Supplemental Figure S1:
Fig. S1: Description and outcomes of confocal fluorescence recovery after photobleaching: The top panel depicts the theoretical outcome of a confocal FRAP experiment conducted using a double bleaching protocol and an interpretation of the corresponding immobile fraction, IF. Within the plasma membrane environment, certain molecules may be tethered, bound, or otherwise immobilized. Such molecules are permanently photobleached (green to black dots) during the first bleach and do not contribute to the fluorescent signal thenceforth. A significant IF is therefore calculated following the first bleach (compare panels t<0 and t=µ for the first bleach). The recovered fluorescent signal after the first bleach forms the baseline signal for the second bleach, therefore, the molecules contributing to the first bleach IF (now shown as brown dots) are disregarded in the second bleach analysis. During the second bleach, the population of newly immobile molecules should therefore be negligible, and the recovery of molecules into the bleached area should approach completion (compare panels t<0 and t=µ for the second bleach); therefore, the second bleach results in a very small or even negligible IF.
The middle panel is a representative FRAP experiment in a prestin-mGFP expressing HEK cell, showing the bleaching and recovery in the region of interest during our double bleaching protocol. The square region of interest (14.3 mm2) in the left image is the area shown in the right three panels. Bleaching occurs within the white circles, and the scale bars are 5 mm.
The bottom panel depicts the FRAP outcome when some membrane molecules are transiently confined in the bleach area within the time course of experimentation. Some molecules that were mobile in the first bleach may become newly immobilized, contributing to incomplete recovery of fluorescence even after the second bleach (compare panels t<0 and t=µ for the second bleach). Thus, significant IF values remain in the calculations from the second bleach data. Therefore, the use of a double-bleaching protocol and analysis of both bleaches permits identification of transiently and newly immobile molecules.