Yuval Garini

Multicolor spectral karyotyping of human chromosomes

The simultaneous and unequivocal discernment of all human chromosomes in different colors would be of significant clinical and biologic importance. Whole-genome scanning by spectral karyotyping allowed instantaneous visualization of defined emission spectra for each human chromosome after fluorescence in situ hybridization. By means of computer separation (classification) of spectra, spectrally overlapping chromosome-specific DNA probes could be resolved, and all human chromosomes were simultaneously identified.

Strange kinetics of single molecules in living cells

The irreproducibility of time-averaged observables in living cells poses fundamental questions for statistical mechanics and reshapes our views on cell biology.The irreproducibility of time-averaged observables in living cells poses fundamental questions for statistical mechanics and reshapes our views on cell biology.

Spectral imaging: principles and applications.

Spectral imaging extends the capabilities of biological and clinical studies to simultaneously study multiple features such as organelles and proteins qualitatively and quantitatively. Spectral imaging combines two well‐known scientific methodologies, namely spectroscopy and imaging, to provide a new advantageous tool. The need to measure the spectrum at each point of the image requires combining dispersive optics with the more common imaging equipment, and introduces constrains as well.

Built-in Quantum Dot Antennas in Dye-Sensitized Solar Cells

A new design of dye-sensitized solar cells involves colloidal semiconductor quantum dots that serve as antennas, funneling absorbed light to the charge separating dye molecules via nonradiative energy transfer. The colloidal quantum dot donors are incorporated into the solid titania electrode resulting in high energy transfer efficiency and significant improvement of the cell stability. This design practically separates the processes of light absorption and charge carrier injection, enabling us to optimize each of these separately. Incident photon-to-current efficiency measurements show a full coverage of the visible spectrum despite the use of a red absorbing dye, limited only by the efficiency of charge injection from the dye to the titania electrode. Time resolved luminescence measurements clearly relate this to Forster resonance energy transfer from the quantum dots to the dye. The presented design introduces new degrees of freedom in the utilization of quantum dot sensitizers for photovoltaic cells. In particular, it opens the way toward the utilization of new materials whose band offsets do not allow direct charge injection.

Single-allele analysis of transcription kinetics in living mammalian cells

We generated a system for in vivo visualization and analysis of mammalian mRNA transcriptional kinetics of single alleles in real time, using single-gene integrations. We obtained high-resolution transcription measurements of a single cyclin D1 allele under endogenous or viral promoter control, including quantification of temporal kinetics of transcriptional bursting, promoter firing, nascent mRNA numbers and transcription rates during the cell cycle, and in relation to DNA replication.

The significance of telomeric aggregates in the interphase nuclei of tumor cells

Telomeres are TTAGGG repetitive motifs found at the ends of vertebrate chromosomes. In humans, telomeres are protected by shelterin, a complex of six proteins (de Lange [ 2005 ] Genes Dev. 19: 2100–2110). Since (Müller [ 1938 ] Collecting Net. 13: 181–198; McClintock [ 1941 ] Genetics 26: 234–282), their function in maintaining chromosome stability has been intensively studied. This interest, especially in cancer biology, stems from the fact that telomere dysfunction is linked to genomic instability and tumorigenesis (Gisselsson et al. [ 2001 ] Proc. Natl. Acad. Sci. USA 98: 12683–12688; Deng et al. [ 2003 ] Genes Chromosomes Cancer 37: 92–97; DePinho and Polyak [ 2004 ] Nat. Genetics 36: 932–934; Meeker et al. [ 2004 ] Clin. Cancer Res. 10: 3317–3326). In the present overview, we will discuss the role of telomeres in genome stability, recent findings on three‐dimensional (3D) changes of telomeres in tumor interphase nuclei, and outline future avenues of research. J. Cell. Biochem. 97: 904–915, 2006.

Fourier Transform Multipixel Spectroscopy and Spectral Imaging of Protoporphyrin in Single Melanoma Cells

Fourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo‐PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge‐coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (103‐−4 × 103 point pixels in a single cell). The net result was the intensity I (x, y, λ), for each pixel of the image (x, y), at any wavelength (λ). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5‐aminolevulinic acid (ALA). The fluorescence image of ALA‐treated cells revealed endo‐PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one sub‐cellular site to another. Photoirradiation of the cells induced point‐specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral‐image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal‐lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo‐products could be visualized at sites of formation in suhcellular compartments.

Characterizing the Three-Dimensional Organization of Telomeres

Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis.

Loss of lamin A function increases chromatin dynamics in the nuclear interior

Chromatin is organized in a highly ordered yet dynamic manner in the cell nucleus, but the principles governing this organization remain unclear. Similarly, it is unknown whether, and how, various proteins regulate chromatin motion and as a result influence nuclear organization. Here by studying the dynamics of different genomic regions in the nucleus of live cells, we show that the genome has highly constrained dynamics. Interestingly, depletion of lamin A strikingly alters genome dynamics, inducing a dramatic transition from slow anomalous diffusion to fast and normal diffusion. In contrast, depletion of LAP2α, a protein that interacts with lamin A and chromatin, has no such effect on genome dynamics. We speculate that chromosomal inter-chain interactions formed by lamin A throughout the nucleus contribute to chromatin dynamics, and suggest that the molecular regulation of chromatin diffusion by lamin A in the nuclear interior is critical for the maintenance of genome organization.

The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells

Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spatial changes in lamina shape were observed before the Hayflick senescence-associated factors, suggesting that the lamina morphology can be used as an early marker to identify senescent cells. Here, we applied quantitative image-processing tools to study the changes in nuclear architecture during cell senescence. We found that centromeres and telomeres colocalised with lamina intranuclear structures, which resulted in a preferred peripheral distribution in senescent cells. In addition, telomere aggregates were progressively formed during cell senescence. Once formed, telomere aggregates showed colocalization with γ-H2AX but not with TERT, suggesting that telomere aggregates are sites of DNA damage. We also show that telomere aggregation is associated with lamina intranuclear structures, and increased telomere binding to lamina proteins is found in cells expressing lamina mutants that lead to increases in lamina intranuclear structures. Moreover, three-dimensional image processing revealed spatial overlap between telomere aggregates and lamina intranuclear structures. Altogether, our data suggest a mechanical link between changes in lamina spatial organization and the formation of telomere aggregates during senescence of hMSCs, which can possibly contribute to changes in nuclear activity during cell senescence.