Figure 5.

GFP-PLCγ2 is recruited at CD59 clusters almost exclusively during the STALL period. (a) An image sequence showing superimposed video frames of simultaneous recordings of a CD59 cluster (green spot, which is the bright-field image of a 50-nm bead) and a single molecule of GFP-PLCγ2 (red spot, with Gaussian spatial smoothing). They were colocalized from frame 7 until frame 16 (pink frame numbers), which are within a STALL period. GFP-PLCγ2 suddenly appears from and returns to the cytoplasm. (b) A typical trajectory of a CD59 cluster (black), which includes three STALL periods (three circles). During one of the STALL periods (the blue part of the trajectory), a GFP-PLCγ2 molecule was recruited (magenta trajectory; the CD59 cluster trajectory during the colocalized period is shown in indigo; see Video 1, available at http://www.jcb.org/cgi/conent/full/jcb.200609175/DC1). The colocalized period is completely included within the CD59 cluster's STALL period. (c) Distribution of the time difference between the beginning of GFP-PLCγ2 recruitment and the onset of STALL (time 0 is set at the first frame of STALL). Note that in this graph, each STALL period starts at time 0 but ends at a different time (the pink shading showing STALL is, thus, graded). For the definition of the time difference (lag time), see Fig. 5 a in Suzuki et al. (2007). The yellow bars represent GFP-PLCγ2 molecules with recruitment periods that were completely included within the STALL period, whereas the black bars represent GFP-PLCγ2 molecules whose recruitment periods are not totally included within the STALL period. (d) Distribution of the period of GFP-PLCγ2 colocalization with CD59 clusters (median of 0.25 s), showing transient recruitment of GFP-PLCγ2. As a negative control, single-molecule recruitment of transferrin receptor to the CD59 clusters was observed. No statistically significant recruitment was detected, as described in Suzuki et al. (2007).