1. Introduction
One of the most ambitious approaches to understand the
miracles of life is to visualize the structure and functional processes in
intact living cells: the basic units of all living organisms. Cells consist of
thousands of different molecules with distinct structure, characteristics and
function. Different cell types have different morphologies and fulfill
different essential functions in multicellular organisms. Cells are able to
grow, replicate, differentiate, sense, respond, communicate, mature, age and,
ultimately, die. Occasionally, cells become dysfunctional causing all kind of
organ malfunctions as basis of disease. Understanding of how cells actually
function, how they respond to changes in their microenvironment, and how cell
functions and dysfunctions can be manipulated and normalized has been occupying
scientists from different fields all over the world since decades.
Since approximately 20 years Fluorescent Protein (FP)
-based tools allow looking at processes within cells with high spatial and
temporal resolution[1-3]. Mainly based on the
pioneering work of Osamu Shimomura, who identified and characterized the Green
Fluorescent Protein (GFP) from the jellyfish Aequoreavictoria, Martin Chalfie,
who was the first expressing GFP successfully in bacteria and worms, and Roger
Y. Tsien, who developed several colorful GFP variants and fluorescent probes, a
new bright era of real time imaging of cell signaling events has been launched
(reviewed in[3]). FPs equipped with distinct
targeting sequences has become powerful tools to label cellular organelles and
structures. Expression of organelle targeted FPs allows imaging of organelle
dynamics[4,5], morphological changes of
organelles and organelle-organelle interactions[5-7] in real time.
FPs fused to proteins of interest are frequently used
to visualize subcellular distribution of proteins[8,9],
protein translocations[8,10]and protein-protein
interactions[8,11] in lifetime on the single
cell level. Using such simple FP-based constructs numerous cellular phenomena
such as the fission and fusion events of mitochondria[5,7], the assembly and disassembly
of elements of the cytoskeleton[12], the Ca2+-induced oligomerization and translocation of
distinct proteins[10,13] and many other cellular
spectacles could be discovered, visualized, quantified and investigated with
high precision and on the molecular level.
2. The Power of Genetically Encoded Fluorescent Probes (GEFPs)
A further sophisticated approach to use FPs is their
implementation as fundamental components of so called Genetically Encoded
Fluorescent Probes (GEFPs) [14,15]. Usually, GEFPs are carefully designed chimeric constructs that are
composed of a naturally-specific sensor domain fused to one or two FPs. Binding
of the natural mediator (e.g. ion, protein, lipid or small molecules like ATP)
to the respective specific sensor (binding) domain or the modification of the
sensor domain by a biochemical process within the cell (e.g.
(de-)phosphorylation, cleavage) affects the spectral properties of the attached
FPs, which can be measured in real time using fluorescence spectroscopy or
microscopy. Accordingly, changes of fluorescence intensities of FPs in GEFPs
report intracellular changes of the concentration, kinetics and/or the activity
of the analyte, which can be an ion[16-19], a metabolite[20-22], a substrate[23], or an enzyme[24-26], and eventually the activity status of certain cell
signaling pathways. As the molecular processes of functioning of all different
GEFPs is plagiarized from nature, every cell signaling event can be principally
visualized with respective GEFPs. Hundreds of different GEFPs have been
developed in order to answer specific questions and to discover complex
phenomena in cell biology, biochemistry and medicine.
GEFPs composed of FPs with different spectral
properties can be combined[27], or combined with small chemical fluorescent sensors[28]in order to record different signaling events
simultaneously in single individual cells. Such simultaneous recordings of
cellular process by distinct sensors with high resolution in time and location
allow multidimensional acquisitions of cell signaling mechanisms. Accordingly,
the usage of GEFPs undoubtedly enables to overcome previous limits and to
explore new frontiers in biology research from different fields. Moreover, such
experiments highly motivate researchers to ask new questions, design novel
informative experiments, and develop further original GEFPs.
3. Improving the usability of
GEFPs
However, the appropriate design, construction and
effective usage of GEFPs are not so trivial[29]. Despite the enormous potential of GEFPs, there are only a limited
number of specialist research groups that continuously develop and improve
GEFPs. In addition, the usage of many sophisticated GEFPs is often restricted
to those scientists who actually have invented the sensor. What are the main
reasons that most of the already available and novel GEFPs are not used as a
matter of routine in most laboratories by many scientists that perform research
related to cell biology? What is actually required to increase the usability
and distribution of GEFPs? GEFPs, as the name implies, are encoded by DNA and
the respective genetic information has to be transferred effectively and
without injury into the cells of interest. While there are several transfection
procedures and transgenic technologies available that basically allow the
insertion of DNA coding for GEFPs, most of these procedures are optimized for a
limited number of cell lines, tissues and whole organisms.
Often scientists consider GEFPs as unusable tools as
they have experienced huge difficulties in transfecting their cells of
interest. Though the viral transfer has been found to overcome limitations of
the gene transfer, in whole animals but also sensitive cells, the application
of viral infection to achieve transfection with the sensor DNA/RNA is limited.
An extension of the spectrum of effective transfection methods and transgenic
technologies will certainly increase the applicability of GEFPs in future. The
coding DNA for many GEFPs is under the control of a strong viral promotor so
that high levels of GEFPs are produced within several hours after incorporation
of the genetic information. However, high expression rates can induce cell
stress and dysfunctions such as the unfolded protein response[30] and, hence, can limit the usability of GEFPs.
The availability of GEFPs under the control of
promotors with different activities and inducible promotors would help in
optimizing the actual concentration of sensors for different cell types. GEFPs
for Ca2+ imaging has been
continuously improved mainly by exchanging classical GFP variants with novel
brighter, more photostable, and less pH-sensitive FPs[2,31,32]. Such developments results in more robust GEFPs with different spectral
properties ranging from blue to red and considerably better signal to noise
ratios. However, often the dynamic range of GEFPs is rather poor with only a
maximal change in the fluorescence signal of 5-10%. Using
such sensors appropriately requires trained users and optimized imaging setups.
In order to expand utilization of these sophisticated tools to laboratories
that are not specialists in fluorescence microscopy but highly benefit from
using GEFPs in their research, affordable and easy-to-use devices with improved
hardware and software components for automatic imaging and analyzes that are
optimized for live cell imaging with GEFPs are urgently required.
Light emitting protein-based sensors have become
indispensable tools in modern cell biology. They enable visualization of cell
signaling with (almost) unlimited spatial and temporal resolution. More than
all other methods GEFPs have taught us so much about the precision and beauty
of cellular processes that maintain life. Future developments in the design and
usage of GEFPs, along with achievements in molecular biology, physics, chemistry,
microscopic techniques, and computer science, these probes will serve among
researchers’ best tools in exploring life’s mysteries.
short commentary