Background

Mitochondria are crucial for cellular survival. Almost all mitochondrial proteins are encoded in the nuclear genome and proper mitochondrial function and biogenesis rely on mitochondrial protein import (Dimogkioka et al., 2021). Multiple mitochondrial protein import machineries exist; each mediates protein translocation into a distinct mitochondrial sub-compartment (Schmidt et al., 2010). Stress conditions, such as the loss of mitochondrial membrane potential, are known to disturb mitochondrial protein import, and defective import can lead to a series of pathologies, such as neurodegenerative and cardiovascular diseases and cancer (Geissler et al., 2000; Palmer et al., 2021).

To investigate mitochondrial protein import, several methods have been established. Radiolabeling of individual mitochondrial proteins followed by incubation with purified mitochondria allows following mitochondrial import of the labeled protein (Murschall et al., 2021; Poveda-Huertes et al., 2021). Besides the technical (e.g., purification of fully active mitochondria) and bureaucratic requirements of performing radioactive lab work, this in vitro approach lacks cellular context and thus does not account for potential contribution of cytosolic factors on protein import. Recently developed proteomics methods allow monitoring the import of newly synthesized proteins into mitochondria on the global level (Schafer et al., 2022). This method is carried out within the cellular context and allows monitoring hundreds of mitochondrial proteins; however, it is a complex proteomics method that requires a mass spectrometry infrastructure not available in most laboratories. Here, we describe a fluorescence microscopy–based approach that allows monitoring mitochondrial import of the previously established mitochondrial targeting sequence (MTS)–enhanced green fluorescent protein (eGFP) reporter protein by live cell imaging (Chen et al., 2003). The reporter consists of eGFP that is selectively targeted to the mitochondrial matrix by means of a N-terminal MTS, which consists of the first 69 amino acids of subunit 9 of the F0-ATPase. Transcription of the reporter gene can be induced with the Tet-On system by treating cells with doxycycline, which allows for a controlled and titratable MTS-eGFP expression. Defective import of the reporter is assessed by its mislocalization to the cytosol, which is monitored by co-staining of mitochondria with MitoTracker^TM Deep Red FM. Subsequent co-localization analysis with the ImageJ-implemented Coloc2 plugin allows assessing mitochondrial protein import defects in a quantitative manner.

Materials and Reagents

1. Cultivating cell lines

   1. 10 cm tissue culture dish (Sarstedt, catalog number: 83.3902)

   2. 6-well cell culture plate (Sarstedt, catalog number: 83.3920.005)

   3. Cell counting slides (dual-chamber) (Bio-Rad, catalog number: 1450015)

   4. 0.45 µm filters (Whatman, catalog number: 10462100)

   5. HeLa FlpIn TRex cell line (Invitrogen, catalog number: R71407)

      Note: This protocol was established for HeLa FlpIn TRex cell lines. It can be transferred to other cell lines that are suitable for microscopy.

   6. HEK 293T cells (human embryonic kidney 293T) (ATCC, catalog number: CRL-3216)

   7. RPMI 1640 medium (+L-Glutamine) (Thermo Fisher Scientific, catalog number: 21875-034)

   8. DMEM medium (+L-Glutamine) (Thermo Fisher Scientific, catalog number 41966029)

   9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 10270-106)

   10. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, catalog number: 25200056)

   11. Dulbecco's phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14190-169)

   12. Trypan blue solution (Sigma-Aldrich, catalog number: T8154)

       
       

2. Stable and inducible MTS-eGFP cell line generation

   1. 15 mL tube (Greiner, catalog number: 188271-N)

   2. Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, catalog number: 11668027)

   3. Puromycin (InvivoGen, catalog number: ant-pr-1)

   4. Opti-MEM I (Thermo Fisher Scientific, catalog number: 31985-047)

   5. Polybrene (Sigma-Aldrich, catalog number: TR-1003-G)

   6. pHDM-VSV-G (Addgene, catalog number: 164440); encodes VSV-G protein

   7. pHDM-Hgpm2 (Addgene, catalog number: 164441); encodes codon-optimized HIV gag-pol

   8. pHDM-tatIB (Addgene, catalog number: 164442); encodes HIV-1 Tat accessory protein

   9. pRC-CMV-revIB (Addgene, catalog number: 164443); encodes HIV-1 Rev accessory protein

   10. pLD-puro-MTS-EGFP (Addgene, catalog number: 190270); contains tetracycline/doxycycline-inducible MTS-eGFP and lentiviral packaging signal

       
       

3. MTS-eGFP mitochondrial import assay

   1. Cell culture microplate, 96-well, black (Greiner, catalog number: 655090)

   2. MitoTracker^TM Deep Red FM (Thermo Fisher Scientific, catalog number: M22426)

   3. Doxycycline (0.25 µg/mL stock in RNAse-free H₂O) (Sigma-Aldrich, catalog number: D9891-10G)

   4. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759)

      Note: CCCP is toxic if swallowed. Avoid skin contact and wash hands thoroughly after handling.

   5. Dimethyl sulfoxide for cell culture (DMSO) (VWR, catalog number: A3672.0100)

   6. Gamitrinib-triphenylphosphonium (GTPP) (custom synthesized)

Equipment

1. -80 °C freezer (Ewald, catalog number: V86-520.1)

2. TC20 automated cell counter (Bio-Rad, catalog number: 1450102)

3. CQ-1 confocal quantitative (Yokogawa) or other live cell microscope with 60× magnification (488 nm, 640 nm laser)

4. Emission filters 525/50 nm for eGFP (CQ-1, Yokogawa)

5. Emission filters 685/40 nm for MitoTracker^TM Deep Red FM (CQ-1, Yokogawa)

6. Incubator for mammalian cell culture (Thermo Fisher Scientific, catalog number: 16416639)

Software

1. ImageJ (NIH, version: 1.53e)

Procedure