Stephen R. Adams

Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors.

Regulation of AMPA receptor (AMPAR) trafficking is important for neural plasticity. Here we examined the trafficking and synthesis of the GluR1 and GluR2 subunits using ReAsH-EDT2 and FlAsH-EDT2 staining. Activity blockade of rat cultured neurons increased dendritic GluR1, but not GluR2, levels. Examination of transected dendrites revealed that both AMPAR subunits were synthesized in dendrites and that activity blockade enhanced dendritic synthesis of GluR1 but not GluR2. In contrast, acute pharmacological manipulations increased dendritic synthesis of both subunits. AMPARs synthesized in dendrites were inserted into synaptic plasma membranes and, after activity blockade, the electrophysiological properties of native synaptic AMPARs changed in the manner predicted by the imaging experiments. In addition to providing a novel mechanism for synaptic modifications, these results point out the advantages of using FlAsH-EDT2 and ReAsH-EDT2 for studying the trafficking of newly synthesized proteins in local cellular compartments such as dendrites.

A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells.

Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein–coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A2A receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66–88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse α2A-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous.

Inhibition of NF-κB Activation by Arsenite through Reaction with a Critical Cysteine in the Activation Loop of IκB Kinase

Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism by which arsenite affects the NF-κB signaling pathway. Activation of transcription factor NF-κB depends on the integrity of the IκB kinase (IKK) complex. We found that arsenite potently inhibits NF-κB and IKK activation by binding to Cys-179 in the activation loop of the IKK catalytic subunits, IKKα/β. The affinity of IKKβ for trivalent arsenic was verifiedin vitro by the ability of IKKβ to enhance the fluorescence of an arsenic-substituted fluorescein dye. The addition of 1,2-dithiol antidotes or replacement of Cys-179 with an alanine residue abolished dye binding to and arsenite inhibition of IKKβ. Overexpression of IKKβ (C179A) protects NF-κB from inhibition by arsenite, indicating that despite the involvement of a large number of distinct gene products in this activation pathway, the critical target for inhibition by arsenite is on the IKK catalytic subunits.

Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity

Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to β-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.

Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator

To investigate the dynamics of guanosine 3′,5′-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1–77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of ≈2 μM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr516 of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5–4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.

Fluorescent labeling of recombinant proteins in living cells with FlAsH.

Publisher Summary FlAsH labeling of recombinant proteins for cellular localization studies can be considered an alternative to the popular method using green fluorescent protein (GFP) fusions, with the FlAsH method having some advantages. The size of the fluorescent tag is considerably smaller: bound FlAsH has a molecular weight of less than 600 and the addition of a FlAsH target site can be as small as four introduced cysteines with an appended peptide adding less than 2 kDa. This compares with a molecular weight of 30,000 for GFP, which is more likely to perturb the native structure and function of the tagged protein. Both FlAsH and GFP tagging generate a fluorescent protein with similar brightness. However, multiple FlAsH sites can be introduced into a protein so that considerably brighter labeling aid in detecting low-abundance proteins. A major advantage of the FlAsH system is the comparative ease of chemical modification of FlAsH. Coupled with the synthetic versatility of organic chemistry, this enables the incorporation of functionalities other than fluorescence into targeted proteins or peptides.

Cytochrome c is released in a single step during apoptosis

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7°C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (ΔΨm). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in ΔΨm preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in ΔΨm.

Genetically targeted chromophore-assisted light inactivation

Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and α1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by ∼90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or α1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.

Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy

Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after cytokinesis. The precise location of Golgi membranes and resident proteins during mitosis remains unclear, partly due to limitations of molecular markers and the resolution of light microscopy. We generated a fusion consisting of the first 117 residues of α-mannosidase II tagged with a fluorescent protein and a tetracysteine motif. The mannosidase component guarantees docking into the Golgi membrane, with the tags exposed in the lumen. The fluorescent protein is optically visible without further treatment, whereas the tetracysteine tag can be reduced acutely with a membrane-permeant phosphine, labeled with ReAsH, monitored in the light microscope, and used to trigger the photoconversion of diaminobenzidine, allowing 4D optical recording on live cells and correlated ultrastructural analysis by electron microscopy. These methods reveal that Golgi reassembly is preceded by the formation of four colinear clusters at telophase, two per daughter cell. Within each daughter, the smaller cluster near the midbody gradually migrates to rejoin the major cluster on the far side of the nucleus and asymmetrically reconstitutes a single Golgi apparatus, first in one daughter cell and then in the other. Our studies provide previously undescribed insights into Golgi disassociation and reassembly during mitosis and offer a powerful approach to follow recombinant protein distribution in 4D imaging and correlated high-resolution analysis.

Fluorescent labeling of tetracysteine-tagged proteins in intact cells

In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed.