Team:Hong Kong HKUST/characterization/mls

Introduction

Mitochondrial Leader Sequence (MLS) helps direct proteins to the mitochondria when this peptide sequence is in front of the N-terminus of the protein of interest. MLS will be removed upon the peptide’s translocation into the mitochondria, but four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein. The CDS of MLS was cloned out from pCMV/myc/mito (Invitrogen, Carlsbard, CA) using PCR.

MLS is submitted in RFC25 standard (BBa_K1119001) to facilitate fusing with other CDS. MLS in RFC10 standard (BBa_K1119001)is submitted as alternative, but it cannot be fused directly to other CDS due to the limitations of RFC10. Users who obtained the part in RFC10 standard can obtain the part by PCR and fuse to other domains using overlapping PCR.

In our characterization, the coding DNA sequence (CDS) of MLS was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format (BBa_K648013). The translation unit was driven by CMV promoter (BBa_K1119006), and terminated by hGH polyA signal (BBa_K404108). The aforementioned construct (BBa_K1119009) was then transfected into HEK293FT cells. Mitochondria were stained with MitoTracker® Red CMXRos dye after transfection and co-localization between the GFP signal and that of the dye was determined as the area of signal overlap. To provide a positive control, the CDS of EGFP from pEGFP-N1 (Clontech) was inserted downstream and in frame with the CDS of the MLS in the commercial plasmid pCMV/myc/mito (Invitrogen, Carlsbard, CA). Our negative control construct was the same as our experimental construct, but minus the MLS CDS. (BBa_K1119008).

The detailed protocols employed for our characterization work can be accessed via the link.

Cell Culture and Transfection

The three constructs were transfected separately into different batches of HEK293FT cells. The protocol for culturing HEK293FT cells was based on American Type Culture Collection resources. The culture medium used was DMEM with 10% FBS and 1% penicillin/streptomycin. For the transfection, we followed the general procedure outlined by Invitrogen LipofectamineTM 2000. Serum-free and antibiotics-free DMEM was used to form DNA-lipofectamine complexes.

Stain with MitoTracker® Dye

In order to test the efficiency of MLS, we need to identify the location of mitochondria. Therefore, rosamine-based MitoTracker® Red CMXRos (Invitrogen, Carlsbard, CA), a red-fluorescent dye, was used to stain the mitochondria in live cells. It can accumulate due to the membrane potential of mitochondria. According to the manual, this dye can be well resolved from the green fluorescence of other probes so it won’t affect the observation of the expression of green fluorescence protein. The protocol we used for staining is from MitoTracker® Mitochondrion-Selective Probes. The working concentration of staining solution made was 0.2μM and the incubation time after staining was 15 minutes.

Fluorescence Microscopy

Since the EGFP, GFP and MitoTracker® Red CMXRos can all be observed under fluorescent microscope, we did fluorescent microscopy in order to obtain results. We observed the transfected cells under confocal microscope.

Data Processing

To quantify the amount of signal overlapped between the GFP signal and the MitoTracker® dye, we adopted the method described by A.P. French et al. in “Colocalization of fluorescent markers in confocal microscope images of plant cells” (French et al., 2008). With the use of the Pearson-Spearman correlation colocalization plugin for ImageJ image processing, scatter plots of the green intensities (y-axis) and red intensities (x axis), Pearson's correlation coefficient and Spearman's correlation coefficient were generated.

Result

Figure 1. MLS directs GFP into mitochondria. When MLS is added to the N-terminus of GFP, the GFP was directed to the mitochondria in the cells, giving patches of GFP signal that overlapped with the signals from MitoTracker®. When MLS is not added to the GFP, the GFP signal can be seen scattered all around inside the cell. Scale bar = 10µm.

Figure 2. Scatter plots of fluorescence intensities of green (y axis) and red (x axis) by construct. It showed that the BioBrick MLS-GFP and commercial MLS-GFP construct had a linear relationship of green intensities and red intensities while the GFP generator alone had no relationship. Pearson's correlation coefficient (rp) and Spearman's correlation coefficient (rs) were determined using the Pearson-Spearman correlation colocalization plugin (French et al., 2008) for ImageJ with a threshold of 0 and listed for each image.

Figure 3. Calculated mean Pearson's (rp) and Spearman's (rs) correlation coefficients for each construct. The coefficients were generated using ImageJ software and specific plugins. For every batch of transfected cells, four samples were used for quantification. Experimental BioBrick MLS-GFP and commercial MLS-GFP: coefficients were close to 1, good colocalization; GFP: Coefficients were close to 0, poor colocalization. Error bars show standard deviation.

Conclusion

The result shows that the colocalization of GFP signal and mitotracking dye is high for the experimental construct of MLS BioBrick. It can be concluded that the MLS can function well and successfully bring recombinant proteins into mitochondria.

Reference

pcmv/myc/mito Invitrogen.(2012).pShooter™ Vector(pCMV/myc vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf
Dean, J., Tran , L., Liao, J., Dipple, K., Reue, K., Tontonoz, P., & Beaven, S. (2009). Resistance to diet-induced obesity in mice with synthetic glyoxylate shunt. Cell Metabolism, 9((6)), 525–536. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19490907
French, A.P., S. Mills, R. Swarup, M.J. Bennett, and T.P. Pridmore. (2008). Colocalization of fluorescent markers in confocal microscope images of plant cells. Nature Protocol. 3:619–628. doi:10.1038/nprot.2008.31.