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Video 2
Single-molecule detection of transient Lyn-GFP recruitment to a CD59 cluster (occurring constitutively) without temporal correlation with the STALL of the CD59 cluster. This video sequence is the original data for Fig. 4 (a and 4 b). A CD59 cluster (green spot) and a single molecule of Lyn-GFP (red spot) were observed simultaneously at video rate. First, the video clip is shown in real time, and then at a 10-fold-slower rate. The STALL part of the trajectory of the CD59 cluster is indicated in blue (otherwise, it is white). Lyn-GFP undergoes Brownian diffusion on the cytoplasmic surface of the plasma membrane (red trajectory). Although some Lyn-GFP (or LynN20-GFP) is located in the cytoplasm, its recruitment to CD59 clusters always takes place from within the membrane. When a Lyn-GFP molecule is recruited at the CD59 cluster, it is indicated by a yellow arrowhead. Colocalization lasted for six video frames (0.2 s). Bar, 500 nm. Note that in all of the experimental results reported here, including the recruitment of Lyn-GFP, Gαi2(YFP), and GFP-PLCγ, the observations were carried out on the upper plasma membrane. This was further confirmed by using 4.5-µm-diameter anti-CD59 IgG-coated beads. These beads bound to the upper surface, being excluded from the space between the bottom membrane and the cover slip because of their large size, and induced frequent transient recruitment of Lyn-GFP, Gαi2(YFP), and GFP-PLCγ, ascertaining that we were able to observe single-molecule events on the upper cell membrane by the present TIRF-related methods, including TIRF at the interface between the medium and the cytoplasm at the upper cell membrane (Sako et al., 2000; Teramura et al., 2006) as well as low-angle oblique (often called LAO) illumination (Tokunaga et al., 1997; Sako, 2006).