Dataset Information

Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.

ABSTRACT: Techniques to analyze human telomeres are imperative in studying the molecular mechanism of aging and related diseases. Two important aspects of telomeres are their length in DNA base pairs (bps) and their biophysical nanometer dimensions. However, there are currently no techniques that can simultaneously measure these quantities in individual cell nuclei. Here, we develop and evaluate a telomere "dual" gold nanoparticle-fluorescent probe simultaneously compatible with both X-ray fluorescence (XRF) and super resolution microscopy. We used silver enhancement to independently visualize the spatial locations of gold nanoparticles inside the nuclei, comparing to a standard QFISH (quantitative fluorescence in situ hybridization) probe, and showed good specificity at ?90%. For sensitivity, we calculated telomere length based on a DNA/gold binding ratio using XRF and compared to quantitative polymerase chain reaction (qPCR) measurements. The sensitivity was low (?10%), probably because of steric interference prohibiting the relatively large 10 nm gold nanoparticles access to DNA space. We then measured the biophysical characteristics of individual telomeres using super resolution microscopy. Telomeres that have an average length of ?10 kbps, have diameters ranging between ?60-300 nm. Further, we treated cells with a telomere-shortening drug and showed there was a small but significant difference in telomere diameter in drug-treated vs control cells. We discuss our results in relation to the current debate surrounding telomere compaction.

SUBMITTER: Jeynes JCG

PROVIDER: S-EPMC5951601 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.

Jeynes J Charles G JCG Geraki Kalotina K Jeynes Christopher C Zhaohong Mi M Bettiol Andrew A AA Latorre Eva E Harries Lorna Wendy LW Soeller Christian C

ACS nano 20171107 12

Techniques to analyze human telomeres are imperative in studying the molecular mechanism of aging and related diseases. Two important aspects of telomeres are their length in DNA base pairs (bps) and their biophysical nanometer dimensions. However, there are currently no techniques that can simultaneously measure these quantities in individual cell nuclei. Here, we develop and evaluate a telomere "dual" gold nanoparticle-fluorescent probe simultaneously compatible with both X-ray fluorescence (X ...[more]

PMID: 29091397

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Project description:Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ∼250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (∼2-6 μm long and 200-300 nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

Dataset Information

Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.

Publications

Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.

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