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. 2007 Nov 9;94(6):2333–2342. doi: 10.1529/biophysj.107.117234

FIGURE 5.

FIGURE 5

Formation of clathrin-coated pits on the plasma membrane of BSC1 cells expressing clathrin LCa Tomato and AP-2 σ2-EGFP. (A) Representative fluorescence snapshots selected from Movie 2 corresponding to an individual coated pit. The panels correspond to alternating TIRF and WF images simultaneously acquired for σ2-EGFP and LCa-Tomato. (B) Fluorescence-intensity plots corresponding to the coated pit shown in A. (C) Model representing sequential stages during the formation of the coated pit. (D, a and b) Fluorescence-intensity plots for the coated pit after normalization of the TIR and WF signals to correct for differences in the WF and TIRF intensities, as outlined in Methods. The traces were filtered with a (0.2,0.6,0.2) wavelet filter. (c) The axial position measurement corresponding to the ensemble of clathrin labeled with LCa-Tomato and AP-2 labeled with σ2-EGFP within the forming pit, as derived using Eq. 5. (d) Relative axial position corresponding to the COF of LCa-Tomato. (E) The projected image of the coated pits forming on the plasma membrane. The image is the time projection of all the pits observed in Supplementary Material (Movie 2). The white circles surround pits that were eliminated from the analysis because they were present at the beginning or end of the time series. None of the pits remained for the whole duration of the movie (210 s). Red circles indicate pits whose traces were analyzed using DiNa. (F) The signal/noise ratio (defined in Methods) for all the pits in the movie whose whole cycle was captured. Red circles mark the pits selected for DiNa analysis because their LCa-Tomato fluorescence signal/noise ratio was >25, measured as the maximum signal divided by the average noise from the whole profile. (G) Distribution of maximum intensities determined for the clathrin LCa-Tomato signal for all the pits (gray) and for those selected for DiNa analysis (red). (H) Δz calculated for the difference in the average axial position between clathrin LCa-Tomato and AP-2 σ2-EGFP from the individual pits selected for DiNa analysis plotted as a function of the fraction of their respective lifetimes. Values outside of the red dotted lines represent meaningful data in excess of 1.5 times the expected calculated resolution of Δz.