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These movies accompany the manuscript Clarke and Maddera, Eur. J. Cell Biol. 85, 1001-1010 (2006). They show the initial steps in the uptake, internalization, and killing of bacteria by Dictyostelium amoebae, strain AX2. The bacteria (E. coli B/r) are expressing GFP or DsRed Express, and the amoebae are expressing fluorescent labels for actin or microtubules. Time series of living cells were collected using a Zeiss LSM 510 confocal microscope.
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Movie
1.
Clarke_Movie1.mov,
431x327 pixels, 4.6MB compressed
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F-actin assembly during the formation and internalization of phagosomes containing bacteria. This cell was expressing mRFP-LimE, which binds to filamentous actin. The cell phagocytosed several bacteria at its leading lamellipodium during this time series, which spans 6 minutes.
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Movie
2.
Clarke_Movie2.mov,
252x248 pixels, 4.8MB compressed
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F-actin dynamics during membrane protrusion, phagocytic cup formation, and phagosome internalization. This cell was expressing mRFP-LimE and coronin-GFP. The latter marker, which may be involved in actin disassembly, labels new actin filaments a few seconds after LimE. Note the transient absence of actin after a phagosome seals, followed by new actin assembly during phagosome internalization.
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Movie
3.
Clarke_Movie3.mov,
321x293 pixels, 2.1MB compressed
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Uptake of bacteria by a cell expressing mRFP-LimE and coronin-GFP. New actin is added close to the phagosome during its internalization.
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Movie
4.
Clarke_Movie4.mov,
339x330 pixels, 4.9MB compressed
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Arp 2/3 dynamics during phagosome internalization. This cell is expressing GFP-Arp3, a subunit of the Arp2/3 complex. The labeling pattern suggests that the Arp2/3 complex is involved in phagosome internalization as well as uptake.
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Movie
5.
Clarke_Movie5.mov,
344x312 pixels, 3.6MB compressed
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Loss of bacterial cytoplasmic fluorescence following uptake. About 3 or 4 minutes after uptake of GFP-E. coli by an AX2 cell, the cytoplasmic GFP signal is lost.
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Movie
6.
Clarke_Movie6.mov,
435x364 pixels, 4.1MB compressed
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Acidification of the cytoplasm of phagocytosed bacteria. The amoebae were pre-incubated with Neutral Red, which stains acidic compartments. Recently-formed phagosomes containing GFP-E. coli were moved to the cell center, where they interacted with pre-existing endosomes and, ~5 to 6 minutes after uptake, became stained with Neutral Red.
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Movie
7.
Clarke_Movie7.mov,
268x291 pixels, 3.6MB compressed
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Microtubule-based phagosome transport. These amoebae, expressing GFP-alpha-tubulin, were pre-incubated with Neutral Red, then mixed with GFP-E. coli. Transport of recently-ingested phagosomes to the cell center took place along microtubules.
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| last
updated:
December 10, 2006
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