Fig. 5. TIL partitions into pre-existing endophilin–LPD clusters on the membrane and further enhances clustering.

a Confocal images of SSBs with conjugated His₆-tagged TIL (Alexa 488) and PRM7 (Alexa 633) in the presence and absence of endophilin (10% Alexa 594 labeled). Scale bar 2.5 µm. b Fluorescence intensity profiles for the images along the dashed yellow lines shown in a demonstrating the extent of co-localization of TIL and PRM7 within the clusters. c Radially averaged normalized autocorrelation functions and its single exponential fits (solid lines) demonstrating the clustering in the TIL, PRM7, and endophilin channels before (left) and after (right) addition of endophilin. d Protein distribution on the SSBs containing tethered PRM7 and endophilin before and 15 min after addition of TIL (50 nM). e Fluorescence intensity profiles for the images along the dashed yellow lines shown in e showing co-localization of TIL and PRM7 into the clusters. f Extent of clustering in the TIL, PRM7 and endophilin channels before (top) and after (bottom) addition of TIL quantified by radially averaged autocorrelation function and its single-exponential fits. g Distribution of tethered LPD^850–1250 and endophilin on SSBs before (top) and after (bottom) addition of TIL (50 nM). h Fluorescence intensity profiles along the yellow dashed lines shown in g. i Radially averaged auto-correlation functions with fits to show the extent of clustering in endophilin, LPD^850–1250, and TIL channels before (top) and after (bottom) addition of TIL. j Left, cross-correlation functions to compare the extent of co-clustering between pre-existing PRM7 and endophilin on a bilayer before (gray) and after (cyan) addition of TIL. The extent of co-clustering between the added TIL with the pre-existing PRM7 is shown on the right. Solid lines are the guide for the eye. k Cross-correlation analysis for the LPD^850–1250/endophilin on a bilayer before (gray) and after (cyan) addition of TIL. On the right, the extent of co-clustering between the added TIL and the pre-existing LPD^850–1250 is shown. All experiments were performed in 20 mM HEPES buffer, 150 mM NaCl, 1 mM TCEP, pH 7.4, and at room temperature.