How have fluorescent proteins contributed to cell biology research?

The 20th century has been witness to our ever growing understanding of life at the molecular level. The biochemical and genetics revolutions have together been responsible for many breakthroughs including whole-genome sequencing of organisms, and protein structure determination by crystallography and nuclear magnetic resonance (NMR) spectroscopy. Neither of the two revolutions, however, has produced the tools necessary for the molecular monitoring of spatio-temporal, intra-, and inter-cellular processes of living systems. Now, a century on, the beginning of a new revolution has been host to the rise of such implements pioneered through the green fluorescent protein (GFP) from the Aequorea victoria jelly fish, relating proteins from other organisms, and engineered derivatives of proteins from the “GFP family”.

Figure A | Cartoon representations showing the characteristic polypeptide fold of a protein from the GFP family (EosFP). The chromophore (white) is included as a stick model (From Wiedenmann et al., 2004)

Figure B | The fluorescence chromophore formed by amino acid residues 65-67 (Ser-Tyr-Gly) in the primary structure of GFP (From Cody et al., 1993)

 

It is the remarkable property of the GFP chromophore, responsible for the fluorescence, which has led to this technical revolution. The GFP polypeptide chain consists of 238 amino acids (Prasher et al., 1992); it folds into a rigid, 11-stranded beta-barrel with a central helix running along its axis (Figure A). A tripeptide (for GFP: Ser65-Tyr66-Gly67) autocatalytically forms a fluorescent chromophore (Figure B), which is held in the middle of the helix (Nienhaus, 2008).

The generally non-toxic features of the GFP allow its expression to high levels in different organisms with very minor physiological effects (Chalfie et al., 1994). When the GFP gene is fused (Figure C) to the gene of a protein being studied, not only does the expressed protein of interest retain its normal activity but the GFP also retains its fluorescence – allowing the location, movement and other activities of the protein to be followed by microscopic monitoring of the GFP fluorescence (Wang and Hazelrigg, 1994).

The potential for such widespread usage and the progressing requirements of researchers led to the engineering and production of a large variety of GFP and related protein mutants with vastly improved brightness, photo-stability, folding properties, and also produced GFP molecules with varying excitation and emission spectra (Tsien, 1998).

Figure C | Schematic depiction of a fusion protein, consisting of a GFP domain (green) connected to another protein (white) (From Nienhaus, 2008).

The cell and protein colouring property of GFP, was incredibly revolutionary, not only for molecular and cell biology, but for all the biosciences. These proteins allowed the monitoring of such living-cell phenomena as gene expression, protein localization and dynamics, protein-protein interactions, cell division, chromosome replication and organization, intracellular transport pathways, organelle inheritance and biogenesis, and many more ever-increasing processes.

Amongst the many applications, the most common use of GFP has been as a genetic fusion partner to host proteins to monitor their localization and fate. A fusion between a cloned gene and GFP can be created using standard subcloning techniques, with the resultant chimera then being expressed in a cell or organism, and this way GFP fusion tags can be used to visualize dynamic cellular events and to monitor protein localization (Zimmer, 2002). The fact that it does not require the presence of any cofactors or substrates makes GFP a very convenient fluorescent fusion protein marker (Zimmer, 2002).

Membrane-bound organelles, including the plasma membrane, nucleus, endoplasmic reticulum, Golgi apparatus, secretory vesicles, mitochondria, peroxisomes, vacuoles, and phagosomes, have also been the subject of study (Tsien, 1998). One key finding is that many of these continuously exchange protein components with each other. For example, the Golgi apparatus, which receives secretory cargo from the Endoplasmic Reticulum (ER), constitutively recycles its components back to the ER and disassembles during mitosis.

The dynamic behaviour of the cytoskeleton in the cell naturally makes GFP a vital part of studies of the cytoskeleton and associated proteins. Amongst all structures of the cytoskeleton, actin is probably the most commonly studied using GFP (Yoon et al., 2002).

Figure D | Reorganization of the actin cytoskeleton in stationary cells. (A-F) Actin stress fibers are clearly visible. (A-E) A series of images with 3 minutes intervals showed the appearance and disappearance of ‘actin clouds’ (arrow in A). (F) The same cell developed new ‘actin clouds’ at a different location by 200 minutes (From Ballestrem et al., 1998)

Figure E | Immobile actin spots in a migrating B16F1 cell. (A-C) Images taken at intervals of 20 seconds. (A’-C’). Migration is illustrated by a static arrowhead given as reference point. Immobile actin spots are marked by circles at identical coordinates (From Ballestrem et al., 1998)

Studies on actin dynamics in living mammalian cells, as that carried out by Ballestrem et al, unveiled the structures involved in the dynamics of the actin cytoskeleton. In the study, an enhanced green fluorescent protein (EGFP) was fused to human beta-actin, so that the actin cytoskeletal dynamics could be explored (Ballestrem et al., 1998). The fusion protein was then incorporated into actin fibres which became depolymerized upon cytochalasin B treatment, and this functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy (Ballestrem et al., 1998). In cells that were stationary, actin-rich, ring-like structured ‘actin clouds’ (Figure D) were observed in addition to stress fibres – and importantly, the ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton (Ballestrem et al., 1998). In migratory cells, EGFP-actin was also found in the advancing lamellipodium, with the immobile actin spots (Figure E) developing in the lamellipodium and thin actin fibres forming parallel to the leading edge (Ballestrem et al., 1998).

GFP and other related proteins may also be used as markers for tumour cells (Figure F). Cell metastasis is a highly dynamic process, occurring in multiple steps, and understanding this process has been limited by the inability to visualize tumour cell behaviour in real time by using animal models. An experiment carried out by Konstantin et al showed that two proteins, RhoC and VEGF, work cooperatively to mediate cancer cell ‘intravasation’ (Konstantin et al., 2007). If these proteins are neutralized, the cells might also be stopped from breaking away from the tumour area (Konstantin et al., 2007).

The universally flexible nature of GFP technology allows its use in other areas of the biosciences such as in neurobiology, where the extensive use of GFP fusions for imaging cells and tissues within multicellular organisms, have become a very important experimental tool. Detailed analysis of neuronal network architecture requires the development of new methods, such visualizing synaptic circuits by genetically labelling neurons with multiple, distinct colours (Livet et al., 2007). In the Brainbow (Figure G) transgenes, Cre/lox recombination is used to create a choice of expression between three or more fluorescent proteins (XFPs), and this ability of the Brainbow system, to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale (Livet et al., 2007).

Figure F | Tumour surrounded by nourishing blood vessels. Breast cancer tumour coloured with DsRED and the surrounding blood vessels with GFP (From Konstantin et al., 2007)

Figure G | The Brainbow – three fluorescent proteins have been used (CFP, YFP and RFP) to colour the brain of a mouse. Different neurons randomly produce different amounts of the proteins. This allows single neurons interlaced within the dense network to be distinguished (From Livet et al., 2007)

 

In addition to these passive applications, GFP can also be utilised as an active indicator of cellular physiology (Chamberlain and Hahn, 2000). The wild-type GFP protein has not been particularly useful as a sensor, but several GFP mutants (principally BFP and YFP) with distinct spectral qualities have been used as environmental sensors in various situations – for example, in monitoring local pH (Pollock and Heim, 1999). The most powerful method of constructing GFP sensors has been to exploit fluorescence resonance energy transfer (FRET), a non-destructive spectroscopic method to measure molecular interactions, between two GFP molecules or between one GFP molecule and a secondary fluorophore (Pollock and Heim, 1999). FRET is a phenomenon that occurs when two fluorophores are in sufficient proximity (<100 Å) and an appropriate relative orientation such that an excited fluorophore (donor) can transfer its energy to a second, longer-wavelength fluorophore (acceptor) in a ‘nonradiative’ manner (Pollock and Heim, 1999). Thus, excitation of the donor can produce light emission from the acceptor, with an associated loss of emission from the donor (Chamberlain and Hahn, 2000).

The many applications of GFP and GFP-like proteins demonstrate the significance of this discovery in research for all the biosciences and not just cell biology. Having said that, however, there is no doubt that the fluorescent protein has revolutionized the way we visualise cells in living organisms, and that without them, none of the applications and breakthroughs discussed above would be possible. The infamously beautiful images produced from the studies certainly have become somewhat iconic. Of course, this is certainly not the end, and we should expect to see more of fluorescent proteins in the coming future. Such intriguing prospects as fusions other than those at the N- or C-Terminus, or alternatives to fluorescence by harnessing chromophores to perform phosphorescence, for example, may just be the appetizers for research’s ever-increasing hunger for this phenomenon.

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References

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