Chandra L. Tucker

Rapid blue-light–mediated induction of protein interactions in living cells

Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase–mediated DNA recombination using light.

Towards an understanding of complex protein networks

Large-scale two-hybrid screens have generated a wealth of information describing potential protein--protein interactions. When compiled with data from systematic localizations of proteins, mutant screens and other functional tests, a network of interactions among proteins and between proteins and other components of eukaryotic cells can be deduced. These networks can be viewed as maps of the cell, depicting potential signaling pathways and interactive complexes. Most importantly, they provide potential clues to the function of previously uncharacterized proteins. Focusing on recent experiments, we explore these protein-interaction studies and the maps derived from such efforts.

An optimized optogenetic clustering tool for probing protein interaction and function

The Arabidopsis photoreceptor cryptochrome 2 (CRY2) was previously used as an optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we report the development of a new CRY2-derived optogenetic module, ‘CRY2olig’, which induces rapid, robust, and reversible protein oligomerization in response to light. Using this module, we developed a novel protein interaction assay, LINC (Light Induced Co-clustering) that can be used to interrogate protein interaction dynamics in live cells. In addition to use probing protein interactions, CRY2olig can also be used to induce and reversibly control diverse cellular processes with spatial and temporal resolution. Here, we demonstrate disrupting clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with light. These new CRY2-based approaches expand the growing arsenal of optogenetic strategies to probe cellular function.

A yeast sensor of ligand binding

We describe a biosensor that reports the binding of small-molecule ligands to proteins as changes in growth of temperature-sensitive yeast. The yeast strains lack dihydrofolate reductase (DHFR) and are complemented by mouse DHFR containing a ligand-binding domain inserted in a flexible loop. Yeast strains expressing two ligand-binding domain fusions, FKBP12-DHFR and estrogen receptor-α (ERα)-DHFR, show increased growth in the presence of their corresponding ligands. We used this sensor to identify mutations in residues of ERα important for ligand binding, as well as mutations generally affecting protein activity or expression. We also tested the sensor against a chemical array to identify ligands that bind to FKBP12 or ERα. The ERα sensor was able to discriminate among estrogen analogs, showing different degrees of growth for the analogs that correlated with their relative binding affinities (RBAs). This growth assay provides a simple and inexpensive method to select novel ligands and ligand-binding domains.

Quantitative proteomics approach for identifying protein–drug interactions in complex mixtures using protein stability measurements

Knowledge about the protein targets of therapeutic agents is critical for understanding drug mode of action. Described here is a mass spectrometry-based proteomics method for identifying the protein target(s) of drug molecules that is potentially applicable to any drug compound. The method, which involves making thermodynamic measurements of protein-folding reactions in complex biological mixtures to detect protein–drug interactions, is demonstrated in an experiment to identify yeast protein targets of the immunosuppressive drug, cyclosporin A (CsA). Two of the ten protein targets identified in this proof of principle work were cyclophilin A and UDP-glucose-4-epimerase, both of which are known to interact with CsA, the former through a direct binding event (Kd ∼ 70 nM) and the latter through an indirect binding event. These two previously known protein targets validate the methodology and its ability to detect both the on- and off-target effects of protein–drug interactions. The other eight protein targets discovered here, which include several proteins involved in glucose metabolism, create a new framework in which to investigate the molecular basis of CsA side effects in humans.

Optogenetic control of cell function using engineered photoreceptors

Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light‐actuated tools allow manipulation of molecular events with ultra‐fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub‐micrometre precision within cells. While light‐actuated chemicals such as photolabile ‘caged’ compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever‐growing optogenetic toolkit.

Quantitative genome-wide analysis of yeast deletion strain sensitivities to oxidative and chemical stress

Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploid Saccharomyces cerevisiae deletion strains (Winzeler et al., 1999). Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. WE carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multichemicalsensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen and 55 specific to mefloquine.We correlate these results with data from other large-scale studies to yield novel insights into cellular function.

Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase

Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use controling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2/CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2/CIB system, to identify versions of CRY2/CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates a α13-α14 turn motif within plant CRYs where perturbations alter signaling state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range allowing robust recombination following exposure to a single, brief pulse of light.

Light-mediated control of DNA transcription in yeast.

A variety of methods exist for inducible control of DNA transcription in yeast. These include the use of native yeast promoters or regulatory elements that are responsive to small molecules such as galactose, methionine, and copper, or engineered systems that allow regulation by orthogonal small molecules such as estrogen. While chemically regulated systems are easy to use and can yield high levels of protein expression, they often provide imprecise control over protein levels. Moreover, chemically regulated systems can affect many other proteins and pathways in yeast, activating signaling pathways or physiological responses. Here, we describe several methods for light mediated control of DNA transcription in vivo in yeast. We describe methodology for using a red light and phytochrome dependent system to induce transcription of genes under GAL1 promoter control, as well as blue light/cryptochrome dependent systems to control transcription of genes under GAL1 promoter or LexA operator control. Light is dose dependent, inexpensive to apply, easily delivered, and does not interfere with cellular pathways, and thus has significant advantages over chemical systems.

In vivo and in vitro examination of stability of primary hyperoxaluria-associated human alanine:glyoxylate aminotransferase.

Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5′-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5′-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.