Fluorescent Microscopy

Fluorescent resonance energy transfer (FRET) can directly measure protein-protein interactions in vivo. Here, we present a tutuorial on our approach to measure FRET between two proteins tagged with CFP and YFP.

Macromolecular complexes can be studied in vivo using color variants of the green fluorescent protein (GFP) to tag proteins under investigation. Originally Wach and coworkers constructed plasmids to label proteins at their carboxy-terminus with GFP. We modified these plasmids to produce the CFP and YFP color variants of GFP, allowing multiple proteins to be labeled concurrently in a single cell. Thus protein interactions predicted by other methods can be tested by determining whether the proteins co-localize to the same region of the cell.

In order to facilitate co-localization studies we constructed a group of benchmark strains. These benchmark strains contain CFP or YFP fusions to proteins previously characterized to reside in specific subcellular domains. Each fusion is expressed under the control of its native promoter by using a PCR method to integrate the GFP variants at the 3-prime end of the targeted yeast ORF. As a demonstration of the use of CFP and YFP for co-labeling studies in live cells, we present a time-lapse movie showing the movement of microtubules, the spindle pole bodies and the nucleus during cell division. Our CFP and YFP plasmids and the benchmark strains are available through our collaborator's website. Please click here to learn how to request strains.

Optical sectioning microscopy and deconvolution greatly improve image resolution. Optical sectioning also makes 3-D rendering possible. The YRC acquires and deconvolves images using the DeltaVision microscope and software system. We present an example of the power of the deconvolution process to visualize the yeast mitochondria. If you have GFP fusions already made, we can quickly produce the initial three-dimensional images of its localization. More extensive collaborations are also possible.