Visualization of The Turnover of Atp Analog in Living Cells

Adenosine 5’-triphosphate (ATP) is a major energy currency of cells and is involved in multiple cellular processes. Monitoring the hydrolytic activity of ATP in cells would be beneficial to understand ATP consuming cellular processes and help in elucidating the mode of action and regulation of the enzymes involved. A number of fluorescence sensors has been reported till date for this purpose but there are no methods available till date for the real-time monitoring of ATP hydrolysis inside living cells. In this regard, a novel fluorogenic ATP probe was designed and synthesized. Upon enzymatic hydrolysis, this molecule displays an increase in fluorescence intensity and fluorescence lifetime which provides a readout of its hydrolysis and thus can be used for monitoring the process involving ATP utilization. We have used confocal fluorescence and fluorescence life time imaging (FLIM) microscopy to monitor the hydrolysis of the ATP analog, Atto 488-adenosine tetraphosphate-Quencher (Ap4), in living cells. Our results demonstrate that the Ap4 is hydrolyzed in lysosomes and autophagosomes. Our studies show that fluorescence microscopy can be directed towards the live-cell imaging of autophagosome-lysosome distribution and autophagic flux using the Ap4 without the need to over-express fluorescently tagged proteins in cells.

Most of the chemical reactions taking place in the biological system are energetically unfavorable and thus they require enzyme catalysts and are coupled to ATP hydrolysis that serves as an energy supply. Besides energy supplier, ATP is required for various other cellular processes. ATP acts as a cofactor for the transfer of phosphate by kinases during the process of protein phosphorylation and provides the energy for the conformational change of motor proteins. ATP is also the starting molecule for the formation of important messengers such as cAMP (cyclic adenosine monophosphate), cyclic-di-AMP, diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A). ATP plays a key role in the energetics, metabolic pathways, enzyme regulation and transduction mechanism of a cell. The amount of ATP is directly proportional to certain physiological states of the cell and also indication of some metabolic disorders. Thus, imaging of ATP in these pathways will provide crucial information for comprehensive understanding of the ATP-related processes and certain physiological disorders. Many different methods have been established for measuring ATP turnover overtime. They are based on the radioactive labeling of ATPs, the spectroscopic detection of the liberated phosphate by the formation of molybdenum blue or formation of complexes with Malachite green. However, these processes require the purification of the reaction products before analysis and thus no real-time and continuous measurement of the ATP hydrolysis is possible.

Also their applications are limited in cells either because they are not accepted by most of the cellular enzymes or they need the overexpression of another fluorescent-tagged protein. For this reason, new methods have been developed that rely on the spectroscopic measurement of the reaction products by enzymatic ATP turnover.

Recently, some novel fluorogenic ATP probes were designed and synthesized. These fluorogenic nucleotide analogs have been used to monitor the enzymatic activity directly without the use of any other reagent. The nucleotide analogs are designed as FRET probes and are labeled with two chemical groups, a fluorescent dye that acts as a FRET donor and another molecule which acts as a FRET acceptor.[12] Thus, in an intact molecule, intramolecular FRET trakes place and upon cleavage of the nucleotide, the donor fluorophore is spatially separated from acceptor fluorophore and the energy transfer is terminated. This leads to the increase in the fluorescence intensity as well increase in the fluorescence life time of the donor fluorophore that is quantified to measure its hydrolysis. This approach has been used successfully to study the activity of the ubiquitin-activating enzyme UBA, phosphodiesterase I of C. adamenteus (SVPD) and to elucidate the ATP-dependent acetone metabolism in bacterial extracts of D. biacutus.

Most of the previous studies have focused on studying ATP hydrolysis in various in vitro systems. We have monitored the cellular ATP consuming pathways in the living cells with high spatial and temporal resolution by using various fluorescence microscopy techniques. We have used confocal and FLIM-FRET microscopy to monitor the hydrolysis of Ap4. Fluorescence lifetime imaging (FLIM) is an approach to measure FRET which detects the time-resolved donor fluorescence signal and the donor lifetime gives a direct measure of energy transfer. The fluorescence lifetime is a characteristic property of a fluorophore and is independent of the excitation intensity, concentration variations, and photobleaching to certain extent. We have demonstrated that Ap4 is utilized in lysosomes as seen from the colocalization studies of the Ap4 fluorescence with lysosome marker. A significant decrease in the hydrolysis of Ap4 was seen when cells were treated with macrolide antibiotic bafilomycin A1, a potent inhibitor of lysosomal H+ ATPase or chloroquine, a lysomotropic weak base that deactivates the lysosomal enzymes. Ap4 hydrolysis activity shows a strong quantitative correlation with the process of cellular autophagy. Our studies indicate the utilization of Ap4 during the process of autophagy as shown by the colocalization of autophagy marker LC3B-RFP and Ap4 hydrolysis puncta. We propose that the Ap4 can be used a chemosensor for monitoring the autophagic flux in living cells.

Having the Ap4 compound synthesized, we visualized its real time hydrolysis by fluorescence lifetime measurements after incorporation into the living cells. This approach is based on the observation of Forster resonance energy transfer (FRET) between two fluorophores. Upon hydrolysis, FRET can be quantified by measuring the decrease in the fluorescence lifetime of donor and this is one of the most efficient and fast methods for measuring FRET. The lifetime was measured on a wide field microscope for each pixel simultaneously. A significant increase in the fluorescence (phase) lifetime was observed over time as a result of enzymatic hydrolysis of the Ap4. To the best of our knowledge, this is the first time that ATP analog hydrolysis has been monitored in living cells with. Ap4 hydrolysis starts as soon as it is introduced into the cells and reaches the steady state in around 60 minutes. However, the actual cellular components and the cellular process that utilized the Ap4 were still elusive. So confocal microscopy was also used later on in order to spatially resolve the cellular components more precisely that utilize this compound.Ap4 hydrolysis localization in lysosomes: In order to ascertain the localization of Ap4 hydrolysis in living cells, organelle stains like mitotracker and lysotracker were used which specifically label the mitochondria and the lysosomes of living cells respectively.

A significant colocalization was found between the Ap4 analog hydrolysis puncta and lysosomes which implies that the hydrolysis of Ap4 analogs takes place in lysosomes. When the cells were treated with β–lapachone which damages the lysosomes and induces necrosis by elevating the levels of free radicles like H2O2 and O2•− , a significant decrease in the hydrolysis activity of Ap4 was seen and also the distribution of the fluorescence in cells increased. An increase in the fluorescence distribution of both lysotracker dye as well as Ap4 could also be seen as a result of lysosomal rupture. This again indicates that the lysosomes are prominently involved in the hydrolysis of the Ap4 analog.

To further confirm the finding, two more lysosomal inhibitors were used. Chloroquine is a lysosomotropic agent that inhibits the enzymatic activity by increasing the pH inside the lysosomes. The monoprotonated form of chloroquine diffuses into the lysosomes where it is diprotanated and is trapped thus changing the lysosomal pH and thereby inhibiting the lysosomal activity. Bafilomycin A1 is a macrolide antibiotic that is used as an inhibitor of lysosomal H+ ATPase. Bafilomycin prevents the acidification of endosomes and lysosomes and thus inhibits lysosomal functioning including autophagic flux. The cells were treated with increasing concentrations of both of these inhibitors overnight and then, cells were imaged after the incorporation of the Ap4 analog. Both of these inhibitors led to a drastic decrease in the hydrolysis of Ap4 in living cells. This shows that Ap4 is utilized in lysosomes. Since lysosomes require an enormous amount of energy to maintain the intraluminal low pH (4.2–5.3) as compared to the cytoplasm, it is highly likely that Ap4 is utilized for the transport of H+ ions and regulation of lysosomal pH by V-ATPases.