MTOC Reorientation Occurs during FcR-mediated Phagocytosis in Macrophages

Supplemental Materials

This article contains the following supporting material:

• Fig1Amerge Movie - MTOC reorientation during phagocytosis in macrophages. Live cell time-lapse imaging of a RAW264.7 cell transfected with γ-tubulin-GFP to visualize the MTOC, overlayed with DIC to visualize the cell. The red arrow tracks the γ-tubulin-GFP. Cells were exposed to IgG-sRBCs in HPMI and images were collected at a rate of 1 frame/2 minutes for 1.5 hours.
• Fig1B Movie - MTOC reorientation during phagocytosis in macrophages. Live cell time-lapse imaging of two RAW264.7 cells transfected with CLIP-170-head-GFP to image the MTs and MTOC. Phagocytosis occurred on the same side of the cell as the MTOC (cell on left) and in a transfected cell where the MTOC was at a location that was already considered partially oriented (cell on right). Cells were exposed to IgG-sRBCs and images were collected at a rate of 1 frame/15 seconds for 20 minutes.
• Figure3 Movie - MTOC reorientation does not occur towards complement-opsonized particles. Live cell time-lapse imaging of a RAW264.7 cell transfected with γ-tubulin-GFP to visualize the MTOC. Fluorescent images were overlayed with DIC images to facilitate visualization of the particle. The red arrow tracks the γ-tubulin-GFP. Cells were exposed to C3bi-sRBCs and images were collected at a rate of 1 frame/minute for 2 hours and 35 minutes.
• Supplemental Figure 1 - Frustrated phagocytosis assay controls. (A) Quantification of fully oriented MTOCs in RAW264.7 cells plated on glass or IgG-coverslips, at extended time intervals. The % of fully oriented MTOCs at 60 minutes for cells plated on glass coverslips reaches the equivalent % level observed in cells plated on IgG-opsonized coverslips for 30 minutes. Data is mean ± S.E.M from 3 experiments (n>30). (B) Quantification of the proximity of the MTOC with respect to the IgG-bead versus the coverslip. RAW264.7 cells were plated on IgG-opsonized coverslips for 30 minutes or glass coverslips for 60 minutes, followed by 30 minutes exposure to 8 µm IgG-beads. Asterisks indicate p<0.05 compared to IgG coverslip group. Data is mean ± S.E.M from 3 experiments (n>15). (C) XZ confocal reconstructions of typical representative RAW264.7 cells after experimentation described in B). Cells were immunostained for γ-tubulin (red). Asterisks indicate location of IgG-bead within cell. Solid line (white) indicates position of coverslip relative to cell. (D) Quantification of the proximity of the MTOC towards the IgG-sRBC versus the coverslip in RAW264.7 cells after internalization of sRBCs while in suspension, followed by plating on IgG- or glass coverslips for 30 or 60 minutes, respectively. Asterisks indicate p<0.05 compared to IgG coverslip group. Data is mean ± S.E.M from 3 experiments (n>15). (E) XZ confocal reconstructions of typical representative RAW264.7 cells after experimentation described in D). Cells were immunostained for γ-tubulin (red) and sRBC (green). Solid line (white) indicates position of coverslip relative to cell.
• Supplemental Figure 2 - Inhibition of dynein and overexpression of mPAR-6-FLAG does not affect phagocytosis. (A and B) Quantification of the phagocytic ability of macrophages overexpressing p50 dynamitin-GFP (A) or mPAR-6-FLAG (B) constructs. Data is mean ± S.E.M from 3 replicate experiments (n>20). (C) Fluorescent and DIC merged image of a RAW264.7 cell transfected with mPAR-6-FLAG (green). After 30 minutes of exposure to 0.8 µm IgG-beads (arrows), cells were fixed and immunostained for pericentrin (red). Asterisks help delineate position of MTOC. Scale bar, 10 µm
• Supplemental Figure 3 - The MTOC does not reorient during Mac-1 mediated phagocytosis in macrophages. DIC and epifluorescent merged images of a RAW264.7 cell transfected with γ-tubulin-GFP (see also Supp. Fig. 3_mov). RAW264.7 cells were exposed to C3bi-sRBCs and time 0 represents time of sRBC attachment. No reorientation of the MTOC was observed throughout the process, as evidenced by its position (arrows) with respect to the nucleus and the sRBC. Asterisk indicates site of sRBC internalization. Scale bars, 10 µm.