Quantitative Live Cell Imaging of Nuclear Shape and Chromatin Dynamics During Development and Environmental Stress in Arabidopsis thaliana Root

Summary

This process uses confocal microscopy in conjunction with a fluorescently labeled nucleus and chromatin marker line in Arabidopsis thaliana to simultaneously record nucleus and chromatin dynamics in Arabidopsis roots and investigate changes in these dynamics in response to developmental and environmental cues.

Abstract

The nucleus is the characteristic organelle of eukaryotic organisms. Unlike the classic textbook view of static nuclei, nuclear shape is dynamic in live cells. Altered or deformed nuclear shape is a hallmark of cancer in animal cells and environmental stress in plants. Nuclear envelope proteins interact with chromatin to regulate gene expression. Unfortunately, little is known about the impact of abiotic stress on nuclear shape, movement, and chromatin dynamics. To confront this issue, we developed a pipeline using confocal microscopy and particle tracking software to quantify nuclear and chromatin dynamics in Arabidopsis roots under control and abiotic stress condition. This confocal imaging method utilizes a dual fluorescently tagged marker line – nuclear envelope protein and chromatin – to perform live cell imaging of the root in model plant Arabidopsis thaliana under control and salt-stressed conditions. These captured movies are analyzed to quantify nuclear and chromatin dynamics using open-source image processing software Fiji/ImageJ with the help of the TrackMate plugin. To validate this method, we imaged and quantified chromatin movement in control and salt-stressed roots, revealing a decrease in chromatin speed under salt-stressed conditions. This method allows for quantitative live cell imaging of root nuclear shape and chromatin dynamics during plant development and environmental stress, thus enabling analysis of changes in nuclear and chromatin dynamics caused by abiotic stressors.

Introduction

Nuclear shape is variable between different cell types and during development in Arabidopsis thaliana roots^1,2. For instance, nuclear shape is spherical in the actively dividing meristematic regions. In contrast to the meristematic region, nuclei in the root transition and elongation zone are less spherical. Furthermore, nuclei in differentiated cells, such as root hairs, pass through the tight root hair base and are actively transformed into elongated nuclei^3,4,5. The dynamic changes in nuclear shape and movement are crucial to the functioning of the cell^6,7. Defects in nuclear shape lead to functional impairment such as reduced nuclear movement^1,3. In mammalian cells, changes in cell and nucleus shape are directly correlated with changes in gene expression and protein synthesis⁸.

During interphase, a cell's DNA is organized as chromatin. The DNA in chromatin is bundled around histones, which can tighten and loosen the DNA to alter expression. Similarly, chromatin positioning relative to the nuclear envelope and proximity to the nuclear periphery are also important for gene expression⁹. Environmental stress, such as osmotic stress, alters nuclear shape and consequently changes the expression of a set of touch-sensitive genes in A. thaliana roots¹⁰. In this context, studying the nuclear shape and chromatin dynamics through live cell timelapse imaging is important. One major aspect of nuclear dynamics is the speed at which nuclei move, as nuclear positioning within the cell drives gene expression, and nuclear repositioning requires nuclear movement¹¹. The speed of chromatin movement within the nucleus is similarly important, as the positioning of genes within the nucleus is critical for determining gene expression¹².

Previously, nuclear shape and chromatin dynamics were imaged and quantified separately; as a result, the relationship between nuclear shape and chromatin dynamics remained unexplored. By imaging nuclei and chromatin simultaneously, their positions relative to each other can be observed, which may reveal movements such as nuclear rotation and intra-nuclear chromatin movement that are otherwise difficult or impossible to demonstrate. In this method, we present a live cell imaging technique to visualize and quantify the nuclear shape via fluorescently tagged outer nuclear envelope protein WPP domain-interacting protein 1 (WIP1)¹³ and chromatin via centromeric histone H3 (CENH3)¹⁴ dynamics simultaneously under control and salt stress conditions, with the goal of enabling the analysis of changes in nuclear and chromatin dynamics caused by abiotic stressors. To enable this method, we have generated a WIP1-GFP/CENH3-mRFP line of A. thaliana. Additionally, we studied the chromatin dynamics in a quantitative manner by using the TrackMate plugin of open-source ImageJ/Fiji software. We tested a dual fluorescent line and chromatin dynamics techniques presented here under salt stress conditions and discovered an alteration in centromeric foci morphology and dynamics due to stress conditions. This simple and reproducible method is ideal for high-throughput tracking of nuclear and chromatin dynamics in Arabidopsis roots under various abiotic stress conditions.

Protocol

1. Growing of A. thaliana seedlings

1. Growth media preparation
   1. Place 800 mL of pure water in a 1 L beaker on a stirring plate. Add a stir bar to the beaker.
   2. Turn on the stir plate at 600 rpm.
   3. Add 10 g of sucrose to the beaker.
   4. Add 2.16 g of Murashige and Skoog (MS) growth medium to the beaker.
   5. Add 5 mg of agar to each of two 1 L media bottles.
   6. Label one bottle "1/2 MS" and label the other "100 mM NaCl".
   7. Label both bottle lids with autoclave tape.
   8. Add 5.84 g of NaCl to the bottle labeled "100 mM NaCl".
   9. Fill the beaker with 1 L of pure water.
   10. Return the beaker to the stir plate and wait until the sucrose and MS have dissolved.
   11. Pour the beaker contents into a 1 L graduated cylinder.
   12. Pour 500 mL of the graduated cylinder contents into each bottle.
   13. Autoclave growth media and 250 mL pure water with a 60 min liquid cycle at 121 °C.
       NOTE: When autoclaving media and water, ensure the lids are loose to avoid potential explosions.
   14. Re-screw growth media lids.
   15. Invert growth media bottles 7 times to ensure agar is thoroughly distributed throughout.
   16. Store growth media in a 4 °C fridge until needed, and microwave to liquify.
2. Setting growth plates and seeds
   1. In a flow hood, use a pipette gun to pipette 50 mL of growth media into each of ten plates (Figure 1A).
   2. While the media solidifies, clean WIP1-GFP x CENH3-mRFP seeds by flooding them in a microcentrifuge tube with 1 mL of 70% ethanol for 10 min (Figure 1B).
      NOTE: When handling ethanol, ensure there are no open flames in the vicinity.
   3. After 10 min, in the flow hood, remove the ethanol and rinse the seeds twice with 1 mL of autoclaved water (Figure 1C).
   4. Once the media has set, use a 1000 µL pipette to place cleaned seeds onto growth plates in two rows of 14 seeds per plate (Figure 1D).
   5. Label the plates with a permanent marker and seal them with micropore tape before leaving the flow hood (Figure 1E).
3. Germinating seedlings
   1. Wrap growth plates in aluminum foil and cold stratify them in a 4 °C fridge for a minimum of 2 days (Figure 1F).
   2. After cold stratification, remove plates from the fridge and leave them vertically under growth lights (~80 µmol/m²/s) for a minimum of 4 days (Figure 1G).
   3. Transfer half of the plates to 100 mM NaCl growth media in the flow hood, then seal and label the plates (Figure 1H).
   4. Grow plates under grow lights for 2 days before imaging (Figure 1I).

2. Live cell imaging of A. thaliana root nuclei and chromatin

1. Slide preparation
   1. Pipette 60 µL of pure water onto a glass slide (Figure 2A).
   2. Lay 3 6-day-old A. thaliana seedling roots into the water (Figure 2B).
   3. Place a 22 mm × 22 mm #1.5 slide cover over the roots without covering the leaves (Figure 2C).
2. Confocal microscope settings
   1. Set laser exposure time to 1000 ms (1 s).
   2. In the laser combiner, set the 488 nm laser to 50% for green fluorescent protein (GFP) and the 561 nm to 50% for red fluorescent protein (RFP) (Figure 2D). Image with 20× objective lens without optical zoom. If recording for longer than 10 min, decrease the intensity to reduce photobleaching.
   3. Set Z-slice step size to 0.3 µm.
   4. Set the interval to 1 min and the total duration to 10 min.
3. Imaging
   1. Locate roots and select up to 20 z-slices and capture 16-bit images.
   2. Start timelapse.
   3. Save timelapse as a TIFF file using lossless compression.
   4. Move data to a computer for analysis.

3. Tracking and analysis of data

NOTE: One will need the Fiji version of the open-source software ImageJ (v. 1.54 recommended), which comes with TrackMate preinstalled.

1. Initialization
   1. Open ImageJ/Fiji.
   2. Click and drag a .vsi file onto the main ImageJ/Fiji application window (Figure 3A).
   3. A prompt titled Bio-Formats Import Options will appear; click Ok in the bottom right (Figure 3B).
   4. A prompt titled Bio-Formats Series Options will appear. Ensure Series 1 is selected and click Ok in the bottom left (Figure 3C).
      NOTE: The file may take a minute to load, especially if it has many Z-layers or time points.
   5. Once the image has opened (Figure 3D), split the signals by navigating on the main ImageJ window to Image > color > split channels (Figure 3E). This will create two separate windows, one for GFP-labeled nuclei and one for RFP-labeled chromatin (Figure 3F).
2. Preprocessing chromatin
   1. Determine which of the two windows corresponds to chromatin.
   2. Click on the chromatin window (Figure 3F) and apply the Z-project operation by navigating on the main ImageJ window to image > stacks > Z project (Figure 4A).
   3. Change projection type to Max Intensity and make sure All Time Frames is active, then click Ok (Figure 4B).
   4. Open the contrast menu by pressing Ctrl+Shift+C or navigating on the main ImageJ window to image > adjust > Brightness/Contrast (Figure 4C).
   5. Slide the second bar (labeled Maximum) to the left until the chromatin is easily visible (Figure 4D).
3. Tracking chromatin
   1. Navigate to plugins (Figure 5A).
   2. Click the down arrow in the plugins menu to scroll to the tracking option (Figure 5A).
   3. Click tracking, then TrackMate.
   4. The initial settings will appear. Confirm that the calibration settings are accurate, then click Next (Figure 5B).
   5. Select Thresholding Detector and click Next (Figure 5C).
   6. On the image window, move to the last frame of the timelapse (Figure 5D).
   7. Next to the intensity threshold, click Auto, then click Preview.
   8. Adjust the intensity threshold and click Preview. Repeatedly adjust and preview the threshold until the preview includes all chromatin and includes minimal noise, then click Next (Figure 5D).
   9. Initial thresholding detection will occur; click Next (Figure 5E).
   10. Manually threshold until only chromatin is visible, then click Next (Figure 5F).
   11. This page allows filtering of spots. Ignore this and click Next (Figure 6A).
   12. Select Simple LAP tracker and click Next (Figure 6B). The LAP tracker is applicable when tracked objects do not merge or split.
   13. Set linking max distance to 5 microns, gap-closing max distance to 15 microns, and gap-closing max frame gap to 1. Click Next (Figure 6C).
   14. Tracking will occur. Tracks will appear in the original image window. Click Next (Figure 6D).
   15. This page allows track filtering. Press the green + button in the bottom left to create a filter, then choose the Number of spots in track mode and press Auto (Figure 6E).
   16. Ensure the mode is set to Above. This will eliminate spots that quickly appear and disappear.
   17. Click Next (Figure 6E).
   18. The display options page allows exporting data as a csv file. At the bottom of the page, click the Tracks button, then Export to CSV (Figure 7A).
   19. This page allows built-in plotting; ignore it and click Next.
   20. On the final page, save the video using Capture overlay and pressing execute. Click it, then navigate to file > save as > GIF (Figure 7B).
4. Basic speed analysis
   1. Open the tracks CSV in a spreadsheet software.
   2. Delete all columns except for TRACK_ID, POSITION_X, POSITION_Y, POSITION_T, FRAME, and RADIUS.
   3. The spots in each track will be out of order. To order them, first freeze the top four rows and then create a new column titled TRACK_FRAME and copy and paste this formula into the column: =concatenate(A4, "_", if(E4>9, E4, concatenate("0", E4))).
   4. Select the TRACK_FRAME column, right-click it, then click Sort sheet A–Z. The spots within each track should now be in order.
   5. To confirm no spots are missing in the middle of any tracks, create a new column and use this formula: =if(E4>E3, E4-E3, "NA"). Ensure the first spot of each track returns "NA" and the rest 1.
   6. Create a column for Speed (micron/frame) and paste the following formula into it: =if(H4="NA", "NA", sqrt((B4-B3)^2 + (C4-C3)^2)). This will use the Pythagorean theorem to calculate the distance between two points.
   7. To translate this to Speed (micron/second), create a new column and paste the following formula into it: =IF(I5="NA", "NA", I5/(D5/E5)). This will find the seconds/frame and translate microns/frame accordingly.

Results

Live cell imaging of nuclear envelope and chromatin dynamics
We generated a dual fluorescent marker line, which includes outer nuclear envelope protein WIP1-GFP and centromeric chromatin CENH3-mRFP (Figure 8). 4-day-old seedlings were transferred to the control plate or 100 mM NaCl-containing plates for 2 days. After 2 days of incubation, there is a clear root growth inhibition observed for salt treatment, as previously reported¹⁵. Both control an...

Discussion

The method presented here is ideal for tracking chromatin dynamics. Compared to past live cell imaging methods with separate imaging, the simultaneous imaging of nuclei and chromatin allows for the exploration of the relationship between nuclear and chromatin dynamics. This may reveal movements such as nuclear rotation and intra-nuclear chromatin movement that are otherwise difficult to demonstrate.

As a live cell imaging method, it is most important to have a fluorescent line with consistent ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
½ Murashige and Skoog (MS) media	bioWorld	30630058
3M micropore tapes	Amazon	A-1530-0
Acqusition software	Olympus/Evident	CellSens
Agar	bioWorld	40100072
CENH3-mRFP	ABRC	CS799459
Cover slip	Genesee	29-116
Glass slide	Genesee	29-101
Image/Fiji	Open-source	https://imagej.net/software/fiji/downloads
Microscope	Olympus/Evident	IX83
Sodium chloride	Fisher Scientific	BP358-212
Spinning disk	Yokogawa	CSU-W1
Square petri dishes	Simport	26-275
Sucrose	bioWorld	41900152
WIP1-GFP	ABRC	CS39987