Erwin Neher

Chemistry Helps Biology: Fluorescent Labels and Caged Compounds


Abstract

Modern imaging techniques have transformed the biosciences. Genetically encoded fluorophores, as well as synthetic fluorescent dyes allow researchers to specifically label proteins and to monitor second messengers, such as the concentration of intracellular Ca++ ions ([Ca++]). Furthermore, novel methods of ultra-resolution light microscopy produce images of the labeled substances at sub-micrometer resolution. Last but not least, light can be used as a tool to manipulate cellular processes by means of caged compounds and light-activated proteins.
I will give a short overview over these recent developments and report on one example from our own work: the use of caged-Ca++ to calibrate the Ca++ dependence of neurotransmitter release.
Synaptic transmission is a complicated process, by which two neurones communicate with each other. The ‘presynaptic’ neuron sends a signal by releasing a substance - the neurotransmitter. This diffuses across a thin gap to the receiving or ‚postsynaptic’ neuron. Binding to special receptors, the neurotransmitter opens ion channels in the membrane, leading again to an electrical signal. This multistep process happens within a fraction of a millisecond.
It has been known since the early 1960s that neurotransmitter release is initiated by an influx of calcium ions into the presynaptic nerve terminal. This leads to an increase in intracellular calcium concentration ([Ca++]i), which - in turn – causes small vesicles (which contain the neurotransmitter) to fuse with the cell membrane and to release their contents. It has also been known for long that the Ca++ influx occurs through ion channels, which are specific for the permeation of Ca++ ions and which open in response to the nerve impulse. These channels must be located very close to synaptic vesicles. Only at close distance can they elicit the release within the shortest time possible. However, it has not been known until recently, how short the distances between ion channels and vesicles are and how much the local Ca++ signal must rise, in order to elicit the proper response.

[Ca++] can be measured using so-called Ca++ indicator dyes, which bind calcium ions and, upon binding, change their fluorescence. Cells can be loaded with such substances and one can observe under the fluorescence microscope localized changes in fluorescence, which mirror local changes in [Ca++]i. This way many features of the Ca++ signal can be studied. However, conventional Ca++ imaging studies are limited by the spatial resolution of light microscopy. This means that no details of Ca++ signals can be observed on the length scale of 100 nm and shorter. This, however, is exactly the range of distances, which are relevant for the assemblies of Ca++ channels and synaptic vesicles.

In order to obtain more insight into the properties of the relevant Ca++ signal we borrowed another tool from chemistry: caged Ca++. Such compounds, e.g. DM nitrophen are chelators, which bind Ca++ tightly, but are light sensitive (Ellis-Davies & Barsotti, 2005). Exposure to a flash of UV-light causes them to disintegrate and to quickly release Ca++. This way we can increase [Ca++]i in a step-like fashion. We use a special large nerve terminal, the Calyx of Held, which can be loaded with both a Ca++ indicator dye and a caged Ca++ compound. [Ca++]i is increased by a flash of UV-light while the response of the postsynaptic cell is measured. The increase of [Ca++] is spatially uniform in this experiment, such that the [Ca++], which we measure by means of the indicator dye is the same, which acts on the vesicle. Thus, we can calibrate the biological sensor for [Ca++], or more precisely: We can establish a quantitative relationship between the speed of the response and the amplitude of the [Ca++] signal. Once we have established such a ‘dose-response-curve‘, we can ask what [Ca++]i is required to achieve a response as fast and large as the physiological one. Such reasoning, together with a more quantitative biophysical model of the release mechanism, allows one to conclude, that the effective Ca++ signal at the location of the vesicle has an amplitude of about 20 µM and lasts for less than a millisecond. Further biophysical modeling shows that such signals are expected to occur at distances of 30 to 50 nm from Ca++ channels, when these open for sub-millisecond periods.

Using caged-Ca++ we were able to learn indirectly about processes, which happen on length-scales below the limit of conventional light microscopy. More recently new methods of ‘super-resolution light microscopy‘ have been introduced, by which objects separated by 50 to 100 nm can be resolved (Berning et al., 2012). Here again advances in chemistry play a major role, since the success of these methods depends critically on extreme photo stability of the chromophores and on a property, which before was of no relevance to fluorescent indicator dyes: For some of these methods the chromophores have to be photo switchable. New chromophores, synthesized during the last few years, have greatly contributed to the success of these exciting new techniques. Last but not least, fluorescent proteins, which can be expressed in a tissue-specific manner and can be linked to other proteins of interest allow for specific labeling of cellular components, opening up new options for the study of biological processes, which were beyond imagination a short while ago.

Ellis-Davies, G.C.R. and R.J. Barsotti (2006). Tuning caged calcium: Photolabile analogues of EGTA with improved optical and chelation properties. Cell Calcium 39, 75-83.
Berning, S., Willig, K.I., Steffens, H., Dibaj, P. and S.W. Hell (2012). Nanoscopy in a living mouse brain. Science 335, 551


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