A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function.


Introduction
Mitochondria play important roles, most notably cellular energy production by oxidative phosphorylation [1], hence the aptly coined phrase "the powerhouse of the cell" [2]. Mitochondria are also involved in Ca 2+ management [3], production of ROS [4], redox signalling [5,6] and apoptosis [7]. Many studies investigating mitochondrial function observe mitochondrial morphology and their dynamics within the cell [8]. Such observations can be achieved by the use of various fluorescent dyes such as Rhodamine 123 (R123) [9], tetramethylrhodamine-methyl-ester (TMRM) [10] and JC-1 (tetraethylbenzimi-dazolylcarbocyanine iodide) [11]. However, these dyes rely upon a mitochondrial membrane potential and can be washed out if the mitochondria experience depolarisation [12]. Furthermore, these dyes are unsuitable for use with aldehyde fixation due to resulting changes in mitochondrial metabolic state [12]. Other fluorescent dyes developed for visualizing mitochondria include the Mitotracker Red and Green dyes. Mitotracker Red binding depends on both the presence of a mitochondrial membrane potential while Mitotracker Green binding does not. These dyes can be used in combination with a number of cell fixation methods, however, they may cause cytotoxic effects following prolonged use. Other methods of real time mitochondrial imaging include the use of fluorescently tagged mitochondrial localised proteins, where the tagged protein is recognised by the mitochondrial 'Translocase of the outer/inner membrane' (TOM/TIM) protein complexes and transported into the mitochondria [13]. The transport of mitochondrial proteins into the mitochondrial matrix is facilitated by an N-terminal pre-sequence [14]. This pre-sequence can consist of 10-80 amino acid residues and is usually cleaved off by the matrix processing peptidase following localization [15]. However, these fluorescent proteins can interfere with the function of the native protein and impede mitochondrial function. As such, non-cytotoxic mitochondrial markers are required.
Dictyostelium discoideum is a tractable model system widely used for research in a range of fields including cell and developmental biology, evolutionary biology, as well as in immunology and molecular pharmacology studies. In cell and developmental biology, D. discoideum is often used to improve our understanding of cell motility [16,17]. In molecular pharmacology research, D. discoideum has been used to investigate mechanisms of action of pharmaceutical drugs including treatments for epilepsy [18][19][20] and neurodegenerative disorders [21,22]. Other studies investigate mechanisms of action of bioactive natural products such as curcumin, naringenin and a range of bitter tastants [23][24][25]. One recent study, identifying a mitochondrial protein, GcvH1, involved in the cellular function of cannabinoids on the glycine cleavage system [26], highlights the presence of an N-terminal mitochondrial localization sequence and thus raises the possibility of using this sequence for mitochondrial tagging in D. discoideum. To further these studies, a non-cytotoxic mitochondrial marker is required that can be used in D. discoideum without affecting cellular respiratory function.
In this study we created a novel expression plasmid (REMIT; red mitochondria) for real time visualization of mitochondria in D. discoideum. The REMIT plasmid allows the expression of an enhanced RFP protein (mRFPmars) [27] with a mitochondrial localization sequence situated at its N-terminus. We show that transfection of REMIT into D. discoideum cells facilitates the localization of mRFPmars to the mitochondrial matrix. We also show that the presence of mRFPmars within the mitochondria has no effect on mitophagy or cellular respiratory function. We therefore present a method that allows the real time visualization of mitochondria within D. discoideum cells with no deleterious effects.

Creation of the REMIT plasmid and over-expression D. discoideum cell lines
Primers (fwd: ATAGAATTCATGTTAAAAACCTTAAGATTTG and rev: TATGGATCCCCATTCATGATCG) complementary to the 5' region of gcvH1 were used to amplify the 99-bp mitochondrial localization sequence (Fig. 1). The PCR product was digested with EcoRI and BamHI and cloned into the extra chromosomal plasmid pDXA-389-2 [27,28].
The PCR product was inserted into the multiple cloning site located immediately 5' of the mRFPmars gene, and sequenced to confirm that no mutations were introduced. Insertion of the PCR product at this location enabled expression of a 27 kDa mRFPmars with a 33 amino acid residue localization sequence linked to its N-terminus via a 6 amino acid residue linker region (Fig. 1). The resulting plasmid was transfected into wildtype (AX3) cells and selected using geneticin (10 μg/ml) to create a REMIT over-expression cell line [29]. The GREMIT plasmid was based upon the same targeting region inserted into the GFP vector pDM1209 [30].
Confocal microscopy was performed at the Bioimaging core facility of the Biomedical Center (LMU Munich) using an inverted Leica TCS SP8 equipped with lasers for 405, 488, 552, and 638 nm excitation.  Images were acquired with a HC PL APO 63x/1.40 oil PH3 objective. Recording was sequential to avoid bleed-through. Alexa-488, and RFP were recorded with the hybrid photo detectors, DAPI with the conventional photomultiplier tube.
High resolution live-cell imaging was performed using an inverted Zeiss LSM 900 Airyscan 2 microscope [33]. Images were acquired with a Plan-Apochromat 63x/1.40 oil DIC objective with a GaASP-PMT detector (450-700 nm) in the MPCX SR-4y modus at an excitation of 558 nm. Z-stacks (25 corresponding to 4.32 μm) were recorded over time (2.55 s per z-stack). 3D reconstructions of single z-stacks were performed using the Imaris software package (Bitplane, Zurich, Switzerland).

Mitochondrial respirometry function
The effects that REMIT expression may have on mitochondrial stress were investigated in real time [35]. In these experiments, a Seahorse XF e24 Extracellular Flux Analyzer was used to measure mitochondrial respirometry within REMIT expressing cells, wildtype cells and cells expressing mRFPmars lacking the mitochondrial localization sequence. Mitochondrial respirometry was measured in terms of the basal O 2 consumption rate, the O 2 consumption rate devoted to the synthesis of ATP, the maximum O 2 consumption rate, the contribution of Complex I to the maximum O 2 consumption rate, the contribution of Complex II to the maximum O 2 consumption rate, and the O 2 consumption rate devoted to mitochondrial function other than ATP synthesis, i.e. "proton leak".

Statistical analysis
The distribution of all experimental data was tested using the Anderson-Darling test for normality. All data that showed a Gaussian distribution were analysed using parametric tests. Data from two groups not showing a Gaussian distribution were analysed using a Mann-Whitney T-test. The one-way analysis of variance (ANOVA) statistical test was used to test for significance between the means of three or more independent groups of normally distributed data.

Results and discussion
The D. discoideum mitochondrial localization sequence (MLS) (Fig. 1) was derived from the GcvH1 protein, a member of the mitochondrial glycine cleavage system enzyme complex [26]. The glycine cleavage system is located within the mitochondrial matrix and has a loose affiliation with the inner mitochondrial membrane. We cloned the GcvH1 MLS onto the N-terminus of mRFPmars [27] to form the REMIT vector that was then transfected into wildtype D. discoideum cells. To validate the mitochondrial localization of mRFPmars in these cells, mRFPmars fluorescence was examined in fixed cells [36] (Fig. 2), showing highly localised distribution in mitochondrial-like structures. Since porin is localised in the outer mitochondrial membrane, we examined this localization, indicating REMIT mRFPmars in the mitochondrial matrix with porin surrounding this labelling (Fig. 2). We then assessed the use of REMIT mRFPmars for labelling mitochondria in live cell imaging experiments. By using live-cell confocal microscopy, REMIT-expressing cells revealed a highly discrete labelling of mitochondria in real time (Fig. 3, and Supplementary movies 1,2), also visible using under-agar inverted fluorescence microscopy (Supplementary movie 3). A similar localization was found using a GFP-encoding vector, GREMIT (Supplementary Fig. 1). We also employed high resolution live-cell imaging to monitor mitochondrial dynamics (Supplementary movie 4), and this enabled 3-dimensional reconstruction of mitochondrial distribution in live cells (Supplementary movie 5).
Supplementary video related to this article can be found at https:// doi.org/10.1016/j.bbrep.2020.100751 In order to maintain a healthy population of mitochondria within the cells, all mitochondria experiencing damage or dysfunction will undergo mitophagy. This process results in defective mitochondria being targeted to the lysosome for autophagic degradation, thereby maintaining cell health [37]. Because transfection with REMIT leads to the localization of mRFPmars to the mitochondria we investigated whether this resulted in mitochondrial damage or dysfunction, resulting in mitophagy, or alternatively increased mitochondrial load (the mitochondrial protein content). To assess mitochondrial loading, we compared levels of a mitochondrial protein, mitochondrial 3-methylcrotonyl-CoA carboxylase α [34], in wildtype cells and cells transfected with REMIT using Western blot analysis (Fig. 4A). From this analysis, no change in mitochondrial load was identified following REMIT transfection.
The requirement of mitochondria to carry out normal respiratory function is fundamental to maintaining a healthy cell. Any deleterious effect on respiratory function as a result of REMIT transfection would result in downstream processes being disrupted. Thus, it is necessary to confirm that mitochondrial respiratory function is not disrupted. Mitochondrial respirometry function was therefore measured in terms of the basal O 2 consumption rate, the O 2 consumption rate devoted to the synthesis of ATP, the maximum O 2 consumption rate, the contribution of complex I to the maximum O 2 consumption rate, the contribution of complex II to the maximum O 2 consumption rate, and the O 2 consumption rate devoted to mitochondrial function other than ATP synthesis, i.e. "proton leak" (Fig. 4B). These data were obtained from cells transfected with REMIT, cells transfected with REMIT lacking the MLS, and untransfected wildtype cells. For all six conditions no significant difference (P > 0.05) was found between the three cell lines. This shows that mitochondrial function is not affected despite the presence of mRFPmars localised within the mitochondrial matrix.
These experiments thus show that transfection of D. discoideum cells with REMIT provides a quick, cheap and convenient method to visualize mitochondria in real time. The use of REMIT would be advantageous in studies involving the need for both visualization and normal respiratory function. These studies can include investigation into mitochondrial fission and fusion events, mitochondrial dynamics and mitochondrial morphology.

Author statement
CJP and RSBW conceived the research and wrote the paper. ECW and JD-O contributed to imaging and measuring mitochondria load. CS, LMF, SJA, PRF analysed mitochondrial function. AM-T provided confocal microscopy and 3D modelling. All authors contributed to editing the paper.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fig. 4. Expression of REMIT within D. discoideum has no effect on mitochondrial load or respiratory function. Mitochondrial load and respiratory function was evaluated in D. discoideum cells transfected with the REMIT plasmid. (A) Western blot analysis was used to assess levels of a mitochondrialspecific marker protein (MCCC1) and total protein levels. Data represent the mean and SEM (P > 0.05), n = 6 (Mann-WhitneyU test). (B) Mitochondrial respiratory function was evaluated in cells transfected with REMIT, with wildtype cells, and with the REMIT plasmid lacking the localization sequence (RFP). Respiratory function was quantified in terms of basal O 2 consumption rate, O 2 consumption rate devoted to the synthesis of ATP, maximum O 2 consumption rate, contribution of complex I to the maximum O 2 consumption rate, contribution of complex II to the maximum O 2 consumption rate, O 2 consumption rate devoted to mitochondrial function other than ATP synthesis, i.e. "proton leak". Data represent the mean and SEM (P > 0.05), n = 6 (One way ANOVA).