Figure 6.

Syt VII–YFP and Lamp1-RFP are sequentially delivered from tubular lysosomes to nascent phagosomes. (A) Selected frames from a wide-field time-lapse video of a BMM transduced with Syt VII–YFP and Lamp1-RFP and exposed to 10 IgG-opsonized zymosan particles/cell (Videos 2–4, available at http://www.jcb.org/cgi/content/full/jcb.200605004/DC1). Frames from 0 min 0 s to 3 min 12 s: initial colocalization of Syt VII–YFP and Lamp1-RFP on tubular lysosomes expanding toward a site of phagosome formation. The arrow on the merged image at 3 min 12 s points to the boxed area reproduced in the enlarged, dissociated images above. Arrows point to tubular lysosomes containing both markers, and arrowheads point to Syt VII–YFP recruited to phagocytic cups (phase-contrast image, top). In the subsequent time points, Lamp1-containing tubules are seen wrapping themselves around Syt VII–YFP-containing phagosomes (frames from 7 min 35 s to 10 min 24 s; Videos 2–4). Dissociated, enlarged images are indicated by white lines originating from the merged image. Arrows point to the site of zymosan uptake. (B) Confocal microscopy images of BMMs transduced with Syt VII–YFP, exposed to IgG-opsonized zymosan for 20 min, fixed, and stained with anti-Lamp1 mAbs (red). In the same BMMs, two patterns of Lamp1 association with phagosomes can be seen (right). (1) Lamp1 patches (white lines) in close association with Syt VII–YFP-containing phagosomes (patches are likely to correspond to Lamp1 tubules surrounding the phagosome; A). (2) Lamp1 and Syt VII–YFP fully colocalize on the phagosome membrane (arrows). Bars (A), 10 μm; (B) 5 μm.