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Endosome fusion in living cells overexpressing GFP-rab5

R. L. Roberts, M. A. Barbieri, K. M. Pryse, M. Chua, J. H. Morisaki and P. D. Stahl

Department of Cell Biology and Physiology and Biochemistry and Molecular Biophysics, Washington University, School of Medicine, St Louis, MO, 63110, USA

TRvB1 cells overexpressing rab5:Q79L by phase contrast videomicroscopy. Photomicrographs show a cluster of docked endosomal vesicles that, over time, participate in multiple "bridge" fusion events. Note that the final "bridge" fusion event is incomplete and results in one giant vesicle as well as one tiny vesicle (arrowheads 2m and 2n). Relative times in seconds are shown. Bar, 2 microns.

TRvB1 cell overexpressing rab5:Q79L recorded by phase contrast videomicroscopy. Photomicrographs show a cluster of giant vesicles that over time show multiple fusion events. The sequence begins with two "bridge" fusion events (black arrowheads 2b, 2c, 2d; white arrowheads 2c, 2d). Arrowheads point to donor vesicles in fusion couplets that show gradual reduction in diameter with incorporation into larger, acceptor vesicles. An "explosive" fusion is shown in 2f through 2l. In "explosive" fusion the "fusion pore" rapidly enlargers (arrows 2f, 2g, and 2h). A third "bridge" fusion event is also displayed (arrowheads 2j, 2k, 2l and 2m). Relative times are shown. Bar, 2 microns.

BHK cell overexpressing GFP-rab5:Q79L. Sequence shows an endosome docking reaction and an "explosive" fusion event. The time interval between frame 2b and 2c is 6 seconds. Note that the vesicles make contact in 5b without an increase in pixel intensity in the "bridge" region while in 2c (six seconds later) there is a nearly 3 fold fluorescence intensity increase in the "bridge" region compared to the rest of the membrane. Bar in 2d, 1.2 microns.

A photobleached fusion couplet in which explosive fusion occurs at t=0s. One portion of the fusion product (top) shows increased fluorescence compared to the bleached portion. The fluorescence intensities over the entire membrane equalize over time.

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