PMC

Scanning electron micrographs and 3D illustrations of PS particles created for phagocytosis experiments. (A) Spheres. (B) Oblate ellipsoids (13%). (C) Prolate ellipsoids (7%). (D) Elliptical disks (9%). (E) Rectangular disks (5%). (F) UFOs (12%). Particles are monodispersed with average standard deviations of measured dimensions for each shape listed in parentheses. A portion of this variation is due to 2–5% standard deviation in the diameter of spheres used as starting materials. (Scale bars: 5 μm.)

Time-lapse video microscopy clips spanning 39 min of macrophages interacting with identical nonopsonized ED particles (major axis 14 μm, minor axis 3 μm) from two different orientations. (A) Cell attaches along the major axis of an ED and internalizes it completely in 3 min. (B) Cell attaches to the flat side of an identical ED and spreads but does not internalize the particle. Continued observation indicated that this particle was not internalized for >110 min. (Scale bars: 10 μm.) (See Movies 1 and 2.) At least three cells were observed for each orientation of each particle type and size. Similar results were observed in all repetitions.

Phagocytosis phase diagram with Ω and dimensionless particle volume V* (particle volume divided by 7.5 μm radius spherical cell volume) as governing parameters (n = 5 for each point). Initialization of internalization is judged by the presence of an actin cup or ring as described in the text. There are three regions. Cells attaching to particles at areas of high Ω, >45°, spread but do not initiate internalization (region C). Cells attaching to particles at areas of low Ω, <45°, initiate internalization (regions A and B). If V* is ≤1, internalization is completed (region A). If V* > 1, internalization is not completed because of the size of the particle (region B). The arrows above the plot indicate the point of attachment for each shape that corresponds to the value of Ω on the x axis. Each case was classified as phagocytosis or no phagocytosis if >95% of observations were consistent. Each data point represents a different shape, size, or aspect ratio particle.

Scanning electron micrographs and actin staining confirm time-lapse video microscopy observations. Micrographs (A–C) of cells and particles were colored brown and purple, respectively. (A) The cell body can be seen at the end of an opsonized ED, and the membrane has progressed down the length of the particle. (Scale bar: 10 μm.) (B) A cell has attached to the flat side of an opsonized ED and has spread on the particle. (Scale bar: 5 μm.) (C) An opsonized spherical particle has attached to the top of a cell, and the membrane has progressed over approximately half the particle. (Scale bar: 5 μm.) (D–F) Overlays of bright-field and fluorescent images after fixing the cells and staining for polymerized actin with rhodamine phalloidin. (D) Actin ring forms as remodeling and depolymerization enable membrane to progress over an opsonized ED by new actin polymerization at the leading edge of the membrane. (E) Actin polymerization in the cell at site of attachment to flat side of an opsonized ED, but no actin cup or ring is visible. (F) Actin cup surrounds the end of an opsonized sphere as internalization begins after attachment. (Scale bars in D–F: 10 μm.) At least five cells were observed for each orientation of each particle. Similar results were observed in all repetitions.

Definition of Ω and its relationship with membrane velocity. (A) A schematic diagram illustrating how membrane progresses tangentially around an ED. T̄ represents the average of tangential angles from θ = 0 to θ = π/2. Ω is the angle between T̄ and membrane normal at the site of attachment, N̄. (B) Membrane velocity (distance traveled by the membrane divided by time to internalize, n ≥ 3; error bars represent SD) decreases with increasing Ω for a variety of shapes and sizes of particles. Nonopsonized particles are indicated by filled circles, and IgG-opsonized particles are indicated by open squares. Each data point represents a different shape, size, or aspect ratio particle. The internalization velocity is positive for Ω ≤ 45° (P < 0.001). Above a critical value of Ω, ≈45°, the internalization velocity is zero (P < 0.001) and there is only membrane spreading after particle attachment, not internalization. The arrows above the plot indicate the point of attachment for each shape that corresponds to the value of Ω on the x axis. Error in Ω is due to the difference in the actual point of contact in time-lapse microscopy from that used to calculate Ω. Only points of contact within 10° of that used to calculate Ω were selected. All data points at the critical point, Ω = 45°, except UFOs, do not have error associated with Ω because of their symmetry.