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. Author manuscript; available in PMC: 2017 Sep 13.
Published in final edited form as: Nat Methods. 2017 Mar 13;14(4):427–434. doi: 10.1038/nmeth.4221

Figure 1. TagRFP-T (TT) red fluorescence fluctuations can be increased by proximity of Dronpa (Dp) in a distance-dependent manner.

Figure 1

(A) Representative images and single-pixel fluorescence intensity traces in HeLa cells expressing DpTT (Dronpa-linker-TagRFP-T) and TagRFP-T alone when excited by 561 nm laser; Scale bar: 10 μm. (B) Aggregated mean normalized autocorrelation function (ACF) of many single molecule fluorescence traces from purified fluorescent DpTT and TagRFP-T. The amplitude increase demonstrates the clear gain in autocorrelation signal from increased millisecond fluctuations of DpTT. (C) Quantified fluctuation in various constructs demonstrating the specific nature of the fluctuation increase; n (cell) numbers are: TT (n = 8), DpTT (n = 7), Dp+TT (n = 9), EGTT (EGFP-linker-TagRFP-T, n = 8), and EGFP-linker-mCherry (EGmCh, n = 9). (D) Quantified fluctuation in various mutant constructs demonstrating Dronpa’s chromophore is not involved but external Dronpa residues are important for the phenomenon. n (cell) numbers are: TT-alone (n = 8), WT (n = 7); chromophore mutants: Dp[S142D]-TT (S142D, n = 8), Dp[C62G/Y63G]-TT (GGG, n = 11); FP surface mutants: Dp[N102I]-TT (N102I, n = 12), Dp[N102I/R149E]-TT (NIRE, n = 9). (E) A variable number of rigid helical repeats permit measurement of distance dependence and sensitivity of the change in fluctuation. Hn represents the number (n) of rigid (EAAAK) repeats; DpTT (short linker) and Dp+TT (not fused) were included as comparisons. H2–H5 linkers are marked with nominal FP distances reported in literature. All constructs in (E) were targeted to the plasma membrane of HeLa cells by the lyn sequence and examined at the n (cell) numbers as follows: DpTT (n = 5), H1 (n = 7), H2 (n = 6), H3 (n = 14), H4 (n = 17), H5 (n = 10), Dp+TT (n = 9). Pair-wise t-test results in (C), (D), and (E) are marked where data were compared with the construct marked “ref”. n.s.: not-significant; *: p<0.05; **: p<0.01; ***: p<0.001 where applicable. In all dot plots and (E), center line and whiskers mark the average and s.e.m. values, respectively.