provides only part of the information about cell–cell interactions
and cellular behaviour. To fully understand these processes, one
must measure the dynamics of the protein of interest in vivo in
living cells and tissues.
Fluorescence recovery after photobleaching (FRAP) is a fluorescent microscopy technique which allows one to do this precisely
and has made a significant impact on the understanding of the
functions of proteins and the regulation of cell adhesion and migration [12–16]. In essence, the technique relies on the disruption of
the signal from a fluorescently tagged protein in a portion of a
sample, which is achieved by bleaching a region with a short,
intense pulse of photons. One then measures the fluorescence in
the bleached area for a period following the bleaching to record the
recovery of the fluorescence signal. Importantly, the proteins in the
bleached area are still present but are merely “dark” due to the loss
of fluorescence. Therefore, what is measured is the exchange of
proteins between the bleached area and the rest of the cell. Carefully designed FRAP experiments not only enable the determination of the overall dynamics of a transmembrane protein, but also
allow one to distinguish the contributions of both the diffusional
and endocytic trafficking processes and to determine the specific
changes in each.
The performance of a FRAP experiment is reliant upon the
following steps: preparation of the sample, calibration of the microscope settings, performing and acquiring the data on the microscope, analysing the data, fitting the recovery curve, and
interpreting the results. Here, we use an example of FRAP performed on the membrane-bound epithelial cadherin (E-cad) molecule which has been tagged with the fluorophore EGFP [17]. The
theoretical aspects of FRAP are extensively described in other publications [18–23]. We therefore focus on the practical aspects of
performing FRAP, which will allow anyone with access to a confocal
microscope to do the whole procedure from sample preparation to
the final result.
We and other research groups have shown that the E-cad signal
recovers in Drosophila and mammalian cells by both diffusional and
endocytic recycling mechanisms, which are kinetically distinct
[1, 15, 24, 25]. Here, we specifically use the example of the
epidermis of the stage 15 Drosophila embryo. However, aside
from sample preparation, the same protocol, calibration, and analysis are applicable for other proteins and cell types. Finally, we
outline the main considerations for designing and analysing FRAP
experiments.
146
Joshua Greig and Natalia A. Bulgakova
and cellular behaviour. To fully understand these processes, one
must measure the dynamics of the protein of interest in vivo in
living cells and tissues.
Fluorescence recovery after photobleaching (FRAP) is a fluorescent microscopy technique which allows one to do this precisely
and has made a significant impact on the understanding of the
functions of proteins and the regulation of cell adhesion and migration [12–16]. In essence, the technique relies on the disruption of
the signal from a fluorescently tagged protein in a portion of a
sample, which is achieved by bleaching a region with a short,
intense pulse of photons. One then measures the fluorescence in
the bleached area for a period following the bleaching to record the
recovery of the fluorescence signal. Importantly, the proteins in the
bleached area are still present but are merely “dark” due to the loss
of fluorescence. Therefore, what is measured is the exchange of
proteins between the bleached area and the rest of the cell. Carefully designed FRAP experiments not only enable the determination of the overall dynamics of a transmembrane protein, but also
allow one to distinguish the contributions of both the diffusional
and endocytic trafficking processes and to determine the specific
changes in each.
The performance of a FRAP experiment is reliant upon the
following steps: preparation of the sample, calibration of the microscope settings, performing and acquiring the data on the microscope, analysing the data, fitting the recovery curve, and
interpreting the results. Here, we use an example of FRAP performed on the membrane-bound epithelial cadherin (E-cad) molecule which has been tagged with the fluorophore EGFP [17]. The
theoretical aspects of FRAP are extensively described in other publications [18–23]. We therefore focus on the practical aspects of
performing FRAP, which will allow anyone with access to a confocal
microscope to do the whole procedure from sample preparation to
the final result.
We and other research groups have shown that the E-cad signal
recovers in Drosophila and mammalian cells by both diffusional and
endocytic recycling mechanisms, which are kinetically distinct
[1, 15, 24, 25]. Here, we specifically use the example of the
epidermis of the stage 15 Drosophila embryo. However, aside
from sample preparation, the same protocol, calibration, and analysis are applicable for other proteins and cell types. Finally, we
outline the main considerations for designing and analysing FRAP
experiments.
146
Joshua Greig and Natalia A. Bulgakova