Nicholas R. Conley

A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells

We have reengineered a red-emitting dicyanomethylenedihydrofuran push-pull fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-molecule level in living cells. This proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.

A Selenium Analogue of Firefly D-Luciferin with Red-Shifted Bioluminescence Emission†

A selenium analogue of amino-D-luciferin, aminoseleno-D-luciferin, is synthesized and shown to be a competent substrate for the firefly luciferase enzyme. It has a red-shifted bioluminescence emission maximum at 600 nm and is suitable for bioluminescence imaging studies in living subjects.

Cy3-Cy5 Covalent Heterodimers for Single-Molecule Photoswitching

Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.

Azido Push-Pull Fluorogens Photoactivate to Produce Bright Fluorescent Labels

Dark azido push-pull chromophores have the ability to be photoactivated to produce bright fluorescent labels suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here, we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push-pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push-pull fluorogens provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that photoactivated push-pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling).

Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution

In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.

Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma

Inhibitors of the kinase CK2 may halt the growth of an aggressive form of medulloblastoma. A targeted, resilient treatment for medulloblastoma Medulloblastoma is an aggressive type of brain tumor that most often arises in children and lacks targeted therapeutic options. The subtypes driven by activity in the sonic hedgehog (SHH) pathway are particularly resistant to current drugs, such as those known as SMO inhibitors, which target this pathway. Purzner et al. used phosphoproteomics to track the development of mouse cells that give rise to medulloblastoma and identified the kinase CK2 as a likely target. CK2 inhibitors blocked the growth of SMO inhibitor–resistant, SHH-type human and mouse medulloblastoma cells and markedly extended the survival of tumor-bearing mice, in which the drug was well tolerated. One of the compounds also blocked the growth of tumors that had mutant CK2, suggesting that it is less susceptible to a common mode of drug resistance. A clinical trial is under way to test this inhibitor in pediatric patients. A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.

Photoactivatable Push-Pull Fluorophores for Single-Molecule Imaging in and out of Cells

We have designed a series of photoactivatable push-pull organic fluorophores, single molecules of which can be imaged in living cells. Photoactivatable probes are needed for superresolution imaging schemes that require active control of single-molecule emission.