Supplemental Material

This article contains the following supporting material:

• Movie 1A - Pressure-induced actin accumulation and phagosome movement viewed by TIRF microscopy. The phagosomes (containing TRITC-labeled yeast) moved in changing directions according to the site of strongest actin deposition (Figure 1A).
• Movie 1B - Although phagosome movement was commonly preceded by an annular accumulation of actin, sometimes a single focus of actin was observed.
• Movie 2 - A TIRF view of the induction of phagosome movement in several adjoining cells by gradual drying of an agar overlay. The phagosomes exhibited multiple cycles of rocketing, with MyoB-GFP accumulating during each pause (Figure 1B).
• Movie 3 - For moving phagosomes, GFP-Arp3 and LimEΔ exhibited almost complete overlap, although the mRFP-LimEΔ signal was slightly enriched close to the phagosome in the deeper focal plane shown here. (Figure 3C)
• Movie 4 - Each time a phagosome paused, a ring of GFP-Arp3 appeared at the plasma membrane. When the phagosome moved away, the ring was left behind and eventually disappeared. (Figure 4A)
• Movie 5A - Back-and-forth phagosome movement in cells expressing GFP-Arp3.
• Movie 5B - Continuing view of a cell from Movie 5A, showing conversion of back-and-forth movement to pinwheeling. The focus was shifted from the plasma membrane to the cell center and back, revealing GFP-Arp3 only near the plasma membrane. (Figure 4B)
• Movie 6 - Rocketing phagosome in a cell expressing mRFP-LimEΔ and coronin-GFP. Each change of direction was accompanied by new actin filaments, which were labeled first by mRFP-LimEΔ, then by coronin-GFP, and finally disappeared. (Figure 5)
• Movie 7 - VatM-GFP was present in the membrane of these rocketing phagosomes, indicating that the phagosomes were in the acidic stage of endocytic transit. (Figure 6A)
• Movie 8 - This rocketing phagosome made three circuits through the cytoplasm during the 232-second time series, traveling at an average velocity of 23 μm/min. It rammed and distorted the nucleus (red) on each circuit. (Figure 7A)
• Movie 9 - This rocketing phagosome, constrained and guided by microtubules, often collided with the centrosome and nucleus, exhibiting sufficient force to displace the nucleus and straighten the microtubules. (Figure 7B)
• Supplemental Movie 1 - Phagosome behavior in a myoB^- mutant. Dictyostelium myoB^- cells (Novak et al., 1995) were transformed with plasmids expressing GFP-α-tubulin and mRFP-LimEΔ. The myoB^- cell shown in this movie was mixed with living S. cerevisiae about 2.5 hours earlier and had phagocytosed two yeast cells. When the myoB^- cell was compressed by an agarose overlay, actin accumulated about the two phagosomes, and one of them began to move. However, the moving phagosome exhibited limited displacement and exerted little apparent force. This movie, like Movie 9, is shown at 24x actual speed.