Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

Summary

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI ¹.

Abstract

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm², which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.

Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the λ repressor CI, and has many other potential applications in systems biology.

Protocol

1. Strain Engineering Workflow

1. Insert sequences encoding (a) a membrane-localization sequence, (b) a fast-maturing fluorescent protein and (c) a protease recognition sequence N-terminal to and in frame with a protein of interest (e.g. a transcription factor). We used the membrane targeted Tsr-Venus reporter ² and fused it to the protease recognition sequence Ubiquitin (Ub) to count the number of expressed bacteriophage λ repressor CI protein molecules. Details on how we constructed the fusion protein Tsr-Venus-Ub-CI, replacing the wild-type CI coding region and incorporating the construct into the E. coli chromosome using λ Red recombination ³, are described in detail in ref. 1.
2. Verify all modifications by sequencing.
3. Transform a plasmid encoding a protease specific to the recognition sequence used above into a strain expressing the fluorescent reporter and protein of interest in translational fusion. We used the protease Ubp1, which cleaves immediately after the C-terminal Ubiquitin residue ⁴.

2. Culture Cells and Prepare Sample

1. Pick one E. coli colony of interest from a freshly streaked agar plate into 1 ml M9 minimal media ⁵ supplemented with 1X MEM amino acids and appropriate antibiotics. Incubate in a shaker at desired temperature long enough to reach OD₆₀₀ > 1.0 (optical density at 600 nm).
2. Dilute the culture into 1 ml fresh M9 medium with appropriate antibiotics to OD₆₀₀ = 0.02. Incubate in a shaker at appropriate temperature. Initial cellular density can be increased if needed for low-temperature growth.
3. When the OD₆₀₀ = 0.2-0.3 (at 37 °C with an initial cell culture at OD₆₀₀ = 0.02, this will take 3-4 hr), start preparing the agarose gel pad (step 3).
4. The cells are ready for imaging when OD₆₀₀ = 0.3-0.4.
5. Transfer 1 ml incubated culture into a 1.5 ml microcentrifuge tube, centrifuge at 10,000 x g for 1 min in a benchtop microcentrifuge.
6. Discard the supernatant and add 1 ml fresh M9 medium and resuspend the pellet gently.
7. Centrifuge at 10,000 x g for 1 min.
8. Repeat step 1.6 and 1.7. Discard the supernatant.
9. Gently resuspend in 1 ml M9 media. Cells can be directly used for microscope imaging or diluted 10- to 100-fold to ensure low cell densities for timelapse imaging. Note that low cell densities are important for extended timelapse imaging. The imaging chamber (step 4) is sealed during the experiment. Due to exponential growth of cells, high initial cell concentrations can significantly deplete oxygen within the chamber after prolonged growth in the gel pad, reducing fluorescent protein maturation and affecting cell growth.

3. Prepare the Agarose Solution

1. Weigh 10-20 mg low-melting-temperature agarose into a 1.5 ml microcentrifuge tube.
2. Add appropriate volume M9 minimal medium without antibiotics to make a 3% agarose solution.
3. Heat the agarose solution for 30 min at 70 °C to melt, inverting the tube to ensure that the solution is completely melted and homogenous. The gel pad can be poured at this point (step 4) or the temperature can be decreased to 50 °C and held for several hours for later use.

4. Prepare Gel Pad on the Chamber

1. About 50 min before imaging, rinse a Microaqueduct slide with water.
2. Rinse one cleaned cover glass (using a stringent cleaning method such as that described in ⁶) and Microaqueduct slide with sterile water and dry by blowing with compressed air.
3. Place the rubber gasket on the Microaqueduct slide so that it covers the inlets and outlets on the glass side of the Microaqueduct slide (this can be modified for applications in which media is perfused through the sample). Apply 50 μl agarose solution (step 3) to the center of the Microaqueduct slide.
4. Top the agarose solution with the cleaned, dry cover glass.
5. Let the agarose solution stand at room temperature for 30 min.
6. Meanwhile, prepare a cell sample (step 2).
7. Carefully peel off the cover glass from the top of gel pad. Add 1.0 μl washed cell culture to the top of the gel pad. Wait for ~2 min for the culture to be absorbed by the gel pad. It is important not to wait so long that the gel pad overdries, but long enough that cells are properly adhered to the gel pad. The Ideal waiting time will vary with temperature and humidity.
8. While waiting, dry another precleaned cover glass.
9. Cover the sample with the new cover glass ensuring that the cover glass and Microaqueduct slide are well aligned.
10. Assemble the temperature controlled growth chamber following the manufacturer's instructions. It can now be used for imaging on microscope (step 5).

5. Imaging and Acquisition of Timelapse Movies

1. Turn on the laser and microscope following manufacturer's instructions (e.g. the laser may need to warmed up for ~0.5 hr before the experiment).
2. Lock the assembled chamber to the stage insert on the microscope. Set the temperature of the growth chamber and the objective heater at 37 °C or other desired growth temperature.
3. If imaging above room temperature, the focus or gel pad will usually drift significantly for ~15 min after the initial temperature shift. In practice, use this time to find regions of interest and modify imaging scripts as necessary for an experiment.
4. Find cells on the microscope and store the position of each cell after centering it within a predefined and aligned imaging region. More than one cell can be imaged in each image acquisition time window. For timelapse imaging over many generations, ensure that imaged cells are initially separated from other cells by at least a few hundred μm so that other colonies will not enter the imaging region during growth.
5. Adjust laser excitation intensity so that almost all fluorescent molecules photobleach within 6 exposures (Figure 5).
6. Using an automated imaging script/journal, acquire timelapse data using the algorithm described in the experimental workflow in Figure 3.

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Results

Typical results from a CoTrAC experiment tracking the production of the λ repressor CI are shown in Figures 4 and 5. In this experiment, 12 colonies were imaged at 5 min intervals. At each time point, the colony was first autofocused and centralized within the imaging region. Next, the centered/focused position was stored and a brightfield image was acquired. The stage was then translocated by ~0.5 μm along the z-axis to move from the brightfield focal plane to the plane bisect...

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Discussion

The CoTrAC method can be generalized to measure the production of other proteins where conventional N- or C-terminal fluorescent protein fusions may disrupt protein activity. The CoTrAC strategy has three unique advantages over current methods. First, co-translational fusion ensures that one molecule of the fluorescent reporter is produced for each molecule of the protein of interest, allowing accurate counting of protein production in real time. Second, the membrane-targeted reporter Tsr-Venus enables single-molecule de...

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Materials

Name	Company	Catalog Number	Comments
Agarose (Low melting temperature)	Lonza	50100
Milli-Q H2O
5×M9 salts			Following recipe described in 9
20% glucose
MgSO4
CaCl2
50×MEM amino acid solution	Invitrogen	11130-051
Temperature-Controlled Growth Chamber Stage adaptor	Bioptechs	FCS-2
Objective Heater	Bioptechs	Model depends on microscope objective
Microaqueduct Slide	Bioptechs	130119-5
Micro cover glasses	VWR	40CIR-1	Can be difficult to source; also available from Bioptechs
Cover glass/slide gasket	Bioptechs	FCS2 0.75 mm
Fluorescence Microscope	Various	Example setup: Coherent Innova 308C Argon-ion laser, Olympus IX-81 microscope, Olympus PlanApo 100X NA 1.45 objective, Metamorph software	Must have laser excitation, automated xyz stage, automation software capable of scripted imaging and autofocus, optics capable of resolving single fluorescent proteins
EM-CCD Camera	Various	Example setup: Andor Ixon DU-898	Must have sufficiently low noise to detect single fluorescent proteins above background