Trends in Neurosciences
It's green outside: tracking cell surface proteins with pH-sensitive GFP
Section snippets
Mutation, mutation, mutation
Engineering of the original wild-type GFP sequence have resulted in general improvements in function. Substitutions of individual or small numbers of amino acid residues have led to increased brightness (e.g. S65T), to removal of the 395 nm excitation peak (e.g. E222G), to reduced thermosensitivity (e.g. V132A), and to more rapid maturation of the fluorophore at the folding (e.g. F64L) and oxidation (e.g. S65T) stages. Currently, the most widely used form of GFP is the commercially available
GFPs as pH sensors – pHluorins
Although wild-type GFP fluorescence is not particularly sensitive to changes in the acidity of its environment in the physiological range [16], the fluorescence of some mutant GFP molecules displays marked pH-dependence 17, 18. The pH sensitivity of GFP is a consequence of the protonation and deprotonation of certain important amino acid residues within the central fluorophore. The characteristics of a GFP molecule depend critically on the state of these amino acids, because H+ transfer within
pHluorins as dynamic markers of exocytosis – watching vesicle fusion
Several studies have used targeted pHluorin fusion proteins to measure dynamically the pH of various intracellular compartments, such as the cytoplasm [22], peroxisomes [23], endosomes and the trans-Golgi network [24]. However, the isolation of pHluorins had a different objective. In the original study [21], pHluorins were fused to the luminal C-terminus of vesicle-associated membrane protein (VAMP)/synaptobrevin to monitor synaptic vesicle exocytosis and recycling – this fusion protein was
Superecliptic pHluorin – brighter is better
The initial screen that identified pHluorins used wild-type GFP, which does not contain the mutations that result in the increased brightness of enhanced variants. However, introduction of two point mutations into the fluorophore yields ‘superecliptic’ pHluorin, which has approximately nine times greater fluorescence than the original ecliptic protein, but which retains the same pH sensitivity [25]. The fluorophore of ecliptic pHluorins has a pKa of ∼7.1. Thus, a pHluorin molecule exposed to
pHluorin as a marker of protein surface expression
Proteins destined for membrane insertion pass through a well-characterized series of membranous compartments, which form the secretory pathway [32]. Proteins are first inserted into the rough endoplasmic reticulum, and then transported through the various organelles of the secretory pathway by a series of vesicular budding and fusion events. Two fundamental properties of the secretory pathway favour the use of pHluorins as investigative tools (Figure 1). First, the interior lumen of the
Putting pHluorins to use
In any experiment using pHluorin-tagged surface proteins, the relative amount of fluorescence from the surface compared to that coming from inside the cell will depend on two factors: first, the proportion of the total protein that is at the surface; second, the luminal pH of the organelles in which the intracellular proteins reside. These characteristics must be defined experimentally (Box 2) and will effectively determine the sensitivity of detection of pHluorin-tagged surface proteins.
Future directions
Naturally, any increase in the efficiency of pH sensors as membrane markers would be welcomed. With this in mind, the new dual emission, pH-sensitive GFP variants that allow ratiometric analysis of pH using single excitation wavelength hold considerable promise [37]. Similarly, by combining a differently coloured, pH-insensitive fluorophore with ecliptic pHluorin, it might prove possible to increase resolution using ratiometric techniques [38]. The principle of multicolour imaging would also be
Summary
There is likely to be an exciting future for pHluorins in neuroscience. Many fundamental questions, for example, regarding exocytosis and endocytosis at both the presynaptic and postsynaptic membrane, and the lifetime and regulation of surface expressed proteins, can now be addressed using pHluorin technology. Continued developments, such as the recent availability of bright superecliptic pHluorin coupled with efficient transgene delivery for efficient neuronal expression, should ensure a new
Acknowledgements
We are grateful to the Medical Research Council, the Wellcome Trust and the European Union (KAR-TRAP 02089) for financial support.
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