A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS.

High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(iii) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample.

Mutations in the X-linked RP2 gene cause intracellular misrouting and loss of the protein.

Mutations in RP2 cause the second most frequent form of X-linked retinitis pigmentosa, a severe retinal degeneration that leads to loss of visual acuity and blindness. The RP2 gene encodes a protein with homology to cofactor C, a tubulin-folding chaperone. By searching protein sequence databases, we identified a whole set of similar molecules from diverse organisms. Protein sequence alignments show that RP2 and cofactor C represent members of two distinct orthologous groups. All previously identified missense mutations affect amino acid residues which are conserved in all RP2 orthologues or both orthologous groups. Intracellular localization of the wild-type protein and mutated variants was determined by fluorescence microscopy of cells expressing RP2 with a green fluorescent protein tag. A mutation in the N-terminus of RP2 abolishes localization to the plasma membrane in HeLa cells. C-terminal protein truncation mutations, which account for 2/3 of the pathogenic RP2 variants, lead to scattered fluorescent foci in the cytoplasm of COS-7 and HeLa cells. Analysis of protein extracts from the respective cells with anti-RP2 antibodies identified truncated proteins of expected size in a low-speed centrifugation pellet while the wild-type protein appeared in the supernatant. Moreover, no protein was detected in immortalized cell lines from patients with protein truncation mutations while mRNA was still present. Thus, loss of the protein and/or aberrant intracellular distribution might be the basis for the photoreceptor cell degeneration in most RP2 cases.