Chapter 13
Fluorescence Recovery After Photobleaching to Study
the Dynamics of Membrane-Bound Proteins In Vivo Using
the Drosophila Embryo
Joshua Greig and Natalia A. Bulgakova
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 to 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.
Key words Protein dynamics, Membrane proteins, E-cadherin, Cell-cell adhesion, FRAP, Diffusion,
Endocytosis
1 Introduction
The maintenance of epithelial tissue, epithelial-to-mesenchymal
transitions (EMTs), and the migration of mesenchymal cells relies
on the dynamic turnover of transmembrane proteins, such as cadherins and integrins [1–5]. These membrane proteins are confined
in space, with diffusion along the plane of the lipid bilayer, but they
can move in and out of the plasma membrane using the specialized
cellular mechanism of endocytic internalization and recycling [6–
9]. Alterations to this dynamic turnover, both diffusional and
endocytic trafficking, result in profound changes in cell behaviour,
and in some cases can induce or prevent EMT [10, 11]. Therefore,
the total amount of a transmembrane protein in a specimen
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_13,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
145
Fluorescence Recovery After Photobleaching to Study
the Dynamics of Membrane-Bound Proteins In Vivo Using
the Drosophila Embryo
Joshua Greig and Natalia A. Bulgakova
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 to 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.
Key words Protein dynamics, Membrane proteins, E-cadherin, Cell-cell adhesion, FRAP, Diffusion,
Endocytosis
1 Introduction
The maintenance of epithelial tissue, epithelial-to-mesenchymal
transitions (EMTs), and the migration of mesenchymal cells relies
on the dynamic turnover of transmembrane proteins, such as cadherins and integrins [1–5]. These membrane proteins are confined
in space, with diffusion along the plane of the lipid bilayer, but they
can move in and out of the plasma membrane using the specialized
cellular mechanism of endocytic internalization and recycling [6–
9]. Alterations to this dynamic turnover, both diffusional and
endocytic trafficking, result in profound changes in cell behaviour,
and in some cases can induce or prevent EMT [10, 11]. Therefore,
the total amount of a transmembrane protein in a specimen
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_13,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
145