A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.

Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells.

Recently, a robust mechanical method has been established to isolate a small subpopulation of highly tumorigenic tumor repopulating cells (TRCs) from parental melanoma cells. In order to characterize the molecular and mechanical properties of TRCs, we utilized the tension gauge tether (TGT) single-molecule platform and investigated force requirements during early cell spreading events. TRCs required the peak single molecular tension of around 40 pN through integrins for initial adhesion like the parental control cells, but unlike the control cells, they did not spread and formed very few mature focal adhesions (FAs). Single molecule resolution RNA quantification of three Rho GTPases showed that downregulation of Cdc42, but not Rac1, is responsible for the unusual biophysical features of TRCs and that a threshold level of Cdc42 transcripts per unit cell area is required to initiate cell spreading. Cdc42 overexpression rescued TRC spreading through FA formation and restored the sensitivity to tension cues such that TRCs, like parental control cells, increase cell spreading with increasing single-molecular tension cues. Our single molecule studies identified an unusual biophysical feature of suppressed spreading of TRCs that may enable us to distinguish TRC population from a pool of heterogeneous tumor cell population.

Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo.

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.

Understanding the photophysics of the spinach-DFHBI RNA aptamer-fluorogen complex to improve live-cell RNA imaging.

The use of aptamer-fluorogen complexes is an emerging strategy for RNA imaging. Despite its promise for cellular imaging and sensing, the low fluorescence intensity of the Spinach-DFHBI RNA aptamer-fluorogen complex hampers its utility in quantitative live-cell and high-resolution imaging applications. Here we report that illumination of the Spinach-fluorogen complex induces photoconversion and subsequently fluorogen dissociation, leading to fast fluorescence decay and fluorogen-concentration-dependent recovery. The fluorescence lifetime of Spinach-DFHBI is 4.0 ± 0.1 ns irrespective of the extent of photoconversion. We detail a low-repetition-rate illumination scheme that enables us to maximize the potential of the Spinach-DFHBI RNA imaging tag in living cells.

Single-molecule analysis reveals three phases of DNA degradation by an exonuclease.

λ exonuclease degrades one strand of duplex DNA in the 5'-to-3' direction to generate a 3' overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer (FRET) to reveal three phases of λ exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3' overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick-paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5' end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.

Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins.

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.

Identifying modulators of protein-protein interactions using photonic crystal biosensors.

Inhibitors and activators of protein-protein interactions are valuable as biological probes and medicinal agents but are often difficult to identify. Herein we describe a high-throughput assay, based upon photonic crystal (PC) biosensors, for the identification of modulators of protein-protein interactions. Through the use of a d-biotin-tris-NTA (BTN) hybrid compound, any His6-tagged protein can be immobilized on the surface of a PC biosensor. Binding of the bound protein to its cognate partner is detected via a shift in the peak wavelength value. We demonstrate this assay with three protein-protein pairs (caspase-9-XIAP, caspase-7-XIAP, FKBP12-FRB) and their small molecule modulators.

Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

Single molecular force across single integrins dictates cell spreading.

Cells' ability to sense and interpret mechanical signals from the extracellular milieu modulates the degree of cell spreading. Yet how cells detect such signals and activate downstream signaling at the molecular level remain elusive. Herein, we utilize tension gauge tether (TGT) platform to investigate the underlying molecular mechanism of cell spreading. Our data from both differentiated cells of cancerous and non-cancerous origin show that for the same stiff underlying glass substrates and for same ligand density it is the molecular forces across single integrins that ultimately determine cell spreading responses. Furthermore, by decoupling molecular stiffness and molecular tension we demonstrate that molecular stiffness has little influence on cell spreading. Our data provide strong evidence that links molecular forces at the cell-substrate interface to the degree of cell spreading.

Crosstalk between the cGAS DNA sensor and Beclin-1 autophagy protein shapes innate antimicrobial immune responses.

Robust immune responses are essential for eliminating pathogens but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) produces the second messenger cGAMP to initiate the stimulator of interferon genes (STING) pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double-stranded DNA (dsDNA) stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNA to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNA. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced antimicrobial immune responses.

Phenylcinnamides as novel antimitotic agents.

Compound 8H is a phenylcinnamide that induces G2/M-phase cell cycle arrest and cell death in cancer cell lines. Here we show that 8H exerts its cytotoxic activity through disruption of microtubule dynamics in vitro and in cell culture. A series of cinnamide derivatives were synthesized and evaluated, and several new compounds were identified that improve on the activity of the parent compound, with IC(50) values for induction of cell death ranging from 1 to 10 microM. Notably, these compounds retain potency in the HL-60/VCR leukemia cell line, which is resistant to antimitotic cancer drugs vincrisitine and paclitaxel through up-regulation of P-glycoprotein drug efflux pumps. As P-glycoprotein expression is often responsible for drug resistance in cancer and the exclusion of compounds from the central nervous system, 8H and its derivatives merit further examination as potential antimitotic therapeutics, specifically for brain cancers and cancers that are resistant to standard antimitotic agents.

Identification of the cellular targets of bioactive small organic molecules using affinity reagents.

The elucidation of molecular targets of bioactive small organic molecules remains a significant challenge in modern biomedical research and drug discovery. This tutorial review summarizes strategies for the derivatization of bioactive small molecules and their use as affinity probes to identify cellular binding partners. Special emphasis is placed on logistical concerns as well as common problems encountered during such target identification experiments. The roadmap provided is a guide through the process of affinity probe selection, target identification, and downstream target validation.

A novel synthetic analogue of a constituent of Isodon excisus inhibits transcription of CYP1A1, -1A2 and -1B1 by preventing activation of the aryl hydrocarbon receptor.

We investigated the effect of a novel synthetic analogue of a constituent from the Chinese medicinal herb Isodon excisus, 3-(3-methoxy-phenyl)-N-(3, 4, 5-trimethoxy-phenyl)-acrylamide (compound 343), on the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in human hepatoma HepG2 cells. We found that compound 343 inhibited the upregulation of cytochrome P-450 (CYP) enzyme activity in cells treated with the AhR ligands and potent carcinogens, dimethylbenz[a]anthracene (DMBA) or 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Compound 343 also inhibited the DMBA- or TCDD-induced increase in CYP1A1, -1A2 and -1B1 mRNA levels. Carcinogen-induced transcription of CYP genes was also suppressed by compound 343, as measured by a reporter gene controlled by the xenobiotic-responsive element (XRE). This was confirmed by measuring the amount of carcinogen-induced CYP1A1 heterogeneous nuclear RNA. Compound 343 blocked the DMBA- or TCDD-induced activation of the AhR DNA-binding capacity for the XRE, as measured by a chromatin immunoprecipitation assay. Compound 343 also inhibited CYP enzyme activity in microsomes isolated from DMBA- or TCDD-treated cells, as well as the activity of recombinant CYP1A1, -1A2 and -1B1, indicating that compound 343 directly inhibits CYP enzymes. These results indicate that compound 343 is both a potent inhibitor of carcinogen-induced CYP enzyme expression, as well as a direct inhibitor of CYP enzymes.

Synthesis and identification of small molecules that potently induce apoptosis in melanoma cells through G1 cell cycle arrest.

Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs) that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are quite potent inducers of apoptotic death in melanoma cell lines (IC(50) approximately 0.5 muM), and importantly, some TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors. Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor kappa-B (NFkappaB) in the cell; NFkappaB is known to be constitutively active in melanoma, and this activity is critical for the proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFkappaB and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and may be effective antimelanoma agents.