Materials and Reagents

Glass-bottom confocal dish, 35 x 10 mm, PP, Sterile (SPL, catalog number: 100350)
Reusable Bottle Top Filter (Tarsons, catalog number: 50060)
Membrane filter with 0.22 µm pore size (Millipore, catalog number: GSWP04700)
Kimwipes disposable wipers (Sigma-Aldrich, catalog number: Z188956)
Schizosaccharomyces pombe cells expressing fluorescently-labeled mitochondria or fission yeast cells stained with MitoTracker dye. Wild-type S. pombe cells can be obtained from National BioResource Project, Japan (https://nbrp.jp)
Lectin from Bandeiraea simplicifolia (Sigma-Aldrich, catalog number: L3759), store at 2-8 °C
Adenine (HiMedia, catalog number: TC082), store below 30 °C
Histidine (HiMedia, catalog number: GRM050), store below 30 °C
Leucine (HiMedia, catalog number: GRM054), store below 30 °C
Uracil (HiMedia, catalog number: GRM264), store below 30 °C
L- Lysine (HiMedia, catalog number: RM3028), store below 30 °C
MitoTracker^TM Orange CMTMRos (Thermo Fisher, catalog number: M7510), store at -5 to -30 °C
MgCl₂·6H₂O (HiMedia, catalog number: MB040), store below 30 °C
CaCl₂·2H₂O (HiMedia, catalog number: MB034), store below 30 °C
KCl (HiMedia, catalog number: GRM3934), store below 30 °C
NaCl (HiMedia, catalog number: MB023), store below 30 °C
Na₂SO₄ (HiMedia, catalog number: MB209), store below 30 °C
Pantothenic acid (HiMedia, catalog number: CMS178), store at 2-8 °C
Nicotinic acid (HiMedia, catalog number: CMS177), store below 30 °C
Inositol (HiMedia, catalog number: GRM102), store below 30 °C
Biotin (HiMedia, catalog number: TC096), store at 2-8 °C
Boric acid (HiMedia, catalog number: MB007), store below 30 °C
MnSO₄ (HiMedia, catalog number: GRM1381), store below 30 °C
ZnSO₄·7H₂O (HiMedia, catalog number: MB233), store below 30 °C
FeCl₂·6H₂O (HiMedia, catalog number: GRM6353), store below 30 °C
Molybdic acid (Sigma-Aldrich, 232084), store at room temperature
KI (HiMedia, catalog number: GRM252), store below 30 °C
CuSO₄·5H₂O (HiMedia, catalog number: GRM630), store below 30 °C
Citric acid (HiMedia, catalog number: GRM1023), store below 30 °C
Yeast extract (HiMedia, catalog number: RM027), store below 30 °C
Glucose (Sigma-Aldrich, catalog number: G8270), store at room temperature
Potassium hydrogen phthalate (HiMedia, catalog number: GRM2405), store below 30 °C
Na₂HPO₄ (HiMedia, catalog number: MB024), store below 30 °C
KH₂PO₄ (Sigma-Aldrich, catalog number: P5655), store at room temperature
NH₄Cl (HiMedia, catalog number: TC092), store below 30 °C
Agar powder, bacteriological (Himedia, catalog number: GRM026), store below 30 °C
NaOH
Phosphate buffered saline (see Recipes)
EMM: Edinburgh Minimal Medium with appropriate supplements (see Recipes)
1. 50x Salt stock
2. 1,000x Vitamin stock
3. 10,000x Mineral stock
YE: Yeast Extract with supplements (see Recipes)

Equipment

Orbital shaking Incubator (Ascension Innovations, model: AI-100)
Laser scanning (point/line) or spinning disk confocal microscope
We used the confocal mode of an InCell Analyzer 6000 (GE Healthcare) Imaging system with 60x/0.7 N.A. objective, fitted with an sCMOS 5.5MP camera having an x-y pixel separation of 108, and 405, 488, 561 and 640 nm laser lines
Standard vertical autoclave (Equitron, model: 7431STWL.AFE.113)
Laminar airflow chamber

Software

IN Cell Analyzer Acquisition Software v.4.6 (GE Healthcare) or any other confocal microscopy system-specific software
Fiji/ImageJ (http://fiji.sc/Fiji, Schindelin et al., 2012)
MATLAB software v. 2015b or above (MathWorks, https://in.mathworks.com)

Procedure

Preparation of S. pombe cells for imaging
1. Culture the yeast cells overnight by adding a loopful of cells (that have grown for at least a day in solid YE media) into 1 ml of liquid YE with appropriate supplements. Grow the cells at 30 °C for 12 h at 200 rpm to OD₆₀₀ of 0.4-0.9.
2. Subculture 200 μl from the overnight culture into 1 ml of fresh medium and grow at 30 °C for ~2 h prior to imaging at 200 rpm.
3. Cover the surface of a 35 mm (No. 1) glass bottom dish with 25% NaOH solution for ~2 h to make the surface hydrophilic. Wash the dish twice with sterilized water and wipe the surface with Kimwipes.
4. Drop 2 μl of lectin (2 mg/ml in PBS) onto the dish and spread evenly using an inoculation loop.
5. Allow the lectin to dry completely inside the laminar airflow at room temperature for 10-15 min.
6. Meanwhile, wash cells once by spinning them down and resuspending them in 1 ml of EMM liquid medium.
7. If cells are already expressing fluorescently-labeled mitochondria (e.g., Cox4-GFP/RFP), proceed directly to Step A10, else follow Steps A7-A9 below to stain mitochondria with MitoTracker dye.
8. Dissolve MitoTracker Orange/DeepRed in DMSO at a concentration of 1 mM and dilute it in liquid EMM to a stock solution of 1 µM. This stock solution can be stored at -20 °C.
9. On the day of the experiment, freshly prepare 200 nM of MitoTracker Orange/DeepRed from the stock solution by diluting with EMM. Incubate cells with 1 ml of 200 nM MitoTracker Orange/DeepRed solution for 15-30 min at 30 °C.
10. After incubation, wash the cells thrice in 1 ml of liquid EMM to eliminate background fluorescence from unincorporated dye, and finally resuspend cells in 1 ml of liquid EMM medium.
11. Transfer 200 μl of cells resuspended in liquid EMM to the lectin-coated glass bottom dish.
12. Incubate the cell suspension on the glass bottom dish for 10 min at room temperature.
13. Wash out unbound cells and add 200 μl of liquid EMM to the dish.
14. Image at room temperature (~25 °C).
Obtaining time-lapse images to observe mitochondrial dynamics
1. Transfer cells to the stage of the confocal microscope.
2. Visualize mitochondria labeled with GFP or RFP using the 488 nm and 561 nm laser lines along with 525/20 and 605/52 nm bandpass emission filters respectively. Alternatively, visualize mitochondria stained with the MitoTracker Orange using the 561 nm laser line along with the 605/52 nm bandpass emission filter. The signal-to-noise ratio must be at least 1.5.
3. Capture mitochondrial dynamics by obtaining time-lapse images that are 12 s apart, for a total time of ~4 min, with Z-stacks for each timepoint with 0.5 µm step-size encompassing the full cell (~5 steps).
4. Save the time-lapse images in an appropriate format, such as TIFF.

Data analysis

Identification of mitochondria and measurement of mitochondrial parameters in Fiji/ImageJ
1. Open time-lapse images obtained using Fiji/ImageJ.
2. Ensure that the pixel to μm conversion for the image files is accurate by checking ‘Set scale’ in Fiji/ImageJ.
3. Crop individual cells in the field of view.
4. Apply ‘Mean’ filter with an appropriate radius which is sufficient to reduce background noise, while still maintaining separation between individual mitochondria. In Figure 1, we used a radius of 2 pixels.
5. Identify mitochondria in the images either manually or based on intensity thresholding (Figure 1).
  
  Figure 1. Analysis of mitochondrial dynamics. Workflow of analysis of the time-lapse images obtained, including cropping, image filtering, identification of mitochondria, measurement of morphology and counting.
6. If identifying mitochondria manually, draw regions of interest (ROIs) around mitochondria in each frame using the freehand selection tool.
7. In the ‘Set measurements’ function of Fiji/ImageJ, check the following options: Shape descriptors, Fit ellipse and Stack Position.
8. If identifying mitochondria using intensity thresholding, use the ‘Analyze particles’ function to display mitochondrial measurements, else run the ‘Measure’ function on each timepoint of the time-lapse images.
9. Save the measurements in appropriate format such as .txt or .csv (Figure 2).
  
  Figure 2. Snapshots of txt files obtained after identification of mitochondria in a single cell for 2 timepoints (‘Slice’ 1 and 2) of a time-lapse movie. Three mitochondria were identified in Timepoint 1 and four in Timepoint 2. The columns ‘Major’, ‘Circ.’ and ‘AR’ are used for quantification of mitochondrial morphology.
Plotting of mitochondrial morphology parameters and estimation of mitochondrial fission/fusion frequencies using MATLAB
1. Import the data saved in .txt or .csv format for each yeast cell into MATLAB.
2. Plot the mitochondrial size distribution after collating the data under the column ‘Major’ of the data files across all timepoints from all the cells. This refers to the length of the ‘Major’ axis of an ellipse fitted to the mitochondria. Use the same dataset to obtain the mean and standard deviation of the mitochondrial sizes across cells.
3. Plot the circularity and aspect ratio of mitochondria using the data under the column ‘Circ.’ and ‘AR’ respectively of the data files across all timepoints from all the cells. Again, obtain the mean and standard deviations of the circularity and aspect ratios across cells using the same data set.
4. Write a script/function to count the number of mitochondria in each frame of the time-lapse movie by enumerating the number of occurrences of each stack position in the first column of the data file.
5. Compare the number of mitochondria in consecutive timepoints to obtain fission and fusion events. For instance, if the number of mitochondria in timepoint 1 is 5 and that in timepoint 2 is 7, then the number of fission events is 7 - 5 = 2. 2 ÷ 12 s = 0.167/s. Conversely, if the number of mitochondria in timepoint 1 is 5 and that in timepoint 2 is 4, then the number of fusion events is 5 - 4 = 1 (Figure 1).
6. Count the total number of fission and fusion events per cell for the entire time-lapse movie to obtain an estimate of fission and fusion frequencies. For example, if the total number of fission and fusion events for a cell in a 228 s time-lapse movie are 8 and 9 respectively, then the fission and fusion frequencies for the cell are 0.035/s and 0.039/s respectively. Obtain the mean and standard deviation of fission and fusion frequencies of all cells.

Statistical analysis of data
The data for fission and fusion frequencies obtained as described above can be represented in box plots with the central line indicating the median (see Mehta et al., 2019). To obtain the 95% confidence interval of the median, perform one-way ANOVA (‘anova1’ in MATLAB) or Kruskal-Wallis Test (‘kruskalwallis’ in MATLAB). The former is used when data is normally distributed and the latter when data are non-normally distributed. The normality of the dataset is tested using ‘chi2gof’ function in MATLAB. Significant difference (P < 0.05) between control and test groups is then be tested using Tukey’s Honestly Significant Difference procedure (‘multcompare’ in MATLAB).

Recipes

Phosphate buffered saline (10x)
Amount
Component
Final concentration
80 g/L
NaCl
0.137 M
2 g/L
KCl
2.7 mM
14.4 g/L
Na₂HPO₄
0.01 M
2.7 g/L
KH₂PO₄
1.8 mM
Adjust the pH to 7.2
Filter sterilize the solution by passing it through a bottle top filter containing a 0.22 µm pore size membrane filter and store at room temperature

EMM: Edinburgh Minimal Medium with appropriate supplements
Note: This recipe is taken from http://www-bcf.usc.edu/~forsburg/media.html.
Depending on the strain, medium with appropriate supplements is used. For imaging, liquid EMM is most ideal (Forsburg and Rhind, 2006)

Amount	Component	Final concentration
3 g/L	potassium hydrogen phthallate	14.7 mM
2.2 g/L	Na₂HPO₄	15.5 mM
5 g/L	NH₄Cl	93.5 mM
20 g/L	glucose	2% w/v
20 ml/L	salts
1 ml/L	vitamins
0.1 ml/L	minerals

Minimal supplemented with amino acids: EMM + 225 mg/L supplements (ade, leu, his, lys, ura...) as required (each amino acid at 225 mg/L)
We make amino acid stock solutions at 7.5 mg/ml (3.75 for uracil) and autoclave the solution for 40 min at 121 °C with 15 psi of pressure

Stock solutions

50x Salt stock
Amount
Component
Final concentration
52.5 g/L
MgCl₂·6H₂O
0.26 M
0.735 g/L
CaCl₂·2H₂O
4.99 mM
50 g/L
KCl
0.67 M
2 g/L
Na₂SO₄
14.1 mM
Filter sterilize the solution by passing it through a bottle top filter containing a 0.22 µm pore size membrane filter and store at 4 °C
1,000x Vitamin stock
Amount
Component
Final concentration
1 g/L
pantothenic acid
4.20 mM
10 g/L
nicotinic acid
81.2 mM
10 g/L
inositol
55.5 mM
10 mg/L
biotin
40.8 μM
Filter sterilize the solution by passing it through a bottle top filter containing a 0.22 µm pore size membrane filter and store at 4 °C

10,000x Mineral stock

Amount	Component	Final concentration
5 g/L	boric acid	80.9 mM
4 g/L	MnSO₄	23.7 mM
4 g/L	ZnSO₄·7H₂O	13.9 mM
2 g/L	FeCl₂·6H₂O	7.40 mM
0.4 g/L	molybdic acid	2.47 mM
1 g/L	KI	6.02 mM
0.4 g/L	CuSO₄·5H₂O	1.60 mM
10 g/L	Citric acid	47.6 mM

Filter sterilize the solution by passing it through a bottle top filter containing a 0.22 µm pore size membrane filter and store at 4 °C

YE: Yeast Extract with supplements
Amount
Component
Final concentration
5 g/L
Yeast extract
0.5% w/v
30 g/L
Glucose
3.0% w/v
Supplements: 225 mg/L adenine, histidine, leucine, uracil and lysine hydrochloride
Autoclave the solution for 40 min at 121 °C with 15 psi of pressure. To make solid medium, add 2% (w/v) Difco Bacto Agar prior to autoclaving

Acknowledgments

This research was supported by the Department of Science and Technology (India)-INSPIRE Faculty Award, the Department of Biotechnology (India) Innovative Young Biotechnologist Award, and the Science and Engineering Research Board (SERB, India) Early Career Research Award awarded to V.A. This protocol was adapted from Mehta et al., 2019.

Competing interests

The authors declare that they have no competing interests with the contents of this article.

References

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