The phenomenon of fluorescence
15 November 2002
Although Krupa Pattni, a biochemistry PhD student, and Sophie Pearn, a biology PhD student, study in different departments, their research is linked by the fascinating phenomenon of fluorescence.
Fluorescing Jellyfish find Invading Salmonella
(Krupa Pattni with George Banting and Mark Jepson)
Aequorea victoria is one of the oldest species of jellyfish on Earth, its origins dating back almost 500 million years. It is brightly luminescent, with light emitted from yellow tissue masses that form glowing points around the margin of the umbrella – each mass consisting of 6,000 - 7,000 photogenic cells. These cells are densely packed with fine granules that contain the components necessary for bioluminescence: a photoprotein called aequorin that emits blue-green light and a green fluorescent protein, or GFP, which accepts energy from aequorin and re-emits it as green light. Extraction, purification and characterisation of GFP from the jellyfish was first carried out in 1962, but it was not until the DNA sequence for GFP was isolated 30 later that it became one of the most widely used tools in molecular and cell biology.
Each mammalian cell contains more than 30,000 different proteins that serve diverse and essential functions within the cell. While scientists know what some of these proteins do, what is not clear is how they all interact with each other. The fact that GFP is highly visible has made it an extremely important tool in such studies, since it is now possible to engineer proteins of interest and tag them with GFP so that they can be seen. In the field of cell biology, biochemists use these fluorescent markers to study what happens to proteins inside normal cells in order to understand their functions better.
One of the best-equipped cell imaging centres in the country is the Cell Imaging Facility at Bristol University, which is funded by the Medical Research Council. There, researchers use imaging techniques to observe the location, movement and interactions of proteins within living mammalian cells. The ability to tag specific proteins with GFP allows the monitoring of many events such as the invasion of a cell by a pathogen.
Each mammalian cell contains more than 30,000 different proteins
Salmonellae bacteria are pathogens which cause a range of diseases from food poisoning to typhoid fever. After food contaminated with Salmonella has been eaten, the bacteria pass through the stomach and gain access to the intestine where there is a low concentration of oxygen. This switches on a number of Salmonella genes that equip the bacteria with proteins required to bind to, and invade, the cells that line the gut. From this sheltered environment inside the cell, the bacteria grow and divide before spreading to other cells. When this happens, about two or three days after ingestion, the carrier experiences the symptoms of diarrhoea, vomiting and nausea commonly associated with food poisoning. In the normal course of events, the carrier’s immune system would swing into action in an attempt to destroy the invading bacteria, but the Salmonellae appear able to manipulate their environment to avoid being destroyed by the host immune system. How do they do it?
Krupa Pattni’s research utilises GFP to focus on early events during the Salmonella invasion of host cells, and how they survive once inside cells. Using the University’s Cell Imaging Facility, it was observed that upon contact with the cell the Salmonellae transport some of their own proteins into it, which then interact with the host’s machinery. One such protein causes a phenomenon known as ‘membrane ruffling’, where the surface of the cell ripples, trapping the Salmonellae bacteria inside the folds. As the membrane closes around them, the bacteria are pinched off into a ‘vesicle’ inside the cell, forming what is known as a ‘Salmonella-containing vacuole’, or SCV. The process is similar to the way in which white blood cells engulf bacteria.The difference here, though, is that white blood cells are specialised cell types whose specific role is to engulf and destroy pathogens, whereas the cells lining the intestine do not usually undergo such processes – although they do possess the machinery to do so. By ‘pushing the right buttons’ (i.e., activating the right proteins), the Salmonellae appear able to use this latent capability of the host cell to their own ends.
Previous work has shown that shortly after formation, the SCV’s morphology is similar to that of an Early Endosome, a normal component of the cell. In the ordinary course of events, an Early Endosome would mature into a Lysosome, which is used for the degradation of any waste matter contained in the cell.To avoid suffering the same fate and being discarded as waste, the Salmonellae inside the SCV must clearly forestall the SCV maturing into a Lysosome. In an attempt to understand how they do this, Pattni has been studying the compositions of the Early Endosome and the SCV. She utilised a protein domain called FYVE that binds to a lipid called PI3P, which is a component of the Early Endosome membrane. Pattni tagged FYVE with the GFP marker so that she could observe the location of PI3P in living cells.
In healthy cells, PI3P is characteristically present only on Early Endosomes, but Pattni has demonstrated that in Salmonellainfected cells it is also present on SCVs, suggesting that the Salmonellae disguise themselves as Early Endosomes in order to hide inside the host cell and avoid destruction. Pattni's hypothesis was that the Salmonellae somehow manipulate the composition of the SCV so that it continues to reflect that of an Early Endosome, thereby ensuring that the Salmonellae remain in a privileged and protected environment.
Consistent with this hypothesis, Pattni observed that in Salmonella-infected cells the SCV appears to ‘flash’ on and off. This is because the FYVE-GFP marker is highly sensitive to levels of PI3P, so as soon as the SCV starts to mature into a Lysosome and the level of PI3P changes, the fluorescent marker no longer recognises it. It therefore detaches from the SCV, rendering the marker invisible. Pattni's theory is that the Salmonellae bacteria then use one of their own proteins to reverse the changes which restores the levels of PI3P, prompting the FYVE-GFP marker to re-attach itself and the SCV to become visible again. Such a hypothesis would explain both the flashing phenomenon and how the Salmonellae remain disguised as an Early Endosome.
The salmonellae disguise themselves as Early Endosomes in order to hide inside the host cell
There is still quite a long way to go to understand fully the exact mechanisms that are occurring here, but continued observation of the normal processes that occur in cells, alongside studying the direct effects of isolated Salmonella proteins on processes inside host cells, may provide an answer. It is hoped that further funding will soon become available for Pattni to continue with this important research that could one day help people suffering from this potentially lethal form of food poisoning. In the longer term, it may be possible to understand how some strains of Salmonella are able to resist antibiotics.
Fluorescing Parrots Flash their Feathers
(Krupa Pattni and Sophie Pearn)
In recent years, biologists have discovered that we do not see the world in the same way that birds do – they have a number of visual characteristics and abilities that humans do not possess. For example, humans see colours as a mixture of three colours – blue, green and red.
Some birds, on the other hand, probably see a mixture of four colours – blue, green, red and ultraviolet. In the past, most research involved judgements about bird coloration that were based on our own colour perception, but colour is not just a wavelength of light; it depends crucially on the nervous system and perception of the receiver. Hence, current work aims to stress the importance of studying bird signals via objective measures of colour, and behavioural manipulations such as mate choice experiments.
Why are parrots so remarkably colourful? This is a question of special interest to biologists as well as to zoos and breeders. Above all, they want to know whether their bright colours are used in communication, particularly sexual signalling. Many parrot feathers, such as the blue cheek patches of the budgerigar, reflect strongly in the ultraviolet (UV) waveband which is visible to birds but invisible to humans. There is, however, a small amount of blue reflectance which gives the patches their human-visible blue colour. In addition, parrots have some plumage patches that are strongly fluorescent, such as the yellow feathers on the crown and cheeks of the budgerigar.
This fluorescence is fascinating. It was first reported in 1937 by Völker, and is thought to occur only in the parrot family, mostly in Australian species. Fluorescence occurs when UV light is absorbed and then reemitted at longer, human-visible wavelengths. It therefore has two components: 1) UV-absorption and 2) yellow emission. This enables biologists to hypothesise that it is either (i) an incidental side effect of feather structure or (ii) involved in signalling in two not necessarily mutually exclusive ways: the yellow emissions may supplement the already yellow pigment of the feather to produce a more saturated colour, and/or the UV absorption may enhance the bird’s conspicuousness by contrasting with nearby UV-reflecting patches, such as the blue cheeks mentioned above. In other words, not only would the blue contrast with the yellow, but also the lack of UV-reflection in the fluorescent pigment (due to UV being absorbed) will stand out against the UV-reflection of the surrounding area.
To determine the answer, and to investigate whether UV reflection is important in signalling, mate choice experiments were performed, using UV-transmitting and UV-blocking Perspex filters to manipulate the wavelengths of light available to female budgerigars when choosing mates. Results revealed that UV reflection from feathers is certainly an important signal when females are choosing a mate, with females preferring UV-reflecting males to non-UV-reflecting males. This is consistent with other research in our department, some of which shows that female blue tits prefer males that exhibit the brightest UV-reflecting blue crest. However, we also found that the yellow glow of fluorescence does not seem to act as a signal. The fact that budgerigars’ plumage appears bright to humans, and can be made to fluoresce with UV lights, need not mean that it is the yellow light visible to humans that is involved in bird signalling.
To supplement these findings, current work involves new methods to quantitatively measure and characterise plumage fluorescence, as well as some objective measurements of other plumage colours in Australian parrots. Also being investigated is the role fluorescence may play in alternative light environments such as dawn, where considerable changes in the ambient light spectrum may affect how plumage colours are perceived.