Fig 1.
C-terminal tagging of Cit1p with mTFP1 does not disrupt localization or function of the protein.
A) BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, or Cit1-mCherry were stained with 1 μg/ml DAPI for 10 min as described in Materials and Methods. DAPI-stained cells were imaged on a wide-field microscope. Z-series were collected through the entire cell at 0.5 μm intervals using a metal halide lamp and appropriate filters at 216 gain and 200 ms exposure time. Cell outlines were drawn over phase images. Scale bar = 1 μm. n = nucleus. mtDNA = mitochondrial DNA. B) BY4741 and cit1∆ cells, and BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, or Cit1-mCherry were grown to mid-log phase in YPD and diluted to OD600 = 0.01 in YPG. 10-fold serial dilutions were performed and 5 μl was placed on solid media (YPG) and grown at 30°C for three days. Images are representative of three independent trials.
Fig 2.
Spectral compatibility of mitochondria-targeted FPs.
BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, and Cit1-mCherry were imaged on a wide-field microscope. Z-series were collected at 0.5 μm intervals throughout the entire cell using a metal-halide lamp with standard GFP, CFP, YFP, and rhodamine filters using 216 gain and 200 ms exposure times. Full filter specifications are listed in Materials and Methods. Only the center wavelengths for our filters for values of excitation and emission filters are listed for ease of readability.
Fig 3.
Perceived brightness and stability of mTFP1 relative to GFP, mCitrine, and mCherry.
BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, or Cit1-mCherry were imaged on a wide-field microscope. Single-plane, time-lapse imaging was performed at the center of the cell at 1 sec intervals for 120 sec using a metal halide lamp and appropriate filters at 216 gain and 200 ms exposure for each fluorophore. A) Integrated fluorescence density at time = 0 was measured using Image J as described in Materials and Methods. *** = p < 0.001. Error bars are SEM. B) Integrated fluorescence density was measured at each time point and normalized to the integrated fluorescence density at time = 0 and graphed as % fluorescence remaining as a function of time. Error bars are SEM. n = 28–44 cells per strain. Data is representative of 3 independent trials.
Table 1.
Properties of fluorescent proteins used in this study.
Full spectra and information for all fluorescent proteins are available at (http://nic.ucsf.edu/FPvisualization/). λex/em are listed as the peak excitation and emission wavelength of the FP. Intrinsic brightness is derived from the product of its extinction coefficient and quantum yield and higher numbers indicate brighter FPs. Bleaching is listed such that higher numbers indicate more photostable FPs. Perceived brightness is defined by relative integrated intensity versus GFP derived from raw data in Figs 3A and S8B. It is important to note that perceived brightness measurements will depend on the choice of filter sets and the light source (full filter specifications can be found in Materials and Methods). Relative bleaching is defined as the percent fluorescence remaining at time = 20 sec in Fig 3B and in S7C Fig as this was the time point where the greatest difference could be visualized between the fluorophores. mTagBFP2 was not characterized in this study, and its full properties can be found here: [2].
Fig 4.
Utilization of mTFP1, mCitrine, mCherry and DAPI for 3- and 4-color, live-cell imaging.
A) BY4741 cells expressing Cit1-mTFP1, Pho88-mCitrine, and Erg6-mCh were imaged either untreated (left panels) or after staining with 1 μg/mL DAPI for 10 min (right panel) at 30°C as described in Materials and Methods. B) Images of BY4741 cells expressing Erg6-mTagBFP2, Cit1-mTFP, Pho88-CmCitrine, and Vph1-mCherry. For A-B, Z-series were collected with wide-field microscopy at 0.5 μm intervals throughout the entire cell using a metal-halide lamp and appropriate filters using 216 gain and the following exposure times: 100 ms for Cit1-mTFP1, 200 ms for Pho88-mCitrine, 200 ms for Erg6-mCherry, 100 ms for DAPI, and 400 ms for mTagBFP2. Cell outlines were drawn over phase images. Scale bar = 1 μm. n = nucleus. mtDNA = mitochondrial DNA. nER = nuclear ER. cER = cortical ER.