Box 1. Techniques for the analysis of protein kinetics in living cells

Fluorescence recovery after photobleaching (FRAP)

The fluorescent signal (blue circle) is bleached in a small intracellular area and the recovery is measured inside this region as a function of time (see figure, top panel). Using this technique, diffusion coefficients (D) and the proportion of molecules that are mobile can be measured. Red curve: 100% of molecules are diffusing (D = 0.5 µm² s^-1); green curve: 80% of molecules are diffusing (D = 0.2 µm² s^-1) and 20% are immobile.

Fluorescence loss in photobleaching (FLIP)

A small area (blue circle) is repeatedly photobleached and the loss in fluorescence intensity is recorded as a function of time in another region of the cell (see figure, second panel). FLIP provides measurements of the number of populations of a particular molecule and their relative proportions. Green curve: a molecule that is present as a single population; red curve: two different populations of a molecule are present, each of which has a different rate constant.

Inverse-FRAP (i-FRAP)

The total fluorescent signal in a cell is bleached (blue circle) except for one area in which the signal is recorded as a function of time (see figure, third panel). This technique is useful for studying small organelles as it gives a direct readout of the residency time of different factors. One of the main limitations of this approach lies in the time that is needed to photobleach the total fluorescent signal that is present in a cell; this makes this technique unsuitable for detecting fast translocations as the signal is lost during the bleaching phase. Red curve: the fluorescent molecule has a half residency time of 15 seconds in the compartment that is being studied.

Photoactivation

A photoactivable version of green fluorescent protein (GFP), which has a decreased absorbance at 488 nm and therefore does not behave as wild-type GFP, is activated in a cell (purple circle) and fluorescence is recorded at the activation site (see figure, bottom panel). Although similar to i-FRAP, the advantage of photoactivation is that it requires less energy than bleaching. Sub-populations that move rapidly and have a short residence time can be detected, whereas in i-FRAP they are bleached and undetectable. Photoactivation allows for the spatial detection of the successive translocations of proteins in a cell, and therefore serves as a visual, pulse-chase, real-time view of protein dynamics. Red curve: the fluorescent molecule has a half residency time of 15 seconds in the activated compartment 1. Green curve: the molecules translocate to compartment 2 after leaving compartment 1.