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JCB performed initial uncaging experiments and performed oxygen dependency experiments. SD and MBH performed and designed other experiments. MBH wrote the paper with JCB and SD..
Photolytic uncaging of biologically-active molecules within cells is a powerful technique. However, the “delivery” of uncaging light into the cytosol can vary between cell types or between individual cells because of optical differences in absorbance or light-scattering properties of the cytoplasm. Here we demonstrate a simple technique for monitoring the magnitude of cytosolic UV delivery during uncaging, which also leaves a quantitative and persistent record of this within the cells.
Photolytic uncaging of compounds within the cytosol of cells is a powerful tool for cell biology, especially for investigating the signaling mechanism of cells1 Photo-release of a biologically-active compound within an individual cell or a population of cells on demand has been used to explore the roles of Ca2+, IP3, ATP, cAMP and other molecules. The list of “caged compounds” is ever increasing as more reagents and chemistries are exploited2. Some of these compounds can also be synthesized as cell permeant esters which permits their loading into cell populations as it negates the need for microinjection into individual cells. The critical part of the photo-release technique is the delivery of light, typically UV light near 360nm, into the cell cytoplasm. This causes photolysis of the light sensitive bond and release of the “caging” moiety and unmasks the required form of the compound in the cytosol. The amount of photolytically generated compound in the cell thus depends on the amount of light (photon flux) which reaches the caged compound within the cytosol. Attempts to quantify the efficacy of uncaging within the cell have been made by coupling a fluorescence change to the uncaging event3 However, this is usually not possible and instead uncaging efficiency is estimated by extrapolating from experiments performed in droplets, in which the environment is controlled and defined, to the cytosol. As uncaging in a droplet allows an estimation of the relative photon flux from the uncaging illumination to be made, the percentage photolysis of caged compound within the cell can then be estimated by taking account of the uncaging parameters 1. However, optically, the cell is rarely as translucent to the uncaging illumination as is the experimental droplet. The optical properties of cytoplasm are complex with light-scattering by small intracellular particles and granules that can attenuate the incident light entering the cell profoundly 4. In addition, there are a number of molecules in the cytosol which absorb light at wavelengths necessary for uncaging. For example, with an extinction coefficient of 6.2 × 103 M−1cm−1 at 339nm 5, and a cytosolic concentration of about millimolar 6, NADH alone will absorb more than 5% of the light passing through a cell 40 μm thick. When one considers that a number of other small molecules and proteins within the cell also absorb light in the UV region, the total absorbance by cell cytosol will be considerably higher. The problem for interpreting the effect of uncaging in different cells is exacerbated by cell-to-cell variation. Even in apparently homogeneous populations of cells, there is rarely uniformity of size, granularity or biochemical parameters7. The number of cytosolic light-scattering granules can also change during the cell cycle or after stimulation and NADH levels also change dramatically during cell activity8. The ability to monitor the relative cytosolic exposure to UV of cells would therefore facilitate the ability to interpret the effects of uncaging with response outcomes from individual cells.
We report here a simple approach which provides a monitor of the exposure of molecules within the cytosol of individual cells to UV illumination. The method can be used to monitor the relative extent of UV exposure of different cells in a population or to compare exposures of cells in different experiments. The method is especially useful as it records the extent of UV exposure with an innocuous but persistent fluorescent marker within cells. This may be especially useful in motile cell populations as the cells exposed to UV illumination carry a record of whether they were exposed and by how much. The future activity of cells can thus be charted even when the cells do not remain at the location of the original UV exposure.
This method relies on the UV-induced photo-oxidation of hydroethidine (also called dihydroethidium) (Fig 1a). There is a single report that UV exposure of hydroethdine (HE) generates a red fluorescent compound9, but we are not aware that this reaction has previously been investigated or exploited. Although hydroethidine has an absorbance maximum at 345nm10, the UV light-induced reaction is unlikely to be the result of a direct photolytic event as we have found that it is dependent on molecular oxygen. It thus more probably follows the pathway established for its reaction with superoxide (Fig 1a) leading to the generation of hydroxyethidium 11,12. The product of the reaction is similar to ethidium, and becomes brightly fluorescent on binding DNA12. The important feature of our method is that the reactant and product have very different water solubilities and consequently have different abilities to cross the cell membrane. The photo-reaction converts the cell permeant hydroethidine (soluble in DMSO) into the membrane-impermeant, water-soluble, charged product, hydroxyethidium (Fig 1b). This change in ability to permeate the membrane is important because it means that photo-generated hydroxyethidium in the extracellular medium does not contaminate the signal as it cannot cross the membrane and gain access to nuclear DNA (Fig 1b). In contrast, intracellularly generated hydroxyethidium will have free access to the nuclear DNA. Nuclear fluorescence thus reports only the UV-induced reaction product which is generated within the membrane diffusion barrier, ie only within the cytosol (Fig 1b) and thus provides a monitor of UV exposure of molecules only within the cytosol.
We demonstrate here the UV light induced photo-conversion of hydroethidine to hydroxyethidium within a number of cell types with different nuclear shapes and with DNA of different degrees of condensation (Fig 1c). The increase in nuclear fluorescence within the cell after UV illumination confirms that a DNA –binding photo-product was generated. The intensity of the nuclear fluorescent signal is linearly related to the accumulated exposure and thus records the total number of UV photons of exposure (Fig 2a). In a given individual cell, the relationship between UV exposure and nuclear fluorescent signal remains constant as can be demonstrated by repeat UV exposures (Fig 2b). However, the effect of UV exposure varies between cell-types (Fig 2a). Since HE diffuses freely into the cells, its concentration is expected to be uniform. Furthermore, each cell has the same amount of DNA, therefore the difference in the rate of rise of fluorescence is attributed to differences in the “delivery” of UV photons to the cytosol. These thus illustrate differences in optical properties of cytosol in different cell-types and different individual cells within a cell population 4. For HE to be a useful marker of UV exposure during uncaging, it is important that it does not interfere with the ability of the uncaging illumination to uncage. As HE has an absorbance maximum at 345nm, it was possible that its absorbance would reduce the efficacy of the uncaging light, However, with an extinction coefficient of 9.75 × 103 M−1cm−1 at 345 nm10, 20 μM cytosolic HE would absorb only 0.2% of the light in a cell 40 μm thick. It therefore adds little to the overall UV absorption of cytosol. This was shown experimentally in neutrophils loaded with caged IP3, uncaging of which elicited a classic Ca2+ signal (Fig 2c). In this system, we have previously found that approximately 3-7 s exposure to UV light from the system were required to elicit the Ca2+ signal13. It was supposed that the generation of IP3 within the cell was slow so that the delay time represented the time required for the concentration of IP3 to reach a threshold for triggering the Ca2+ signal. The explanation for the variable delay between cells13,14 was less clear, but it was possible that it resulted artefactually from differences in the delivery of UV to individual cells.. Using HE however, it was found that the variable time delays did not result from variations in the optical properties of individual cells as the rate of HE oxidation on UV exposure was synchronous in individual cells within a microscopic field (Fig 2d). Instead the delay must originate within the signaling mechanism of the cell.
The simple method outlined here therefore provides a useful monitor of the delivery of UV light to molecules within the cytosol and provides an essential measure which is required for the interpretation of uncaging experiments.
This research was funded by the Danish Natural Science Research Council (grant no 272-06-0345) and the Wellcome Trust (WT079962AIA). The authors thank Prof. Lars Folke Olsen, University of Southern Denmark, for useful discussions.