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. Author manuscript; available in PMC: 2018 Mar 9.
Published in final edited form as: Cell. 2017 Mar 9;168(6):1041–1052.e18. doi: 10.1016/j.cell.2017.02.011

Figure 4. Dimerization of mKit quantified by dual-color single molecule TIRF imaging.

Figure 4

(A) Single molecule dimerization of mXFP-mKit expressed in HeLa cells and labelled by anti-GPF nanobodies conjugated with Rho11 (red) and DY647 (blue), respectively. Magenta represents co-localization. Top: cartoon of the assay; bottom: raw image series of mXFP-mKit in presence of SCF and trajectory of a dimer. Yellow lines indicate the co-trajectories of mKit dimers. Scale bar: 1 μm. (B) Trajectories (from 100 frames, 3.2 s) of individual Rho11-labeled (red) and Dy647-labeled mKit (blue) and co-trajectories (magenta) for an unstimulated cell, as well as cells after stimulation with SCF (100 nM) and S4-3a (100 nM), respectively. Scale bar: 5 μm. (C) Relative numbers of co-trajectories for mXFP-mKit in absence of ligand (unstim.) and after stimulation with indicated concentrations of SCF or S4-3a, compared to negative and positive control experiments. (D) A close-up comparison of mXFP-mKit dimerization shown in (C). (E) Formation of an individual mKit dimer in the presence of SCF probed by co-tracking Rho11 (red), Dy647 (blue), co-trajectory (magenta). Scale bar: 1 μm. (F) Distance plot of (E) identifying the formation of an individual mKit dimer. See also Figure S3.