Supplemental Materials Molecular Biology of the Cell Gilberti et al.
SUPPLEMENTAL FIGURE LEGENDS: Figure S1: The effect of pharmacological inhibitors on particle uptake. The data presented in Figure 1 was replotted as phagocytic index as described in Methods. Unlike in Figure 1, nocodazole appears to inhibit the uptake of opsonized particles. However, this is due to a lower number of particles bound to cells in the presence of nocodazole (ie. total particles per cell). Of the opsonized particles that bind to cells, the same proportion is taken up in the presence and absence of nocodazole. Error bars represent SEM of at least three experiments performed in duplicate on separate days. Figure S2: Depolymerization of microtubules by nocodazole in MHS macrophages. Cells were treated with increasing concentrations of nocodazole and then fixed and stained with anti-tubulin antibody. 800 nm is the lowest concentration of nocodazole that completely inhibits polymerization. Figure S3: Actin-rich membranes form around COZ particle phagosomes. (A) PMA-stimulated GFP-actin-expressing macrophages were exposed to complementopsonized zymosan (COZ). After several seconds, actin associates with particles. (B) Time course of actin association with and dissociation from COZ particle phagosomes. Time zero represents maximum actin localization after particle-cell interaction. N=10. Error bars represent SEM. Figure S4: The localization of cortical actin changes when microtubules are depolymerized. (A, C) GFP-actin expressing macrophages were treated with 800 nm nocodazole (Noc) while imaging. Time zero represents the first frame after addition of the compound. There was a rapid increase in the localization of the probe to the peripheral actin cortex. (B, D) The same phenomenon was observed when macrophages were treated with 800 nm Noc and then fixed and stained with rhodamine phalloidin. Error bars represent SEM. Figure S5: Actin polymerization stalls at different stages of internalization in the presence of pharmacological inhibitors. Stably-transfected GFP-actin-expressing macrophages were treated with (A) 500 nm Cyto-D or (B) 500 nm latrunculin prior to exposure to Ab-opsonized (top time montage of each panel) or nonopsonized (bottom time montage of each panel) particles. In the presence of either of the inhibitors, some actin accumulates at Ab-opsonized-particle phagosomes (top panels), but no accumulation occurs at nonopsonized-particle phagosomes in the presence of latrunculin. Figure S6: Microtubule repolymerization stimulates new phagocytosis but not uptake of previously bound particles. Latex and silica particles can be distinguished
in the microscope because they interact with transmitted light differently (DIC panel). Macrophages treated with 800 nm nocodazole were exposed to Ova-Ltx (arrowheads) for 10 minutes before washing out the nocodazole and adding Ova-ssi particles (arrows). All of the Ova-ssi particles (9/9) added after release from nocodazole were rapidly internalized (black holes in the cytoplasm in the GFP-actin panel, but none (0/19) of the Ova-Ltx particles bound while microtubules were depolymerized were internalized. Reversing the order of particle addition produced the same result; 8/8 Ova- Ltx particles were internalized post-nocodazole, but 0/7 Ova-ssi particles were internalized. The Texas Red anti-tubulin panel displays immunostaining of microtubules that have re-polymerized after nocodazole washout. Figure S7: Pharmacological inhibitors are not toxic to MH-S macrophages. Cells were treated with nocodazole (Noc), cytochalasin-d (C-D), latrunculin-b (LatB), LY294002 (LY), or wortmannin (wort), at the concentrations used to inhibit phagocytosis, in the presence of propidium iodide to measure the extent of membrane permeable cells at 10 hours. Error bars represent SEM. Video 1: Phagocytosis of Ab-opsonized silica does not require intact microtubules. MH-S cells stably expressing GFP-actin were treated with 800 nm nocodazole and then exposed to Ab-opsonized 3-µm spherical silica. Ten z sections through the cell volume were collected at approximately one minute intervals. The movie shows a maximum intensity projection of the z slices and a single plane of the DIC images. In the presence of nocodazole, uptake of Ab-opsonized silica occurs in an actin-dependent manner. Video 2: Non-opsonized silica is internalized in a microtubule-dependent fashion. Stably-transfected GFP-actin MH-S cells were treated as in Video 1 except exposed to nonopsonized silica particles. In the presence of nocodazole, particles bind to cells, but GFP-actin does not associate with those sites and the particles are not internalized. Video 3: Actin associates with and dissociates from particle phagosomes before endo-lysosomal vesicles fuse with phagosomes. MH-S cells stably expressing GFP-actin were loaded with a red fluorescent dextran to label endolysosomal compartments and then exposed to nonopsonized silica particles. Images were acquired in multiple z sections at each time point. A single z section is shown. GFP-actin (green) rapidly associates with particle phagosomes upon particle binding to the cell. After actin dissociates, an increase in phagosomal fluorescence (red dextran) is observed, which is an indication of endolysosomal vesicle fusion with phagosomes. The dextran labeling of phagosomes is strongest for the first particles taken up and decreases in intensity as more particles are internalized by the same cell. There is no F-actin ring associated with the particle phagosome (lower middle of the cell) that appears as a black hole in the cytoplasm at 19 minutes because the actin ring is in a different focal plane. 2
Video 4: Actin associates with phagosomes before the early endosome marker 2xFYVE. MH-S cells were transiently, dual-transfected with 2xFYVE-GFP and Ruby-Lifeact and then exposed to non-opsonized 3-µm spherical silica. Images were acquired simultaneously in both fluorescence channels and DIC in a single Z plane. Ruby-lifeact associates with nascent particle phagosomes before 2xFYVE is recruited to the phagosomal membrane. 3
A 3 Phagocytic Index 2 1 0 B 4 3 2 1 Ab-ssi Ab-ssi + PP2 Ab-ltx Ab-ltx + PP2 Ova-ssi Ova-ssi + PP2 Ova-ltx Ova-ltx + PP2 0 6 4 2 0 Ab-ssi Ab-ssi + Noc Ab-ltx Ab-ltx + Noc Ova-ssi Ova-ssi + Noc Ova-ltx Ova-ltx + Noc Phagocytic Index C Ab-ssi Ab-ssi + LY Ab-ssi + Wort Ab-ssi + Lat Ab-ssi + CytoD Ab-ltx Ab-ltx + LY Ab-ltx + Wort Ab-ltx + Lat Ab-ltx + CytoD Ova-ltx Ova-ltx + LY Ova-ltx + Wort Ova-ltx + Lat Ova-ltx + CytoD Ova-ssi Ova-ssi + LY Ova-ssi + Wort Ova-ssi + Lat Ova-ssi + CytoD Phagocytic Index
A -360" -240" -120" 0" 120" B Phagosomal GFP-Actin (arb. unit) 300 200 100 COZ -400-200 0 200 400 Time (sec)
A 0 1' 2' 3' B -Noc +Noc C Cortical GFP-actin (arb. unit) 900 850 800 750 700 650 0 2 4 6 8 10 12 Time (min) D Cortical Phalloidin (arb. unit) 800 600 400 200 0 -Noc +Noc
100 PI Stained Nuclei at 10h (%) 80 60 40 20 0 Cells alone Cells alone + Noc Cells alone + wort Cells alone + LY Cells alone + C-D Cells alone + LatB