Measurement and perturbation of morphogen lifetime: effects on gradient shape.
Protein lifetime is of critical importance for most biological processes and plays a central role in cell signaling and embryonic development, where it impacts the absolute concentration of signaling molecules and, potentially, the shape of morphogen gradients. Early conceptual and mathematical models of gradient formation proposed that steady-state gradients are established by an equilibration between the lifetime of a morphogen and its rates of synthesis and diffusion, though whether gradients in fact reach steady state before being read out is a matter of controversy. In any case, this class of models predicts that protein lifetime is a key determinant of both the time to steady state and the spatial extent of a gradient. Using a method that employs repeated photoswitching of a fusion of the morphogen Bicoid (Bcd) and the photoconvertible fluorescent protein Dronpa, we measure and modify the lifetime of Dronpa-Bcd in living Drosophila embryos. We find that the lifetime of Bcd is dynamic, changing from 50 min before mitotic cycle 14 to 15 min during cellularization. Moreover, by measuring total quantities of Bcd over time, we find that the gradient does not reach steady state. Finally, using a nearly continuous low-level conversion to the dark state of Dronpa-Bcd to mimic the effect of increased degradation, we demonstrate that perturbation of protein lifetime changes the characteristic length of the gradient, providing direct support for a mechanism based on synthesis, diffusion, and degradation.
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