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.2007 May 21;177(4):731-42.
doi: 10.1083/jcb.200609175.

Dynamic recruitment of phospholipase C gamma at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2

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Dynamic recruitment of phospholipase C gamma at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2

Kenichi G N Suzuki et al. J Cell Biol..

Abstract

Clusters of CD59, a glycosylphosphatidylinositol-anchored receptor (GPI-AR), with physiological sizes of approximately six CD59 molecules, recruit Galphai2 and Lyn via protein-protein and raft interactions. Lyn is activated probably by the Galphai2 binding in the same CD59 cluster, inducing the CD59 cluster's binding to F-actin, resulting in its immobilization, termed stimulation-induced temporary arrest of lateral diffusion (STALL; with a 0.57-s lifetime, occurring approximately every 2 s). Simultaneous single-molecule tracking of GFP-PLCgamma2 and CD59 clusters revealed that PLCgamma2 molecules are transiently (median = 0.25 s) recruited from the cytoplasm exclusively at the CD59 clusters undergoing STALL, producing the IP(3)-Ca(2+) signal. Therefore, we propose that the CD59 cluster in STALL may be a key, albeit transient, platform for transducing the extracellular GPI-AR signal to the intracellular IP(3)-Ca(2+) signal, via PLCgamma2 recruitment. The prolonged, analogue, bulk IP(3)-Ca(2+) signal, which lasts for more than several minutes, is likely generated by the sum of the short-lived, digital-like IP(3) bursts, each created by the transient recruitment of PLCgamma2 molecules to STALLed CD59.

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Figures

Figure 1.
Figure 1.
Increase of the cytoplasmic IP3 concentrations induced by CD59 clusters, as observed by the epifluorescence imaging of the PH domain (from PLCδ1)–GFP expressed in T24 cells. Typical results are shown. CD59 clustering was induced by the addition of IgG-gold particles or the natural ligand, C8, whereas Fab-gold is a non–cross-linking control. Pretreatment of cells is summarized in the legend to Fig. 2 and Materials and methods.
Figure 2.
Figure 2.
Relative concentration of IP3 in the cytoplasm versus PIP2 on the cytoplasmic surface of the plasma membrane. (a) The fluorescence intensity of the PH domain–GFP, measured along the white lines in Fig. 1 (IgG-gold at 0 [before] and 15 min after IgG-gold addition). The definitions of ICyt and IPM are also shown. (b) The ICyt/IPM ratio provides a convenient measure of the increase of cytoplasmic IP3 versus membrane PIP2. Pretreatment of cells: 1 μM U73122 (PLC blocker), 10 μM PP2, 1.7 nM PTX, 4 mM MβCD, and 50 nM latrunculin B (for 15, 5, 1,320, 30, and 10 min, respectively; 37°C) blocked the increase in the cytoplasmic IP3 concentration, but a 15-min preincubation with 1 μM U73433 (negative control for U73122) did not. The PLC activator, chlorpromazine (CPZ; 30 μM), increased the cytoplasmic IP3 concentration, which was inhibited with U73122, but not with U73343. To avoid overcrowding, the largest error bar (15 min after IgG-gold application; ± SEM) found in the whole set of experiments is shown. Chlorpromazine-related results are only shown up to 5 min because the value is already near the maximum in this parametrization (IPM cannot be defined when IPM is approximately equal to ICyt). The result with IgG beads (50-nm-diameter latex) indicates that these beads used for observing single-molecule recruitment of Lyn, Gαi2, and PLCγ2 are as signal capable as IgG-gold.
Figure 3.
Figure 3.
Typical time-dependent changes of the cytoplasmic Ca2+ concentrations after the addition of IgG-gold particles or C8 (via the release of Ca2+ from ER through IP3 receptor), as observed by epifluorescence microscopy of fluo-4 in live T24 cells. (a) Typical epifluorescence images of Fluo-4 in the cytoplasm after the addition of IgG-gold. The box in the first image indicates the region of interest (24 × 24 pixels representing 250 μm2), from which the second trace in panel b was obtained. The numbers in the images correspond to the numbers shown in the second trace. (b) The fluorescence intensity of Fluo-4 measured in the box of 24 × 24 pixels, plotted as a function of time. See the legend to Fig. 2 and the Materials and Methods for the various pretreatments of cells. Rhod-2 was used for DOPE cross-linking experiments, to avoid the signal from fluorescein-DOPE. Calcium signals in intact cells and the cells that experienced cholesterol depletion and the subsequent repletion are different in complex and subtle ways. The calcium signal for the latter cells starts at about the same time after stimulation, but the initial rate of the increase of the intracellular Ca2+ concentration ([Ca2+]i)is low, and thus the calcium oscillation starts at ∼3 min later. As usual with pharmacological experiments, the mechanisms for the details of the cellular reactions are difficult to understand. The fourth trace from the bottom is after the application of IgG beads, indicating that these beads are as signal capable as IgG-gold.
Figure 4.
Figure 4.
Clusters of CD59, DAF, and PLAP exhibited STALL in both T24 and NRK cells, within a 48-nm-diameter area. Typical trajectories of IgG-gold–induced clusters, recorded at video rate (33-ms resolution, at which hop movement across the compartment boundaries cannot be resolved), in T24 and NRK cells in culture. Some of the STALL parts of the trajectories are enlarged (bottom). The STALL area sizes are substantially smaller than the median compartment sizes of T24 (110 nm) and NRK (230 nm) cells, shown as square lattices.
Figure 5.
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 athttp://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).
Figure 6.
Figure 6.
Immunofluorescence colocalization of IgG-gold–induced CD59 clusters and PLCγ2 spots. (a) Some of the CD59 clusters formed by IgG-gold particles (green spots; stained with fluorescein-conjugated secondary antibodies to the mouse anti-CD59 antibody attached to the IgG-gold) are colocalized with PLCγ2 spots (red spots). Lookup tables similar to that used for Fig. 1 c in Suzuki et al. (2007) were used. Yellow arrowheads indicate colocalized IgG-gold and anti-PLCγ2 spots. Light green and pink arrowheads indicate IgG-gold and PLCγ2 spots, respectively, which do not show colocalization with each other. (b) The fraction of IgG-gold particles colocalized with the PLCγ2 spots after various pretreatments (mean ± SEM).
Figure 7.
Figure 7.
A working model showing how STALL of CD59 clusters may be induced and how it may function as a transient platform to transduce the extracellular CD59 signal to the intracellular IP3–Ca2+ signal (via PLCγ2 recruitment to STALL). The numbers in parentheses correspond to the signaling steps described in the Discussion section.
Figure 8.
Figure 8.
Prolonged bulk signal may be produced by the addition of extremely short, pulse-like recruitment of single molecules. Model A, called the complex integration model, describes the case where each individual signaling event lasts for a length comparable to that for the bulk signaling, which would necessitate a complicated integration of these signals to realize the desired level of the bulk signal. Model B, called the simple summation model, describes the case where each single-molecule event occurs like a digital pulse, as compared with the duration of the bulk signal, which then can simply be added up to generate the desired bulk signal, without the need for complicated integration.
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