Laimonas Kelbauskas

Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.

Quantitative characterization of preneoplastic progression using single-cell computed tomography and three-dimensional karyometry

The development of morphological biosignatures to precisely characterize preneoplastic progression necessitates high‐resolution three‐dimensional (3D) cell imagery and robust image processing algorithms. We report on the quantitative characterization of nuclear structure alterations associated with preneoplastic progression in human esophageal epithelial cells using single‐cell optical tomography and fully automated 3D karyometry. We stained cultured cells with hematoxylin and generated 3D images of individual cells by mathematically reconstructing 500 projection images acquired using optical absorption tomographic imaging. For 3D karyometry, we developed novel, fully automated algorithms to robustly segment the cellular, nuclear, and subnuclear components in the acquired cell images, and computed 41 quantitative morphological descriptors from these segmented volumes. In addition, we developed algorithms to quantify the spatial distribution and texture of the nuclear DNA. We applied our methods to normal, metaplastic, and dysplastic human esophageal epithelial cell lines, analyzing 100 cells per line. The 3D karyometric descriptors elucidated quantitative differences in morphology and enabled robust discrimination between cell lines on the basis of extracted morphological features. The morphometric hallmarks of cancer progression such as increased nuclear size, elevated nuclear content, and anomalous chromatin texture and distribution correlated with this preneoplastic progression model, pointing to the clinical use of our method for early cancer detection.

Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Förster resonance energy transfer measurements

Since its discovery, green fluorescent protein (GFP) and its variants have proven to be a good and convenient fluorescent label for proteins: GFP and other visible fluorescent proteins (VFPs) can be fused selectively to the protein of interest by simple cloning techniques and develop fluorescence without additional cofactors. Among the steadily growing collection of VFPs, several pairs can be chosen that can serve as donor and acceptor fluorophores in Forster resonance energy transfer (FRET) experiments. Among them, the cyan fluorescent proteins (ECFP/Cerulean) and the enhanced yellow fluorescent protein (EYFP) are most commonly used. We show that ECFP and Cerulean have some disadvantages despite their common use: Upon irradiation with light intensities that are commonly used for intensity- and lifetime-based FRET measurements, both the fluorescence intensity and the fluorescence lifetime of ECFP and Cerulean decrease. This can hamper both intensity- and lifetime-based FRET measurements and emphasizes the need for control measurements to exclude these artifacts.

Visualization of SHP-1-target interaction

Signaling of receptor tyrosine kinases (RTKs) is regulated by protein-tyrosine phosphatases (PTPs). We previously discovered the efficient downregulation of Ros RTK signaling by the SH2 domain PTP SHP-1, which involves a direct interaction of both molecules. Here, we studied the mechanism of this interaction in detail. Phosphopeptides representing the SHP-1 candidate binding sites in the Ros cytoplasmic domain, pY2267 and pY2327, display high affinity binding to the SHP-1 N-terminal SH2 domain (Kd=217 nM and 171 nM, respectively). Y2327 is, however, a poor substrate of Ros kinase and, therefore, contributes little to SHP-1 binding in vitro. To explore the mechanism of association in intact cells, functional fluorescent fusion proteins of Ros and SHP-1 were generated. Complexes of both molecules could be detected by Förster resonance energy transfer (FRET) in intact HEK293 and COS7 cells. As expected, the association required the functional SHP-1 N-terminal SH2 domain. Unexpectedly, pY2267 and pY2327 both contributed to the association. Mutation of Y2327 reduced constitutive association in COS7 cells. Ligand-dependent association was abrogated upon mutation of Y2267 but remained intact when Y2327 was mutated. A phosphopeptide representing the binding site pY2267 was a poor substrate for SHP-1, whereas Ros activation loop phosphotyrosines were effectively dephosphorylated. We propose a model for SHP-1-Ros interaction in which ligand-stimulated phosphorylation of Ros Y2267 by Ros, phosphorylation of Y2327 by a heterologous kinase, and inactivation of Ros by SHP-1-mediated dephosphorylation play a role in the regulation of complex stability.

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the systems capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.

Nucleosomal stability and dynamics vary significantly when viewed by internal versus terminal labels.

Nucleosomes are a major impediment to regulatory factor activities and therefore to the operation of genomic processes in eukaryotes. One suggested mechanism for overcoming in vivo nucleosomal repression is factor-mediated removal of H2A/H2B from nucleosomes. Using nucleosomes labeled internally with FRET fluorophores, we previously observed significant, DNA sequence-dependent variation in stability and dynamics under conditions (subnanomolar concentrations) reported to produce H2A/H2B release from nucleosomes. Here, the same analytical approaches are repeated using 5S and MMTV-B nucleosomes containing FRET labels that monitor the terminal regions. The results show that stability and dynamics vary significantly within the nucleosome; terminally labeled constructs report significantly reduced stability and enhanced DNA dynamics compared to internally labeled constructs. The data also strongly support previous suggestions (1) that subnanomolar concentrations cause H2A/H2B release from nucleosomes, including the 5S, and (2) that stabilities in the internal regions of 5S and two promoter-derived nucleosomes (MMTV-B, GAL10) differ. Sequence-dependent nucleosome stability/dynamics differences could produce inherent variations in the accessibility of histone-associated DNA in vivo. Such intrinsic variation could also provide a mechanism for producing enhanced effects on specific nucleosomes by processes affecting large chromatin regions, thus facilitating the localized targeting of alterations to nucleosomes on crucial regulatory sequences. The results demonstrate clearly the importance of studying physiologically relevant nucleosomes.

High resolution TCSPC lifetime imaging

Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging in laser scanning microscopes can be combined with a multi-detector technique that allows to record time-resolved images in several wavelength channels simultaneously. The technique is based on a multi-dimensional histogramming process that records the photon density versus the time within the fluorescence decay function, the x-y coordinates of the scanning area and the detector channel number. It avoids any time gating or wavelength switching and therefore yields a near-ideal counting efficiency. We show an instrument that records dual wavelength lifetime images with up to 512 x 512 pixels, and single wavelength lifetime images with up to 1024 x 1024 pixels. It resolves the components of double-exponential decay functions down to 30 ps, and works at the full scanning speed of a two-photon laser scanning microscope. The performance of the instrument is demonstrated for simultaneous lifetime imaging of the donor and acceptor fluorescence in CFP/YFP FRET systems and for tissue samples stained with several fluorophores.

DNA sequence-dependent variation in nucleosome structure, stability, and dynamics detected by a FRET-based analysis.

Förster resonance energy transfer (FRET) techniques provide powerful and sensitive methods for the study of conformational features in biomolecules. Here, we review FRET-based studies of nucleosomes, focusing particularly on our work comparing the widely used nucleosome standard, 5S rDNA, and 2 promoter-derived regulatory element-containing nucleosomes, mouse mammary tumor virus (MMTV)-B and GAL10. Using several FRET approaches, we detected significant DNA sequence-dependent structure, stability, and dynamics differences among the three. In particular, 5S nucleosomes and 5S H2A/H2B-depleted nucleosomal particles have enhanced stability and diminished DNA dynamics, compared with MMTV-B and GAL10 nucleosomes and particles. H2A/H2B-depleted nucleosomes are of interest because they are produced by the activities of many transcription-associated complexes. Significant location-dependent (intranucleosomal) stability and dynamics variations were also observed. These also vary among nucleosome types. Nucleosomes restrict regulatory factor access to DNA, thereby impeding genetic processes. Eukaryotic cells possess mechanisms to alter nucleosome structure, to generate DNA access, but alterations often must be targeted to specific nucleosomes on critical regulatory DNA elements. By endowing specific nucleosomes with intrinsically higher DNA accessibility and (or) enhanced facility for conformational transitions, DNA sequence-dependent nucleosome dynamics and stability variations have the potential to facilitate nucleosome recognition and, thus, aid in the crucial targeting process. This and other nucleosome structure and function conclusions from FRET analyses are discussed.

On-chip isotachophoresis separation of functional DNA origami capture nanoarrays from cell lysate

AbstractScaffolded DNA origami, a versatile method to construct high yield selfassembled DNA nanostructures, has been investigated to develop water-soluble nanoarrays for label free RNA detection, drug delivery, molecular positioning and recognition, and spatially ordered catalysis of single molecule chemical reactions. Its attributes that facilitate these applications suggest DNA origami as a candidate platform for intracellular targeting. After the interaction with targeted proteins in cell lysate, it is critical to separate and concentrate DNA origami nanoarrays from the crude cell lysate for further analysis. The recent development of microchip isotachophoresis (ITP) provides an alternative robust sample preconcentration and electrophoretic separation method. In this study, we present online ITP for stacking, separation and identification of aptamer-functionalized DNA origami and its thrombin complex in a simple cross-channel fused silica microfluidic chip. In particular, the method achieved separation of a binding complex in less than 5 min and 150-fold signal enhancement. We successfully separated and analyzed the thrombin bound origami-aptamer spiked into cell lysate using on-chip ITP. Our results demonstrate that origami/thrombin nanostructures can be effectively separated from cell lysate using this method and that the structural integrity of the concentrated binding complex is maintained as confirmed by atomic force microscopy (AFM). An ITP-based separation module can be easily coupled to other microchip pre- and post-processing steps to provide an integrated proteomics analysis platform for diagnostic applications.

A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis.