Ion imaging is a powerful methodology to assess fundamental biological processes in live cells. The limited efficiency of some ion-sensing probes and their fast leakage from cells are important restrictions to this approach. In this study, we present a novel strategy based on the use of dendrimer nanoparticles to obtain better intracellular retention of fluorescent probes and perform prolonged fluorescence imaging of intracellular ion dynamics. A new sodium-sensitive nanoprobe was generated by encapsulating a sodium dye in a PAMAM dendrimer nanocontainer. This nanoprobe is very stable and has high sodium sensitivity and selectivity. When loaded in neurons in live brain tissue, it homogenously fills the entire cell volume, including small processes, and stays for long durations, with no detectable alterations of cell functional properties. We demonstrate the suitability of this new sodium nanosensor for monitoring physiological sodium responses such as those occurring during neuronal activity.
[Show abstract][Hide abstract] ABSTRACT: Stimulus-dependent elevation of intracellular Ca(2+) ([Ca(2+)](i)) affects the expression of numerous genes--a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na(+)](i) trigger c-Fos expression via a novel Ca(2+) (i)-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na(+)](i)/[K(+)](i)-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na(+)](i) and reduce [K(+)](i), cells were treated for 3 hrs with the Na(+),K(+)-ATPase inhibitor ouabain or placed for the same time in the K(+)-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p<0.0001; R(2)>0.62). Among these Na(+) (i)/K(+) (i)-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca(2+)](i), we performed identical experiments in Ca(2+)-free media supplemented with extracellular and intracellular Ca(2+) chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na(+) (i)/K(+) (i)-sensitive genes. Among the ubiquitous Na(+) (i)/K(+) (i)-sensitive genes whose expression was regulated independently of the presence of Ca(2+) chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca(2+)-depleted cells. Overall, our findings indicate that Ca(2+) (i)-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na(+)](i)/[K(+)](i) ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders.
PLoS ONE 05/2012; 7(5):e38032. DOI:10.1371/journal.pone.0038032 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendrimers are characterized, among other quite interesting properties, by heterogeneity of zones inside their molecules and for a high local concentration of functionalities. These two particularly properties of dendrimers confer them a potential selective and sensitive recognition of guest molecules. On the other side, semiconductor nanoparticles (quantum dots), which are highly fluorescent nanomaterials, can be conjugated with numerous molecules including dendrimers, just by chemically modification of their surface. The coupling of dendrimers and quantum dots result in hybrids organic/inorganic nanocomposites, which can alter the charge, functionality and reactivity, based, for example, in the fluorescence changes induced by molecular recognition at their surfaces. This can simultaneously enhance the stability and dispersion of the nanocomposites. Here we review the advantages of dendrimers modified quantum dots as nanoprobes, to investigate their feasibility to be used as analytical tool in a wide variety of ions and molecules.
Dendrimers: Synthesis, Applications and Role in Nanotechnology, Edited by Heather B. Harris, Brian L. Turner, 01/2013: chapter Dendrimers Quantum Dots Nanocomposites for Chemical Sensing; Nova Publishers., ISBN: 978-1-62808-604-1
[Show abstract][Hide abstract] ABSTRACT: Fluorescent compounds based on synthetic small molecules are powerful tools to visualize biological events in living cells and organisms. Ever since the discovery of organic fluorescent compounds in the late nineteenth century, efforts have been made to "see" the behaviors of specific biomolecules in living systems by using these dyes as labels. Also, following the development of fluorescent Ca(2+) indicators in the 1980s, many fluorescent probes or biosensors, which are defined as molecules that show a change in fluorescence properties in the presence of their target molecule, have been reported and applied in biological research. Today, a variety of probes are available that target metal ions, pH, enzyme activities, and signaling molecules. In this review, we first consider the history of organic fluorescent molecules and discuss their utility for labeling biomolecules and staining cells. Then, we review recent progress in small-molecule fluorescent probes for metal ions and reactive oxygen species, focusing on representative work in each category. Finally, we briefly discuss attempts to create novel kinds of probes, including hybrids of small molecules and genetically encoded proteins, with the potential to overcome some of the limitations of current probes.
Pflügers Archiv - European Journal of Physiology 03/2013; 465(3):347-59. DOI:10.1007/s00424-013-1234-z · 4.10 Impact Factor
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