View online magazine
These new tools are photoactivatable fluorescent proteins (PAFPs) and other advanced fluorescent proteins (FPs), several of which have been developed by Vladislav Verkhusha, Ph.D., associate professor of anatomy & structural biology at Einstein, and a member of the Biophotonics Center. PAFPs and FPs allow scientists to noninvasively visualise the structures and processes in living cells at the molecular level. It is now possible, for example, to follow cancer cells as they seek out blood vessels and spread throughout the body or to watch how cells manage intracellular debris, preventing premature aging.
Scientist Live discussed Prof. Verkhusha's research with him as well as its implications.
How long have you been involved in the development of PAFPs and FPs? How did you first come to work on them and why?
I have been involved in the improvement of existing and in the development of new FPs since 2000; and in the development of new PAFPs since 2004. However, I have started to work with FPs in 1997. The first FP I used in my studies was a jellyfish's GFP, which is now well known. We have applied
GFP to study intercellular adhesion and cytoskeleton dynamics in Drosophila flies. In fact, we were the first who have made the transgenic fly lines expressing not simply cytosolic GFP but the chimeric
beta-actin-GFP and D-cadherin-GFP constructs, which were absolutely functional during all stages of fly development. This paper has been published in 1999.
How do you generally approach researching new fluorescent proteins? What are some of obstacles you face during your research?
To develop new FPs we need to understand the formation mechanism of the chromophore, at least qualitatively, which we plan to improve or develop. We also need to investigate will be our future FP or PAFP similar to the existing or it will be unique for some potential application to be solved. Lastly, we need to choose the appropriate original FP, which we will use as a template for our mutagenesis.
The main obstacle is still limited capabilities of the existing high-throughput approaches, which we use to screen the libraries of mutants to find the best FP or PAFP variant.
Please describe your more recent findings and how they add to the current crop of fluorescent proteins? What are their advantages?
(a) The photoactivatable red FP, we named PA-mCherry, is the first PAFP that converts from a dark to the red fluorescent state. The closest analogues are already originally fluorescing in green and then convert to the red. (in other words, for PA-mCherry it is a dark-->red light (photo) induced conversion, but for the available closest analogue proteins that is a green-->red photoconversion). The absence of green fluorescence and single-molecule behavior make PA-mCherry a preferred tag for two-color
diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two- color photoactivated localization microscopy (PALM).
(b) The blue FP, mTagBFP, is currently the brightest monomeric FP in the blue range of the spectrum. It provides the highest signal-to-noise ratio in the imaging. mTagBFP is currently also the best donor for the Forster Resonance Energy Transfer (FRET) cellular applications.
(c) The Fluorescent Timers (FTs), which change their fluorescence from blue to red over time, for the first time allow to study long-term (up to tens of hours) trafficking of the newly synthesized different cellular proteins and provide precise timing of various intracellular processes. I have to note that PAFPs can be used to study trafficking and intracellular dynamics too but the tracking time in their case is usually limited to tens minutes or so.
If you were to pick one standout with the most promise, which would you choose and why?
I would pick PA-mCherry because this is the probe, which is necessary for the new revolutionary super-resolution optical technique such as PALM and its derivatives. In five years the fluorescence microscopes allowing the PALM imaging will be widely available, like currently available laser scanning confocal microscopes. Today every optical microscopy lab or facility has the confocal microscope. In five years they all will have the super-resolution microscopes. As we now have the whole pallet of multi-color permanently fluorescent FPs for confocal microscopy, we similarly need multicolor probes for PALM. And PA-mCherry provides the second photoactivatable (i.e., red) color for this future microscopy (the first photoactivatable, green, color is provided by developed several
years ago PA-GFP and PS-CFP proteins).
Finally, what is next for your laboratory?
The next step is to develop the new photoactivatable colors (i.e., the new PAFPs) such as blue, cyan, yellow, orange and far-red for the super-resolution imaging of live cells.
Reporting by Marc Landas