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. Author manuscript; available in PMC: 2019 Jul 5.
Published in final edited form as: Mol Cell. 2018 Jul 5;71(1):155–168.e7. doi: 10.1016/j.molcel.2018.06.016

Figure 1. Distributed Amphifluoric FRET (DAmFRET) reveals nucleation barriers to self-assembly in vivo.

Figure 1.

(A) Proteins of interest were tagged to mEos3.1, which is photoconvertible by violet light and in a highly controlled and reproducible manner.

(B) The 2μ-origin of the plasmid, along with strong selection enables variable and high copy numbers in a population of cells. The Gal promoter ensures high concentrations of protein in an inducible manner.

(C) Cells were genetically engineered to undergo cell cycle arrest upon Gal induction, thereby eliminating propagation via cell division and ensuring each nucleation event is an independent one in a de facto closed reaction vessel. Non-collinearity of the 488 nm (in blue) and 561 nm (in green) lasers in the ImageStream®x MkII ensures that direct and sensitized emission (FRET) of the red molecules can be distinguished. See also Method S1.

(D) DAmFRET plot of human ASC. The dashed line approximates the mean AmFRET value of cells expressing fully monomeric protein. The blue curve to the right represents the fit of the DAmFRET plot to a Weibull distribution (see Method Details). The parameter δ relates to the sharpness of the transition and describes the persistence of the low population with respect to concentration. The green and red curves represent hypothetical distributions with values of δ that are lower or higher, respectively, than that of the blue curve.

(E) Montage of cells expressing ASC protein, showing switch-like acquisition of puncta. Images represent sum projection of confocal slices.