h. Membrane rafts and Fas were analyzed by confocal microscopy using FITC-CTx (green fluorescence) and anti-Fas mAb followed CY3-conjugated anti-mouse antibody (red fluorescence), respectively. Images shown are representative of three independent experiments. Bar, 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results reported here demonstrate the formation of clusters of lipid rafts that serve as platforms where death receptors, including Fas, DR5, and TNF-R1, membrane-bound FasL, and downstream signaling molecules, including FADD, procaspases 8 and 10, JNK, and Bid, are concentrated. Very recently, we have found that the antitumor ether lipid ET-18-OCH3 is able to concentrate Fas as well as downstream signaling molecules into lipid rafts and this clustering process is required for its apoptotic activity (7). Our present results with the antitumor drug Aplidin further expand this finding, showing that three major death receptors and membrane-bound FasL can be also translocated and concentrated into lipid rafts. To our knowledge, this is the first report showing the clustering of three major death receptors in lipid rafts and the concentration of both a death receptor and its corresponding ligand in specific areas of the cell surface that can act as potent apoptosis promoters. Taken together, these data lead us to define a new way by which apoptosis can be triggered through the concentration of death receptors, adaptors, and downstream signaling molecules in clusters of lipid rafts that trigger a potent cell death signal. The translocation of Bid to lipid rafts following drug treatment suggests an early link between death receptor (extrinsic)- and mitochondrial (intrinsic)-signaling pathways in apoptosis. In fact, we have found that overexpression of Bcl-2 in leukemic cells blocks mitochondrial-mediated apoptotic signaling and prevents cell death induced by Aplidin and by the anti-Fas CH-11 agonistic antibody (14). The clustering of apoptotic signaling molecule-enriched rafts described here seems to play a major role in the extrinsic death receptor signaling and in coupling of both extrinsic and intrinsic mitochondrial pathways of apoptosis.

We hypothesize that accumulation of Fas into aggregates of stabilized membrane lipid domains from a highly dispersed distribution may represent a general mode of regulating Fas activation and apoptosis. Membrane rafts could also serve to generate high local concentration of Fas as platforms for coupling adaptor and effector proteins required for Fas downstream signaling, facilitating and amplifying signaling processes by transient local assembly of various cross-interacting signaling molecules. This is of particular importance in Fas-mediated signal transduction because death receptors lack enzymatic activity and the initial triggering events depend largely on protein/protein interactions. Interestingly, there is an increasing number of agents that promote Fas clustering (3) as well as cocapping of Fas and membrane rafts, such as ET-18-OCH3, resveratrol, and cisplatin (6, 7, 27, 28). This suggests that Fas capping is a general and efficient process in the triggering of apoptosis. However, efficiency in promoting the concentration of death receptors described here is largely stimulus-dependent. Aplidin is considered the most potent proapoptotic agent ever described (14); accordingly, the present data indicate the unprecedented concentration of such a high number of death receptors and apoptotic signaling molecules in membrane rafts that could explain the extraordinary ability of this antitumor drug in promoting apoptosis. In this regard, Aplidin is able to promote the translocation of three major death receptors, namely Fas, DR5, and TNF-R1, in clusters of lipid rafts.

Interestingly, the present data show for the first time the translocation of membrane-bound FasL into membrane rafts. This concentration of Fas and membrane-bound FasL into specific and small areas of the cell membrane may lead to an increase in cell-killing competence and suggests a regulatory mechanism by which cells concentrate receptors and ligands at specific regions of the cell surface, leading to a more effective cell response.

We have also found that Aplidin is rapidly incorporated into lipid rafts. This process is readily detected after only a few minutes of incubation with tumor cells and precedes Fas clustering. Thus, it is suggested that Aplidin could rearrange membrane rafts, promoting their clustering and redistributing the protein content. Our data indicate that membrane rafts are critical for both Aplidin uptake and concentration of death receptors, membrane-bound FasL, and downstream signaling molecules that eventually lead to apoptosis. In this regard, the antitumor ether lipid ET-18-OCH3 has been also found to be incorporated into membrane rafts (7, 29) and to trigger the concentration of apoptotic proteins into lipid rafts (7), although to a lesser level than Aplidin. These results highlight the importance of membrane rafts in the regulation of apoptosis as well as in cancer chemotherapy; therefore, lipid rafts represent a potential target for therapeutic intervention.

Our findings on the concentration of both Fas and membrane-bound FasL in lipid rafts indicate that Aplidin can act through FasL-independent activation of Fas as well as through Fas/FasL interaction. This translocation of Fas and membrane-bound FasL into clusters of lipid rafts gives an explanation for the long-standing dilemma on the involvement of the Fas/FasL system in cancer chemotherapy first postulated by Friesen et al. (1) in the mid 1990s. De novo FasL synthesis is not essential for the induction of apoptosis upon treatment with chemotherapeutic agents, contrary to previous suggestions (1), but a redistribution of pre-existing membrane-bound FasL into clusters of membrane rafts furnishes small areas of the cell surface as potent promoters of cell death. Fas could induce cell death independently of FasL, once clustered in membrane rafts together with downstream signaling molecules. Fas/FasL interactions may enhance this deadly response through binding of the concentrated Fas and membrane-bound FasL in lipid rafts in an autocrine way between adjacent cells. Interaction of the corresponding Fas/FasL pairs in both adjoining cells would lead to their apoptotic cell death. Our data show that raft association concentrates apoptotic molecules into membrane microdomains, allowing efficient death receptor and death ligand presentation and triggering a potent apoptotic response. The clusters of apoptotic-signaling molecule-enriched rafts described here act as scaffolds of Fas downstream signaling, providing a new framework in death receptor-mediated apoptosis and cancer chemotherapy. This can lead to a new way to elicit an efficient apoptotic response that could also sensitize cells to a variety of extracellular insults, including death receptor ligands.


   FOOTNOTES
 
* This work was supported by Fondo de Investigación Sanitaria Grants FIS04/0843 and FIS02/1199. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 34-923-294806; Fax: 34-923-294795; E-mail: fmollin{at}usal.es.

1 The abbreviations used are: FasL, Fas ligand; CTx, cholera toxin B subunit; DR5, death receptor 5; FADD, Fas-associated death domain-containing protein; JNK, c-Jun amino-terminal kinase; mAb, monoclonal antibody; MCD, methyl-{beta}-cyclodextrin; TNF, tumor necrosis factor; TNF-R1, TNF receptor 1; TRAIL, TNF-related apoptosis-inducing ligand; FITC, fluorescein isothiocyanate; rh, recombinant human; GM1, Gal{beta}1,3GalNAc{beta}1,4(NeuAc{alpha}2,3)-Gal{beta}1,4Glc-ceramide. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Friesen, C., Herr, I., Krammer, P. H., and Debatin, K. M. (1996) Nat. Med. 2, 574-577[CrossRef][Medline] [Order article via Infotrieve]
  2. Aragane, Y., Kulms, D., Metze, D., Wilkes, G., Poppelmann, B., Luger, T. A., and Schwarz, T. (1998) J. Cell Biol. 140, 171-182[Abstract/Free Full Text]
  3. Mollinedo, F., and Gajate, C. (2004) in Fas Signaling (Wajant, H., ed), pp. 1-15, Landes Bioscience, Georgetown, TX
  4. Peter, M. E., and Krammer, P. H. (2003) Cell Death Differ. 10, 26-35[CrossRef][Medline] [Order article via Infotrieve]
  5. Peter, M. E., and Krammer, P. H. (1998) Curr. Opin. Immunol. 10, 545-551[CrossRef][Medline] [Order article via Infotrieve]
  6. Gajate, C., and Mollinedo, F. (2001) Blood 98, 3860-3863[Abstract/Free Full Text]
  7. Gajate, C., Del Canto-Janez, E., Acuna, A. U., Amat-Guerri, F., Geijo, E., Santos-Beneit, A. M., Veldman, R. J., and Mollinedo, F. (2004) J. Exp. Med. 200, 353-365[Abstract/Free Full Text]
  8. Scheel-Toellner, D., Wang, K., Singh, R., Majeed, S., Raza, K., Curnow, S. J., Salmon, M., and Lord, J. M. (2002) Biochem. Biophys. Res. Commun. 297, 876-879[CrossRef][Medline] [Order article via Infotrieve]
  9. Grassme, H., Cremesti, A., Kolesnick, R., and Gulbins, E. (2003) Oncogene 22, 5457-5470[CrossRef][Medline] [Order article via Infotrieve]
  10. Hueber, A. O., Bernard, A. M., Herincs, Z., Couzinet, A., and He, H. T. (2002) EMBO Rep. 3, 190-196[CrossRef][Medline] [Order article via Infotrieve]
  11. Varma, R., and Mayor, S. (1998) Nature 394, 798-801[CrossRef][Medline] [Order article via Infotrieve]
  12. Simons, K., and Toomre, D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 31-39[CrossRef][Medline] [Order article via Infotrieve]
  13. Geldof, A. A., Mastbergen, S. C., Henrar, R. E., and Faircloth, G. T. (1999) Cancer Chemother. Pharmacol. 44, 312-318[CrossRef][Medline] [Order article via Infotrieve]
  14. Gajate, C., An, F., and Mollinedo, F. (2003) Clin. Cancer Res. 9, 1535-1545[Abstract/Free Full Text]
  15. Cuadrado, A., Gonzalez, L., Suarez, Y., Martinez, T., and Munoz, A. (2004) Oncogene 23, 4673-4680[CrossRef][Medline] [Order article via Infotrieve]
  16. Gajate, C., Fonteriz, R. I., Cabaner, C., Alvarez-Noves, G., Alvarez-Rodriguez, Y., Modolell, M., and Mollinedo, F. (2000) Int. J. Cancer 85, 674-682[CrossRef][Medline] [Order article via Infotrieve]
  17. Cheng, P. C., Dykstra, M. L., Mitchell, R. N., and Pierce, S. K. (1999) J. Exp. Med. 190, 1549-1560[Abstract/Free Full Text]
  18. Schon, A., and Freire, E. (1989) Biochemistry 28, 5019-5024[CrossRef][Medline] [Order article via Infotrieve]
  19. Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998) J. Cell Biol. 141, 929-942[Abstract/Free Full Text]
  20. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[CrossRef][Medline] [Order article via Infotrieve]
  21. Xavier, R., Brennan, T., Li, Q., McCormack, C., and Seed, B. (1998) Immunity 8, 723-732[CrossRef][Medline] [Order article via Infotrieve]
  22. Giurisato, E., McIntosh, D. P., Tassi, M., Gamberucci, A., and Benedetti, A. (2003) J. Biol. Chem. 278, 6771-6778[Abstract/Free Full Text]
  23. Larbi, A., Douziech, N., Dupuis, G., Khalil, A., Pelletier, H., Guerard, K. P., and Fulop, T., Jr. (2004) J. Leukocyte Biol. 75, 373-381[Abstract/Free Full Text]
  24. Harder, T., and Simons, K. (1999) Eur. J. Immunol. 29, 556-562[CrossRef][Medline] [Order article via Infotrieve]
  25. Parlato, S., Giammarioli, A. M., Logozzi, M., Lozupone, F., Matarrese, P., Luciani, F., Falchi, M., Malorni, W., and Fais, S. (2000) EMBO J. 19, 5123-5134[CrossRef][Medline] [Order article via Infotrieve]
  26. Bubb, M. R., Spector, I., Beyer, B. B., and Fosen, K. M. (2000) J. Biol. Chem. 275, 5163-5170[Abstract/Free Full Text]
  27. Delmas, D., Rebe, C., Lacour, S., Filomenko, R., Athias, A., Gambert, P., Cherkaoui-Malki, M., Jannin, B., Dubrez-Daloz, L., Latruffe, N., and Solary, E. (2003) J. Biol. Chem. 278, 41482-41490[Abstract/Free Full Text]
  28. Lacour, S., Hammann, A., Grazide, S., Lagadic-Gossmann, D., Athias, A., Sergent, O., Laurent, G., Gambert, P., Solary, E., and Dimanche-Boitrel, M. T. (2004) Cancer Res. 64, 3593-3598[Abstract/Free Full Text]
  29. van der Luit, A. H., Budde, M., Ruurs, P., Verheij, M., and van Blitterswijk, W. J. (2002) J. Biol. Chem. 277, 39541-39547[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
C. S. Mitsiades, E. M. Ocio, A. Pandiella, P. Maiso, C. Gajate, M. Garayoa, D. Vilanova, J. C. Montero, N. Mitsiades, C. J. McMullan, et al.
Aplidin, a Marine Organism-Derived Compound with Potent Antimyeloma Activity In vitro and In vivo
Cancer Res., July 1, 2008; 68(13): 5216 - 5225.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Mikhailov, A. Sokolovskaya, G. G. Yegutkin, H. Amdahl, A. West, H. Yagita, R. Lahesmaa, L. F. Thompson, S. Jalkanen, D. Blokhin, et al.
CD73 Participates in Cellular Multiresistance Program and Protects against TRAIL-Induced Apoptosis
J. Immunol., July 1, 2008; 181(1): 464 - 475.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
M. Beneteau, M. Pizon, B. Chaigne-Delalande, S. Daburon, P. Moreau, F. De Giorgi, F. Ichas, A. Rebillard, M.-T. Dimanche-Boitrel, J.-L. Taupin, et al.
Localization of Fas/CD95 into the Lipid Rafts on Down-Modulation of the Phosphatidylinositol 3-Kinase Signaling Pathway
Mol. Cancer Res., April 1, 2008; 6(4): 604 - 613.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. J. Munoz-Alonso, L. Gonzalez-Santiago, N. Zarich, T. Martinez, E. Alvarez, J. M. Rojas, and A. Munoz
Plitidepsin Has a Dual Effect Inhibiting Cell Cycle and Inducing Apoptosis via Rac1/c-Jun NH2-Terminal Kinase Activation in Human Melanoma Cells
J. Pharmacol. Exp. Ther., March 1, 2008; 324(3): 1093 - 1101.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Moretti, A. Procopio, R. Lazzarini, M. R. Rippo, R. Testa, M. Marra, L. Tamagnone, and A. Catalano
Semaphorin3A signaling controls Fas (CD95)-mediated apoptosis by promoting Fas translocation into lipid rafts
Blood, February 15, 2008; 111(4): 2290 - 2299.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Sharif-Askari, D. Gaucher, R. Halwani, J. Ma, K. Jao, A. Abdallah, E. K. Haddad, and R.-P. Sekaly
p56Lck Tyrosine Kinase Enhances the Assembly of Death-inducing Signaling Complex during Fas-mediated Apoptosis
J. Biol. Chem., December 7, 2007; 282(49): 36048 - 36056.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Park, K. Teja, J. J. O'Shea, and R. M. Siegel
The Yersinia Effector Protein YpkA Induces Apoptosis Independently of Actin Depolymerization
J. Immunol., May 15, 2007; 178(10): 6426 - 6434.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. DeMorrow, S. Glaser, H. Francis, J. Venter, B. Vaculin, S. Vaculin, and G. Alpini
Opposing Actions of Endocannabinoids on Cholangiocarcinoma Growth: RECRUITMENT OF Fas AND Fas LIGAND TO LIPID RAFTS
J. Biol. Chem., April 27, 2007; 282(17): 13098 - 13113.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Gajate and F. Mollinedo
Edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts
Blood, January 15, 2007; 109(2): 711 - 719.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. Hunter and G. F. Nixon
Spatial Compartmentalization of Tumor Necrosis Factor (TNF) Receptor 1-dependent Signaling Pathways in Human Airway Smooth Muscle Cells: LIPID RAFTS ARE ESSENTIAL FOR TNF-{alpha}-MEDIATED ACTIVATION OF RhoA BUT DISPENSABLE FOR THE ACTIVATION OF THE NF-{kappa}B AND MAPK PATHWAYS
J. Biol. Chem., November 10, 2006; 281(45): 34705 - 34715.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
Y. Suarez, L. Gonzalez-Santiago, N. Zarich, A. Davalos, J. F. Aranda, M. A. Alonso, M. A. Lasuncion, J. M. Rojas, and A. Munoz
Plitidepsin Cellular Binding and Rac1/JNK Pathway Activation Depend on Membrane Cholesterol Content
Mol. Pharmacol., November 1, 2006; 70(5): 1654 - 1663.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Bari, P. Spagnuolo, F. Fezza, S. Oddi, N. Pasquariello, A. Finazzi-Agro, and M. Maccarrone
Effect of Lipid Rafts on Cb2 Receptor Signaling and 2-Arachidonoyl-Glycerol Metabolism in Human Immune Cells
J. Immunol., October 15, 2006; 177(8): 4971 - 4980.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Nieto-Miguel, C. Gajate, and F. Mollinedo
Differential Targets and Subcellular Localization of Antitumor Alkyl-lysophospholipid in Leukemic Versus Solid Tumor Cells
J. Biol. Chem., May 26, 2006; 281(21): 14833 - 14840.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
H.-L. Hsiao, W.-S. Wang, P.-M. Chen, and Y. Su
Overexpression of thymosin {beta}-4 renders SW480 colon carcinoma cells more resistant to apoptosis triggered by FasL and two topoisomerase II inhibitors via downregulating Fas and upregulating Survivin expression, respectively
Carcinogenesis, May 1, 2006; 27(5): 936 - 944.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
U. Nachbur, D. Kassahn, S. Yousefi, D. F. Legler, and T. Brunner
Posttranscriptional regulation of Fas (CD95) ligand killing activity by lipid rafts
Blood, April 1, 2006; 107(7): 2790 - 2796.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
N. Cahuzac, W. Baum, V. Kirkin, F. Conchonaud, L. Wawrezinieck, D. Marguet, O. Janssen, M. Zornig, and A.-O. Hueber
Fas ligand is localized to membrane rafts, where it displays increased cell death-inducing activity
Blood, March 15, 2006; 107(6): 2384 - 2391.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Yu, E. Davicioni, T. J. Triche, and G. Merlino
The Homeoprotein Six1 Transcriptionally Activates Multiple Protumorigenic Genes but Requires Ezrin to Promote Metastasis
Cancer Res., February 15, 2006; 66(4): 1982 - 1989.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Abouzahr, G. Bismuth, C. Gaudin, O. Caroll, P. Van Endert, A. Jalil, J. Dausset, I. Vergnon, C. Richon, A. Kauffmann, et al.
Identification of target actin content and polymerization status as a mechanism of tumor resistance after cytolytic T lymphocyte pressure
PNAS, January 31, 2006; 103(5): 1428 - 1433.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11641    most recent
M411781200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gajate, C.
Right arrow Articles by Mollinedo, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gajate, C.
Right arrow Articles by Mollinedo, F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement