Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to ...stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.
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. Ann Neurol 2014;75:303–308
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 β 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.
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.
The scanning model of RNA translation proposes that highly stable secondary structures within mRNAs can inhibit translation, while structures of lower thermal stability also affect translation if ...close enough to the 5' methyl G cap. However, only fragmentary information is available about the dependence of translation efficiency in live mammalian cells on the thermodynamic stability, location, and GC content of RNA structures in the 5'-untranslated region. We devised a two-color fluorescence assay for translation efficiency in single live cells and compared a wide range of hairpins with predicted thermal stabilities ranging from -10 to -50 kcal/mol and 5' G cap-to-hairpin distances of 1-46 bases. Translation efficiency decreased abruptly as hairpin stabilities increased from deltaG = -25 to -35 kcal/mol. Shifting a hairpin as little as nine bases relative to the 5' cap could modulate translation more than 50-fold. Increasing GC content diminished translation efficiency when predicted thermal stability and cap-to-hairpin distances were held constant. We additionally found naturally occurring 5'-untranslated regions affected translation differently in live cells compared with translation in in vitro lysates. Our study will assist scientists in designing experiments that deliberately modulate mammalian translation with designed 5' UTRs.
Engineering a memory with LTD and LTP Nabavi, Sadegh; Fox, Rocky; Proulx, Christophe D ...
Nature (London),
07/2014, Letnik:
511, Številka:
7509
Journal Article
Recenzirano
Odprti dostop
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 A(2A) 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 alpha(2A)-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.
Imaging reporter gene expression in living subjects with various imaging modalities is a rapidly accelerating area of research. Applications of these technologies to cancer research, gene therapy, ...and transgenic models are rapidly expanding. We report construction and testing of several triple fusion reporter genes compatible with bioluminescence, fluorescence and positron emission tomography (PET) imaging. A triple fusion reporter vector harboring a bioluminescence synthetic Renilla luciferase (hrl) reporter gene, a reporter gene encoding the monomeric red fluorescence protein (mrfp1), and a mutant herpes simplex virus type 1 sr39 thymidine kinase HSV1-truncated sr39tk (ttk); a PET reporter gene was found to preserve the most activity for each protein component and was therefore investigated in detail. After validating the activities of all three proteins encoded by the fusion gene in cell culture, we imaged living mice bearing 293T cells transiently expressing the hrl-mrfp-ttk vector by microPET and using a highly sensitive cooled charge-coupled device camera compatible with both bioluminescence and fluorescence imaging. A lentiviral vector carrying the triple fusion reporter gene was constructed and used to isolate stable expressers by fluorescence-activated cell sorting. These stable 293T cells were further used to show good correlation (R(2) approximately 0.74-0.85) of signal from each component by imaging tumor xenografts in living mice with all three modalities. Furthermore, metastases of a human melanoma cell line (A375M) stably expressing the triple fusion were imaged by microPET and optical technologies over a 40-50-day time period in living mice. Imaging of reporter gene expression from single cells to living animals with the help of a single tri-fusion reporter gene will have the potential to accelerate translational cancer research.
DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential ...expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.
The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation of protein kinase and phosphatase activities, yet current relevant ...imaging methods such as phosphorylation-specific antibodies or fluorescent peptide substrates require fixation or microinjection and lack temporal or spatial resolution. We present a genetically encoded fluorescent reporter for protein kinase A (PKA) consisting of fusions of cyan fluorescent protein, a phosphoamino acid binding domain (14-3-3τ), a consensus substrate for PKA, and yellow fluorescent protein, cAMP elevations cause 25-50% changes in the ratios of yellow to cyan emissions in live cells caused by phosphorylation-induced changes in fluorescence resonance energy transfer. The reporter response was accelerated by tethering to PKA holoenzyme and slowed by localization to the nucleus. We demonstrate that deliberate redistribution of a substrate or colocalizing a substrate and PKA can modulate its susceptibility to phosphorylation by the kinase. The successful design of a fluorescent reporter of PKA activity and its application for studying compartmentalized and dynamic modulation of kinases lays a foundation for studying targeting and compartmentation of PKA and other kinases and phosphatases.
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