Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) Schematic depicts the supported lipid bilayer system used to study phagocytosis. Silica beads are coated with a supported lipid bilayer containing fluorescent atto647 lipids. Anti-biotin IgG binds to biotinylated lipids in the bilayer. 10xHis-tagged CD47 extracellular domain is attached to beads via a Ni 2+ -chelating DGS-NTA lipid, so that the SIRPα binding domain is positioned outwards. (B) Beads (magenta) conjugated with IgG or IgG+CD47 were incubated with mouse bone marrow derived macrophages (BMDMs) expressing membrane tethered GFP (GFP-CAAX; green) for 30 minutes then imaged with confocal microscopy. The average number of beads engulfed per macrophage was counted and normalized to the maximum average number of beads engulfed per macrophage for that experiment to control for batch to batch variability in macrophage appetite. (C) Schematic depicts the stages of phagocytosis: target particle binding, initiation of phagocytosis, and completion (phagocytic cup closure). (D) Representative images of each stage. BMDMs are expressing GFP-CAAX (green, top; greyscale, bottom) and the bead supported lipid bilayer is labeled with atto647 (magenta, top). Box in the top panel shows area of inset below. Timelapse confocal microscopy was used to quantify the fraction of beads bound to a macrophage (E); the time from binding to initiation of phagocytosis, if it occurred (F); the fraction of bound beads that proceed to the initiation step (G); the engulfment time, which is the time from initiation to completion, if it completed (H); and the fraction of beads that proceeded from the initiation to completion (I). For (F) and (H), the large filled data points represent the mean of an independent replicate, while the smaller unfilled data points of the same shape indicate individual cells quantified on the same day for that replicate. For (B) each dot represents an independent replicate composed of at least 100 macrophages per condition; data was compared using an unpaired t test. For (E)-(I) each independent replicate composed of at least 15 engulfment events. The means from four independent replicates were compared using an unpaired t test. In all graphs, bars denote the mean ± SEM. * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Binding Assay, Incubation, Derivative Assay, Expressing, Membrane, Confocal Microscopy, Control, Labeling

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) Timelapse images show reaching phagocytosis of an IgG bead target by a BMDM. White arrowheads point to extension of phagocyte membrane (GFP-CAAX, green) out around the target (atto647 lipid; magenta). White arrow denotes subsequent closure of the phagocytic cup while the bead remains outside the phagocyte cortex. Images correspond to Video S1 (B) Timelapse images show sinking phagocytosis of IgG+CD47 bead. Yellow arrows show closure of the phagocytic cup when the bead is within the macrophage. Images correspond to Video S2. (C) Graph depicts the fraction of successful phagocytosis that occurred via sinking phagocytosis based on the apparent morphology in timelapse confocal microscopy data. (D) Graph depicts the position of the bead centroid relative to the phagocyte cortex when phagocytosis was completed. (E) Graph depicts the fraction of retracted reaching cups out of the total number of initiated reaching cups. Retracted cups were defined as cases in our timelapse data set where membrane extensions grew around the target but were subsequently disassembled. (F) Images show an example of failed reaching phagocytosis of an IgG+CD47 bead. White arrowheads highlight extension of the macrophage membrane, which is subsequently retracted. Images correspond to Video S3. For (C) and (E), the averages from four independent experiments were plotted and compared using an unpaired t test. Bars represent the mean ± SEM. For (D), each data point represents an individual bead ( n =60 targets per condition from 4 independent experiments), and data was compared using an unpaired t test. ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Membrane, Confocal Microscopy

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A, B) BMDMs were incubated with IgG or IgG+CD47 beads then fixed and stained with 488 phalloidin (greyscale) to visualize F-actin during phagocytosis. Enrichment of F-actin at late-stage (>50% completed) phagocytic cup rims was measured by comparing the mean fluorescent intensity (MFI) of phalloidin at the cup rim (red dot) to the MFI of phalloidin at the cell cortex (blue line). Bead position is indicated with a dashed yellow line. (B) Graph depicts data quantification described in (A). The large filled data points represent the mean of an independent replicate, while the smaller unfilled data points of the same shape indicate individual cells collected on that day for that replicate. Dashed line at 1 corresponds to no F-actin enrichment at cup rims. (C) BMDMs were treated with pharmacological inhibitors of Rac and Cdc42 (NSC23766 and MBQ-167) or Rho (C3 transferase) for 24 hours, then incubated with IgG, IgG+CD47, or unopsonized supported lipid bilayer coated beads. Phagocytosis was measured by confocal microscopy. The average number of beads phagocytosed was normalized to the maximum phagocytosis in that replicate. (D) BMDMs were treated with pharmacological inhibitors of Rac and Cdc42 (NSC23766 and MBQ-167) or Rho (C3 transferase) for 24 hours, then the inhibitors were removed and replaced with fresh media. WT or CD47 KO mouse L1210 leukemia cells were dyed with CellTrace Far Red then opsonized with an anti-murine CD20 monoclonal antibody and added to the BMDMs. Phagocytosis was monitored for 10 hours via timelapse microscopy. The percent of BMDMs that engulfed was quantified, and data was normalized to maximum for that experiment. For (B), the means of 4 independent experiments were compared using an unpaired t test. For (C) and (D), each data point represents an independent experiment including quantification of at least 100 macrophages. Data was compared using one-way ANOVA with Holm Sidak multiple comparison test. In all graphs, bars represent the mean ± SEM. * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005, **** denotes p<0.00005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Incubation, Staining, Confocal Microscopy, Microscopy, Comparison

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A-E) WT or CD47 KO mouse L1210 leukemia cells were dyed with CellTrace Far Red (magenta) then opsonized with an anti-murine CD20 monoclonal antibody and added to the BMDMs (green) from WT or Rac2 E62K/+ mice. (A) Stills from a representative timelapse showing WT BMDMs incubated with CD47 KO L1210 cells. Yellow arrow indicates phagocytosis. (B) Stills from a representative timelapse showing Rac2 E62K/+ BMDMs incubated with CD47 KO L1210 cells. Yellow arrow indicates phagocytosis. (C) Stills from a representative timelapse showing WT BMDMs incubated with CD47-positive WT L1210 cells. (D) Stills from a representative timelapse showing Rac2 E62K/+ BMDMs incubated with CD47-positive WT L1210 cells. Yellow arrow indicates phagocytosis. (E) Graphs show the phagocytosis of L1210 cells during a 10 hour timelapse, normalized to the maximum phagocytosis on each day. Data compared using one-way ANOVA with Holm Sidak multiple comparison test. Line and bars denote mean and SEM. * denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Incubation, Comparison

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) BMDMs were incubated with unopsonized (--), IgG, or IgG+CD47 beads for 10 minutes, then lysed directly in 2X Laemmli sample buffer. Whole cell lysates were immunoblotted with the indicated antibodies. Representative of n = 3 independent replicates. (B, C) The ratio of phosphorylated Syk to total Syk or phosphorylated Vav1 to total Vav1 was quantified and normalized to the IgG condition for that replicate. (D) BMDMs were incubated with unopsonized (--), IgG, or IgG+CD47 beads for 10 minutes, then lysed in LB1 lysis buffer. Vav1 was immunoprecipitated, then probed for tyrosine phosphorylation. Representative of n = 2 independent immunoprecipitations. For (B) and (C), data was compared with one-way ANOVA with Holm Sidak multiple comparison test. Error bars denote SEM. * denotes p<0.05.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Incubation, Lysis, Immunoprecipitation, Comparison

Journal: bioRxiv

Article Title: CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

doi: 10.1101/2025.02.11.637707

Figure Lengend Snippet: (A) TIRF images show IgG (AlexaFluor 488-IgG, green) and mCherry-Vav1 (magenta) as BMDMs interact with an IgG (top) or IgG+CD47 (bottom) bilayer. The linescan shows the fluorescent intensity of AlexaFluor 488-IgG and mCherry-Vav1 at the indicated position (white arrow in images). Intensity was normalized so that 1 is the highest observed intensity and 0 is the lowest observed intensity. (B) The mean fluorescent intensity of mCherry-Vav1 at IgG clusters (left) and the area of the IgG clusters (right) was measured for n =30 cells landing on IgG or IgG+CD47 bilayers, pooled from 3 independent experiments. (C) Images from TIRF microscopy timelapse show IgG (black) clustering as BMDMs land and spread on a bilayer with IgG (top) or IgG+CD47 (bottom). Graph depicts the average area of contact from n ≥16 cells pooled from 3 separate experiments. (D) The Pearson’s Correlation Coefficient was calculated for mCherry-Vav1 and AlexaFluor 488-IgG in the footprint of n ≥38 cells from 3 separate experiments landing on IgG or IgG+CD47 bilayers. (E) BMDMs expressing membrane-tethered mCherry (mCherry-CAAX) or a hyperactive Vav1 mutant (mCherry-Vav3F) were incubated with unopsonized (--), IgG, or IgG+CD47 beads for 30 minutes, then visualized via confocal microscopy. The average number of beads engulfed per macrophage was quantified and normalized to the highest average for that experiment. Points denote averages from each of the 3 replicates comprising at least 100 macrophages. (F) Diagram shows the proposed pathway for CD47 signaling. Data was compared using an unpaired t test (B, D) or one way ANOVA with Holm Sidak multiple comparison test. (E). Line and error bars denote mean and SEM.* denotes p<0.05, ** denotes p<0.005, *** denotes p<0.0005. Scale bars are 10µm.

Article Snippet: Anti-biotin IgG (Jackson ImmunoResearch Laboratories Cat# 200-002-211) was added at 1 nM, and 10x-His tagged CD47 (Sino Biological, Cat# 57231-M49H-B) was added at 10 nM.

Techniques: Microscopy, Expressing, Membrane, Mutagenesis, Incubation, Confocal Microscopy, Comparison