RICERCA ITALIANA     

Research program

ADVANCED TOOLS OF MOLECULAR IMAGING AND GENETIC ENGINEERING TO STUDY DISTRIBUTION AND DYNAMICS OF CELL MAGNESIUM: NOVEL APPROACHES TO LINK MAGNESIUM HOMEOSTASIS AND CELL FUNCTIONS.

University Co-ordinator

Università Cattolica del Sacro Cuore - Patologia generale - ()

Research Unit Leader

Federica Wolf

Description

The research program consists in the application of the new class of fluorescent probes derived from a diaza-18-crown-6 molecule conjugated to two hydroxyquinoline groups (DCHQ) to assess differences in magnesium compartimentalisation and movements by live cell imaging. The scientific background for this approach stands in a previuosly published work (Farruggia and Wolf, 2006), where we showed that a rapid intracellular magnesium transient occurs in response to stimuli that affect mitochondrial function. To further characterise the origin and the consequences of such magnesium transient, we plan to perform studies on isolated mitochondria. This approach is advantageous as it allows to focus on a single compartment whose response can be modulated differently by various classes of compounds known to affect specific functions, e.g. oxidative phosphorylation uncouplers, or respiration inhibitors for the different complexes (I, II, III, IV, V) of the electron transport chain, or able to perturb the matrix ion composition, e.g. inhibitors of specific ion transporters or of the permeability transition pore. First of all, we aim to understand the relationship between the magnesium transient previously shown (Farruggia, 2006) and the mitochondrial membrane potential by live imaging and flow cytometry of the mitochondrial probe JC-1. Then, we intend to examine whole live cells, where the consequences of the magnesium transient will be investigated. In particular, we will attempt to relate the magnesium rise and other events associated with apoptosis, by comparing the effect of uncouplers (FCCP, CCCP, etc.) to that of apoptotic stimuli acting either on death receptors (Fas, TNFalpha;, etc.) or on the mitochondrial permeability transition pore (arachidonic acid, bongkrekic acid, atractyloside). Subsequently, the extent and timing of the magnesium transient will be evaluated in comparison with other apoptotic endpoints (phosphatidylserine exposure, cytochrome c release, caspase activation, etc.), by using specific fluorochromes, flow cytometry, immunofluorescence and western blotting. Finally, we also intend to study apoptosis-resistant cells, for example cisplatin-resistant tumour cells, to determine whether the magnesium transient is modified in these cells and might be thus necessary for the execution of the apoptotic programme. To this end, the correlation between magnesium release and activation of Ca/Mg dependent endonucleases and endonuclease G might be explored.
The synthesis of an ester derivative that can be trapped intracellularly upon cleavage of the ester moiety has already been designed and work is in progress in Prof. Savage's laboratory, in Brigham Young University, Provo, UT, USA. Prof. Savage has been collaborating with us so far, and will continue to give his support regarding the chemical synthesis of new derivatives. The ester derivative of DCHQ probes will be employed to investigate magnesium fluxes through membranes (plasmatic, microsomial) and put them in correlation with the expression of TRPM6/7 channels; these data should further clarify the mechanism by which these channels control cellular magnesium homeostasis, as suggested by recent findings (Nadler, 2001; Schmitz, 2003). In a second phase of the project, more specific cellular models will be evaluated: our cell strains adapted to grow in very high and very low magnesium (45 and 0.05 mM respectively) which should have a deranged expression of magnesium-specific transport systems, and endothelial cells genetically modified to express or not TRPM6 and 7, obtained by the RU of Milano. The availability of a confocal microscope equipped with a two-photon pulsed laser makes possible to perfom FLIM (Tadrous, 2000) and FRAP experiments, and thus to define the distribution and photodynamic properties of DCHQ probes. The findings of these in vivo experiments will be compared to the data in vitro obtained in Bologna, providing further biological information.
In parallel, we will collaborate with the RU of CNR to set up a protocol for the preparation of cell samples to be examined for magnesium intracellular distribution by synchrotron-based X-ray fluorescence microscopy (SXRFM). Cell samples will need to be fixed and positioned on synthetic support (e.g. formvar layer) which do not interfere with SXRFM measurements. The fixing procedure could be performed by organic solvent (such as methanol/acetone) or by freeze drying. Based on the literature which describes similar approaches to study intracellular distribution of iron or copper (Yang et al., 2005), we will discriminate the most appropriate treatment. In order to localize or co-localize signals and to compare data obtained by fluorescent magnesium probe and by SXRFM, we will attempt to stain the cells prepared for SXRFM with a magnesium specific fluorchrome of DCHQ series or of other kind if those are not suitable to this samples.

Project phases.
1semester:
1. Study of intracellular magnesium on isolated mitochondria or whole cells upon specific stimuli (FCCP, AA, PTpore inhibitors, homones or PKA)
2. Characterization and characteristics of DCHQ2 for live cell imaging. Comparison with DCHQ1
3. Preparation of cell samples for SXRFM.
2nd and 3rd semesters:
1. Application of the ester-derivative for the intracellular entrapment of DCHQ1allwing to study magnesium fluxes.
2. Study of magnesium fluxes through the plasma membrane following stimulation with hormones and growth factors. Association with cellular proliferation
3. Analysis of magnesium fluxes in correlation with an altered expression of ion specific channels such as HC11 grown in high or low magnesium.
4. Continuing collaboration with RU of CNR for the preparation of cell samples for SXRFM.
4th semester:
1.Study of magnesium fluxes with intracellular DCHQ1 on genetically modified epithelial cells for TRPM6 and 7 provided by the RU of Milano.
2. Comparing results on intracellular magnesium distribution by the different new approaches used.
1. Design and characterization of DCHQ derivatives targeted to specific subcellular organelles: mitochondria, endoplasmic reticulum, nucleus. Confirmation of the subcellular localization by simultaneous imaging with fluorescent markers specific for each organelle (mitotrackers, ER enzymes, nuclear proteins.

Duties of RU:
1. Application of DCHQ probes to the study of cellular magnesium homeostasis by live cell imaging.
2. With intracellularly entrapped derivative of DCHQ (ester form, studying cytoplasmic fluxes of magnesium in response to mitogenic stimuli and to altered expression of magnesium transporters.
3.Testing other DCHQ derivatives targeted to specific subcellular compartments or magnesium pools.
4. Providing cells and elaborating the new protocol of sample preparation for SXRFM in strict Collaboration with RU from CNR.