Severing of budded yeast. Amoebae (D. discoideum) were plated with yeast (S. cerevisiae) in a glass bottom chamber and viewd by confocal microscopy. The amoebae were expressing mRFP-LimEdelta (red) to label actin filaments and PHcrac-GFP (green) to label the membrane of the nascent phagocytic cup, which is rich in phosphatidylinositol (3,4,5) trisphosphate. The phagocytic cup partially engulfs both lobes of a budded yeast; the cup initially extends beyond the mother-bud neck. The cup then retracts back to the neck, and actin filaments strongly accumulate there, applying force. This situation persists for six minutes, ending with severing of the yeast, sealing of the cup, and ingestion of one lobe of the severed yeast. The time series was collected at 3.94-second intervals using a Zeiss LSM 510 laser scanning confocal microscope equipped with a 63x 1.4 NA DIC objective lens. Pixel size 0.12 x 0.12 microns.
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