Fig. 3.

Moesin signaling is downstream of PIP2 sorting driven by solid structures. a PIP2 (green) was visualized with PH-PLCδ-GFP alongside actin cytoskeleton by SIM on RAW264.7 cells incubated with 3 μm naked polystyrene beads. Line profiles corresponding to fluorescence intensities of PIP2 and actin were generated across the indicated phagocytic cups. N = 5. Scale bars, 5 µm. b Identical to a except that pERM (T558) and actin were analyzed. Phospho-ERM were visualized with anti-pERM antibody. N = 4. Scale bars, 5 µm. c PIP2 (green), PC (red), and PE (red) were visualized with PH-PLCδ-GFP, TopFluor TMR-PC or TopFluor TMR-PE on RAW264.7 cells incubated with 3 μm biotin-BSA-coated polystyrene beads as in a. N = 3. Scale bars, 5 µm. d Schematic of fluorescence imaging of bead/cell contact by a bead delivered with AFM. e PH-PLCδ-mCherry and Moesin-EGFP were co-expressed in RAW264.7 cells. A polystyrene bead was used to contact the cell surface. Images were taken at a 6 s interval for 500 s. Localization of PIP2 and Moesin at the site of contact (indicated with “*”) was examined with kymographs generated from the indicated line. The normalized fluorescence is defined as the ratio of the fluorescence intensity of PIP2 or Moesin at the site of contact over non-contact regions on the cell membrane (right). N = 3. Scale bar, 5 μm. f Phagocytosis efficiency was examined for both RAW264.7 and DC2.4 cells in the presence of 0.1 and 10 mM Geneticin. n = 50, N = 4. ****p < 0.0001 by one-way ANOVA with Scheffé post hoc. g PH-PLCδ-GFP-expressing RAW264.7 or HEK293T cells were touched with a single polystyrene bead recorded with a 6 s interval for 900 and 1200 s, respectively. Polarized distribution of PIP2 upon touching at the site of contact (indicated with “*”) on cell membrane was examined with kymograph. The normalized fluorescence of PIP2, PC, and PE was calculated for RAW264.7 and HEK293T cells (right). n ≥ 10, N = 3. Scale bars, 5 µm. ***p < 0.001 by one-way ANOVA with Scheffé post hoc