Real-time quantification of nuclear RNA export using an intracellular relocation probe

Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions. Despite the broad availability of nucleic acid dyes, tracking and quantifying the highly dynamic process of RNA export in live cells is challenging. When dye-labeled RNA enters the cytoplasm, the dye molecules are released upon degradation of the RNA, allowing them to re-enter the cell nucleus. As a result, the ratio between the dye exported with RNA into the cytoplasm and the portion staying inside the nucleus cannot be determined. To address this common limitation, we report the design of a smart probe that can only check into the nucleus once. When adding to cells, this probe rapidly binds with nuclear RNAs in live cells and reacts with intrinsic H₂S. This reaction not only activates the fluorescence for RNA tracking but also changes the structure of probe and consequently its intracellular localization. After disassociating from exported RNAs in cytoplasm, the probe preferentially enters lysosomes rather than cell nucleus, enabling real-time quantitative measurement of nuclear RNA exports. Using this probe, we successfully evaluated the effects of hormones and cancer drugs on nuclear RNA export in live cells. Interestingly, we found that hormones inhibiting RNA exports can partially offset the effect of chemotherapy.     

Label-free molecular imaging of immunological synapses between dendritic and T cells by Raman micro-spectroscopy

Confocal Raman micro-spectroscopy (CRMS) was used to measure spectral images of immunological synapse formation between dendritic and T cells without using molecular labels or other invasive procedures. The purpose-built inverted CRMS instrument integrated an environmental enclosure and a near-infrared laser to allow measurements on live cells maintained under physiological conditions. The integration of the wide-field fluorescence also enabled viability assays and direct comparison between Raman spectral images and gold-standard immuno-fluorescence images for specific molecules. Raman spectral images of nucleus and proteins were built by fuzzy c-mean clustering method. The Raman images were found to be in good correspondence with the immuno-fluorescence images of DNA and actin. These results indicate that actin is a main contributor to the Raman spectrum of the cytoplasm of dendritic and T cells. While for control cells the Raman spectral images of proteins indicated a more homogeneous distribution of proteins in the cytoplasm of dendritic cells, they indicated a higher accumulation of proteins at the immunological synapses when dendritic cells were pre-treated with laminin. These conclusions were also supported by confocal immuno-fluorescence imaging after cell fixation and labelling. This study demonstrates the potential of CRMS for label-free non-invasive imaging of junctions between live cells. Therefore, this technique may become a useful tool for studying cellular processes in live cells and where non-invasive molecular specific imaging is desirable, such as cell-cell interactions.     

Quantum dot-insect neuropeptide conjugates for fluorescence imaging, transfection, and nucleus targeting of living cells

We identified an insect neuropeptide, namely, allatostatin 1 from Drosophila melanogaster, that transfects living NIH 3T3 and A431 human epidermoid carcinoma cells and transports quantum dots (QDs) inside the cytoplasm and even the nucleus of the cells. QD-conjugated biomolecules are valuable resources for visualizing the structures and functions of biological systems both in vivo and in vitro. Here, we selected allatostatin 1, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2, conjugated to streptavidin-coated CdSe-ZnS QDs. This was followed by investigating the transfection of live mammalian cells with QD-allatostatin conjugates, the transport of QDs by allatostatin inside the nucleus, and the proliferation of cells in the presence of allatostatin. Also, on the basis of dose-dependent proliferation of cells in the presence of allatostatin we identified that allatostatin is not cytotoxic when applied at nanomolar levels. Considering the sequence similarity between the receptors of allatostatin in D. melanogaster and somatostatin/galanin in mammalian cells, we expected interactions and localization of allatostatin to somatostatin/galanin receptors on the membranes of 3T3 and A431 cells. However, with QD conjugation we identified that the peptide was delivered inside the cells and localized mainly to the cytoplasm, microtubules, and nucleus. These results indicate that allatostatin is a promising candidate for high-efficiency cell transfection and nucleus-specific cell labeling. Also, the transport property of allatostatin is promising with respect to label/drug/gene delivery and high contrast imaging of live cells and cell organelles. Another promising application of allatostatin is that the transport of QDs inside the nucleus would lift the limit of general photodynamic therapy to nucleus-specific photodynamic therapy, which is expected to be more efficient than photosensitization at the cell membrane or in the cytoplasm as a result of the short lifetime of singlet oxygen.     

Solvent-sensitive dyes to report protein conformational changes in living cells

Covalent attachment of solvent-sensitive fluorescent dyes to proteins is a powerful tool for studying protein conformational changes, ligand binding, or posttranslational modifications. We report here new merocyanine dyes that make possible the quantitation of such protein activities in individual living cells. The quantum yield of the new dyes is sharply dependent on solvent polarity or viscosity, enabling them to report changes in their protein environment. This is combined with other stringent requirements needed in a live cell imaging dye, including appropriate photophysical properties (excitation >590 nm, high fluorescence quantum yield, high extinction coefficient), good photostability, minimal aggregation in water, and excellent water solubility. The dyes were derivatized with iodoacetamide and succinimidyl ester side chains for site-selective covalent attachment to proteins. A novel biosensor of Cdc42 activation made with one of the new dyes showed a 3-fold increase in fluorescence intensity in response to GTP-binding by Cdc42. The dyes reported here should be useful in the preparation of live cell biosensors for a diverse range of protein activities.     

Serum Albumin Binding Inhibits Nuclear Uptake of Luminescent Metal‐Complex‐Based DNA Imaging Probes

The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir^IIIRu^II tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru^IIRu^II analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell‐free studies reveal that the Ir^IIIRu^II complex binds bovine serum albumin, BSA, in Sudlow’s Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum‐free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.     

Synthesis of chloro-substituted analogs of Thiazole orange - Fluorophores for flow cytometric analyses

Synthesis, absorption and fluorescence properties of a series of asymmetrical monomethine cyanine dyes, chloro-containing analogs of Thiazole orange, are reported. Their staining ability was studied by flow cytometry. The saturating concentrations of each dye that gives a stable staining intensity have been determined. The ability of dyes B9, B11, B13 to stain live macrophages and apoptotic splenocytes was investigated. Positive signal in nucleus of adherent macrophages detected by fluorescent microscopy showed good specificity of B9, B11 and B13 dyes for DNA. In apoptotic assay cells positive for Annexin V were stained more brightly with the dyes B9, B11 and B13 than with propidium iodide. Despite that B13 showed high DNA selectivity it induces apoptosis of splenocytes and it is not suitable for detection of dead cells. The other synthesized chloro-containing analogs of Thiazole orange B9 and B11 can be successfully used for flow cytometric analyses of DNA content in live cells and for analyses of cell apoptosis.     

Aggregation-induced Emission Fluorogen as Mammalian Cell Cytoplasmic Tracker with Long Retention Time and High Photo-stability

Fluorescent probes used for cell imaging are powerful tools in cell-based assays and research.In this study,we exhibited a water-soluble aggregation-induced emission fluorogen(AIEgen),BSPO-TPE,specifically stained cytoplasm in live cells and had an excellent photostability when compared to that of two widely used commercial fluorescent dyes.The long cytoplasm retention time of BSPO-TPE demonstrated its suitability as a live cell cytoplasm tracker.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—VIII. CELLULAR DISTRIBUTION INV–79 CHINESE HAMSTER CELLS AND PHOTOTOXICITY OF SELECTIVELY SULFONATED ALUMINUM PHTHALOCYANINES

Abstract— Water soluble chloro aluminum phthalocyanines sulfonated to different degrees are studied for phototoxicity and cellular distribution inV–79 Chinese hamster cells. The more hydrophobic disulfonated dyes, with sulfonate substituents on adjacent benzyl groups of the phthalocyanine ring structure, exhibited the best cell penetrating properties and the highest phototoxicity. Fluorescence microscopy revealed that the dye was uniformly distributed in the cytoplasm but absent in the nucleus. The greater cell membrane penetrating properties of the lower as compared to the higher sulfonated dyes are attributed to the amphiphilic nature of the former.     

A highly stable RNA aptamer probe for the retinoblastoma protein in live cells

Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable in vivo. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity (K_d = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.

An RNA G-quadruplex aptamer, specific for the human retinoblastoma protein (RB) and highly stable inside cells, is selected and its application to live cell probing of the protein illustrated.     

Tracking the Dynamic Folding and Unfolding of RNA G‐Quadruplexes in Live Cells

Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1 , for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes.     

Analysis of heterogeneous mitochondria distribution in somatic cell nuclear transfer porcine embryos

We previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.     

Live‐Cell Imaging of Endogenous mRNAs with a Small Molecule

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non‐engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene‐specific RNA aptamer, combined with a cell‐permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live‐cell imaging of the endogenous mRNA of β‐actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin‐2, cortactin, and cytoplasmic FMR1‐interacting protein 2. The RNA‐imaging technology and its further optimization might permit live‐cell imaging of any RNA molecules.     

Styryl molecules light-up RNAs

A combinatorial library of 1336 fluorescent styryl molecules was synthesized aiming to select dyes that are photostable, non-toxic, and specific for RNA molecules in living cells . These dyes are potentially important to the study of gene expression in live cells.     

Multicolor, one- and two-photon imaging of enzymatic activities in live cells with fluorescently Quenched Activity-Based Probes (qABPs)

Fluorescence imaging provides an indispensable way to locate and monitor biological targets within complex and dynamic intracellular environments. Of the various imaging agents currently available, small molecule-based probes provide a powerful tool for live cell imaging, primarily due to their desirable properties, including cell permeability (as a result of their smaller sizes), chemical tractability (e.g., different molecular structures/designs can be installed), and amenability to imaging a wide variety of biological events. With a few exceptions, most existing small molecule probes are however not suitable for in vivo bioimaging experiments in which high-resolution studies of enzyme activity and localization are necessary. In this article, we reported a new class of fluorescently Quenched Activity-Based Probes (qABPs) which are highly modular, and can sensitively image (through multiple enzyme turnovers leading to fluorescence signal amplification) different types of enzyme activities in live mammalian cells with good spatial and temporal resolution. We have also incorporated two-photon dyes into our modular probe design, enabling for the first time activity-based, fluorogenic two-photon imaging of enzyme activities. This, hence, expands the repertoire of 'smart', responsive probes currently available for live cell bioimaging experiments.     

Live-cell-permeant thiophene fluorophores and cell-mediated formation of fluorescent fibrils

In our search for thiophene fluorophores that can overcome the limits of currently available organic dyes in live-cell staining, we synthesized biocompatible dithienothiophene-S,S-dioxide derivatives (DTTOs) that were spontaneously taken up by live mouse embryonic fibroblasts and HeLa cells. Upon treatment with DTTOs, the cells secreted nanostructured fluorescent fibrils, while cell viability remained unaltered. Comparison with the behavior of other cell-permeant, newly synthesized thiophene fluorophores showed that the formation of fluorescent fibrils was peculiar to DTTO dyes. Laser scanning confocal microscopy of the fluorescent fibrils showed that most of them were characterized by helical supramolecular organization. Electrophoretic analysis and theoretical calculations suggested that the DTTOs were selectively recognized by the HyPro component of procollagen polypeptide chains and incorporated through the formation of multiple H-bondings.     

Multiplexed imaging detection of live cell intracellular changes in early apoptosis with aggregation-induced emission fluorogens

Apoptosis is an important process for maintaining tissue homeostasis and eliminating abnormal cells in multicellular organisms. Abnormality in apoptosis often leads to severe diseases such as cancers. Better understanding of its mechanisms and processes is therefore important. Accompanying molecular biology events of apoptosis is a series of cellular morphology changes: nucleus condensation, cell shrinkage and rounding, cell surface blebbing, dynamic blebbing, apoptotic membrane protrusions and nucleus fragmentations and finally, the formation and release of apoptotic bodies. It is difficult to detect cellular changes in the early phase of apoptosis due to the subtle changes at this phase. In the current study, we induced apoptosis in HeLa cells with H₂O₂ and used nuclear dye Hoechst 33258, mitochondria, lysosome and cytoplasmic protein specific aggregation-induced emission fluorogens (AIEgens), TPE-Ph-In, 2M-DABS and BSPOTPE to successfully perform live cell multiplexed imaging to investigate early apoptosis cellular events. We showed the gradual dissipation of mitochondria membrane potential until it is nondetectable by TPE-Ph-In. Increased mitophagy detected by TPE-Ph-In and 2M-DABS, condensed nucleus detected by Hoechst 33258, increased permeability and/or reduced integrity of nuclear membrane, and increased intracellular vesicles detected by 2M-DABS are some of the early events of apoptosis.     

Gold Nanocage Assemblies for Selective Second Harmonic Generation Imaging of Cancer Cell

Second harmonic generation (SHG) imaging using near infrared laser light is the key to improving penetration depths, leading to biological understanding. Unfortunately, currently SHG imaging techniques have limited capability due to the poor signal‐to‐noise ratio, resulting from the low SHG efficiency of available dyes. Targeted tumor imaging over nontargeted tissues is also a challenge that needs to be overcome. Driven by this need, in this study, the development of two‐photon SHG imaging of live cancer cell lines selectively by enhancement of the nonlinear optical response of gold nanocage assemblies is reported. Experimental results show that two‐photon scattering intensity can be increased by few orders of magnitude by just developing nanoparticle self‐assembly. Theoretical modeling indicates that the field enhancement values for the nanocage assemblies can explain, in part, the enhanced nonlinear optical properties. Our experimental data also show that A9 RNA aptamer conjugated gold nanocage assemblies can be used for targeted SHG imaging of the LNCaP prostate cancer cell line. Experimental results with the HaCaT normal skin cell lines show that bioconjugated nanocage‐based assemblies demonstrate SHG imaging that is highly selective and will be able to distinguish targeted cancer cell lines from other nontargeted cell types. After optimization, this reported SHG imaging assay could have considerable application for biology.     

Filming a live cell by scanning electrochemical microscopy: label-free imaging of the dynamic morphology in real time

ABSTRACT: The morphology of a live cell reflects the organization of the cytoskeleton and the healthy status of the cell. We established a label-free platform for monitoring the changing morphology of live cells in real time based on scanning electrochemical microscopy (SECM). The dynamic morphology of a live human bladder cancer cell (T24) was revealed by time-lapse SECM with dissolved oxygen in the medium solution as the redox mediator. Detailed local movements of cell membrane were presented by time-lapse cross section lines extracted from time-lapse SECM. Vivid dynamic morphology is presented by a movie made of time-lapse SECM images. The morphological change of the T24 cell by non-physiological temperature is in consistence with the morphological feature of early apoptosis. To obtain dynamic cellular morphology with other methods is difficult. The non-invasive nature of SECM combined with high resolution realized filming the movements of live cells.     

AIEgen based light-up probes for live cell imaging

Fluorescent light-up probes comprising a tetraphenylethene unit with aggregation-induced emission (AIE) characteristics and a water-soluble peptide have been designed and synthesized which provide cell membrane and nuclear permeability to live cells. This strategy has offered new opportunities for the development of probes with light-up ability and good signal-to-noise ratio. The selectivity or targeting specificity is determined by the peptide sequence, i.e. a nuclear localization signal that leads to nucleus imaging and a cell biomarker targeting peptide that offers specific light-up imaging of HT-29 cells.