Figure 1.
Design and reporting principle of NANOMS.
FRET-biosensor design of the three different NANOMS. (A) The myristoylated N-terminal membrane-targeting motifs of mouse Gαi2 (residues 1–35), human Yes (1–17)- and human Src (1–16)-kinases were genetically fused to the N-terminus of fluorescent proteins mCFP or mCit. The sequence of the employed membrane-targeting motifs can be found in Table S2. (B) Intracellular processing involves cleavage of the N-terminal methionine (grey) by methionine amino-peptidase (Met-AP), NMT-mediated myristoylation on glycine 2 (yellow) and depending on the motif cysteine-palmitoylation (red). (C) Lipid modified reporters spontaneously organize into plasma membrane nanocluster. Tight packing of membrane targeted donor (mCFP)- and acceptor (mCit)-fluorophores (blue and yellow squares, respectively) in nanocluster leads to FRET. FRET can decrease due to loss of nanoclustering or cytoplasmic redistribution of the NANOMS after inhibitor treatment. As membrane anchorage is required for the functioning of myristoylated proteins, NANOMS report on functional membrane anchorage.
Figure 2.
NANOMS report on chemical inhibition of NMT.
(A) FRET-responses of Yes-, Src- and Gi2-NANOMS transfected BHK cells treated with 4 µM of the specific NMT inhibitor DDD85646. The error bars denote the s.e.m (n = 5). Samples were statistically compared with the untreated control. See Methods for more information on statistical analysis. (B) Confocal sensitized acceptor FRET-imaging of Yes-NANOMS expressed in BHK cells. Cells were treated as indicated. Top row shows acceptor channel images, and bottom row FRET images. The look-up table shows the FRET-index FR, color coded with high FRET levels in black and yellow (value 1) indicating no FRET. Scale bar is representative for all images and corresponds to 10 µm. (C) Dose-response curves of the effect of DDD85646 on the Emax values of Yes- and Src-NANOMS expressed in BHK cells (n = 6).
Figure 3.
NANOMS reports on RNAi-mediated depletion of NMT.
(A) HEK293 EBNA cells transiently expressing Yes-NANOMS and (B) HEK293 cells transiently expressing Gi2-NANOMS were treated with NMT1 or NMT2 specific siRNAs or control siRNA. Knock-down efficiencies are shown in Figure S4. The characteristic Emax-value was determined on flow cytometric FRET data. The error bars denote the s.e.m (n = 4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.
Figure 4.
Cherry-picked chemical compound library screen with Yes-NANOMS.
(A) Chemical structures of chemical compounds that were included in the cherry-picked chemical library. (B) BHK21 cells were transfected with Yes-NANOMS and screened with shown chemical compounds at a final concentration of 10 µM/mL. FRET-response of Yes-NANOMS to the chemical compounds is represented with Emax values. Block line indicates the average Emax and the error bars denote the s.e.m (n≥4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.