To the Editor: In their recent article, Schwarzenbacher et al.1 use an elegant micropatterning technique to demonstrate the effect of immobile CD4 receptors on the mobility of the interacting protein Lck. One powerful feature of protein immobilization methods is the ability to extract binding and unbinding parameters using fluorescence recovery after photobleaching (FRAP)2,3. However, in their calculation of CD4-Lck unbinding rates, the authors made a simplifying assumption that introduces a serious risk of error.

Binding of fluorescent molecules to immobile sites will slow recovery after photobleaching provided the binding events are sufficiently frequent and long-lived. The effect of binding on FRAP depends on the relative rates of binding and diffusion. At the extreme where diffusion is much more rapid than binding, fluorescence recovery reflects only unbinding. These circumstances are referred to as diffusion-uncoupled, to distinguish them from instances in which fluorescence recovery is a complex function of both binding and diffusion (diffusion-coupled)3,4.

Schwarzenbacher et al.1 derive the lifetime of the CD4-Lck interaction after concluding that Lck contrast recovery is diffusion-uncoupled. They made this conclusion after comparing the time required for an Lck molecule to diffuse across a bleached region (the characteristic diffusion time) and the time required for recovery of fluorescence contrast. The diffusion time for full-length Lck (with a diffusion coefficient of ∼0.2–1.0 μm2 s−1) across 20 × 5 μm rectangles will be ∼6–30 s. The authors argue that this is sufficiently rapid compared to the 160 s required for recovery after photobleaching that diffusion is negligible and thus that the recovery rate corresponds to the unbinding rate. Although this makes intuitive sense, the diffusion-coupled and diffusion-uncoupled regimes cannot be distinguished simply by comparing diffusion and recovery rates4. A necessary preliminary step is to determine empirically whether diffusion has an impact on recovery. This is most commonly done by measuring recovery as a function of bleached region size.

Notably, diffusion can be important even for cases in which recovery after photobleaching is extremely slow3,4. Estimates have also suggested that diffusion-coupled recovery will be more common than diffusion-uncoupled recovery for most binding partners3. Moreover, inappropriately ignoring diffusion can have serious consequences, as derived dissociation rates may be in error by as much as two orders of magnitude5. Schwarzenbacher et al.1 calculated unbinding rates for full-length (membrane-associated) and truncated (cytosolic) Lck that differed by this amount, and concluded that the Lck membrane anchor has a profound influence on the stability of the protein-protein interaction. The micropatterning method described by the authors represents a valuable addition to the inventory of live-cell protein immobilization techniques. However, as is the case for other photobleaching techniques, extraction of accurate binding and unbinding parameters using this method will require experimental assessment of the impact of diffusion on fluorescence or contrast recovery.