Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae

Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule analysis. Here we present a method to characterise maturation of GFP and mCherry genetic protein fusions in budding yeast Saccharomyces cerevisiae.


Background
Single-molecule fluorescence microscopy enables sensitive quantification of molecular stoichiometry, mobility and copy number, not only on a cell-by-cell basis but also precisely to individual sub-cellular compartments (Leake, 2012; Wollman and Leake, 2015; Shashkova et al., 2017). The technique relies on endogenously expressed fluorescent protein fusions of the wild type protein of interest such that there is one-to-one labelling. However, all fluorescent proteins have an in vivo maturation time varying from a few minutes to several tens of minutes before entering a bright fluorescing state (Badrinarayanan et al., 2012). It is therefore of upmost importance to measure any maturation effects and quantify if there is any immature 'dark fraction' of labelled protein. These measurements are also particularly relevant to fluorescence recovery after photobleaching (FRAP). FRAP can be This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). used to study molecular turnover in living cells (Beattie et al., 2017). FRAP is based on photobleaching of a cell region where a fluorescently labelled component is localized, followed by quantification of any fluorescence recovery in that region over time. The measured relation between the fluorescence intensity as a function of time following an initial photobleach can be used to determine molecular mobility and kinetics parameters, such as the rate of dissociation of a particular fluorescent component from a molecular complex (Leake et al., 2006). Therefore, any 'new' fluorescence coming from fluorescent protein maturation might affect this apparent result. We present here a protocol to characterise the maturation of Mig1-GFP and Nrd1-mCherry fusion proteins in living yeast Saccharomyces cerevisiae cells used in our single-molecule studies .

1.
Streak cells from a frozen stock, using a sterile pipette tip on a freshlyprepared YPD agar plate (see Recipes), and incubate at 30 °C for at least 24 h.

2.
Set an overnight culture in a 14 ml tube by inoculating 3 ml of YPD with cells grown on a YPD plate. Single colonies are not needed for genomically integrated strains. Incubate at 30 °C, 180 rpm.

3.
In the morning exchange the YPD medium (see Recipes) to YNB medium (see Recipes) supplemented with 4% glucose:

a.
Pellet the cells by centrifugation at 1,000 x g for 3 min, remove the supernatant.

b.
Resuspend the cells in 3 ml of YNB medium without any carbon source.

c.
Pellet the cells by centrifugation at 1,000 x g for 3 min, remove the supernatant.

d.
Suspend the cells in 3 ml of YNB supplemented with 4% glucose and incubate at 30 °C, 180 rpm, for ~4 h.

e.
Wash the culture by centrifugation (1,000 x g, 3 min) and resuspend in 2 ml of YNB with 4% glucose. Incubate at 30 °C, 180 rpm, for about 10 min.
f. Add 2 μl of 100 mg/ml cycloheximide solution (see Recipes) to the final concentration of 100 μg/ml. Incubate for 1 h at room temperature, without shaking, protect from light.

g.
Place 5 μl of the culture on a microscope slide and cover with a 22 x 22 mm coverslip. Avoid any air under the coverslip.

B.
Data acquisition

1.
Place the sample under the microscope, coverslip on the objective and find a region of interest containing 5-10 cells (100x magnification) which appear stationary and firmly anchored to the glass surface.

2.
Optimize exposure times for in vivo imaging of both GFP and mCherry fluorescent proteins to be able to detect a clear signal without saturating the detector. Under our microscope: GFP exposure time-22 sec, mCherry-7 sec.

3.
Find another region with 5-10 cells positioned far away from the previous one to avoid any potential bleaching from previous illumination exposure.

4.
Take a brightfield and a fluorescence image, by pressing the 'snap' button, with both channels using chosen exposure times, opening the mercury lamp shutter for only the length of exposure.

5.
Photobleach GFP or mCherry by continuous illumination of the appropriate wavelength until the region appears completely dark. Continue for 1 min longer. With our settings the total exposure time is: 3 min 40 sec for GFP and 4 min for mCherry. Immediately after, begin timing and acquire a picture of the bleached fluorescent protein with an appropriate channel and a brightfield image. This is denoted time point 0 min.

7.
As simultaneous photobleaching of GFP and mCherry is not possible under this microscope, the time points were staggered for GFP and mCherry as listed in Table 1.

C.
Data analysis

1.
Images are converted into open standard tiff files from zvi by AxioVision software.

2.
Further analysis is performed using ImageJ.

a.
Open the first unbleached brightfield image.

b.
By choosing an 'oval' selection tool, define a region of interest (ROI), an area around a cell as shown in Figure 1. It does not matter how much of non-cell area is included as every cell will be background-correct during the analysis.

c.
Open a fluorescence image of the same set, and define the same area of the same cell by simultaneously choosing 'Shift' and 'E' keys on the keyboard (Selection → Restore).

d.
From the menu bar select: Analyze → set measurements. Pick 'area' (represents a number of pixels, N) and 'integrated density' (sum intensity for the cell, S cell ). Press 'OK'.

e.
To obtain numeric values press 'Ctrl' + 'M' (Analyse → Measure). Record the result in Excel.

f.
Repeat throughout the entire data set for both channels keeping the same ROI.

g.
Repeat the entire procedure for all cells.

h.
Background correction: Choose random background areas around cells ( Figure 2) and obtain numerical results for sum intensity (S bg ).

i.
Find the average (S Abg ) and multiply by the number of pixels from cell (N) measurements. This is the intensity of the background represented within the cell area (I bg ).

j.
Subtraction of the average background sum intensity (I bg ) from the total intensity of the cell (S cell ) represents I cell , the cellular fluorescence intensity with background correction.

k.
The average of fluorescence intensity of all cells analysed within the data set gives the final value of the fluorescence intensity (I final ) with appropriate estimation of SD and/or SE.

l.
Plot the final fluorescence intensity (I final ) vs. experimental time ( Figures 3A and 3B) for fluorescently labelled cells and wild type autofluorescent cells. Any signal above autofluorescence is due to fluorescent protein maturation. For GFP ( Figure 3A), no maturation was detected so it can be assumed that all of the fluorescent protein was mature in the cells and there is no 'dark' fraction. For mCherry ( Figure 3B), some fluorescence recovery was measured. The following steps outline quantification of the maturation time and dark fraction.

m.
Subtract the autofluorescence from the mean fluorescent protein intensity at each time point ( Figure 3C).

n.
Export the intensity and time values after the bleach by copying and pasting into two new variables in MATLAB, called x (for the time values ) and y (intensity values): i. Right click on the Workspace → New. Name it x or y. Press 'Enter' on the keyboard. ii.

Double click on this new variable opens a table in the
Editor where values of time (for x) or intensity (for y) can be pasted.

o.
Open the curve fitting toolbox from the Apps menu.

p.
Select x for 'X data' and y for 'Y data'.

q.
Choose custom equation and type: where, I bleach is the remaining intensity after the bleach, I rec is the recovered intensity above I bleach and t mat is the maturation time.

r.
If 'Auto fit' is ticked, fitting will be automatic.

s.
If the fit has not converged correctly, adjust the 'Start point' parameters in 'Fit Options' to reasonable estimates from the data i.e., y at x = 0 for I bleach and y at x = end minus I bleach for I rec .

t.
If the fit has converged record the fit and goodness of fit parameters from the 'Results' panel. For Figure 3C, I rec = 4.3 x 104 ± 3 x 104 counts, I bleach = 5.2 x 104 ± 2.5 x 104 counts, t mat =17 ± 10 min with R 2 = 0.7.

u.
To calculate the proportion dark, immature protein; divide I rec by the initial, autofluorescence corrected pre-bleach intensity.

Data analysis
Data

Notes
Autofluorescence is calculated as indicated in the protocol above but using a wild type yeast strain (i.e., without any fluorescent proteins present).

1.
Glucose 50% w/v Weigh 500 g of glucose Order of photobleaching and data acquisition using two channels