GPI-anchored receptor clusters transiently recruit Lyn and G alpha for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

Abstract

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold-induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Galphai2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein-protein and lipid-lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP(3) production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Galphai2. Because both Galphai2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Galphai2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

Figure 1.

Probes used in this work. (a) Nonstimulated CD59 was observed using Cy3-conjugated anti-CD59 Fab (Fab-Cy3) and 40-nm-diameter gold particles conjugated with small numbers of anti-CD59 Fab fragments (Fab-gold), which hardly induced CD59 cross-linking. To observe CD59 engaged in signaling, CD59 was first tagged with a Fab-gold particle and then the ligand (C8) was added, or CD59 was cross-linked by 40-nm-diameter gold particles conjugated with anti-CD59 whole IgG (IgG-gold). (b) MycCD59TM, a transmembrane mutant of CD59, and fluorescein-labeled DOPE, an unsaturated phospholipid, were introduced in the plasma membrane, and the effects of cross-linking were examined. (c) The distribution of IgG-gold on the cell surface 5 min after IgG-gold addition (arrowheads). IgG-gold was stained with fluorescein-labeled secondary antibodies to enhance its image, to avoid nondetected IgG-gold and, hence, large variations in the signal intensity for each IgG-gold. Background subtraction was done as shown in the lookup table.

Figure 2.

Clustering of CD59 by IgG-gold or C8 induced phosphorylation of SFKs, including Lyn, in their activation loops. (a) The level of SFK phosphorylation after IgG-gold addition, as shown in a Western blot with anti–pY418-Src antibodies (recognizing Lyn phosphorylation at Y397). The bottom bands are Lyn, and the top bands are p60SFKs (c-Src, Yes, and Fyn), as identified by reprobing with each respective antibody. (b) The ratio of activated Lyn to total Lyn (immunoblotting with anti-pY418 versus that with anti-Lyn; arbitrary units) plotted against the incubation time. (c) Time courses of Lyn phosphorylation after IgG-gold addition after various pretreatments of cells at 37°C: 50 nM latrunculin B, 4 mM MβCD (and also the subsequent cholesterol replenishment with 10 mM MβCD–cholesterol), 10 μM PP2 (SFK blocker), and 1.7 nM PTX (Gα blocker; 10 min, 30 min, 5 min, and 22 h, respectively). All of the SEMs for the points plotted here are within 30% of the given value. The overall amounts of cholesterol per cell after cholesterol depletion and after the subsequent repletion were found to be 66 and 118% of the original amount (SD of ±6%).

Figure 3.

CD59 clusters undergo alternating periods of STALL and apparently simple diffusion. (a, top) Typical trajectories of IgG-gold recorded at video rate. CD59 clusters exhibit alternating periods of apparently simple Brownian diffusion (black trajectories) and STALL (magenta trajectories in the circular blue areas). See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200609174/DC1) for the trajectory on the right. (bottom) A representative trajectory of Fab-gold recorded at video rate, shown for comparison with those of IgG-gold. (b and c) Histograms showing the distributions of the STALL and diffusing periods, respectively, of IgG-gold (left) and Fab-gold + C8 (right; magenta curves indicate the best exponential fits).

Figure 4.

Single-molecule detection of the transient recruitment of Lyn-GFP and Gαi2(YFP) to CD59 clusters. (a and c) Image sequences showing superimposed video frames of simultaneous recordings of a CD59 cluster (induced by a 50-nm latex particle, which was observed by a bright-field microscopy; green) and single molecules of Lyn-GFP (a) or Gαi2(YFP) (c; red). Gaussian spatial smoothing was applied to the images. A single Lyn-GFP (Gαi2[YFP]) molecule was colocalized from frame 7 until 12 in panel a (frame 4 until 6 in c; indicated by pink frame numbers). (b and d) A typical trajectory of a CD59 cluster (black), including STALL periods (the blue parts of the trajectories) and a period (the indigo parts of the trajectories) in which it was colocalized with Lyn-GFP or Gαi2(YFP) (magenta part in the otherwise orange trajectory; it overlaps with a STALL site but at different times). See Videos 2 and 3 (available at http://www.jcb.org/cgi/content/full/jcb.200609174/DC1) for the original video sequences for Lyn-GFP (a and b) and Gαi2(YFP) (c and d) shown here, respectively. Colocalization was defined by Koyama-Honda et al. (2005).

Figure 5.

Lyn-GFP is dynamically recruited at CD59 clusters, without any time correlation with STALL, whereas Gαi2(YFP) colocalization, which is also transient, occurs right before the onset of STALL. (a) The definition of the time difference (lag time = ΔT) between the recruitment of a single molecule of Lyn-GFP (or Gαi2[YFP]) and the onset of STALL (time = 0). It was defined as the first frame of Lyn-GFP (or Gαi2[YFP]) recruitment to a CD59 cluster (T_Rec) minus the first frame of the nearest STALL (T_STALL). (b and d) The distribution of the time difference (ΔT) between the recruitment of a single molecule of Lyn-GFP (b) (or Gαi2[YFP]; panel d) and the onset of STALL. Each STALL period starts at time 0 but ends at different times (the pink shade showing STALL is, thus, graded). The frequency of incidental colocalization events was evaluated by artificially shifting the CD59 cluster video sequence by 50 frames (1.67 s) and was subtracted (see Materials and Methods). (c and e) The distribution of the colocalization duration of single molecules of Lyn-GFP (c) or Gαi2(YFP) (e) at CD59 clusters (medians are shown).

Figure 6.

Immunofluorescence colocalization of Lyn and Gαi2 with CD59 cross-linked by primary and secondary antibodies. After CD59 was extensively cross-linked by antibodies, cells were fixed with 4% paraformaldehyde and stained with anti-Lyn and/or anti-Gαi2 antibodies.

Figure 7.

Immunofluorescence colocalization of IgG-gold particles with immunostained spots formed by anti-pY418 (actually Y397 for Lyn, the phosphorylation of which enhances its activity), anti-Lyn, and anti-Gαi2 antibodies. (a, top) Some of the CD59 clusters formed by IgG-gold particles (green; stained with fluorescein-conjugated secondary antibodies) are colocalized by anti-pY418 spots (red). Lookup tables similar to that in Fig. 1 c were used. Yellow arrowheads indicate colocalized IgG-gold and anti-pY418 spots. Light green and pink arrowheads indicate IgG-gold and anti-pY418 spots, respectively, that do not show colocalization with each other. (bottom) Fluorescein-DOPE is not concentrated at the IgG-gold particles. (b) The fraction of IgG-gold particles colocalized by the anti-pY418, -Lyn, or -Gαi2 spots before and after various pretreatments. (c) Some of IgG-gold particles (green) are colocalized by both anti-Lyn and -Gαi2 spots (red and blue, respectively). Yellow arrowheads indicate colocalized IgG-gold, Lyn, and Gαi2 spots. (d) The fraction of IgG-gold particles colocalized by both anti-Lyn and anti-Gαi2 spots before and after the pretreatment with MβCD. Error bars indicate SEM.

Figure 8.

A model showing how the recruitment of Lyn and Gαi2 at a CD59 cluster might induce its STALL. The recruitment of Gαi2 and Lyn to the same CD59 cluster induces the binding of Gαi2 and Lyn, activating Lyn. This might lead to phosphorylation of as-yet-unknown proteins, X or Y (transmembrane or cytoplasmic surface associated), triggering the binding of the CD59 cluster to an actin filament and/or an actin-dependent membrane domain, inducing STALL.