Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) Schematic representation of a 3-plex SILAC approach for profiling phosphorylation dynamics in resting and BCR-stimulated DG75 cells. DG75 cells were cultured in SILAC medium as indicated and were left untreated, or were BCR-stimulated, for 2, 5, 10, or 20 min. Daudi cells were stimulated for 2 and 10 min. Lysates were mixed in a 1:1:1 ratio and digested with trypsin. Resulting phosphopeptides were enriched by either SCX/TiO2 chromatography (global phosphoproteome analysis; GPome) or phosphotyrosine immunoprecipitation (pY-IP; pYome analysis), and analyzed by LC-MS/MS. For analysis of protein expression levels, proteins were separated by 1D-PAGE, digested with trypsin, and analyzed by LC-MS/MS (see SI Materials and Methods for details). (B) Schematic representation of a 2-plex SILAC approach for profiling phosphorylation changes upon inducible CD79a knockdown or upon SYK inhibition. DG75 cells were cultured in SILAC medium and treated as indicated. Lysates were processed as described in A. (C) DG75 and Daudi cells were loaded with the ratiometric Ca2+-chelator INDO-1-AM and subjected to BCR-induced Ca2+ flux analysis by flow cytometry.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Cell Culture, Chromatography, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: Tonic BCR signaling. (A) CD79a shRNAs are toxic for BL cell lines. The figure shows the fraction of GFP-positive, shRNA-expressing cells relative to the GFP-negative, shRNA-negative fraction at the times indicated (normalized to day 0). Data are representative of three experiments. (B) CD79a and actin immunoblots of lysates derived from DG75 cells that were treated with doxycycline for 18 h to express either unspecific shRNAs (Control) or shRNAs targeting CD79a. (C) BCR cell surface expression in DG75 control cells or CD79a knockdown cells was monitored by flow cytometry 18 h after shRNA induction. (D and E) Unsupervised clustering analysis of all p-sites that were regulated upon BCR stimulation/CD79a knockdown/SYK inhibition. Values for each p-site (row) in all conditions (columns) are colored based on the z-score of the log2-transformed SILAC ratios.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: shRNA, Expressing, Western Blot, Derivative Assay, Flow Cytometry, Inhibition, Transformation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) BCR cell surface expression was monitored by flow cytometry (red line, DG75; blue line, Daudi). (B) Scatter plots showing the fold-change of p-sites on mainly serines/threonines (GPome, Left) and mainly tyrosines (pYome, Right) as determined by quantitative MS upon BCR stimulation versus CD79a knockdown and SYK inhibition. Selected phosphorylated proteins and p-sites are highlighted.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Expressing, Flow Cytometry, Inhibition

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ reduces cSMAC formation and proximal BCR signaling at the immune synapse. Raji D1.3 B cells were transfected with either control siRNA or GMFγ siRNA and added to COS-7 APCs expressing the mHEL-HaloTag Ag (magenta). The cells were fixed at the indicated times and stained with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79, cyan). The B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images from one of five independent experiments. Scale bars: 5 μm. (B) The total fluorescence intensity of the mHEL-HaloTag Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each B cell and the median values were calculated for each time point. Each symbol on the graph represents the median value for the GMFγ knockdown cells, expressed as a percent of the median value for the control siRNA-transfected cells for the same time point in the same experiment. The differently shaped symbols represent five independent experiments. Paired t -tests were used to calculate p -values. (C) The percent of cells that had formed a cSMAC, defined as > 90% of the total Ag fluorescence intensity being contained in one or two clusters, is graphed. The different symbols represent independent experiments. Paired t -tests were used to calculate p -values. (D) The total fluorescence intensity of pCD79 that was present in clusters at the B cell-APC contact site was quantified for each B cell. The left panel shows representative data from one experiment. Each dot is one cell. n > 31 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. The Mann-Whitney U -test was used to calculate p -values. The right panel shows the results from five independent experiments, presented as in (B) , with n > 30 cells per condition in each experiment. Each symbol represents a single experiment in which the median pCD79 fluorescence intensity for GMFγ-depleted cells is expressed as a percent of the corresponding median value for the control cells. Paired t -tests were used to calculate p -values. (E) For each B cell represented in (D) , the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of the clustered mHEL-HaloTag Ag. The median (blue line) and interquartile ranges (black box) are shown. The data are presented as in (B , D) . **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p ≤ 0.05; ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Staining, Microscopy, Fluorescence, Knockdown, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ does not impair CD79 phosphorylation induced by soluble anti-Ig antibodies. Cells were transfected with either control siRNA or GMFγ siRNA. (A) Raji D1.3 B cells were stimulated with 20 μg/mL goat anti-mouse IgM to initiate signaling through the D1.3 BCR. (B) Ramos B cells were stimulated with donkey anti-human IgM. Cell lysates were analyzed by immunoblotting with antibodies that recognize the phosphorylated CD79a ITAM (pCD79a), actin (loading control), or GMFγ. The left panels show a representative experiment. The right panels show results from 4 independent experiments. For each sample, the pCD79a band intensity was divided by the corresponding actin band intensity (loading control). The resulting ratios were normalized to that for the 0 min control siRNA cell sample (defined as 1.0) in the same experiment. On the graph, each of the four experiments is indicated by a different symbol. The bars show the mean ± SEM. Paired t -tests were used to calculate p -values. ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Phospho-proteomics, Transfection, Control, Western Blot

Journal: eLife

Article Title: N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters

doi: 10.7554/eLife.87833

Figure Lengend Snippet: Wild-type (WT) splenic B-cells were pre-labeled with Cy3-Fab fragment of goat anti-mouse IgM + G at a concentration of 2.5 µg per 10 6 cells at 4 °C for 30 min, followed by incubation with Fab’-PLBs or Tf-PLBs for 5 min at 37 °C. Cells were fixed, permeabilized, stained for pCD79a, and imaged using interference reflection microscopy (IRM) and total internal reflection fluorescence microscopy (TIRF). Shown are representative IRM and TIRF images from three independent experiments. Scale bar, 2 µm.

Article Snippet: Antibody , Rabbit IgG anti-mouse pCD79a (Y182) (rabbit monoclonal) , Cell Signaling Technology , 14732 S , IF (1:100).

Techniques: Labeling, Concentration Assay, Incubation, Staining, Microscopy, Fluorescence

Journal: eLife

Article Title: N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters

doi: 10.7554/eLife.87833

Figure Lengend Snippet: ( A–G ) Flox control and B-cell-specific N-wasp knockout mouse (cNKO) B-cells incubated with AF546-Fab’-PLB were fixed at 1, 3, 5, and 7 min, permeabilized, stained for phosphorylated CD79a (pCD79a, Tyr182), and imaged using total internal reflection fluorescence microscopy (TIRF) and interference reflection microscopy (IRM). ( A ) Representative IRM and TIRF images of a flox control versus a cNKO B-cell at 7 min. Scale bars, 2 µm. ( B–D ) Ratios of pCD79a MFI relative to AF546-Fab’ MFI were plotted against AF546-Fab’ peak FI in individual AF546-Fab’ clusters in the contact zone of flox control ( B ), cNKO B-cells ( C ), or flox control and cNKO B-cells overlay ( D ). AF546-Fab’ clusters were identified as described in and . Blue dots represent individual AF546-Fab’ clusters with an equal number of clusters from the 4 time points. The black line and diamond symbols represent the average ratios of pCD79a MFI to Fab’ MFI in individual BCR-Fab’ clusters within the indicated Fab’ peak FI range. The brown line and square symbols represent the fraction of the AF546-Fab’ clusters out of the total, within the indicated Fab’ peak FI range. Clusters were divided into three populations based on their peak AF546-Fab’ FI, relatively low (<190), medium (190-280), and high (>280, detected only in contracted cells), and the pCD79a to AF546-Fab’ MFI ratios of the three populations were compared ( B and C ). Data were generated from three independent experiments with ~20 cells and ≥125 clusters per condition per experiment. *p<0.05, ***p<0.001, by non-parametric student’s t -test. The p -values in ( D ) were corrected using the Benjamini-Hochberg/Yekutieli method for false discovery rate control. ( E–G ) The mean fluorescence intensity (MFI) (± SEM) of AF546-Fab’ ( E ) and pCD79a ( F ) and the MFI ratio (± SEM) of pCD79a relative to AF546-Fab’ ( G ) in individual AF546-Fab’ clusters at indicated times were compared between flox control and cNKO B-cells and between different times. ( H–K ) WT B-cells treated with CK-689 or CK-666 after 2 min-incubation with AF546-Fab’-PLB. ( H ) Representative IRM and TIRF images of a CK-689- versus a CK-666-treated B-cell at 7 min. Scale bars, 2 µm. ( I–K ) The MFI (± SEM) of AF546-Fab’ ( I ) and pCD79a ( J ) and the MFI ratio (± SEM) of pCD79a relative to AF546-Fab’ ( K ) in individual AF546-Fab’ clusters were compared between CK-689- and CK-666-treated B-cells after 7 min stimulation. Data points represent individual clusters. Horizontal solid lines in the violin plots represent the mean, while the dotted lines represent the quartiles of the distribution. Data were generated from three independent experiments with ~20 cells per condition per experiment. *p<0.05, ***p<0.001, by non-parametric student’s t -test. MATLAB codes were used for detecting and quantifying AF546-Fab’ clusters .

Article Snippet: Antibody , Rabbit IgG anti-mouse pCD79a (Y182) (rabbit monoclonal) , Cell Signaling Technology , 14732 S , IF (1:100).

Techniques: Control, Knock-Out, Incubation, Staining, Fluorescence, Microscopy, Generated

Journal: eLife

Article Title: N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters

doi: 10.7554/eLife.87833

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit IgG anti-mouse pCD79a (Y182) (rabbit monoclonal) , Cell Signaling Technology , 14732 S , IF (1:100).

Techniques: Control, Liposomes, Antibody Labeling, Software

Journal: bioRxiv

Article Title: Epstein–Barr Virus BALF0/1 Subverts the Caveolin and ERAD Pathways to Target B-cell Receptor Complexes for Degradation

doi: 10.1101/2024.01.04.574276

Figure Lengend Snippet: BALF0/1 mediated BCR degradation requires the Ig heavy chain cytoplasmic tail (A) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79A targeting sgRNA, 4-HT induced into lytic cycle for 24 h. (B) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79B sgRNA, 4-HT induced into lytic cycle for 24 h. (C) Immunoblot analysis of WCL from Cas9+ P3HR-1 cells expressing the indicated CD79A and CD79B sgRNA, 4-HT induced into lytic cycle for 24 h. (D) Mean ±SEM percentage of cells with PM IgM signals as in Figure S6 of P3HR-1 cells expressing the indicated control or dual CD79A/CD79B sgRNA, uninduced or 4-HT induced, as indicated, using data from 12 randomly selected panels of 240 cells from n=3 replicates, analyzed using the ImageJ ComDet plugin. (E) Immunofluorescence analysis of CD79A, CD79B, IgM and calnexin in P3HR-1 cells expressing control sgRNA and 4-HT induced for lytic replication in the presence of bortezomib for 24h. (F) Fluorescence intensity line scanning for calnexin (magenta) and IgM (green) signals from the white rectangle region (E). (G) Immunofluorescence analysis of IgM, calnexin, CD79A and CD79B in Cas9+ P3HR-1 cells expressing CD79A and CD79B sgRNA. Cells were 4-HT induced for lytic replication and treated with bortezomib for 24 h. (H) Fluorescence intensity line scanning for calnexin (magenta) and IgM (green) signals from the white rectangle region in (G). (I) Mean ±SEM percentage of cells with overlapping calnexin and IgM signals as in panel E, G and Figure S6 of P3HR-1 cells expressing the indicated control or dual CD79A/CD79B sgRNA, uninduced or 4-HT induced in the absence or presence of bortezomib as indicated, using data from 12 randomly selected panels of 240 cells from n=3 replicates, analyzed using the ImageJ ComDet plugin. (J) Schematic model of the BCR complex immunoglobulin heavy (IgH) chain, light chain and the CD79A and CD79B signaling chains. The C’ IgM cytoplasmic tail residues are shown. (K) Immunoblot analysis of WCL from 293T cells transiently expressing wildtype (WT) or cytoplasmic tail deleted (ΔKVK) EGFP-tagged IgM heavy chain alone or together with either BXLF1 or BALF0/1. (L) Immunoblot analysis of WCL from 293T transiently expressing the IgA heavy chain, alone or together with BALF0/1 or BXLF1. Statistical analysis was performed with Student’s t-test unless otherwise specified. ns p > 0.05. White bars indicate scale. See also Figure S 6. Blots are representative of at least n=2 replicates.

Article Snippet: Antibodies used for immunoblot analysis in this study were: anti-Human IgM goat polyclonal antibody (Southern Biotech #2020-01), anti-Human IgG goat polyclonal antibody (Southern Biotech #2040-01), anti-HA-Tag (C29F4) rabbit mAb (Cell Signaling #3724), anti-GAPDH (D16H11) XP® rabbit mAb (Cell Signaling #5174), anti-CD79A rabbit polyclonal antibody (Proteintech #22349-1-AP), anti-CD79B (D7V2F) rabbit mAb (Cell Signaling #96024), anti-EBV BALF0/1 rabbit mAb (generated by Genscript for this study), anti-EBV ZEBRA Mouse mAb (BZ1) (Santa Cruz# sc-53904), anti-EBV Ea-D mouse mAb (1108– ) (Santa Cruz #sc-69679), anti-EBV p18 goat polyclonal antibody (Invitrogen #PA1-73003), anti-calnexin rabbit mAb (Cell Signaling #2433), anti-HRD1/SYVN1 rabbit polyclonal antibody (Proteintech #13473-1-AP), anti-BAP31 rabbit polyclonal antibody (Proteintech #11200-1-AP), anti-caveolin 1 mouse mAb (7C8) (Thermo Fisher #MA3-600), anti-GFP tag rabbit polyclonal antibody (Proteintech #50430-2-AP), goat anti-rabbit IgG, HRP-linked antibody (Cell Signaling #7074), goat anti-mouse IgG, HRP-linked antibody (Cell Signaling #7076) and bovine anti-goat IgG (H+L) HRP-linked antibody (Jackson ImmunoResearch Laboratory #805-035-180).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Fluorescence

Journal: Nature Communications

Article Title: NFAT2 is a critical regulator of the anergic phenotype in chronic lymphocytic leukaemia

doi: 10.1038/s41467-017-00830-y

Figure Lengend Snippet: Nfat2 ablation leads to histologic transformation of CLL to aggressive disease. a H&E staining of paraffin-embedded spleen sections of one representative TCL1 Nfat2 +/+ and TCL1 Nfat2 −/− mouse at 12 and 28 weeks of age ( upper panels ) and immunohistochemical staining for CD3, B220 and Ki-67 ( lower panels ). For higher magnification of B220 and Ki-67 staining and CD79a staining in TCL1 Nfat2 −/− mice and Staining of Nfat2 +/+ control mice see also Supplementary Fig. . b H&E staining of paraffin-embedded spleen sections of representative TCL1 Nfat2 +/+ and TCL1 Nfat2 −/− mice and immunohistochemical staining for CD3, B220 and CD79a at an age of 36 weeks

Article Snippet: Antibodies for CD79a (Abbexa, 1:100), LYN (clone 11A7, Abcam, 1:100) or CD79a (NSJ, 1:200) and LCK (Cell signalling, 1:50 and a conventional DAPI staining were used for immunofluorescence and co localisation analysis.

Techniques: Transformation Assay, Staining, Immunohistochemical staining, Control

Journal: Nature Communications

Article Title: NFAT2 is a critical regulator of the anergic phenotype in chronic lymphocytic leukaemia

doi: 10.1038/s41467-017-00830-y

Figure Lengend Snippet: Lck is a direct target gene of NFAT2 and co localises with the BCR in CLL cells. a Schematic illustration of the Lck gene with a putative NFAT binding site. b Chromatin immunoprecipitation (ChIP) with CLL patient cells stimulated for 16 h with PMA/ionomycin. Cells were fixed with paraformaldehyde and DNA content was sheared by sonication. ChIP was performed with NFAT2 (7A6) and IgG control antibodies. The number of immunoprecipitated regions for each gene was calculated and normalised to the respective IgG control. One representative experiment of three independent experiments is shown. c LCK mRNA expression in untouched isolated physiological B cells from healthy volunteers ( n = 6) and human CLL patients ( n = 11) with indolent CLL ( n = 6) and aggressive CLL ( n = 5) normalised to GAPDH assessed by RT-PCR (Welch’s t -test, mean ± S.E.M., * P < 0.05, ** P < 0.01, *** P < 0.001). d Representative LCK protein expression and its activating phosphorylation at Tyr394 in physiological B cells ( n = 2), indolent CLL ( n = 3) and aggressive CLL ( n = 3) cells assessed by western blotting. e , f Quantitative analysis of LCK e and P-LCK (Tyr394) f expression on western blots with physiological B cells ( n = 8), indolent CLL ( n = 8) and aggressive CLL ( n = 8) cells (Welch’s t -test, mean ± S.E.M., ** P < 0.01). g Co localisation assay: Proteins were labelled with monoclonal antibodies from different species (1) and subsequently incubated with oligonucleotide-coupled secondary antibodies (2). In the case of close proximity of both proteins, the oligonucleotides on the secondary antibodies are able to ligate (3). A subsequent polymerisation and DNA amplification initiates the development of a red fluorescent signal (4), which can be detected in the fluorescence microscope. h Cytospins of PBMCs from 20-week-old TCL1 Nfat2 +/+ and TCL1 Nfat2 −/− mice were either prepared without stimulation or after treatment with 20 µg/ml αIgM F(ab’) 2 fragments for 10 min. Cells were stained with antibodies for CD79a and LCK. Co localisation of CD79a and LYN was used as a positive control. For negative controls, the primary antibody against CD79a was not added. DAPI was used for nuclear staining (630×)

Article Snippet: Antibodies for CD79a (Abbexa, 1:100), LYN (clone 11A7, Abcam, 1:100) or CD79a (NSJ, 1:200) and LCK (Cell signalling, 1:50 and a conventional DAPI staining were used for immunofluorescence and co localisation analysis.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Sonication, Control, Immunoprecipitation, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Bioprocessing, Incubation, DNA Amplification, Fluorescence, Microscopy, Staining, Positive Control

Journal: ACS sensors

Article Title: Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-μFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

doi: 10.1021/acssensors.3c01060

Figure Lengend Snippet: Figure 4. Uni-μFCM quantified single-cell membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes with correspondingly quantitative identification systems established. (A) Scatter plots of single-cell expressions of CD3, CD79a, and LAMP1 from a T-lymphocytic cell line Jurkat, a B-lymphocytic cell line BALL-1, and a myeloid cell line HL-60. (B) Quantitative identifications of Jurkat, BALL-1, and HL-60 mainly based on single-cell expressions of CD3 and CD79a. (C) Scatter plots of single-cell expressions of CD3, CD79a, and LAMP1 from a healthy donor and a patient blood sample. (D) Quantitative identifications of T-lymphocytes, B-lymphocytes, and leukocytes with the myeloid source based on single-cell expressions of CD3 and CD79a as well as cell diameters.

Article Snippet: Fluorescence-labeled antibodies used for cell staining included antibodies of OCT4-Alexa 488 purchased from Thermo Fisher, USA, CD3-Alexa 488, XRCC3-Alexa 488, c-Myc-PE, CD79a-PE, Cdc20-PE, KLF4-PerCP, LAMP1-PerCP, and BubR1-PerCP purchased from NOVUS, USA.

Techniques: Membrane