Single Live Cells Research Articles

PML‐Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2‐Nrf2 in the Nucleus of Single Live Cells

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a basic leucine‐zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1‐Cullin3/RING box 1 E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2‐Nrf2 in single live cells. Using this approach, we show that the half‐life (t1/2) of Dendra2‐Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG‐132 or induction of oxidative stress with tert‐butylhydroquinone (tBHQ) extended the half‐life of Dendra2‐Nrf2 by 6‐ and 28‐fold, respectively. By inhibiting nuclear export using leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML‐NBs. We further demonstrate that co‐expression of Dendra2‐Nrf2 and Crimson‐PML‐I lacking two PML‐I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2‐Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Altogether, these data suggest that functional PML‐NBs alter the rate of degradation of nuclear Nrf2, which underscores the importance of these structures in Nrf2‐mediated response to oxidative stress.Support or Funding InformationHHS |National Institutes of Health (NIH):, SC1CA143985; HHS |National Institutes of Health (NIH):, SC1CA211030; HHS |National Institutes of Health (NIH):, UL1TR000445; HHS |National Institutes of Health (NIH):, U54MD007593; HHS |National Institutes of Health (NIH):, G12MD007586; HHS |National Institutes of Health (NIH):, U54CA163069; HHS |National Institutes of Health (NIH):, R24DA036420; HHS |National Institutes of Health (NIH):, S10RR0254970; HHS |National Institutes of Health (NIH):, 5R25GM059994; HHS |National Institutes of Health (NIH):, 5T32GM007628‐37This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

The effects of long duration chronic exposure to hexavalent chromium on single live cells interrogated by scanning electrochemical microscopy

Chromium is a useful heavy metal which has been employed in numerous industry and house applications. However, there are several known health risks associated with its uses. Cr (VI) is a toxic heavy metal format which serves no essential biological role in humans. It has been associated with oxidative stress, cytotoxicity, and carcinogenicity. Contamination of groundwater or soil due to improper handling lead to long term environmental damage. This study explores the effects of long duration chronic exposure to Cr (VI) on live human cells. Herein, scanning electrochemical microscopy (SECM) depth scan imaging was employed to monitor the membrane permeability of single live human bladder cancer (T24) cells following incubation with various Cr (VI) concentration stimuli. SECM was used to provide insights into the long duration effects on membrane homeostasis of individual cells exposed to constant levels of Cr (VI). Further investigation of total population viability was performed by MTT assay. Dependent on the exposure time, transition between three distinct trends was observed. At short incubation times (≤1–3 h) with low concentrations of Cr (VI) (0–10 μM), membrane permeability was largely unaffected. As time increased a decrease in membrane permeability coefficient was observed, reaching a minimum at 3–6 h. Following this a dramatic increase in membrane permeability was observed as cell viability decreased. Higher concentrations were also found to accelerate the timeframe at which these trends occurred. These findings further demonstrate the strength of SECM as a bioanalytical technique for monitoring cellular homeostasis.

The integrated lensing effect and its application in single cell refractive index measurement in a dual-fiber optical trap

We have fully investigated the integrated lensing effect that the beam suffers in a dual-fiber optical trap. A modified model for coupling of a beam to the opposing fiber has been built in consideration of the integrated lensing effect. We discussed the effect of z-axial position, radius and refractive index of the trapped sphere on the coupling efficiency using the modified coupling coefficient model. The measured and calculated coupling efficiency as a function of z-axial position have the same tendency in the allowable error. Comparison among above simulation and experiment has made clear that the magnitude relation between the smallest spot size and the mode field radius decide whether the spot size matching or wavefront phase matching dominate the coupling coefficient. What’s more, we point out that the dependence of relative coupling coefficient on the refractive index has knee point. That is an obstacle to the application of single live cell refractive index measurement method based on lensing effect. Nevertheless, this method is absorbing and promising because there is a linear range for the known experimental parameters in an optical trap, and it needs no additional accessory.

Monitoring the Tagged mRNA Export Rate via Nuclear Pore Complex in Live Cells with a Snapshot

As mRNA conveys genetic information from DNA to the ribosome, its export rate changes under DNA manipulation and further affects hence protein translation. In live cells, the rate of a specific kind of mRNA imply the cells’ future growth tendency or even differentiation direction on molecular level. Due to its easy degradation, the gross of mRNA was usually semi-quantified from the extracts of a large amount of cells by using techniques like RT-PCR. However, monitoring a specific mRNA rate requires single-live-cell synchronous quantification. Therefore, in this study, we developed an intravital fluorescent detectable system for the export of mRNA through nuclear pore complexes (NPC) with laser scan confocal microscopy. By adapting a mRNA motion model and NPC active transportation model to the fluorescence images, tagged mRNA transport rate was calculated for each single live cell. With this method, it would help us to better understand the regulation of gene expression and even to visualize cells’ differentiation direction.

Recent advances in understanding vertebrate segmentation.

Segmentation is the partitioning of the body axis into a series of repeating units or segments. This widespread body plan is found in annelids, arthropods, and chordates, showing it to be a successful developmental strategy for growing and generating diverse morphology and anatomy. Segmentation has been extensively studied over the years. Forty years ago, Cooke and Zeeman published the Clock and Wavefront model, creating a theoretical framework of how developing cells could acquire and keep temporal and spatial information in order to generate a segmented pattern. Twenty years later, in 1997, Palmeirim and co-workers found the first clock gene whose oscillatory expression pattern fitted within Cooke and Zeeman’s model. Currently, in 2017, new experimental techniques, such as new ex vivo experimental models, real-time imaging of gene expression, live single cell tracking, and simplified transgenics approaches, are revealing some of the fine details of the molecular processes underlying the inner workings of the segmentation mechanisms, bringing new insights into this fundamental process. Here we review and discuss new emerging views that further our understanding of the vertebrate segmentation clock, with a particular emphasis on recent publications that challenge and/or complement the currently accepted Clock and Wavefront model.

Single gold nanoparticle plasmonic spectroscopy for study of chemical-dependent efflux function of single ABC transporters of single live Bacillus subtilis cells.

ATP-binding cassette (ABC) membrane transporters serve as self-defense transport apparatus in many living organisms and they can selectively extrude a wide variety of substrates, leading to multidrug resistance (MDR). The detailed molecular mechanisms remain elusive. Single nanoparticle plasmonic spectroscopy highly depends upon their sizes, shapes, chemical and surface properties. In our previous studies, we have used the size-dependent plasmonic spectra of single silver nanoparticles (Ag NPs) to study the real-time efflux kinetics of the ABC (BmrA) transporter and MexAB-OprM transporter in single live cells (Gram-positive and Gram-negative bacterium), respectively. In this study, we prepared and used purified, biocompatible and stable (non-aggregated) gold nanoparticles (Au NPs) (12.4 ± 0.9 nm) to study the efflux kinetics of single BmrA membrane transporters of single live Bacillus subtillis cells, aiming to probe chemical dependent efflux functions of BmrA transporters and their potential chemical sensing capability. Similar to those observed using Ag NPs, accumulation of the intracellular Au NPs in single live cells (WT and ΔBmrA) highly depends upon the cellular expression of BmrA and the NP concentration (0.7 and 1.4 nM). The lower accumulation of intracellular Au NPs in WT (normal expression of BmrA) than ΔBmrA (deletion of bmrA) indicates that BmrA extrudes the Au NPs out of the WT cells. The accumulation of Au NPs in the cells increases with NP concentration, suggesting that the Au NPs most likely passively diffuse into the cells, similar to antibiotics. The result demonstrates that such small Au NPs can serve as imaging probes to study the efflux function of the BmrA membrane transporter in single live cells. Furthermore, the dependence of the accumulation rate of intracellular Au NPs in single live cells upon the expression of BmrA and the concentration of the NPs is about twice higher than that of the same sized Ag NPs. This interesting finding suggests the chemical-dependent efflux kinetics of BmrA and that BmrA could distinguish nearly identical sized Au NPs from Ag NPs and might possess chemical sensing machinery.

PML-Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2-Nrf2 in the Nucleus

Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Methods: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. Results: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Conclusion: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.

Static droplet array for culturing single live adherent cells in an isolated chemical microenvironment.

We present here a new method to easily and reliably generate an array of hundreds of dispersed nanoliter-volume semi-droplets for single-cells culture and analysis. The liquid segmentation step occurs directly in indexed traps by a tweezer-like mechanism and is stabilized by spatial confinement. Unlike common droplet-based techniques, the semi-droplet wets its surrounding trap walls thus supporting the culturing of both adherent and non-adherent cells. To eliminate cross-droplet cell migration and chemical cross-talk each semi-droplet is separated from a nearby trap by an ∼80 pL air plug. The overall setup and injection procedure takes less than 10 minutes, is insensitive to fabrication defects and supports cell recovery for downstream analysis. The method offers a new approach to easily capture, image and culture single cells in a chemically isolated microenvironment as a preliminary step towards high-throughput single-cell assays.

Spectromicroscopic observation of a live single cell in a biocompatible liquid-enclosing graphene system.

On-the-spot visualization of biochemical responses of intact live cells is vital for a clear understanding of cell biology. The main obstacles for instant visualization of biochemical responses of living cells arise from the lack of a sophisticated detecting technique which can simultaneously provide chemical analysis tools and the biocompatible wet conditions. Here we introduce scanning transmission X-ray microscopy (STXM) combined with a liquid-enclosing graphene system (LGS), offering biocompatible conditions and improved X-ray absorption spectra to probe the chemical responses of live cells under wet conditions. This set-up enables us to probe a subtle change in absorption spectra depending on the oxidation state of a miniscule amount of oxygen in the functional groups present in each cell and its surroundings containing a minimal amount of liquid water. As an example of in situ biochemical responses of wet cells, chemical responses of a single Colo 205 cell are visualized and analyzed using X-ray absorption near the oxygen K-edge. This spectromicroscopic method using LGS can be applied to diverse biological samples under wet conditions for the analysis of their biochemical responses.

Development and characterization of a scintillating cell imaging dish for radioluminescence microscopy.

Radioluminescence microscopy is an emerging modality that can be used to image radionuclide probes with micron-scale resolution. This technique is particularly useful as a way to probe the metabolic behavior of single cells and to screen and characterize radiopharmaceuticals, but the quality of the images is critically dependent on the scintillator material used to image the cells. In this paper, we detail the development of a microscopy dish made of a thin-film scintillating material, Lu2O3:Eu, that could be used as the blueprint for a future consumable product. After developing a simple quality control method based on long-lived alpha and beta sources, we characterize the radioluminescence properties of various thin-film scintillator samples. We find consistent performance for most samples, but also identify a few samples that do not meet the specifications, thus stressing the need for routine quality control prior to biological experiments. In addition, we test and quantify the transparency of the material, and demonstrate that transparency correlates with thickness. Finally, we evaluate the biocompatibility of the material and show that the microscopy dish can produce radioluminescent images of live single cells.

3D super-localization of intracellular organelle contacts at live single cell by dual-wavelength synchronized fluorescence-free imaging.

A fluorescence-free real-time three-dimensional (3D) super-localization method for the analysis of 3D structure of organelles (e.g., mitochondria-associated endoplasm reticulum [mito-ER] contacts) in live single cells under physiological conditions was developed with dual-wavelength enhanced dark-field microscopy. The method was applied to live single cells under physiological conditions to analyze the complex 3D mito-ER contact region by choosing an optimum nanotag with distinct scattering properties. Combining dual-view with enhanced dark-field microscopy provided concurrent images of different scattering wavelengths of nanotag-labeled mitochondria and ER. The reconstructed super-localized images resolved controversy over the distance between the intracellular organelles at functional contacts. The distance between mitochondria and ER was measured to be 45nm, which was ~ 50% greater than in a previous report using electron microscopic tomography, and was a better fit for the likely features of these structures. These results indicate that this method was a reliable and convenient approach for investigating the 3D structure of organelles, such as mito-ER contacts in live single cells, and provided accurate information under physiological conditions. Graphical abstract Fluorescence-free enhanced dark-field 3D super-resolution microscopy (3D SRM) method, with dual-wavelength simultaneous imaging (DWSI) for 3D analysis of mitochondria-endoplasmic reticulum (Mito-ER) at their functional contact site.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

Dual Inhibition of Mdmx and Mdm2 Using an Alpha-Helical P53 Stapled Peptide (ALRN-6924) As a Novel Therapeutic Strategy in Acute Myeloid Leukemia

While the TP53 tumor suppressor gene is mutated in more than 50% of human tumors, in Acute Myeloid Leukemia (AML) TP53 mutations are rare, occurring in less than 10% of cases. Yet, functional inactivation of wild-type p53 due to non-mutational abnormalities occurs frequently in AML and other hematological malignancies. A major mechanism of p53 inactivation results from the overexpression of its endogenous inhibitors MDMX (also known as MDM4, HDMX, and HDM4) and MDM2 which are frequently overexpressed in various p53 wild-type human cancers, including AML. Strikingly, MDMX has been reported to be overexpressed in over 92% of AML cases, while MDM2 overexpression is less frequent. Pharmacological disruption of both these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in AML and other cancers with wild-type p53. Nonetheless, selective targeting of this pathway has thus far been limited to MDM2-only small molecule inhibitors which lack affinity for MDMX. ALRN-6924 is an optimized alpha helical p53 stapled peptide and first-in-class dual MDMX/MDM2 inhibitor which has recently entered phase I/II clinical testing (NCT02264613, NCT02909972) in solid tumors and lymphomas with, thus far, excellent tolerability and objective responses as single agent1. The goal of our study was to evaluate the molecular, cellular, and biochemical mechanisms of action of ALRN-6924 in AML.We used biochemical affinity studies as well as single molecule FISH and live single cell imaging to assess MDMX/MDM2 binding as well as p21 transactivation by p53 in response to ALRN-6924. Effects on cellular proliferation, apoptosis, DNA repair, cell cycle, clonogenic capacity, and serial replating were determined using AML cell lines and primary human AML patients' cells, including in leukemic stem (CD34+ CD38-) and progenitor (CD34+CD38+) cells. Genome-wide molecular effects were determined by RNA seq. P53 activity in a patient undergoing treatment with ALRN-6924 was measured by intracellular staining of p53 and its target gene p21 in CD34+ cells by flow cytometry. Furthermore, we evaluated ALRN-6924 activity in a xeno-transplantation model of human AML in NSG mice.We found that MDMX is significantly overexpressed in highly fractionated leukemic stem (Lin-CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in AML patients compared to identically sorted, age-matched healthy controls (p<0.0001). Dual MDMX/MDM2 inhibition using ALRN-6924 led to striking anti-leukemic effects in suspension culture of AML cell lines and primary cells, with negligible effects on healthy controls. ALRN-6924 robustly activated p53-dependent transcription at the single cell and single molecule level, and exhibited strong biochemical and molecular biological on-target activity in AML cell lines and primary cells in vitro, as well as in a patient who received ALRN-6924 treatment. Co-immunoprecipitation experiments demonstrated that ALRN-6924 disrupts both MDMX/P53 as well as MDM2/P53 interactions at relevant therapeutic dosages in AML cells. RNA seq data showed that dual MDMX/MDM2 inhibition led to global transcriptional activation of p53-dependent pathways in AML cells. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibited cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patients' cells, including in leukemic stem cell-enriched populations. Furthermore, ALRN-6924 disrupted functional clonogenic and serial replating capacity of AML cells, and showed superiority over MDM2-only inhibition. Most strikingly, ALRN-6924 led to highly significant improved survival in an AML xenograft model in vivo (p<0.0001).Our study provides insight into the molecular, cell biological, and in vivo effects of pharmacological dual MDMX/MDM2 inhibition in AML, which may have important implications for other MDMX/MDM2-related cancers. Our findings provide proof-of-concept for ALRN-6924 as a novel therapeutic in p53-wildtype AML, and provide a rationale for its further preclinical and clinical development in AML and other cancers.Furthermore, the success of targeting p53 raises the intriguing prospect that the same development path is possible for other helix-in-groove targets, and may thus pave the way for a new class of targeted therapeutics.1 ASCO 2017 annual meeting: J Clin Oncol 35, 2017 (suppl; abstr 2505) DisclosuresGuerlavais:Aileron: Employment. Annis:Aileron Therapeutics: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Aivado:Aileron: Employment. Steidl:Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.

Clickable Multifunctional Dumbbell Particles for in Situ Multiplex Single-Cell Cytokine Detection.

We report a novel strategy for fabrication of multifunctional dumbbell particles (DPs) through click chemistry for monitoring single-cell cytokine releasing. Two different types of DPs were prepared on a large scale through covalent bioorthogonal reaction between methyltetrazine and trans-cyclooctene on a microchip under a magnetic field. After collection of the DPs, the two sides of each particle were further functionalized with different antibodies for cell capturing and cytokine detection, respectively. These DPs labeled with different fluorescent dyes have been used for multiplex detection and analysis of cytokines secreted by single live cells. Our results show that this new type of DPs are promising for applications in cell sorting, bioimaging, single-cell analysis, and biomedical diagnostics.

DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores.

Red fluorescent proteins (RFPs) have emerged as valuable biological markers for biomolecule imaging in living systems. Developing artificial fluorogenic systems that mimic RFPs remains an unmet challenge. Here, we describe the design and synthesis of six new chromophores analogous to the chromophores in RFPs. We demonstrate, for the first time, that encapsulating RFP chromophore analogues in canonical DNA G-quadruplexes (G4) can activate bright fluorescence spanning red and far-red spectral regions (Em = 583−668 nm) that nearly match the entire RFP palette. Theoretical calculations and molecular dynamics simulations reveal that DNA G4 greatly restricts radiationless deactivation of chromophores induced by a twisted intramolecular charge transfer (TICT). These DNA mimics of RFP exhibit attractive photophysical properties comparable or superior to natural RFPs, including high quantum yield, large Stokes shifts, excellent anti-photobleaching properties, and two-photon fluorescence. Moreover, these RFP chromophore analogues are a novel and distinctive type of topology-selective G4 probe specific to parallel G4 conformation. The DNA mimics of RFP have been further exploited for imaging of target proteins. Using cancer-specific cell membrane biomarkers as targets, long-term real-time monitoring in single live cell and two-photon fluorescence imaging in tissue sections have been achieved without the need for genetic coding.

Multimodal hyperspectral optical microscopy

We describe a unique approach to hyperspectral optical microscopy, herein achieved by coupling a hyperspectral imager to various optical microscopes. Hyperspectral fluorescence micrographs of isolated fluorescent beads are first employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements. Different science applications of our instrument are then described. Spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE) layer reveals that optical densities on the order of 10−3 can be resolved by spatially averaging the recorded optical signatures. This is followed by three applications in the general areas of plasmonics and bioimaging. Notably, we deploy hyperspectral absorption microscopy to identify and image pigments within a simple biological system, namely, a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal spectral imaging measurements spanning the realms of several scientific disciplines.

Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamicsthrough a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.