Fig. 7.

Myo1e and myo1f promote local membrane lifting around phagocytic adhesion sites. a Cytoskeletal tension of WT and dKO BMDM (mean ± SD) measured using atomic force microscopy (n = 30 WT and 33 dKO cells, p = 0.0026, unpaired t-test). b Membrane tension (mean ± SD) measured in WT and dKO BMDM while resting (data pooled from two independent experiments, n = 69 WT and 75 dKO cells, p = 0.0003, unpaired t-test) and performing phagocytosis (data pooled from two independent experiments, n = 48 WT and 46 dKO cells, ****p < 0.0001, unpaired t-test). c Schematic of membrane tension measurement by the tether-pulling assay during phagocytosis. Beneath, representative bright-field image of assay. Scale bar, 10 µm. d Membrane lifting observed around phagocytic adhesions. TIRFM imaging of frustrated phagocytosis in RAW macrophage expressing mEmerald-myo1f and mScarlet-PM (plasma membrane marker) (inverted intensity for myo1f and PM). Yellow arrowheads in the zoomed image of the boxed region denote sites of membrane lifting around phagocytic adhesions. Scale bar, 10 µm; zoom scale bar, 2 µm. e Examples of myo1e/f placement at membrane lifting sites. TIRFM images of frustrated phagocytosis in RAW macrophages transfected with membrane marker and myo1e/f (inverted intensity for myo1 and PM). Top row contains schematic representations corresponding to merged panels. Scale bar, 1 µm. f, g Membrane lifting around phagocytic adhesions observed in WT (f), but not dKO (g) BMDM. WT and dKO BMDM with EGFP-actin and stained with membrane label FM 4-64 were imaged by TIRFM during frustrated phagocytosis (inverted intensity for actin and PM). Scale bar, 5 µm; inset scale bar, 2 µm. h Inverted fluorescence intensity maps obtained by averaging multiple phagocytic adhesions in WT and dKO BMDM and showing membrane (FM 4-64) relative to F-actin. Data from three independent experiments (n = 107 WT and 102 dKO adhesions from at least 15 cells). Scale bar, 1 µm. i Graphical model: myo1e/f tether membrane at phagocytic adhesion sites. Without these linkages, local membrane tension is altered, allowing unrestricted actin polymerization causing enlarged phagocytic adhesions. This results in an actin-dense phagocytic cup that completes closure at a slower rate