Self-organizing actin waves that simulate phagocytic cup structures

Abstract

This report deals with actin waves that are spontaneously generated on the planar, substrate-attached surface of Dictyostelium cells. These waves have the following characteristics. (1) They are circular structures of varying shape, capable of changing the direction of propagation. (2) The waves propagate by treadmilling with a recovery of actin incorporation after photobleaching of less than 10 seconds. (3) The waves are associated with actin-binding proteins in an ordered 3-dimensional organization: with myosin-IB at the front and close to the membrane, the Arp2/3 complex throughout the wave, and coronin at the cytoplasmic face and back of the wave. Coronin is a marker of disassembling actin structures. (4) The waves separate two areas of the cell cortex that differ in actin structure and phosphoinositide composition of the membrane. The waves arise at the border of membrane areas rich in phosphatidylinositol (3,4,5) trisphosphate (PIP3). The inhibition of PIP3 synthesis reversibly inhibits wave formation. (5) The actin wave and PIP3 patterns resemble 2-dimensional projections of phagocytic cups, suggesting that they are involved in the scanning of surfaces for particles to be taken up.PACS Codes: 87.16.Ln, 87.19.lp, 89.75.Fb.

Figure 1

Shape dynamics of actin waves viewed on a substrate-attached cell surface. Wave images have been recorded from a single cell of Dictyostelium discoideum at the indicated times by spinning-disc confocal microscopy. During propagation of the wave, the cell showed negligible net movement. The images are color-coded and superimposed on top of each other. Shape changes within a period of less than 2 minutes are best recognized by comparing the green and white images. Waves can fuse or they may split into two as in the red image at the end of the recorded sequence. The images are taken from Figure 1 in [5].

Figure 2

Spatial organization of actin waves and the temporal pattern of actin polymerization and depolymerization. Left panel: schematic cross-section through an actin wave showing the region at the front and close to the membrane where myosin-IB is enriched (green) and the sloping roof of the wave to which coronin is recruited (yellow). Vertical arrows indicate up and down regulation of actin polymerization. Data suggest two gradients of actin polymerization, one falling from the substrate-attached membrane to the top of the wave, the other from its front to the tail. Right panel: translation of the spatial profile into a temporal sequence of actin net polymerization and depolymerization. The profile illustrates the sequence of changes that occur when a wave passes over a point on the membrane. The data published in [4] suggest that an initial phase of high-rate actin polymerization turns into a longer phase of depolymerization.

Figure 3

Spatial relationship of actin organization to the presence or absence of PIP3 in the underlying membrane. The diagram represents a line scan through an actin wave pattern on a planar glass surface. On top, the relationship of actin- and PIP3-labels is shown. The fluorescence intensity of actin (red) peaks at the position of the wave formed at the border of a PIP3-rich area of the cell membrane (green). Horizontal arrows point into the changing directions of wave propagation. On the bottom, actin structures are shown to differ between the PIP3-rich area circumscribed by the wave and the external area depleted of PIP3. The Arp2/3 complex known to nucleate branches of actin filaments dominates in the PIP3-rich area, while proteins associated with anti-parallel bundles of filaments are prevailing in the external area. This diagram summarizes data published in [5] and [7].

Figure 4

Reversible suppression of actin wave formation by the PI3-kinase inhibitor LY-294002. Cells recovering from LatA treatment were allowed to form waves before 50 μM LY-294002 were added. In (A to D) time is indicated in seconds before and after addition of the inhibitor (t = 0), in (E) before and after its removal. In (A, B, and E) the LimEΔ-GFP label for actin (green) is superimposed on phase-contrast images showing cell shape (red). In (C and D) cells are double-labeled with mRFP-LimEΔ for actin (red) and GFP-clathrin light chains for clathrin-coated structures (green). A, B, actin wave formation is suppressed by LY-294002 while small actin patches are persisting. C, co-localization of the actin and clathrin labels in six patches at the substrate-attached surface of an LY-treated cell. D, clathrin and actin dynamics in an LY-treated cell. Arrowheads point to clathrin-coated structures (44 s) that recruit actin thus turning from green to red (49 s) and subsequently disappear from the membrane (55 s). E, recovery of wave formation in a big cell after the removal of Ly-294002. Bars, 10 μm in (A, B, and E); 5 μm in (C and D).

Figure 5

Comparison of actin-PIP3 patterns on a planar surface and in phagocytic cups. Left: autonomous actin waves (red) circumscribe a PIP3-rich area of the substrate-attached cell membrane (green). Right: similarly, an actin ring at the rim of a phagocytic cup surrounds the PIP3-rich membrane area invaginated in contact with a particle. Dotted red lines indicate accumulation of filamentous actin on top of PIP3-enriched membrane areas. This diagram is based on data reported in [7].

Figure 6

Arrangement of actin-associated proteins in phagocytic cups. During the uptake of yeast particles, cells were imaged to localize GFP-tagged myosin-IB, Arp2/3, or coronin (green) in combination with filamentous actin (red). Centers of the particles are indicated by asterisks. A, bright-field image of a late phagocytosis stage showing a particle being engulfed. B, double-fluorescence recording of the same cell showing GFP-myosin-IB enriched at the border of the cup close to the plasma membrane. C, uptake of a particle by a cell expressing GFP-Arp3, indicating coincident localization of the Arp2/3 complex and the labeled actin. D, the phagocytic cup in a cell expressing GFP-coronin shows coronin remote from the membrane at the interface between the actin layer and the cytoplasmic space. The progressing edge of the cup is free of coronin. Bar, 10 μm.