Particle rigidity influences F-actin flashing dynamics on phagosomes. RAW 264.7 macrophages stably transfected with LifeAct-RFP or LifeAct-CFP were challenged with either C3bi-opsonized sRBC or C3bi-opsonized polystyrene beads (3.87 μm) and monitored with epifluorescence. (A) Duration of F-actin recruitment on phagosomes from the initial increase in LifeAct signal, back to cytosolic levels (n=87 F-actin flashes from C3bi-sRBC phagosomes, n=50 F-actin flashes from C3bi-beads phagosomes). (B) Sum of the time taken for all F-actin recruitment events on a given CR-phagosome (n=15 C3bi-sRBC phagosomes, n=14 C3bi-beads phagosomes). (C) Time interval between two F-actin flashes on a given CR-phagosome (n=72 intervals from C3bi-sRBC phagosomes, n=36 from C3bi-beads phagosomes). (D) Time for first F-actin flash on phagosomes from phagocytic cup formation (n=15 phagosomes for each treatment). (E) Total number of F-actin flashes on a given CR-phagosome (n=15 phagosomes for each treatment). (F) Frequency of F-actin flashes on CR-phagosomes (n=3, averages within a given field of view at 40× magnification, from a total of 67 flashing CR-phagosomes and 204 non-flashing CR-phagosomes). (G,H) Representation of F-actin flashing dynamics on C3bi-sRBCs (G) and C3bi-beads (H). Error bars represent s.e.m. *P<0.05, **P<0.01.

CR3 integrin and focal adhesion proteins are recruited to F-actin flashes on phagosomes. (A) Immunofluorescence images of fixed RAW 264.7 macrophages challenged with C3bi-opsonized beads for 45 min. External particles were immunostained prior to cell permeabilization. F-actin was labeled with rhodamine phalloidin and cells were immunostained for CR3, talin, FAK or phospho-FAK (Tyr397). Arrows indicate F-actin flashing phagosomes and arrowheads indicate non-flashing phagosomes. (B) Quantification of recruitment of CR3, talin, FAK and phospho-FAK to flashing or non-flashing phagosomes after 45 min of internalization. CR3 recruitment to phagosomes was also analyzed at 20 min post-internalization. Numbers of phagosomes quantified were as follows: talin, n=50; FAK and phospho-FAK, n=90; CR3, n>150. (C) Live imaging of RAW 264.7 cells co-transfected with mEmerald-talin and LifeAct-RFP. Arrows indicate flashing phagosomes and time is indicated in minutes from the start of imaging. (D,E) Quantification of LifeAct (LA)-RFP flashing dynamics on 15 phagosomes from four independent movies. In mEmerald-talin transfected cells, the duration of LA-RFP recruitment to phagosomes was similar (D), but there was a significant increase in time between flashes on phagosomes, compared with cells transfected with LA-RFP alone (E). (F) Representative live-cell fluorescence images of RAW 264.7 cells stably transfected with LifeAct-RFP and transiently transfected with PH-PLCδ-GFP. Time is indicated in minutes, beginning from phagocytic cup formation. Arrowhead indicates a F-actin cup and arrows indicate a flashing phagosome that did not recruit the PI(4,5)P₂ probe. Error bars represent s.e.m. **P<0.01. Scale bars: 10 μm.

RhoA and Arp2/3 effectors are recruited and required for F-actin flashes on phagosomes. (A) Immunofluorescence images of fixed RAW 264.7 macrophages challenged with C3bi-opsonized beads for 45 min and stained with phalloidin for F-actin and antibodies against RhoA, p190RhoGAP, p34 Arp2/3 or WASP. Arrows indicate F-actin flashing phagosomes and arrowheads indicate non-flashing phagosomes. (B) Quantification of recruitment of RhoA, p190RhoGAP, p34 Arp2/3 or WASP to F-actin flashing or non-flashing phagosomes after 45 min of internalization. Numbers of phagosomes quantified were as follows: RhoA and WASP, n=20; p190RhoGAP, n>250; p34, n>350. (C) Live-cell imaging of RAW 264.7 cells co-transfected with GFP-rGBD and LifeAct-RFP. Arrow indicates a flashing phagosome after uptake of multiple particles and time is indicated in minutes from the start of imaging. Dashed boxes in the right-hand images indicate the region shown at higher magnification. (D,E) Quantification of GFP-rGBD flashing dynamics on seven phagosomes from four independent movies. In GFP-rGBD-transfected cells, the duration of LifeAct (LA)-RFP recruitment to phagosomes was similar (D), with a slight, but not significant, increase in time between flashes on phagosomes, compared with cells transfected with LA-RFP alone (E). (F) For ROCK inhibition, cells were pretreated with 10 μM Y-27632 ROCK inhibitor for 8 h prior to phagocytosis and fixation. F-actin was stained using rhodamine phalloidin to detect the number of flashing phagosomes in untreated cells, which was significantly reduced in Y-27632-treated cells. Three biological replicates were performed. (G) Spinning disk confocal imaging of LifeAct-RFP-expressing RAW 264.7 cells after uptake of C3bi-sRBCs and treatment with 150 μM CK-666 to inhibit Arp2/3. Arrow indicates a flashing phagosome that persisted for an extended period. Time is indicated in minutes from the start of imaging. (H) Number of phagosomes that exhibited F-actin flashes (LifeAct-RFP recruitment) in CK-666-treated cells, compared with DMSO-treated cells. (n>200 phagosomes). (I–K) Quantification of flashing dynamics and particle deformation in LifeAct-RFP-positive phagosomes in CK-666-treated macrophages; 30 flashing phagosomes from ten independent movies were analyzed. (I) Average flashing time and time between flashes in cells treated with CK-666. (J) Of the phagosomes that recruited LifeAct-RFP, frequent phenotypes included protracted (extended) flashes on phagosomes and long flashes with extended time between flashes. (K) sRBC morphology changes observed by DIC imaging in CK-666-treated cells. The majority (21/30) of sRBCs in LifeAct-RFP-positive phagosomes had no detectable change in morphology in Arp2/3-inhibited cells, with only nine out of 30 sRBCs showing detectable deformation in the presence of the drug. Error bars represent s.e.m. **P<0.01. Scale bars: 10 μm.

Non-muscle myosin IIA is present on flashing phagosomes and its inhibition attenuates particle deformation in phagosomes. (A) Immunofluorescence images of fixed RAW macrophages challenged with C3bi-beads for a 10 min pulse, followed by washing and allowing phagocytosis to proceed for 30 min. External beads were stained with Cy5. F-actin was labeled with rhodamine phalloidin and NMIIA was immunostained with a polyclonal antibody (Biolegend). Arrows indicate a F-actin flashing phagosome that was positive for NMIIA, and arrowheads indicate a non-flashing phagosome that did not recruit myosin. (B) To test for myosin involvement in F-actin flashing and particle deformation, cells were pretreated, or not, with 100 μM blebbistatin prior to C3bi-sRBC ingestion and live fluorescent imaging. Arrows indicate a LifeAct-RFP-positive phagosome in control (DMSO) cells where particle deformation was observed using DIC imaging. Arrowheads indicate a LifeAct-RFP-positive phagosome in blebbistatin-treated cells, where no visible changes in sRBC morphology were observed by DIC microscopy. (C) Frequency of F-actin flashes for phagosomes in untreated cells compared with treated cells. (D) Frequency of phagosome deformation observed through DIC in F-actin-positive phagosomes (n>50 phagosomes from three replicates of each treatment). Error bars represent s.e.m. **P<0.01. Scale bars: 10 μm.

F-actin flashes delay EEA1 recruitment and reduces LAMP accumulation on phagosomes. (A) RAW 264.7 cells challenged with C3bi-beads were fixed every 5 min after particle internalization for a total of 60 min and stained for EEA1 and F-actin. Arrows indicate a flashing phagosome and arrowheads indicate a phalloidin-negative phagosome. (B) EEA1 recruitment frequency to CR-phagosomes was determined at every time point (n=229 F-actin flashing CR-phagosomes across three biological replicates; n=1259 phalloidin-negative CR-phagosomes across three biological replicates). (C) To analyze the effects of F-actin flashing on phagolysosome formation, RAW 264.7 cells were fixed after 30 min of phagocytosis of C3bi-sRBCs. Cells were stained for LAMP1, F-actin and sRBCs. Arrows and arrowheads indicate flashing and non-flashing phagosomes, respectively. (D) The frequency of LAMP1-positive phagosomes was determined (n=24 flashing CR-phagosomes across three biological replicates; n=173 non-flashing phagosomes across three biological replicates). Error bars represent s.e.m. *P<0.05, **P<0.01. Scale bars: 10 µm.