Panel A shows flow cytometry dot plots of surface and total anti-Immunoglobulin M F(ab′)₂ in NTg and SORL1 knockout cells. Panel B shows a bar graph quantifying surface B-cell receptor internalization relative to NTg. Panel C shows a bar graph quantifying anti-Immunoglobulin M internalization relative to surface B-cell receptor. Panel D shows a dot plot comparing surface B-cell receptor internalization between wildtype and Sorl1 knockout cells. Panel E shows a bar graph quantifying transferrin internalization relative to NTg. Panel F shows confocal fluorescence microscopy images of anti-Immunoglobulin M F(ab′)₂ and Cluster of Differentiation 19 with merged and side-view images in NTg and SORL1 knockout cells. Panel G shows a dot plot quantifying anti-Immunoglobulin M positive vesicles per cell. Panel H shows a dot plot quantifying mean anti-Immunoglobulin M fluorescence intensity per vesicle. Panel I shows a dot plot quantifying anti-Immunoglobulin M internalization. Panel J shows a dot plot quantifying mean anti-Immunoglobulin M fluorescence intensity per cell. Panel K shows a dot plot comparing synapse area between NTg and SORL1 knockout cells. Panel L shows a line graph of phosphorylated Syk mean fluorescence intensity over time. Panel M shows a bar graph comparing phosphorylated ERK1/2 fold change in unstimulated and anti-Immunoglobulin M-treated cells. Panel N shows a bar graph comparing phosphorylated Akt1 Ser473 fold change between NTg and SORL1 knockout cells.
SorLA regulates BCR uptake in Ramos-Cas9 and primary murine B cells. (A) Representative flow cytometry dot plots showing anti-IgM F(ab′)2 internalization in NTg and SORL1KO Ramos-Cas9 cells. Surface (streptavidin fluorescence) and total surrogate antigen (anti-IgM fluorescence) stains of single live cells analyzed by flow cytometry following 15-min incubation on ice (blue) or at 37°C (red). (B) Quantification of soluble anti-IgM uptake using % Internalization calculation in NTg and SORL1KO Ramos-Cas9 cell lines relative to mean NTg % anti-IgM internalization per experiment. n = 10–14 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by different symbols) from five independent experiments. (C) Soluble anti-IgM surrogate antigen uptake in NTg and SORL1KO Ramos-Cas9 cell lines normalized to the initial surface IgM level (MFI at time = 0 on ice). n = 10–14 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by different symbols) from five independent experiments. (D) Soluble anti-IgM surrogate antigen uptake in WT and Sorl1−/− primary murine B cells (CD43−). Quantified using % internalization calculation and normalized relative to mean WT % anti-IgM internalization per experiment. P value shown calculated by Mann–Whitney U test, n = 19–20 mice from four independent experiments. (E) Soluble transferrin internalization using % internalization calculation for NTg and SORL1KO Ramos-Cas9 cell lines relative to mean NTg % transferrin internalization per experiment. n = 6–8 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by different symbols) from three independent experiments. (F) Representative fluorescence micrographs showing NTg (top) and SORL1KO (bottom) Ramos-Cas9 anti-IgM F(ab′)2 surrogate antigen uptake from PMS. Anti-IgM-loaded PMS (left), Ramos-Cas9 cells interacting with PMS (center), merge (right), magnified side view 3D projection (blue box). 100× magnification; scale bars = 10 μm. (G) Number of anti-IgM+ vesicles per cell in NTg and SORL1KO Ramos-Cas9 cell lines relative to mean NTg per experiment. (H) Mean anti-IgM fluorescence per anti-IgM+ vesicle per cell for NTg and SORL1KO Ramos-Cas9 cell lines relative to mean NTg per experiment. (I) Anti-IgM internalization as a percentage of total anti-IgM at the synapse per cell for NTg and SORL1KO Ramos-Cas9 cell lines relative to mean NTg cell lines. (J) Mean total anti-IgM fluorescence representing total amount internalized per cell for NTg and SORL1KO cell lines relative to mean NTg per experiment. (K) Synapse area of NTg and SORL1KO Ramos-Cas9 cells at contact with PMS at 15 min, measured using Analyze particles macro (ImageJ). n = 74–95 cells in three independent experiments. (L) Syk phosphorylation over 15 min at 37°C per phospho-flow time course experiment, n = 2–3 per experiment, two independent experiments. Background MFI subtraction using secondary only control. (M) p-Erk1/2 fold change in NTg and SORL1KO Ramos-Cas9 B cell lines, relative to unstimulated. p-Erk1/2 fold change quantified by densitometry of a p-Erk1/2 immunoblot. n = 4. (N) p-Akt1 Ser473 phospho-flow. p-Akt1 Ser473 fold change in NTg and SORL1KO Ramos-Cas9 B cell lines at 15 min, relative to 0-min time point. P value shown calculated by Mann–Whitney U test, n = 5–8 from three independent experiments. Data in B, C, E, L, and M show mean ± SEM. P values are calculated by unpaired t test. Data in D and G–K are shown as box and whisker plots, where the line indicates the median, the box represents the interquartile range, and whiskers denote the minimum and maximum values. P values calculated by Mann–Whitney U test, n = 100 cells from two independent experiments.