A CD59 cluster (green) transiently recruits (left to right) a Lyn-GFP molecule (red).

Extracellular receptors anchored in the plasma membrane outer leaflet somehow entice intracellular proteins to the cytosolic side of the membrane for activation. Companion studies by Suzuki et al. (pages 717 and 731) give a peek at these dynamics at the level of single molecules.

The team shows that liganded clusters of the GPI-anchored receptor (GPI-AR) CD59 undergo temporary immobilizations, called STALLs (stimulation-induced temporary arrest of lateral diffusion), which serve as fleeting platforms for activating a signaling cascade. How a signal gets from the outside in without a transmembrane stretch has been intensely investigated.

When GPI-ARs come together, a slew of events take place inside the cell: the cluster can associate with Gα proteins, activate Src-family kinases (such as Lyn), and trigger the IP3/calcium signaling cascade. Previous studies based on large aggregates of GPI-ARs pointed to the involvement of raft microdomains for regulating the interactions.

To show how these events occur over space and time, the authors chose a single-molecule approach that used a more physiological clustering of just three to nine CD59 receptors. The CD59 clusters recruited both Lyn and Gαi2 frequently and transiently (100–200 ms). The resulting meeting between Gαi2 and Lyn activated Lyn and led to a CD59 STALL, which lasted for about half a second.

The STALL may be the result of Lyn's phosphorylating an unknown protein that hooks the CD59 receptors to actin filaments. However it happens, the STALL created a temporary landing platform for PLCγ, which converts PIP2 to IP3 and thereby releases calcium from the ER. Treatments that interfered with STALLs also blocked the calcium signal.

Each PLCγ molecule hovered at the membrane for only ∼0.25 s, but the total IP3 signal has been measured to last for ∼15 min. The authors suggest that the bulk signal is produced by the summation of thousands of digital bursts from individual PLCγ molecules. A transmembrane mutant of CD59 also induced Lyn recruitment, albeit at lower levels, leading this group to conclude that both protein- and lipid raft–based interactions are at work. Exactly how a raft microdomain might draw this molecular crowd remains up for grabs.