Protocol for rapid manipulation of mitochondrial morphology in living cells using inducible counter mitochondrial morphology (iCMM)

Summary Disruption of mitochondrial morphology occurs during various diseases, but the biological significance is not entirely clear. Here, we describe a detailed step-by-step protocol for a chemically inducible dimerization system-based synthetic protein device, termed inducible counter mitochondrial morphology. This system allows artificial manipulation of mitochondrial morphology on a timescale of minutes in living mammalian cells. We also describe an AI-assisted imaging processing approach. For complete details on the use and execution of this protocol, please refer to Miyamoto et al., 2021.

Disruption of mitochondrial morphology occurs during various diseases, but the biological significance is not entirely clear. Here, we describe a detailed step-by-step protocol for a chemically inducible dimerization system-based synthetic protein device, termed inducible counter mitochondrial morphology. This system allows artificial manipulation of mitochondrial morphology on a timescale of minutes in living mammalian cells. We also describe an AI-assisted imaging processing approach.

SUMMARY
Disruption of mitochondrial morphology occurs during various diseases, but the biological significance is not entirely clear. Here, we describe a detailed step-bystep protocol for a chemically inducible dimerization system-based synthetic protein device, termed inducible counter mitochondrial morphology. This system allows artificial manipulation of mitochondrial morphology on a timescale of minutes in living mammalian cells. We also describe an AI-assisted imaging processing approach. For complete details on the use and execution of this protocol, please refer to Miyamoto et al., 2021.

BEFORE YOU BEGIN
In this paper, we describe a step-by-step protocol for manipulating mitochondrial morphology in living cells at any given time point within a few minutes using iCMM, a Boolean YES logic gate-based synthetic protein device based on a Chemically Inducible Dimerization (CID) system. When this protocol is followed, mitochondrial morphological changes are observed within 1-5 min of actuating the iCMM. It employs the CID method with rapamycin as a chemical dimerizer; however, the fundamental experimental procedure is the same when other CID methods are used.
The iCMM consists of a functional iCMM effector (FiCE), which can induce changes in mitochondrial morphology, and a mitochondrial outer membrane-specific anchor protein that tethers the effector to the mitochondria in the presence of a chemical dimerizer ( Figure 1A). In contrast, an effector that does not cause mitochondrial morphological changes is termed a control iCMM effector (CiCE), and a synthetic protein device consisting of a CiCE and a mitochondria-specific anchor is referred to as a negative iCMM (NiCMM; Figure 1A).
The main characteristic of iCMM is that it can induce morphological changes in mitochondria on a minute timescale at any time point. Thus, changes that occur in the cell immediately after induction of mitochondrial morphological changes can be observed.

Plasmid preparation for transfection
Timing: 3 days 1. Expression plasmids encoding effectors and anchor should be prepared in advance. These plasmids are available from Addgene (Watertown, MA, USA; https://www.addgene.org/). 2. Day 1: transform plasmids into competent cells as follows: a. Thaw ECOS Competent E. coli DH5a cells (NIPPON GENE, Tokyo, Japan) on ice. b. Add 10 ng of plasmid to 20 mL competent cells in a 1.5-mL microcentrifuge tube and mix gently. c. Incubate the mixture on ice for 20 min. d. Heat shock at 42 C for 45 s. e. Put the mixture on ice for 3 min. f. Add 50 mL LB liquid medium without antibiotics to the mixture. g. Incubate the mixture at 37 C for 60 min. h. Warm LB agar plates containing 50 mg/mL kanamycin at 37 C for 60 min. i. Spread all of the mixture onto the LB agar plates containing 50 mg/mL kanamycin. j. Incubate the plates at 37 C overnight (16-18 h).
Alternatives: Other competent cells, such as the E. coli DH5a Competent Cells (TaKaRa, 9057), can be used instead of ECOS Competent E. coli DH5a cells.
3. Day 2: select a single clone and culture it in 100 mL LB medium containing 50 mg/mL kanamycin in a 200-mL flask. 4. Shake at 37 C and 160 rpm overnight (16-18 h). 5. Day 3: transfer the bacterial culture to two 50-mL conical tubes and centrifuge at 4,000 3 g and 4 C for 10 min. 6. Remove the supernatant.
Pause point: Bacterial pellets can be stored at À80 C until purification.

Timing: 3 days
Human cervical adenocarcinoma HeLa cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% ZellShield (Minerva Biolabs, Berlin, Germany) at 37 C in 5% CO 2 .
Alternatives: iCMM can be used with cells other than HeLa cells. We confirmed that iCMM can induce mitochondrial morphological changes in Hep 3 B human hepatoma and U-2 OS human osteogenic sarcoma cells (Miyamoto et al., 2021).
8. Day 1: thaw cells stored in a À80 C freezer or in liquid nitrogen were transfered to a 15-mL conical tube containing 10 mL prewarmed culture medium.
Note: CELLBANKER (Nippon Zenyaku Kogyo, Tokyo, Japan), a ready-to-use cell cryopreservation medium, may be used for cell stocks.
9. Centrifuge the tube with swing buckets at 161 3 g and at room temperature (15 C-25 C) for 2 min.
Note: Sorvall ST 8 (Thermo Fisher Scientific) may be used for centrifugation.
10. Remove the supernatant and resuspended precipitated cells in 10 mL prewarmed culture medium. 11. Plate the cell suspension in a 10-cm cell culture dish.
Note: After plating cells in the culture dish, use a microscope to ensure that the cells are not dead. Furthermore, 24 h after plating verify that confluency is < 50%; if not, then proceed to Step 13 (Day 3).
12. Incubate cells at 37 C and 5% CO 2 for 48 h. 13. Day 3: remove the culture medium. 14. Add 4 mL prewarmed phosphate-buffered saline (PBS) at the periphery of the dish and slowly tilt to gently wash the cells. 19. Add 7 mL prewarmed culture medium and suspend the cells by gentle pipetting. 20. Transfer the cells to a sterilized 15-mL conical tube. 21. Centrifuge the tube with swing buckets at 161 3 g and at room temperature (15 C-25 C) for 2 min. 22. Add 7 mL prewarmed culture medium and suspend the cells by gentle pipetting. 23. To count cells, mix 40 mL of the cell suspension with 40 mL trypan blue in a 1.5-mL microcentrifuge tube. Pipette up and down gently several times to mix. 24. Use an automated cell counter with 10 mL of the mixture.
Alternatives: A hemocytometer may be used for cell counts.
Note: Verify, at least qualitatively, that cell viability, shape, and size are not atypical. If the cell counter used can measure quantitative information about viability, size, among others, then it is recommended to record such numerical information.
Note: In this case (1 3 10 6 cells in a 10-cm dish), seeded HeLa cells need to be passaged after 48 h. To passage cells at 72-h intervals, plate 0.5 3 10 6 cells in a 10-cm dish. The number of cells to be plated should be changed according to the cells' growth rate. Confluency of 80% is recommended as a standard for cell passaging. To prevent damage to the cells, we do not recommend passaging at 24-h intervals.
CRITICAL: Before starting the experiment, infection of the cells with Mycoplasma should be excluded using PCR and Hoechst staining. To examine whether cells are infected with Mycoplasma by PCR, we recommend using ready-to-use kits such as e-MycoTM Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Cat#25235), e-MycoTM VALiD Mycoplasma PCR Detection Kit (iNtRON Biotechnology, Cat#25239), TaKaRa PCR Mycoplasma Detection Set (Takara, Cat#6601), or LookOutâ Mycoplasma PCR Detection Kit (Sigma-Aldrich, Cat#MP0035-1KT). For Hoechst staining-based Mycoplasma contamination test, please refer to the technical article at the following site: https://www.sigmaaldrich.com/ JP/en/technical-documents/technical-article/microbiological-testing/mycoplasma-testing/ testing-for-mycoplasma.
Note: The LB agar plates can be stored at 4 C away from light until use. They should be used within a month.
Note: The medium can be stored at room temperature (15 C-25 C) without antibiotics until use. Thus, the LB liquid medium can be prepared a day before its intended use.

Maintenance of HeLa cells
Culture medium (DMEM supplemented with 10% FBS and 1% ZellShield) Note: The culture medium can be stored at 4 C away from light until use.

Live cell imaging
-To prepare a stock solution of rapamycin, dissolve rapamycin in dimethyl sulfoxide to a final concentration of 100 mM. Stock solutions may be stored at À30 C until use. -For live cell imaging, prepare phenol red-free DMEM supplemented with 10% FBS, 4 mM Lglutamine, and 1% penicillin and streptomycin (referred to as imaging medium).
Note: The imaging medium can be stored at 4 C away from light until use.

STEP-BY-STEP METHOD DETAILS
Seeding and culturing of the cells (day 1)

Timing: 3 h
Before starting this protocol, cells should be cultured for at least one week after thawing; additionally, the confluency of the cells should not exceed 80%.
1. Add 4 mL prewarmed PBS at the periphery of the dish and slowly tilt to gently wash the cells.
2. Remove PBS. 3. Add 1 mL 0.05% trypsin-EDTA solution to the dish. 4. Incubate the cells at 37 C for 4 min in a 5% CO 2 incubator. 5. Gently tap the dish to completely detach the cells. 6. Add 5 mL prewarmed fresh culture medium and suspend the cells by gentle pipetting. 7. Transfer the cells to a sterilized 15-mL conical tube. 8. Centrifuge the tube with swing buckets at 161 3 g and at room temperature (15 C-25 C) for 2 min. 9. Add 7 mL prewarmed culture medium and suspend the cells by gentle pipetting. 10. To count cells, mix 40 mL of the cell suspension with 40 mL trypan blue in a 1.5-mL microcentrifuge tube. Gently pipette up and down several times to mix. 11. Use an automated cell counter with 10 mL of the mixture.
Alternatives: A hemocytometer may be used for cell counts.
Note: Verify, at least qualitatively, that cell viability, shape, and size are not atypical. If the cell counter used can measure quantitative information about viability and size, among others, it is recommended to record such numerical information.
Alternatives: A 3.5-cm poly-lysine-coated glass-bottom dish can be substituted for either glass cover slips coated with poly-D-lysine or a 35-mm imaging dish with a polymer coverslip bottom (e.g., iBidi, Fitchburg, WI, USA; cat# 81156). Notably, as changes in cell adhesion conditions may alter cellular responses to various factors, it is recommended that cell adhesion conditions be kept consistent.
13. After plating, culture cells at 37 C and 5% CO 2 in an incubator for 4 h (until cells attach well) and then transfect. Transfection (day 1)

Timing: 30 min
14. Transfect as follows: a. Add 0.5 mg of each plasmid-encoding effector and anchor (1:1 ratio; total 1 mg) to 100 mL Opti-MEM (Thermo Fisher Scientific) in a 5-mL polystyrene round-bottom tube. b. Add 3 mL FuGENE HD (Promega) and mix gently to avoid introducing bubbles. c. Incubate the transfection mixture at room temperature (15 C-25 C) for 10 min. d. Add 100 mL of the mixture to the cells that are plated on a 3.5-cm poly-lysine-coated glassbottom dish and mix gently.
Note: The amount and proportion of plasmids need to be optimized according to the used cell line, the transfection reagents, and the purpose of the experiment. Live cell imaging using epifluorescence microscopy (day 2)

Timing: 2 h
Cells were viewed using a 403 objective (Plan Apochromat Lambda Series, Nikon) mounted on an inverted Eclipse Ti2-E microscope (Nikon) and imaged using a Zyla 4.2 PLUS sCMOS camera (Oxford Instruments). Imaging data were processed using the NIS-Elements AR imaging software (Nikon).
16. Two hours before live cell imaging, replace culture medium containing the transfection mixture with 2 mL prewarmed fresh culture medium.
Note: To reduce cytotoxicity of transfection reagents, the culture medium should be replaced at least 12-16 h after transfection.
17. Activate the STX stage top incubator system (Tokai Hit, Fujinomiya, Japan) to maintain a temperature of 37 C in 5% CO 2 for 1 h before live cell imaging. 18. Add 1 mL 1 mM MitoTracker Red CMXRos (Thermo Fisher Scientific) into the glass-bottom dish (final concentration: 500 nM) 30 min before live cell imaging.
Note: To stain the cells uniformly, 1 mL MitoTracker Red CMXRos may be aliquoted to a 1.5-mL microcentrifuge tube and suspended in 100 mL culture medium. The suspension may then be added to the dish followed by gentle agitation. Additionally, the concentration of MitoTracker Red CMXRos should be optimized for each cell line used according to the manufacturer's protocol (https://www.thermofisher.com/document-connect/document-connect.html?url=https %3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fmp07510.pdf& title=VXNlciBHdWlkZTogTWl0b1RyYWNrZXIgTWl0b2Nob25kcmlvbi1TZWxlY3RpdmUgUHJ vYmVz).

OPEN ACCESS
19. During the staining process, start the microscopy system according to the manufacturer's instructions (https://www.microscope.healthcare.nikon.com/products/inverted-microscopes/eclipse-ti2-series/eclipse-ti2-e). 20. Thirty minutes after adding MitoTracker Red CMXRos, remove the culture medium and wash twice using 1 mL prewarmed imaging medium. 21. Add 2 mL prewarmed imaging medium. 22. Find and focus cells expressing NiCMM/iCMM. 23. Select five positions for live cell imaging. 24. Set the imaging parameter of the NIS-Elements AR imaging software (Nikon) (Figure 2) Note: Although we do not have strict criteria for determining the imaging positions, we believe it is important to select the imaging positions so that we can capture iCMM/ NiCMM-expressing cells without bias as much as is possible.
Note: Live cell imaging was performed under the same imaging conditions for each experiment. Representative imaging conditions are described below.

General information
Camera Name: Andor Zyla VSC-07685 Numerical Aperture: 0.95 Note: To treat the cells uniformly with rapamycin, aliquot 1 mL 100 mM rapamycin into a 1.5-mL microcentrifuge tube and suspend in 100 ml imaging medium. Add the suspension so that it is spread evenly over the entire culture dish and is not accumulated in only one place.
Note: The addition of rapamycin does not necessarily have to be performed in the dark. However, it is recommended that imaging is performed in the dark.
Note: It is essential to use imaging conditions that do not cause changes in mitochondrial morphology unless the iCMM is activated. To optimize imaging conditions, it is recommended to perform time-lapse imaging without adding rapamycin and confirm that mitochondrial morphology does not change (no fragmentation or aggregation of mitochondria).
27. Process the imaging data using NIS-Elements AR imaging software (Nikon).
Note: For image processing, it is necessary to follow the Data and Image Processing Policy of each journal. We show in Figure 4 the image processing to be performed for a representative mitochondrial image acquired by epifluorescence microscopy. Note: When performing quantitative analysis of mitochondrial morphology, it is necessary to perform image processing according to the method used. Methods using Image J (Valente et al., 2017) and machine learning (Leonard et al., 2015;Zahedi et al., 2018) have been developed for mitochondrial morphology analysis.

EXPECTED OUTCOMES
iCMM facilitates inducing mitochondrial morphology changes in a time scale of minutes. Mitochondrial morphology changes induced by iCMM depend on the used FiCE. Specifically, YF induces a large punctate structure of mitochondria (due to clustering rather than fusion of mitochondria), while YF-Cav1s and mActZ-FY induce a small punctate structure ( Figure 5). In contrast, NiCMM, which is the technical counterpart of iCMM, does not induce any mitochondrial morphological changes, even when CiCE is translocated to the mitochondria ( Figure 5). We have so far confirmed that the induction of mitochondrial morphological changes by iCMM occurs similarly in HeLa human cervix adenocarcinoma cells, Hep 3B human hepatoma cells, and U-2 OS human bone osteosarcoma cells (Miyamoto et al., 2021).
In our experiments, we confirmed that iCMM-induced mitochondrial morphology changes do not affect mitochondrial membrane potential or reactive oxygen species production over a 120-min observation window as judged by tetramethylrhodamine ethyl ester (Thermo Fisher, T669) and MitoSox (Thermo Fisher, M36008), respectively. In contrast, the oxygen consumption and extracellular acidification rates were decreased only when mitochondrial morphological changes were induced by mActZ-FY.

LIMITATIONS
It should be noted that as iCMM artificially induces changes in mitochondrial morphology, comparisons of the respective results with physiological mitochondrial morphological changes should be made with caution. However, detailed observation of cellular responses to iCMM-induced alterations may yield novel insights. Thus, an appropriate combination of iCMM and conventional genetic approaches (e.g., modification of genes involved in mitochondrial morphology) is required to elucidate the operating mechanism(s) of living organisms, with a focus on mitochondrial morphology.
The current protocol employs rapamycin; however, it is important to note that rapamycin is a potent inhibitor of mammalian/mechanistic targeting of rapamycin complex 1, a serine/threonine kinase. If the use of rapamycin is not suitable, then other CID methods or optogenetics may be used instead (Stanton et al., 2018). However, optimization of the effectors and anchor is necessary.

Problem 1
No or very few cells express iCMM/NiCMM (steps 14 and 22 in step-by-step method details).

Potential solution
With respect to cell lines that are used for the first time, it is recommended to transfect only mYF in order to optimize the transfection protocol. The amount of plasmid to be transfected (0.1-5 mg), the ratio of plasmid to transfection reagent (1:1-1:5), and the time between transfection and experiment (12-72 h) are the three most crucial points to consider. It is also possible that performing transfection 18-24 h after seeding the cells may improve the results. If there are no cells expressing iCMM/ NiCMM at all, we recommend changing the transfection reagent or the transfection method (e.g., electroporation and lentivirus transfection).
By contrast, if the efficiency of gene expression is low in a cell line that has been expressing genes successfully in the past, first check the quality of the plasmid: ensure that the A260/A280 ratio is < 1.8, and that the A260/A230 ratio is between 1.8 and 2.2. In addition, run 500 ng plasmid on a 1% agarose gel by electrophoresis to ensure that the plasmid is not degraded. Next, check cell morphology and proliferation rate to determine whether cell characteristics are changed. If there are no respective issues, it is recommended to individually change the conditions and factors (Opti-MEM or transfection reagent) for stepwise examination. New reagents or media may also be tested.

Problem 2
The mitochondrial structure is aggregated in cells expressing iCMM/NiCMM before starting timelapse imaging ( Figure 6) (step 22 in step-by-step method details).

Potential solution
If expression levels of Tom20-CR are excessively high, mitochondria may not be able to form a normal network structure. This can be improved by reducing the amount of plasmid introduced into the cells or by reducing the time between transfection and experiment. In addition, this may also be improved by using a mitochondria-targeting signal (MTS) other than Tom20-derived MTS or by changing the length of the linker connecting the functional domains.

Problem 3
The number of cells adhering to the glass bottom dish is low (step 22 in step-by-step method details).

Potential solution
It is possible that cells detach during washing. Although the proteins constituting iCMM/NiCMM do not induce cell death, transfection-induced cell death occurs at a certain rate. Optimization of cell confluence is also recommended as a potential solution.

Problem 4
The mitochondrial structure does not change after adding chemical dimerizer (step 26 in step-bystep method details).

Potential solution
Ensure that expression levels of FiCEs and of the anchor (Tom20-CR) are adequate. For example, YF may not induce mitochondrial morphological changes effectively at low expression levels. mActZ-FY has lower expression levels than other FiCEs, however, lower expression levels are more effective for inducing mitochondrial morphology changes.
If expression of FiCE and of the anchor is sufficient, add additional chemical dimerizers. If an additional chemical dimerizer induces mitochondrial morphological changes, it is possible that the previous chemical dimerizer was not added adequately. Further, ensure that the chemical dimerizer had been stored properly.

Problem 5
Fluorescence intensity of mitochondria stained with MitoTracker Red CMXRos decrease during time-lapse imaging (step 27 in step-by-step method details).

Potential solution
Change the exposure time and ND filter to achieve the lowest fluorescence intensity at which mitochondria can be observed or reduce the number of imaging positions. As MitoTracker Red CMXRos stains mitochondria in a mitochondrial membrane potential-dependent manner, it is also necessary to avoid imaging under conditions in which the membrane potential is reduced.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Takafumi Miyamoto (takmi565@md.tsukuba.ac.jp).

Materials availability
Plasmids generated in this study have been deposited to Addgene (see key resources table).

Data and code availability
This study did not generate any datasets or code.