Fluorescence Recovery After Photobleaching to Study the Dynamics of Membrane-Bound Proteins In Vivo Using the Drosophila Embryo

Abstract

The epithelial-to-mesenchymal transition is a highly dynamic cell process and tools such as Fluorescence recovery after photobleaching (FRAP), which allow the study of rapid protein dynamics, enable the following of this process in vivo. This technique uses a short intense pulse of photons to disrupt the fluorescence of a tagged protein in a region of a sample. The fluorescent signal intensity after this bleaching is then recorded and the signal recovery used to provide an indicator of the dynamics of the protein of interest. This technique can be applied to any fluorescently tagged protein, but membrane-bound proteins present an interesting challenge as they are spatially confined and subject to specialized cellular trafficking. Several methods of analysis can be applied which can disentangle these various processes and enable the extraction of information from the recovery curves.Here we describe this technique when applied for the quantification of the plasma membrane-bound E-cadherin protein in vivo using the epidermis of the late embryo of Drosophila melanogaster (Drosophila) as an example of this technique.

Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins

“…9M). In wildtype, recovery was roughly similar to what others have observed (Cliffe et al ., 2004; Greig and Bulgakova, 2021)—recovery plateaued after 400-500 seconds and the mobile fraction was about 75%. We then repeated this analysis in M/Z pyd mutants.…”

Multifaceted control of E-cadherin dynamics by Adaptor Protein Complex 1 during epithelial morphogenesis

“…We found that AP-1 depletion halved the halftime of the slow component’s recovery ( Figure 7B ). E-cad recovery following AP-1 depletion was best-fitted by a single exponential model, likely as the slow halftime reduction made it impossible to confidently separate the components ( Greig and Bulgakova, 2021 ). The halftime of a diffusion-uncoupled recovery depends only on the reaction off-rate (internalization rate in this case) ( Sprague and McNally, 2005 ; Iyer et al.…”

“…The recovery curves were obtained by manually measuring intensities of background, control region, and photobleached region using 2-µm-diameter circular regions for each time point in Fiji. The raw data were processed as in Greig and Bulgakova (2021) to obtain the normalized recovery curves, namely following background subtraction the intensity at the bleached spot was normalized to the intensities of a control area at the same time point and the bleached area before bleaching.…”

Multifaceted control of E-cadherin dynamics by the Adaptor Protein Complex 1 during epithelial morphogenesis

“…We found that AP-1 depletion halved the halftime of the slow component's recovery (Fig 7B). Ecad recovery following AP-1 depletion was best-fit by a single exponential model, likely as the slow halftime reduction made it impossible to confidently separate the components [61].…”

“…The recovery curves were obtained by manually measuring intensities of background, control region, and photobleached region using 2-µm diameter circular regions for each time point in Fiji. The raw data was processed as in [61] to obtain the normalized recovery curves, namely following background subtraction the intensity at the bleached spot was normalized to the intensities of a control area at the same time point and the bleached area before bleaching.…”