Azzurra Sargenti

Quantitative chemical imaging of the intracellular spatial distribution of fundamental elements and light metals in single cells.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/μm(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.

Synthesis of a highly Mg2+-selective fluorescent probe and its application to quantifying and imaging total intracellular magnesium

Magnesium plays a crucial role in many physiological functions and pathological states. Therefore, the evolution of specific and sensitive tools capable of detecting and quantifying this element in cells is a very desirable goal in biological and biomedical research. We developed a Mg2+-selective fluorescent dye that can be used to selectively detect and quantify the total magnesium pool in a number of cells that is two orders of magnitude smaller than that required by flame atomic absorption spectroscopy (F-AAS), the reference analytical method for the assessment of cellular total metal content. This protocol reports itemized steps for the synthesis of the fluorescent dye based on diaza-18-crown-6-hydroxyquinoline (DCHQ5). We also describe its application in the quantification of total intracellular magnesium in mammalian cells and the detection of this ion in vivo by confocal microscopy. The use of in vivo confocal microscopy enables the quantification of magnesium in different cellular compartments. As an example of the sensitivity of DCHQ5, we studied the involvement of Mg2+ in multidrug resistance in human colon adenocarcinoma cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin, and found that the concentration was higher in LoVo-R cells. The time frame for DCHQ5 synthesis is 1–2 d, whereas the use of this dye for total intracellular magnesium quantification takes 2.5 h and for ion bioimaging it takes 1–2 h.

Intracellular magnesium content decreases during mitochondria-mediated apoptosis induced by a new indole-derivative in human colon cancer cells

A newly synthesized indole-derivative is able to induce cytostatic and cytotoxic effects in the colon cancer cells HT29, effecting apoptosis by activation of an intrinsic pathway. Magnesium is involved in both cell growth and apoptosis even though its role in the latter process is not well defined. The aims of this work were: firstly, to verify if magnesium content is related to the proliferative rate in HT29 cells; secondly, to assess the involvement of the cation in mitochondria-mediated apoptosis triggered by the new antiproliferative molecule. The effects of the indole-derivative in treated cells included cell-cycle arrest in the G2/M phase, and apoptotic death confirmed by release of cytochrome c from the mitochondrial compartment. Moreover, we demonstrated that the basal content of magnesium in HT29 cells inversely correlates with cell saturation density. In addition, a decrease in both free and intracellular total magnesium concentration was observed along with the induced apoptosis. Taken together, these data suggest that magnesium participates in the complex signaling network of cell proliferation and apoptosis.

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution

AbstractThe quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstractSingle cells or large population analysis this is the question!

The different expression of TRPM7 and MagT1 impacts on the proliferation of colon carcinoma cells sensitive or resistant to doxorubicin

The processes leading to anticancer drug resistance are not completely unraveled. To get insights into the underlying mechanisms, we compared colon carcinoma cells sensitive to doxorubicin with their resistant counterpart. We found that resistant cells are growth retarded, and show staminal and ultrastructural features profoundly different from sensitive cells. The resistant phenotype is accompanied by the upregulation of the magnesium transporter MagT1 and the downregulation of the ion channel kinase TRPM7. We demonstrate that the different amounts of TRPM7 and MagT1 account for the different proliferation rate of sensitive and resistant colon carcinoma cells. It remains to be verified whether they are also involved in the control of other “staminal” traits.

p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of the cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity.

Magnesium Deprivation Potentiates Human Mesenchymal Stem Cell Transcriptional Remodeling

Magnesium plays a pivotal role in energy metabolism and in the control of cell growth. While magnesium deprivation clearly shapes the behavior of normal and neoplastic cells, little is known on the role of this element in cell differentiation. Here we show that magnesium deficiency increases the transcription of multipotency markers and tissue-specific transcription factors in human adipose-derived mesenchymal stem cells exposed to a mixture of natural molecules, i.e., hyaluronic, butyric and retinoid acids, which tunes differentiation. We also demonstrate that magnesium deficiency accelerates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. We argue that magnesium deprivation generates a stressful condition that modulates stem cell plasticity and differentiation potential. These studies indicate that it is possible to remodel transcription in mesenchymal stem cells by lowering extracellular magnesium without the need for genetic manipulation, thus offering new hints for regenerative medicine applications.

Repeatability and reproducibility of intracellular molar concentration assessed by synchrotron-based x-ray fluorescence microscopy

Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the major obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.

Expanding the targets of the diaza-18-crown-6 hydroxyquinoline derivatives family to Zn(II) ions for intracellular sensing

We have recently developed an optimised synthetic strategy for the preparation of chemosensors based on the diaza-18-crown-6 hydroxyquinoline structure. This flexible approach allows to obtain a variety of substituted derivatives with improved fluorescence, metal ion binding, uptake and localisation properties. In this context, we have focused our attention on the design of specific chemosensors for intracellular Zn2+ ions, because of their important role in many biological processes. Interestingly, among them one in particular shows the properties of making it a promising candidate for effective Zn(II) ions detection in biological environments. It presents, in fact, a very low toxicity, Zn2+/Mg2+ selectivity and a very large fluorescence enhancement on complexation.

FLIM of a novel intracellular magnesium probe with an excitation source-based supercontinuum laser (Conference Presentation)

Advanced microscopy requires excitation light with coherence, monochromaticity and control of pulse duration. The ideal laser source should be tuneable with continuity over a wide spectral range, capable of emitting short pulses and amenable to fibre coupling. All these requirements are met by supercontinuum lasers that can generate highly coherent picosecond pulses at wavelengths spanning from blue light to NIR and beyond. A supercontinuum laser (SuperK EXTREME - NKT photonics, Denmark) was used to perform fluorescence lifetime imaging of human osteosarcoma cells labelled with a new probe (DCHQ5). DCHQ5, in addition to quantify the total intracellular Mg concentration in cell populations, allows one to visualize the intracellular Mg distribution in single cells. Using an acousto-optic filter, a 10 nm spectral band, centred at 480 nm was sliced from the white light exiting the supercontinuum source. The excitation light, made by a 20 MHz train of few-picosecond-long pulses, was coupled to a 400 μm fibre using a wide aperture microscope objective in order to excite all modes. The flat top light source consisting in the distal end of the fibre was imaged onto the object plane of the microscope (Leica DM-RM) to provide uniform illumination over the whole field of view. This approach, which required a home-made optical system, implemented the epifluorescence scheme with a 63X immersion objective lens, used to deliver the excitation light and collect the fluorescence signal through a dichroic mirror @500 nm and a long pass filter @505 nm. The fluorescence images were acquired with a fast picosecond camera (Picostar, LaVision Germany) made by a light intensifier coupled to a low noise CCD. A sequence of images was collected at different delays (in the interval 0-60 ns) with respect to the laser pulses, from each microscope field. Data analysis was performed using a custom written software based on the Matlab engine. The dataset, made by all the gated images, was processed using a non-linear algorithm for bi-exponential fit, operated in parallel mode. Amplitude and lifetime maps were recovered for both the fluorescent components that characterise the emission of the DCHQ5 probe. Additionally, to better estimate a possible variation of the decay dynamics of the probe in different cell comportments with alike magnesium concentration, two AOIs were isolated on the basis of the fluorescence intensity. All the pixels within the AOIs were binned to a single decay curve, which was fitted with a non-linear algorithm based on the NAG mathematical package. The fluorescence lifetime maps show a uniform pattern over the whole cell, for both the short and long living components of the fluorescence, while the amplitude maps reveal a minimum in correspondence of the nucleus and a high value in the cytoplasm. This result is confirmed by the analysis of the AOIs. Therefore, the intracellular distribution of the fluorescence intensity depends on amplitude variations of both DCHQ5 fluorescent components, and not on lifetime variations. Thus, fluorescence intensity maps reflect the concentration of the complex DCHQ5-Mg, and can be effectively used to quantify intracellular Mg2+.