2 Materials and Methods
2.4 Confocal Microscopy
Live cells in 35 mm Fluorodishes (World Precision Instruments) transfected with fluorescent constructs or with added fluorophores were imaged on either a Leica SP5 with a 63× 1.4 NA oil-immersion objective lens or a Zeiss 780 with a 63×
1.2 NA water-immersion objective. Both microscopes were in temperature- controlled environmental chambers that were warmed to 37 °C. Before imaging, culture media was replaced with 1 mL pre-warmed HEPES-buffered DMEM (Sigma) to maintain pH outside of a CO2-controlled environment. Fluorophore
excitation was achieved using either 405, 458, 488 514, or 564 nm laser lines. Cellular compartments were visualised using the following fluorophores: plasma membrane – FM4-64 (Life Technologies); nucleus – Hoechst (Sigma Aldrich); endoplasmic reticulum – CellLight ER tracker (Life Technologies); F-actin – CellLight LifeAct-RFP plasmid DNA transfected at 1 µg/35 mm plate (Ibidi).
2.4.1
Tonicity-mediated translocation
Live cells were exposed to tonicity changes whilst on the stage of the microscope to allow imaging of the same cells before and after tonicity changes. This was done by adding treatment solutions designed to give the desired final ion concentrations and osmolality to 1 mL of culture media already on the cells. Images were analysed by extraction of fluorescence line profiles covering the membrane and avoiding the nucleus using ImageJ. The profiles were processed using homemade software in Matlab. Background was subtracted using the extracellular regions of the profile. Average membrane expression was
calculated as the mean of the maximal intensity at the two membrane peaks and cytosol expression was calculated as the mean intensity in the region between the membrane peaks starting 10 pixels in. Relative membrane expression (RME) was then calculated as (Im - Ic)/Im, where Im and Ic are the average membrane and
cytosol intensities respectively as described by Conner et al [169]. This gives an RME of 1 if the entire signal is at the membrane and an RME of 0 if there are equal average intensities in membrane and cytosol pixels. Five profiles from different regions were used for each cell and the resulting RME values were averaged to give an estimate of the RME for that cell. One experimental repeat consisted of analysis of 5 profiles each from at least three different cells in the same image. Results from this software were validated by comparison to results
using the non-automated procedure previously described [169]. Figure 2-6 shows a schematic of this analysis method.
2.4.2
Fluorescence recovery after photobleaching (FRAP)
FRAP was performed using a circular bleaching area of radius 1 µm. Bleaching was performed at 100% laser power, which was maintained until the
fluorescence in the bleach region was reduced to <10% of the pre-bleach
fluorescence. After bleaching, fluorescence from the bleached area was measured every 5 seconds and recovery curves were fitted to a single phase exponential recovery function. Diffusion coefficients were calculated using the approach and equation of Kang et. al. [208]:
! =!!+!! 8!!/!
Where D is the diffusion constant, rn is the bleaching radius (set by the
experimenter, 1 m in our experiments), !!/! is the half-time of recovery from
the exponential fit and re is an effective bleaching radius, measured from a post-
bleach image. This effective radius corrects for the fact that bleaching is not instantaneous and therefore during bleaching some bleached fluorophores will diffuse out of the bleaching region and some unbleached fluorophores will diffuse into the bleaching region, tending to make the actual bleached area larger than that set by the experimenter. Recovery curves were collected from 5
different cells on the same plate per experiment in 6 experimental repeats.
2.4.3
Forster resonant energy transfer (FRET)
AQP4-Turquoise2 and AQP4-Venus constructs were generated by site-directed mutagenesis of the AQP4-GFP construct described in section 2.1.1. Turquoise2
Figure 2-6 Calculation of the relative membrane expression of GFP fusion proteins from confocal fluorescence micrographs.
0 10 20 30 40 50 60 70 0 5 10 F lu or es ce n ce / a.u . distance / µm 0 10 20 30 40 50 60 0 5 10 F lu or es ce n ce / a.u . distance / µm background subtraction and assignment of membrane peaks and intracellular region M1 M2 C1:CN RME= M1+M2 2 − Ci i=1 N
∑
N M1+M2 2 calculation of relative membrane expression (RME) extraction of fluorescence profiles from confocal micrographsis a cyan fluorescent protein (CFP) derivative [209] and Venus is a yellow fluorescent protein (YFP) derivative [210].
CFP has an excitation maximum at ~425 nm and an emission maximum at 474. Venus has an excitation maximum at 515 nm and emission maximum at 528 nm. The microscope system used for this work had a 405 nm diode laser and 458 nm Argon laser line which could both be used to excite CFP. Both lasers gave similar fluorescence intensity for CFP at the same laser power, however the 405 nm laser excited Venus much less efficiently, so was chosen to minimize direct excitation of Venus in the FRET experiments.
FRET experiments were performed using the sensitized emission methodology with the FRET signal corrected for donor emission in the acceptor channel and direct excitation of the acceptor, following van Rheenen et al [211] and
normalised to the acceptor emission to give a normalized apparent FRET efficiency:
!= !!"−!!!−(!−!")!!
1−!" !!
where FIA is the measured indirect (i.e. with donor excitation) acceptor
fluorescence (the FRET signal), FD is the direct donor emission, FA is the direct
acceptor emission and the greek letters are correction factors for acceptor fluorescence excited by the donor laser and detected at through the donor filters (α), donor fluorescence detected through the acceptor filters (β), excitation of the acceptor excited by the donor laser and measured through the acceptor filters (γ), and FRET signal measured through the donor filters (δ). The correction factors were obtained by imaging samples containing donor only or acceptor only before performing full FRET experiments.