A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors.

Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins.

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

Engineering a memory with LTD and LTP.

It has been proposed that memories are encoded by modification of synaptic strengths through cellular mechanisms such as long-term potentiation (LTP) and long-term depression (LTD). However, the causal link between these synaptic processes and memory has been difficult to demonstrate. Here we show that fear conditioning, a type of associative memory, can be inactivated and reactivated by LTD and LTP, respectively. We began by conditioning an animal to associate a foot shock with optogenetic stimulation of auditory inputs targeting the amygdala, a brain region known to be essential for fear conditioning. Subsequent optogenetic delivery of LTD conditioning to the auditory input inactivates memory of the shock. Then subsequent optogenetic delivery of LTP conditioning to the auditory input reactivates memory of the shock. Thus, we have engineered inactivation and reactivation of a memory using LTD and LTP, supporting a causal link between these synaptic processes and memory.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization.

Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41's binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue-NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10-7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve "ghosts" months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.

Click-EM for imaging metabolically tagged nonprotein biomolecules.

EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.

Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship.

Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.

Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging.

Fluorine-19 magnetic resonance imaging ((19)F MRI) probes enable quantitative in vivo detection of cell therapies and inflammatory cells. Here, we describe the formulation of perfluorocarbon-based nanoemulsions with improved sensitivity for cellular MRI. Reduction of the (19)F spin-lattice relaxation time (T1) enables rapid imaging and an improved signal-to-noise ratio, thereby improving cell detection sensitivity. We synthesized metal-binding ß-diketones conjugated to linear perfluoropolyether (PFPE), formulated these fluorinated ligands as aqueous nanoemulsions, and then metallated them with various transition and lanthanide ions in the fluorous phase. Iron(III) tris-ß-diketonate ('FETRIS') nanoemulsions with PFPE have low cytotoxicity (<20%) and superior MRI properties. Moreover, the (19)F T1 can readily be reduced by an order of magnitude and tuned by stoichiometric modulation of the iron concentration. The resulting (19)F MRI detection sensitivity is enhanced by three- to fivefold over previously used tracers at 11.7 T, and is predicted to increase by at least eightfold at the clinical field strength of 3 T.

Tunable and reversible drug control of protein production via a self-excising degron.

An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe small molecule-assisted shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, resulting in the production of an untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized copies of the protein. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto an RNA virus for which no licensed inhibitors exist. As SMASh does not require the permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh involves only a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.

PKMζ, but not PKCλ, is rapidly synthesized and degraded at the neuronal synapse.

Synthesizing, localizing, and stabilizing new protein copies at synapses are crucial factors in maintaining the synaptic changes required for storing long-term memories. PKMζ recently emerged as a molecule putatively responsible for maintaining encoded memories over time because its presence correlates with late LTP and because its inhibition disrupts LTP in vitro and long-term memory storage in vivo. Here we investigated PKMζ stability in rat neurons to better understand its role during information encoding and storage. We used TimeSTAMP reporters to track the synthesis and degradation of PKMζ as well as a related atypical PKC, PKCλ. These reporters revealed that both PKMζ and PKCλ were upregulated after chemical LTP induction; however, these new PKMζ copies exhibited more rapid turnover than basally produced PKMζ, particularly in dendritic spines. In contrast to PKMζ, new PKCλ copies exhibited elevated stability. Stable information storage over long periods of time is more challenging the shorter the metabolic lifetime of the candidate molecules.

New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation from the Solid Phase.

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.

Molecular targeting of papillary thyroid carcinoma with fluorescently labeled ratiometric activatable cell penetrating peptides in a transgenic murine model.

BACKGROUND AND OBJECTIVES: Molecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC. METHODS: Thirteen BRAFV600E mice with PTC were studied-seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy. RESULTS: The average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/- 0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals. CONCLUSIONS: RACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone.

Very long-term memories may be stored in the pattern of holes in the perineuronal net.

A hypothesis and the experiments to test it propose that very long-term memories, such as fear conditioning, are stored as the pattern of holes in the perineuronal net (PNN), a specialized ECM that envelops mature neurons and restricts synapse formation. The 3D intertwining of PNN and synapses would be imaged by serial-section EM. Lifetimes of PNN vs. intrasynaptic components would be compared with pulse-chase (15)N labeling in mice and (14)C content in human cadaver brains. Genetically encoded indicators and antineoepitope antibodies should improve spatial and temporal resolution of the in vivo activity of proteases that locally erode PNN. Further techniques suggested include genetic KOs, better pharmacological inhibitors, and a genetically encoded snapshot reporter, which will capture the pattern of activity throughout a large ensemble of neurons at a time precisely defined by the triggering illumination, drive expression of effector genes to mark those cells, and allow selective excitation, inhibition, or ablation to test their functional importance. The snapshot reporter should enable more precise inhibition or potentiation of PNN erosion to compare with behavioral consequences. Finally, biosynthesis of PNN components and proteases would be imaged.

Gibberellins accumulate in the elongating endodermal cells of Arabidopsis root.

Plant hormones are small-molecule signaling compounds that are collectively involved in all aspects of plant growth and development. Unlike animals, plants actively regulate the spatial distribution of several of their hormones. For example, auxin transport results in the formation of auxin maxima that have a key role in developmental patterning. However, the spatial distribution of the other plant hormones, including gibberellic acid (GA), is largely unknown. To address this, we generated two bioactive fluorescent GA compounds and studied their distribution in Arabidopsis thaliana roots. The labeled GAs specifically accumulated in the endodermal cells of the root elongation zone. Pharmacological studies, along with examination of mutants affected in endodermal specification, indicate that GA accumulation is an active and highly regulated process. Our results strongly suggest the presence of an active GA transport mechanism that would represent an additional level of GA regulation.

In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation.

Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high- and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensory-deprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development.

Improved PeT molecules for optically sensing voltage in neurons.

VoltageFluor (VF) dyes have the potential to measure voltage optically in excitable membranes with a combination of high spatial and temporal resolution essential to better characterize the voltage dynamics of large groups of excitable cells. VF dyes sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. We show that tuning the driving force for PeT (ΔGPeT + w) through systematic chemical substitution modulates voltage sensitivity, estimate (ΔGPeT + w) values from experimentally measured redox potentials, and validate the voltage sensitivities in patch-clamped HEK cells for 10 new VF dyes. VF2.1(OMe).H, with a 48% ΔF/F per 100 mV, shows approximately 2-fold improvement over previous dyes in HEK cells, dissociated rat cortical neurons, and medicinal leech ganglia. Additionally, VF2.1(OMe).H faithfully reports pharmacological effects and circuit activity in mouse olfactory bulb slices, thus opening a wide range of previously inaccessible applications for voltage-sensitive dyes.

Improving FRET dynamic range with bright green and red fluorescent proteins.

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

Ratiometric activatable cell-penetrating peptides label pancreatic cancer, enabling fluorescence-guided surgery, which reduces metastases and recurrence in orthotopic mouse models.

BACKGROUND: The aim of this study was to evaluate the efficacy of using matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)-cleavable ratiometric activatable cell-penetrating peptides (RACPPs) conjugated to Cy5 and Cy7 fluorophores to accurately label pancreatic cancer for fluorescence-guided surgery (FGS) in an orthotopic mouse model. METHODS: Orthotopic mouse models were established using MiaPaCa-2-GFP human pancreatic cancer cells. Two weeks after implantation, tumor-bearing mice were randomized to conventional white light reflectance (WLR) surgery or FGS. FGS was performed at far-red and infrared wavelengths with a customized fluorescence-dissecting microscope 2 h after injection of MMP-2 and MMP-9-cleavable RACPPs. Green fluorescence imaging of the GFP-labeled cancer cells was used to assess the effectiveness of surgical resection and monitor recurrence. At 8 weeks, mice were sacrificed to evaluate tumor burden and metastases. RESULTS: Mice in the WLR group had larger primary tumors than mice in the FGS group at termination [1.72 g ± standard error (SE) 0.58 vs. 0.25 g ± SE 0.14; respectively, p = 0.026). Mean disease-free survival was significantly lengthened from 5.33 weeks in the WLR group to 7.38 weeks in the FGS group (p = 0.02). Recurrence rates were lower in the FGS group than in the WLR group (38 vs. 73 %; p = 0.049). This translated into lower local and distant recurrence rates for FGS compared to WLR (31 vs. 67 for local recurrence, respectively, and 25 vs. 60 % for distant recurrence, respectively). Metastatic tumor burden was significantly greater in the WLR group than in the FGS group (96.92 mm(2) ± SE 52.03 vs. 2.20 mm(2) ± SE 1.43; respectively, χ (2) = 5.455; p = 0.02). CONCLUSIONS: RACPPs can accurately and effectively label pancreatic cancer for effective FGS, resulting in better postresection outcomes than for WLR surgery.

Early detection of thrombin activity in neuroinflammatory disease.

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes.

Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.