Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: GeCKO library essential gene survival scores. The plot shows mean CRISPR survival scores of sgRNAs targeting a set of top 200 essential genes (Wang et al, ). Correlation of the CRISPR survival score and sgRNA on‐target efficiency for a set of sgRNAs targeting the essential genes from the GeCKO library. On‐target efficiency was calculated using “Rule Set 2” algorithm (Doench et al, ). Brunello library essential gene survival scores. Correlation of survival score and sgRNA on‐target efficiency in Brunello library. Custom minilibrary essential gene survival scores.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: CRISPR

Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: Western blot showing loss of endophilin A2 protein after SH3GL1 targeting in Ramos cells using 2 independent sgRNAs, compared with 2 non‐targeting sgRNAs. Soluble anti‐IgM F(ab') 2 antigen internalization at 20 min in Ramos cells targeted with indicated sgRNAs. P , significance in one‐way ANOVA versus control. Plot shows mean and SEM of 7 independent sgRNA infections. Validation of gene candidates affecting soluble antigen uptake in primary mouse B cells. Follicular B cells isolated from CRISPR‐targeted bone marrow chimera mice internalize anti‐mouse IgM F(ab') 2 . Data show mean and SEM of N = 3 mice. P , statistics from 2‐way ANOVA; at 5 min, all 3 genotypes show P < 0.0005 versus control; at 15 min P values are indicated in corresponding color. Antigen internalization from PMS in SH3GL1 ‐targeted Ramos cells. **** P < 0.0001 in one‐way ANOVA. N = 547, 389 and 136 cells, respectively, from 2 experiments. Internalization of soluble Fab or F(ab') 2 fragments in Ramos cells targeted with indicated sgRNAs. * P = 0.0124 ** P = 0.0092 *** P = 0.0004 using 2‐way ANOVA. Data show mean ± SEM, from 6 biological replicates across two experiments. TIRF images of Ramos cells transduced with endophilin A2‐GFP and mCherry‐ClathrinLC on antigen‐loaded PMS. Scale bar = 5 μm. White arrowheads highlight endophilin A2 colocalization with antigen clusters. See also Movie . Percent of endophilin A2 and clathrinLC spots colocalizing with each other. Levels of random spot colocalization were calculated using randomized spot coordinates. Data show mean and SEM from 107 cells analyzed from 4 experiments; statistics from one‐way ANOVA. Percentage of surrogate antigen spots colocalizing with endophilin A2, clathrin LC or both markers. Levels of random spot colocalization were calculated using randomized spot coordinates. Data show mean and SEM from 107 cells analyzed from 4 experiments; statistics from one‐way ANOVA. Colocalization of surrogate antigen clusters with endophilin A2‐GFP following deletion of clathrin adaptors EPN1 or PICALM1. Data show mean and SEM from 23–29 cells. IgM‐BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs. Anti‐endophilin A2 blots detect overexpressed endophilin A2‐GFP in streptavidin immunoprecipitates and total supernatants from anti‐IgM‐biotin‐stimulated Ramos and DG75 cells. Representative experiment and quantification from three independent pulldowns in WT, BLNK ‐, or GRB2 ‐knockout (KO) DG75 cells. Pulldown intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment. Data show mean and SEM; P values calculated using one‐way ANOVA. Recruitment of endophilin A2 to antigen clusters quantified as mean antigen intensity in endophilin A2 clusters relative to mean antigen intensity in the entire synapse. N = 30, 29, and 36 cells, respectively, from 2 experiments. P , significance in Kruskal–Wallis test with multiple comparisons.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: Western Blot, Control, Biomarker Discovery, Isolation, CRISPR, Transduction, Immunoprecipitation, Knock-Out

Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: CRISPR‐targeted bone marrow chimera workflow, showing flow cytometry detection of donor‐derived (GFP + ), sgRNA‐containing (mCherry + ) cells. Development of B‐cell populations in chimeras' bone marrow and spleen from Cd4 ‐ or Sh3gl1 ‐targeted HSCs. Percentage of mCherry + cells at each stage is normalized to initial infection rate of transferred lineage ‐ cells, and shown as mean + SEM. N = 11–12 mice. * P = 0.0286, *** P < 0.0001 using two‐way ANOVA. MZ development in mCherry + B‐cell compartment. Representative stain of surface CD86 and CD69 on adoptively transferred HEL‐specific follicular B cells 24 h post‐SRBC‐HEL immunization. Quantification of surface CD86 and CD69 mean fluorescence intensity (MFI). Data show mean and SEM from six mice across two experiments. Fractions of mCherry + cells in GC, PC, IgG1 + class‐switched and follicular B cells at 4‐, 8‐, and 14 days post‐SRBC‐HEL immunization. N = 8–10 mice; data shows mean and SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001 using 2‐way ANOVA with multiple comparisons.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: CRISPR, Flow Cytometry, Derivative Assay, Infection, Staining, Fluorescence

Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: A Surveyor assay detecting genomic DNA editing. 200bp genomic region around the targeted PAM site in Sh3gl1 is amplified from cells expressing Sh3gl1 , Cd4 , or non‐targeting sgRNA. PCR amplicons from targeted and wild‐type DNA are mixed, denatured, and re‐annealed. CRISPR‐mediated editing results in mis‐matched DNA loops, which are cleaved by an endonuclease and visualized as two separate bands. B Sh3gl1 transcript levels in sorted donor B‐cell populations from chimeric mice with mCherry expression between 40 and 60%. Data show mean + SEM from four mice in two experiments. C–F Total antibody levels measured by clonotyping ELISA using capture antibody. One representative experiment showing measurements from 4 mice. Data show mean + SEM. (C) IgM (D) IgG1 (E) IgG3 (F) IgA. G CRISPR‐targeted Ramos, activated with 2 μg/ml of anti‐human IgM F(ab') 2 . Data show mean and SEM from 4 independent infections. H Primary B cells from Sh3gl1 −/− mice or WT littermates, activated with 2 μg/ml anti‐mouse Igκ F(ab') 2 , for 380 seconds, followed by addition of ionomycin (5 μg/ml) at 300 s. N = 3 mice. Data show mean + SEM. I Western blot detection of total and phosphorylated Syk, Erk, and Akt in primary B cells from Sh3gl1 −/− mice or WT littermates, activated with 2 μg/ml anti‐mouse Igκ F(ab') 2 . J Chemokine‐induced migration through ICAM‐1 or anti‐Igκ‐coated transwells. mCherry + percentage is normalized to input population. N = 2 mice.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: Amplification, Expressing, CRISPR, Enzyme-linked Immunosorbent Assay, Western Blot, Migration

Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: Soluble anti‐IgM internalization in CRISPR‐targeted follicular B cells. N = 11–12 mice. Data show mean ± SEM. P , statistical significance from two‐way ANOVA. Soluble anti‐IgD internalization. N = 3 mice. Data show mean ± SEM. P , statistical significance from two‐way ANOVA. Soluble anti‐IgG internalization in GC B cells from SRBC‐immunized mice. N = 4 mice. Data show mean ± SEM. P , statistical significance from two‐way ANOVA. Internalization of anti‐IgM from PMS quantified in fixed naïve B cells at 10‐min post‐synapse formation. N = 172 and 97 cells analyzed in one out of two representative experiments. Data show mean ± SEM. P = 0.0001 in unpaired t ‐test. Surface IgM or IgD MFI relative to WT in naïve splenic follicular B cells. N = 3 mice. Data show mean ± SEM. IgM degradation following total cell surface biotinylation, measured as loss of biotin‐labeled membrane IgM (mIgM) in cell lysates over time. Western densitometry quantifying IgM degradation assay. Proportion of biotin detected is normalized to the initial WT condition (left) or to the initial amount for each genotype (right). Data show mean ± SEM from three independent tests. P , statistical significance from two‐way ANOVA. MHCII antigen presentation in SWHEL B cells 24 h post‐adoptive transfer and immunization with Ea‐HEL‐SRBC conjugate as detected by anti‐Y‐Ae surface stain. Relative anti‐Y‐Ae MFI in CRISPR‐targeted B cells normalized to HEL‐only immunized mice. N = 10 immunized mice across 3 experiments. Data show mean ± SEM. In vitro antigen presentation in CRISPR‐targeted B cells measured by anti‐Y‐Ae. N = 8 independent cultures in 2 experiments. Data show mean ± SEM. Proliferation (CFSE dilution) of OT‐II CD4 cells in co‐cultures with anti‐Igκ‐OVA pulsed B cells; and quantification from 6–8 mice. Data show mean ± SEM. Relative anti‐Y‐Ae MFI in GC from immunized wild‐type and Sh3gl1 −/− mice. N = 11 from two experiments. Data show mean ± SEM.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: CRISPR, Labeling, Membrane, Western Blot, Degradation Assay, Immunopeptidomics, Adoptive Transfer Assay, Staining, In Vitro

Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: A CRISPR‐targeted follicular B‐cell proliferation after 3 days of CD40L stimulation. mCherry + populations were quantified within individual DDAO peaks. N = 3 mice. P , statistical significance calculated by two‐way ANOVA reached P < 0.0001 for all proliferation peaks. B CD80 mRNA levels in B cells following 24 h of CD40L culture, relative to untreated B cells. Data from RNAseq of N = 3 mice. C, D Isolated naïve B cells from CRISPR‐targeted chimeras after 3‐day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. * P < 0.05, ** P < 0.01, **** P < 0.0001 using two‐way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3–4 mice. E mCherry + cell percentage in CRISPR‐targeted Ramos cells over time. N = 6 independent experiments. F Cell counts at passage 3–6 of CRISPR‐targeted puromycin‐selected Ramos cells. N = 4 independent infections. P , statistical significance from unpaired t ‐test. G Oxygen consumption rate (OCR) in CRISPR‐targeted Ramos cells. N = 2 independent infections. P , two‐way ANOVA of basal and maximum OCR. H OCR in B cells isolated from Sh3gl1 −/− and WT littermates. N = 9 mice. P , two‐way ANOVA of basal and maximum OCR. I OCR in T cells isolated from Sh3gl1 −/− and WT littermates. N = 4 mice. J–L Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO 4 or 5 μM hemin chloride. (J) SH3GL1 ‐targeted Ramos cells. N = 4 independent infections. P , statistical significance from two‐way ANOVA from both treatments versus RPMI. (K) Control sgRNA‐targeted Ramos cells. (L) Sh3gl1 −/− isolated B cells, maintained in CD40L culture. N = 3 mice. P , significance calculated by two‐way ANOVA. M B cells isolated from WT littermates, maintained in CD40L culture. N OCR in Sh3gl1 −/− or WT B cells after overnight culture in CD40L, supplemented with 5 μM hemin chloride. N = 6 mice. P , two‐way ANOVA with matched samples for basal and maximum OCR. O TfR internalization assay in Ramos cells using biotinylated anti‐CD71. Four independent CRISPR infections in two experiments. P , significance calculated using two‐way ANOVA and indicated in corresponding color. P, Q Mouse transferrin internalization in B cells from SRBC‐immunized WT and Sh3gl1 −/− littermates. N = 7 mice per genotype in two experiments. P , statistical significance analyzed using two‐way ANOVA. (P) Naïve B cells. (Q) GC B cells. Data information: All data show means ± SEM.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: CRISPR, Isolation, Staining, Control

Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: Heat map of TPM values from RNAseq comparing follicular B cells from WT and Sh3gl1 −/− littermates. Shown are the top 20 genes upregulated in WT B cells 24 h after CD40L activation. Numbers of viable cells in 3‐day culture of follicular B cells in specified cytokines. N = 3 mice. Data show mean and SEM; P values are calculated using two‐way ANOVA. AnnexinV stain of B‐cell cultures in (B). Data show mean and SEM; P values are calculated using two‐way ANOVA. Endophilin A2, EPN1, and PICALM survival scores in hematopoietic cell lines from our screens using the Brunello library in Ramos cells, together with CRISPR scores from published genome‐wide screens (Wang et al, ; Wang et al, ; Phelan et al, ). Scores are normalized using the mean value of the top 200 essential genes in each screen. Dotted line indicates typical CRISPR score threshold for gene essentiality. Decreased percentage of SH3GL1 ‐targeted Ramos population in experiments with indicated initial percentage of targeted cells. Data show one representative experiment out of 3. BrdU pulse‐chase assay in Ramos cells. Clockwise from top left: equal BrdU incorporation at time = 0 h; slower progression of BrdU‐labeled cells into G2 phase in SH3GL1‐ compared with CTRL‐targeted cells at time = 4 h; slower progression into G1 of following cycle while greater proportion of SH3GL1 cells remain in initial S phase at time = 8 h; slower progression between G2 and M phase in SH3GL1 ‐targeted cells resulting in significantly greater numbers in G2 and less in G1 or S phase of subsequent cycle. N = 4 independent infections. Data show mean + SEM. * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 using 2‐way ANOVA with multiple comparisons. GSEA of RNAseq analysis comparing WT and Sh3gl1 −/− sorted follicular B cells. Three mice of each genotype were used for RNAseq and top 10 significantly enriched gene sets up‐ or down‐regulated in Sh3gl1 −/− B cells are shown. Expression of genes upregulated in Sh3gl1 −/− B cells in GO: 0006879 cellular_iron_ion_homeostasis. N = 3 mice. Data show mean ± SEM.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: Activation Assay, Staining, CRISPR, Genome Wide, Pulse Chase, BrdU Incorporation Assay, Labeling, Expressing

Journal: EMBO Reports

Article Title: Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

doi: 10.15252/embr.202051328

Figure Lengend Snippet: mCherry percentage in SH3GL1 ‐targeted Ramos cells over time, supplemented with 5× normal concentration of l ‐Glutamine or NEAA. N = 5 independent infections. Data show mean ± SEM. mCherry percentage in CTRL‐targeted Ramos cells over time, supplemented with 5× normal concentration of l ‐Glutamine or NEAA. N = 5 independent infections. Data show mean ± SEM. Surface GLUT1 stain in CRISPR‐targeted Ramos cells, N = 8 across 3 experiments. Data show mean ± SEM. Accumulation of 6‐NBDG (fluorescent glucose analog) after 10‐min internalization in Ramos cells; N = 4. Data show mean ± SEM. Anti‐TUFM immunofluorescence showing total mitochondrial mass and organization in CRISPR‐targeted Ramos cells. Scale bar = 5 μm. MitoTracker Red CMXRos stain for active mitochondria in primary B cells from WT and Sh3gl1 −/− B cells. Human transferrin uptake in targeted Ramos cells. Data show mean ± SEM from six independent CRISPR infections in two experiments; significance calculated using two‐way ANOVA. Ramos expressing clathrinLC‐mCherry and endophilinA2‐GFP internalize Atto647N‐labeled human transferrin. Quantification of transferrin clusters with either clathrin spots, endophilinA2 spots, or both. N = 25–30 cells across two experiments. Significance calculated using two‐way ANOVA.

Article Snippet: CRISPR sgRNA sequences were designed using the Broad Institute's sgRNA Designer.

Techniques: Concentration Assay, Staining, CRISPR, Immunofluorescence, Expressing, Labeling